AU718378B2 - LYST1 and LYST2 gene compositions and methods of use - Google Patents

Description

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DESCRIPTION

LYST1 AND LYST2 GENE COMPOSITIONS AND METHODS OF USE 1 1.1 .Field of the Invention Thie present invention relates generally to the field of molecular biology. More particularly, certain embodiments concern methods and compositions comprising novel DNA segments, and proteins derived from mammalian species. More particularly, the invention provides Lystl and Lyst2 gene compositions from murine origins and the homologous LYST1 and LYST2 gene 20 compositions from human origins. Various methods for making and using these LYST/Lyst DNA segments, native peptides and synthetic protein derivatives are disclosed, such as, for example, the use of DNA segments as diagnostic probes and templates for protein production, and the use of LYST1, Lystl, LYST2, and Lyst2 proteins, fusion protein carriers and Lyst-derived peptides in various pharmacological and immunological applications.

25 1.2 Description of the Related Art 1.2.1 Chediak-Higashi (CH) Syndrome Chediak-Higashi syndrome (CHS) is an autosomal recessive, immune deficiency disease that maps on chromosome (Chr) lq42-q43 (Goodrich and Holcombe, 1995; Barrat et al. 1996; Fukai et atl., 1996). Affected individuals have giant, perinuclear lysosomes, defective granulocyte, NK and cytolytic T cell function, and die prematurely of infection or malignancy (Beguez Cesar, 1943; ,RA Blume et al., 1968; Wolff et al, 1972; Blume and Wolff, 1972; Root et al., 1972; Roder et a., 'T 1982; Baetz et al., 1995). CHS patients also exhibit partial oculocutaneous albinism, platelet storage WO 97/28262 PCTfUS97/01748 pool deficiency and neurologic defects such as peripheral neuropathy and ataxia (Windhorst et al., 1968; Meyers e al., 1974; Maeda etal., 1989; Pettit and Berdal, 1984; Misra etal., 1991). Recently it was demonstrated that intracellular protein transport to and from the lysosome is disordered in CHS (Baetz et al., 1995; Brandt et al., 1975; Burkhardt et al., 1993; Zhao et al., 1994). Such functional defects in secretory lysosomes of granular cells (leukocytes, melanocytes, megakaryocytes and cerebellar Purkinje cells) provide a unifying hypothesis that can explain the diverse clinical features ofCHS (Griffiths, 1996).

As an antecedent to identification of the human CHS gene, the inventors undertook positional cloning of the mouse mutation beige which had long been considered homologous to CHS. The clinical and pathologic features of CHS and bg are very similar and bg maps on proximal mouse Chr 13 within a linkage group conserved with human chromosome 1q42-q43 (the position of the CHS locus) (Jenkins etal., 1991). Additional evidence that human CHS and bg mice were homologous disorders came from interspecific genetic complementation studies, which demonstrated that fusion of bg mouse and human CHS fibroblasts failed to reverse lysosomal morphologic abnormalities (Penner and Prieur, 1987).

Recently the inventors' group and one other succeeded in identifying the gene that is defective in bg mice (Perou et al., 1996a). However, the reported bg candidate cDNA sequences (Lyst and BG) were different. Both sequences were isolated from the same yeast artificial chromosome (YAC) clone. This YAC had been authenticated by mapping within the bg critical region and by restoration of normal lysosomal morphology to bg fibroblasts upon transfection (Perou et al., 1996a; Perou et al., 1996b). Furthermore, both of the candidate gene sequences contained mutations in different bg alleles.

1.3 Deficiencies in the Prior Art Methods for the treatment and diagnosis of Chediak-Higashi Syndrome have not been developed because the sequence of the CH gene has not been identified in mice or humans.

Despite some recent studies in mice, there is only speculation that a linkage similar to that found in beige mice might exist in the human gene (Owen, et al., 1986). There is some evidence that indicate that the CH mutation is located in the same gene in mouse, mink and human (Perou and Kaplan, 1993); however, except for the beige mouse, the locus of the mutation has not been identified.

WO 97/28262 PCTIS97/01748 CHS patients have been reported to suffer from several serious medical conditions, including impaired natural killer cell activity (Haliotis et al., 1980) and defective lymphocytemediated antibody dependent cell mediated leukocyte mediated ADCC against tumor cell targets (Klein, et al., 1980). Despite the recognition of these deficiencies, little progress in treatment has been achieved, mainly because the gene harboring the mutation leading to these impairments has not yet been identified.

Chediak-Higashi Syndrome occurs only in a small minority of the population. However, there is a growing realization of the potential role of the CH gene product (LYST1) in developing treatments for conditions such as systemic autoimmune disease and possibly certain types of malignancy related to the regulation of protein trafficking within cells by the CH gene (LYST1).

Therefore, what is lacking in the prior art is the isolation and characterization of the CH gene from mice and humans, useful in the development of treatments and assays for autoimmune diseases such as CHS and certain forms of cancer.

2. Summary of the Invention Positional cloning of the mouse CHS homologous is facilitated by the existence of numerous remutations at the bg locus. All have arisen spontaneously, with the exception of the SB/LeJ-bg allele, which was induced by radiation. The present invention addresses one or more of the foregoing or other problems associated with the detection of Chediak-Higashi Syndrome in humans. Both the mouse gene and the homologous human have been cloned and sequenced.

The isolation and sequencing of the Chediak-Higashi gene (LYST1) from both murine and human sources has now provided methods of detecting CHS at the gene level, such as by various assays making use of the gene, gene segments and/or the encoded proteins or polypeptides. In addition to the practical value, the gene provides a tool for understanding and controlling mechanisms of regulation of protein trafficking to lysosomes, and particularly to the contribution of vesicular sorting to diverse cellular functions. An immediate result of the identification of the LYST1 gene is the ability to perform linkage analysis and to identify individuals at risk to have progeny carrying the mutated gene. The inventors have shown that the murine gene, Lystl, and BG sequences are derived from a single gene with alternatively spliced mRNAs. In an important embodiment, the inventors have also identified the human homolog of the bg gene (Lyst LYST1.

3 0 LYST1 maps within the CHS critical region and is mutated in several CHS patients.

WO 97/28262 PCT/S97/01748 2.1 LYST and Lyst Gene Compositions As used herein, the term "DNA segment" refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding LYST/Lyst refers to a DNA segment that contains LYST or Lyst coding sequences yet is isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Included within the term "DNA segment", are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like. Preferred LYSTgenes are the LYST1 and LYST2 genes from human origin, while preferred Lyst genes are the Lysti and Lyst2 genes from murine origin.

Similarly, a DNA segment comprising an isolated or purified LYST/Lyst gene refers to a DNA segment including a LYST or Lyst coding sequence and, in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring genes or protein encoding sequences. In this respect, the term "gene" is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides. Such segments may be naturally isolated, or modified synthetically by the hand of man. Preferred DNAs are those which comprise one or more LYST genes, with human LYST1 and LYST2 genes being particularly preferred, or one or more Lyst genes, with murine Lystl and Lyst2 genes being particularly preferred.

"Isolated substantially away from other coding sequences" means that the gene of interest, in this case, a gene encoding a LYST/Lyst protein or peptide, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.

In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that encode a LYST/Lyst species that includes within its amino acid sequence an amino acid sequence essentially as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID WO 97/28262 PCTIUS97/01748 NO: 14. In other particular embodiments,.the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that include within their sequence a nucleotide sequence essentially as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:13.

The term "a sequence essentially as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14" means that the sequence substantially corresponds to a portion of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO: 12, or SEQ ID NO: 14, and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO: 14. The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein (for example, see Illustrative Embodiments). Accordingly, sequences that have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids that are identical or functionally equivalent to the amino acids SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14 will be sequences that are "essentially as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, or SEQ ID NO: 14".

In certain other embodiments, the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:13. The term "essentially as set forth in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, or SEQ ID NO: 13" is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:13 and has relatively few codons that are not identical, or functionally equivalent, to the codons of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:13. Again, DNA segments that encode proteins exhibiting LYST, Lyst, LYST-like, or Lyst-like activity will be most preferred.

It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet WO 97/28262 PCTIUS97/01748 still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various upstream or downstream regulatory or structural genes.

Naturally, the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO:l, SEQ ID NO:3, SEQ ID SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, or SEQ ID NO: 13. Nucleic acid sequences that are "complementary" are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term "complementary sequences" means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO: 13 under relatively stringent conditions such as those described herein.

The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments may be prepared that include a short contiguous stretch identical to or complementary to SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, or SEQ ID NO: 13, such as about 14 nucleotides, and that are up to about 10,000 or about 5,000 base pairs in length, with segments of about 3,000 being preferred in certain cases. DNA segments with total lengths of about 2,000, about 1,000, about 500, about 200, about 100 and about 50 base pairs in length (including all intermediate lengths) are also contemplated to be useful.

It will be readily understood that "intermediate lengths", in these contexts, means any length between the quoted ranges, such as 14, 15, 16, 17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31, WO 97/28262 PCT/US97/01748 7 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through the 200-500; 501-1,000; 1,001-2,000; 2,001-3,000; 3,001-5,000; 5,001-10,000 ranges, up to and including sequences of about 12,001, 12,002, 12,003, 13,001, 13,002 and the like.

It will also be understood that this invention is not limited to the particular nucleic acid sequences disclosed in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO;11, or SEQ ID NO:13, or to the particular amino acid sequences as disclosed in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14. Recombinant vectors and isolated DNA segments may therefore variously include the LYST or Lyst coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include LYST, Lyst, LYST-like, or Lyst-like coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.

If desired, one may also prepare fusion proteins and peptides, where the LYST or Lyst coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively).

Recombinant vectors form further aspects of the present invention. Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length protein or smaller peptide, is positioned under the control of a promoter. The promoter may be in the form of the promoter that is naturally associated with a LYST1, Lystl, LYST2, or Lyst2 gene, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment, for example, using recombinant cloning and/or PCR" technology, in connection with the compositions disclosed herein.

In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a LYST/Lyst gene in its natural environment. Such promoters may include LYST or Lyst promoters normally associated with other genes, and/or 3 0 promoters isolated from any bacterial, viral, eukaryotic, or mammalian cell. Naturally, it will be WO 97/28262 PCTfUS90148 important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al., 1989. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides.

Prokaryotic expression of nucleic acid segments of the present invention may be performed using methods known to those of skill in the art, and will likely comprise expression vectors and promotor sequences such as those obtained from tac, trp, lac, lacUV5 or T7. When expression of the recombinant LYST1 LYST2, Lysti or Lyst2 proteins is desired in eukaryotic cells, a number of expression systems are available and known to those of skill in the art. An exemplary eukaryotic promoter system contemplated for use in high-level expression is the Pichia expression vector system (Pharmacia LKB Biotechnology).

In connection with expression embodiments to prepare recombinant recombinant

LYSTI

LYST2, Lystl or Lyst2 proteins and peptides, it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire LYST1 LYST2, Lystl or Lyst2 or functional domains, epitopes, ligand binding domains, subunits, etc. being most preferred.

However, it will be appreciated that the use of shorter DNA segments to direct the expression of LYST1 LYST2, Lyst or Lyst2 peptides or epitopic core regions, such as may be used to generate anti-LYST or Lyst antibodies, also falls within the scope of the invention.

DNA

segments that encode peptide antigens from about 15 to about 100 amino acids in length, or more preferably, from about 15 to about 50 amino acids in length are contemplated to be particularly useful.

The LYST or Lyst genes and DNA segments may also be used in connection with somatic expression in an animal or in the creation of a transgenic animal. Again, in such embodiments, the use of a recombinant vector that directs the expression of the full length or active LYST/Lyst protein is particularly contemplated. Expression of a LYST/Lyst transgene in animals is particularly contemplated to be useful in the production of anti-LYST/Lyst antibodies for use in passive immunization methods, the detection of LYST/Lyst proteins, and the purification of LYST/Lyst protein in large quantity.

WO 97/28262 PCTfUS97/01748 q In addition to their use in directing the expression of LYST/Lyst, the nucleic acid sequences disclosed herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that nucleic acid segments that comprise a sequence region that consists of at least a 14 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 14 nucleotide long contiguous sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, or SEQ ID NO: 13 will find particular utility. Longer contiguous identical or complementary sequences, those of about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) and even up to full length sequences will also be of use in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize to LYST/Lyst-encoding sequences will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200 nucleotides or so, identical or complementary to SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, or SEQ ID NO: 13 are particularly contemplated as hybridization probes for use in, Southern and Northern blotting. This would allow LYST/Lyst structural or regulatory genes to be analyzed, both in diverse cell types and also in various bacterial cells. The total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 14 and about 100 nucleotides, but larger contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect.

The use of a hybridization probe of about 14-25 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than 14 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality WO 97/28262 PCT/UJS97/01748 and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to 25 contiguous nucleotides, or even longer where desired.

Hybridization probes may be selected from any portion of any of the sequences disclosed herein. All that is required is to review the sequence set forth in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, or SEQ ID NO: 13 and to select any continuous portion of the sequence, from about 14-25 nucleotides in length up to and including the full length sequence, that one wishes to utilize as a probe or primer. The choice of probe and primer sequences may be governed by various factors, such as, by way of example only, one may wish to employ primers from towards the termini of the total sequence.

The process of selecting and preparing a nucleic acid segment that includes a contiguous sequence from within SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, or SEQ ID NO: 13, may alternatively be described as preparing a nucleic acid fragment. Of course, fragments may also be obtained by other techniques such as, by mechanical shearing or by restriction enzyme digestion. Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCR T M technology of U.S. Patent 4,683,202 (incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.

Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of the entire LYST/Lyst gene or gene fragments. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCI at temperatures of 501C to 70'C. Such selective conditions tolerate little, if any, mismatch between WO 97/28262 PCT/tJS97/01748

/I

the probe and the template or target strand, and would be particularly suitable for isolating LYST or Lyst genes.

Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate LYST or Lyst sequences from related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as about 0.15 M to about 0.9 M salt, at temperatures ranging from 20 0 C to 55 0 C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.

In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acidcontaining samples.

In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization WO 97/28262 PCT/US97/01748 17-.

probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantitated, by means of the label.

2.2 Recombinant Host Cells and Vectors Particular aspects of the invention concern the use ofplasmid vectors for the cloning and expression of recombinant peptides, and particular peptide epitopes comprising either native, or site-specifically mutated LYST or Lyst proteins, peptides, or epitopes. The generation of recombinant vectors, transformation of host cells, and expression of recombinant proteins is wellknown to those of skill in the art. Prokaryotic hosts are preferred for expression of the peptide compositions of the present invention. An example of a preferred prokaryotic host is E. coli, and in particular, E. coli strains JM101, XL1-BlueTM, RR1, LE392, B, X1776 (ATCC31537), and W3110 1, prototrophic, ATCC273325). Alternatively, other Enterobacteriaceae species such as Salmonella typhimurium and Serratia marcescens, or even other Gram-negative hosts including various Pseudomonas species may be used in the recombinant expression of the genetic constructs disclosed herein.

In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli may be typically transformed using vectors such as pBR322, or any of its derivatives (Bolivar et al., 1977). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins.

In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts.

For example, bacteriophage such as ?GEM1I- 1 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.

Those promoters most commonly used in recombinant DNA construction include the Plactamase (penicillinase) and lactose promoter systems (Chang et al., 1978; Itakura et al., 1977; Goeddel et al., 1979) or the tryptophan (trp) promoter system (Goeddel et al., 1980). The use of WO 97/28262 PCTUS97/01748 13 recombinant and native microbial promoters is well-known to those of skill in the art, and details concerning their nucleotide sequences and specific methodologies are in the public domain, enabling a skilled worker to construct particular recombinant vectors and expression systems for the purpose of producing compositions of the present invention.

In addition to the preferred embodiment expression in prokaryotes, eukaryotic microbes, such as yeast cultures may also be used in conjunction with the methods disclosed herein.

Saccharomyces cerevisiae, or common bakers' yeast is the most commonly used among eukaryotic microorganisms, although a number of other species may also be employed for such eukaryotic expression systems. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al., 1980). This plasmid already contains the trpL gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC44076 or PEP4-1 (Jones, 1977). The presence of the trpL lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for 3phosphoglycerate kinase (Hitzeman et al., 1980) or other glycolytic enzymes (Hess et al., 1968; Holland et al., 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.

Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing a yeast-compatible promoter, an origin of replication, and termination sequences is suitable.

In addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts in the routine practice of the disclosed methods. In principle, any such cell WO 97/28262 PCTIUS97/01748 culture is workable, whether from vertebrate or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years. Examples of such useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7, 293 and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expression vectors are often obtained from viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early and late promoters of virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al., 1978). Smaller or larger fragments may also be used, provided there is included the approximately 250 bp sequence extending from the HindIIl site toward the BglI site located in the viral origin of replication.

Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.

The origin of replication may be obtained from either construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral Polyoma, Adeno, VSV, BPV) source, or may be obtained from the host cell chromosomal replication mechanism.

If the vector is integrated into the host cell chromosome, the latter is often sufficient.

It will be further understood that certain of the polypeptides may be present in quantities below the detection limits of the Coomassie brilliant blue staining procedure usually employed in 2 5 the analysis of SDS/PAGE gels, or that their presence may be masked by an inactive polypeptide of similar Although not necessary to the routine practice of the present invention, it is contemplated that other detection techniques may be employed advantageously in the visualization of particular polypeptides of interest. Immunologically-based techniques such as Western blotting using enzymatically-, radiolabel-, or fluorescently-tagged antibodies described herein are considered to be of particular use in this regard. Alternatively, the peptides of the present invention may be detected by using antibodies of the present invention in combination WO 97/28262 PCT/US97/0748 with secondary antibodies having affinity for such primary antibodies. This secondary antibody may be enzymatically- or radiolabeled, or alternatively, fluorescently-, or colloidal gold-tagged.

Means for the labeling and detection of such two-step secondary antibody techniques are wellknown to those of skill in the art.

2.3 Recombinant Expression of one or more LYST Gene Products As used throughout, a "LYST/Lyst" gene is intended to mean a LYST or Lyst gene from a mammalian source, with human LYST and murine Lyst genes being most preferred. In keeping with the genetic nomenclature schemes known to those of skill in the art, "LYST' genes are those genes derived from human sources while "Lyst" genes are those genes derived from murine sources. Thus, LYST1 and LYST2 genes are two genes of the "LYST/Lyst" family which are isolated from humans, while Lyst] and Lyst2 represent two genes of the "LYST/Lyst" family which are their murine homologs, respectively.

In analogous fashion, a "LYST/Lyst" protein is intended to mean a LYST or Lyst protein isolated from a mammalian source, with human and murine peptides being most preferred. In keeping with the genetic nomenclature schemes known to those of skill in the art, "LYST" proteins are those proteins encoded by LYST genes derived from human sources while "Lyst" proteins are those proteins encoded by Lyst genes derived from murine sources. Thus, LYST1 and LYST2 are the proper designations of two proteins of the "LYST/Lyst" protein family which are isolated from humans, while Lysti and Lyst2 represent the two homologous proteins of the LYST/Lyst protein family isolated from murines.

Because there are long and short isoforms of these proteins, the inventors have referred throughout the specification to "Lystl isoform "Lystl isoform II," and so forth to distinguish between the two isoforms. Such isoform designations may also be abbreviated as "Lyst or "Lystl-II," and so forth. Human protein isoforms may be referred to in corresponding manner: "LYST1-I" and "LYSTI-isoform I" describe the long isoform of the human protein, while "LYST -II" and "LYSTI-isoform II" are terms used to described the short isoform of the human proteins. Therefore, Lyst I-I and Lyst 1-II are terms used to represent two isoforms of the murine isoforms of Lyst and LYST1-I and LYSTI-II are terms used to represent two isoforms of the human LYST1. Similarly, Lyst2-I and Lyst2-II would represent two isoforms of the murine Lyst2 protein, while LYST2-I and LYST2-II would represent two isoforms of the human LYST2 protein.

WO 97/28262 PCT/US97/01748 ic The present invention also concerns recombinant host cells for expression of an isolated LYST1, Lystl, LYST2, or Lyst2 gene. It is contemplated that virtually any host cell may be employed for this purpose, but certain advantages may be found in using a bacterial host cell such as E. coli, S. typhimurium, B. subtilis, or others. Expression in eukaryotic cells is also contemplated such as those derived from yeast, insect, or mammalian cell lines. These recombinant host cells may be employed in connection with "overexpressing" the LYST1, Lystl, LYST2, or Lyst2 protein, that is, increasing the level of expression over that found naturally in mammalian cells. As is well known to those of skill in the art, many such vectors and host cells are readily available for the recombinant expression of proteins, one particular detailed example of a suitable vector for expression in mammalian cells is that described in U. S. Patent 5,168,050, incorporated herein by reference. However, there is no requirement that a highly purified vector be used, so long as the coding segment employed encodes a protein or peptide of interest the LYST1, Lystl, LYST2, or Lyst2 protein) and does not include any coding or regulatory sequences that would have an adverse effect on cells. Therefore, it will also be understood that useful nucleic acid sequences may include additional residues, such as additional non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various regulatory sequences.

After identifying an appropriate epitope-encoding nucleic acid molecule, it may be inserted into any one of the many vectors currently known in the art, so that it will direct the expression and production of the protein or peptide epitope of interest the LYST1, Lystl, LYST2, or Lyst2 protein) when incorporated into a host cell. In a recombinant expression vector, the coding portion of the DNA segment is positioned under the control of a promoter. The promoter may be in the form of the promoter which is naturally associated with a LYST1-, Lyst LYST2-, or Lyst2-encoding nucleic acid segment, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment, for example, using recombinant cloning and/or PCR T technology, in connection with the compositions disclosed herein. Direct amplification of nucleic acids using the PCR M technology of U.S. Patents 4,683,195 and 4,683,202 (herein incorporated by reference) are particularly contemplated to be useful in such methodologies.

In certain embodiments, it is contemplated that particular advantages will be gained by positioning the LYSTI-, Lystl-, LYST2-, or Lyst2-encoding DNA segment under the control of a WO 97/28262 PCT/US97/01748 recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a LYST1, Lystl, LYST2, or Lyst2-encoding DNA segment in its natural environment. Such promoters may include those normally associated with other genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the particular cell containing the vector comprising the LYSTI-, Lystl-, LYST2-, or Lyst2-encoding nucleic acid segment.

The use of recombinant promoters to achieve protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al., (1989). The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level or regulated expression of the introduced DNA segment. For eukaryotic expression, the currently preferred promoters are those such as CMV, RSV LTR, the promoter alone, and the SV40 promoter in combination with the SV40 enhancer. In certain embodiments, the expression of recombinant LYST1, Lystl, LYST2, or Lyst2 protein is carried out using prokaryotic expression systems, and in particular bacterial systems such as E.

coli. Such prokaryotic expression of nucleic acid segments of the present invention may be performed using methods known to those of skill in the art, and will likely comprise expression vectors and promotor sequences such as those obtained from tac, trp, lac, lacUV5 or T7 promotors.

For the expression of the LYST1, Lysti, LYST2, or Lyst2 protein and LYST1-, Lystl-, LYST2-, or Lyst2-derived epitopes, once a suitable clone or clones have been obtained, whether they be native sequences or genetically-modified, one may proceed to prepare an expression system for the recombinant preparation of the LYSTI, Lysti, LYST2, or Lyst2 protein or peptides derived from one or more of the LYSTI, Lystl, LYST2, or Lyst2 proteins. The engineering of DNA segment(s) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. It is believed that virtually any expression system may be employed in the expression of LYSTI, Lystl, LYST2, or Lyst2 proteins or epitopes derived from such proteins.

Alternatively, it may be desirable in certain embodiments to express the gene products or derived epitopes in eukaryotic expression systems. The DNA sequences encoding the desired WO 97/28262 PCT/US97/01748 ,y epitope (either native or mutagenized) may be separately expressed in various eukaryotic systems as is well-known to those of skill in the art.

It is proposed that transformation of host cells with DNA segments encoding such epitopes will provide a convenient means for obtaining the protein or peptide of interest.

Genomic sequences are suitable for eukaryotic expression, as the host cell will, of course, process the genomic transcripts to yield functional mRNA for translation into protein.

It is similarly believed that almost any eukaryotic expression system may be utilized for the expression of the proteins of the present invention, or of peptides or epitopes derived from such proteins, baculovirus-based, glutamine synthase-based or dihydrofolate reductase-based systems may be employed. In preferred embodiments it is contemplated that plasmid vectors incorporating an origin of replication and an efficient eukaryotic promoter, as exemplified by the eukaryotic vectors of the pCMV series, such as pCMV5, will be of most use.

For expression in this manner, one would position the coding sequences adjacent to and under the control of the promoter. It is understood in the art that to bring a coding sequence under the control of such a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame of the protein between about 1 and about 50 nucleotides "downstream" of(i.e., 3' of) the chosen promoter.

Where eukaryotic expression is contemplated, one will also typically desire to incorporate into the transcriptional unit which includes nucleic acid sequences encoding the LYST/Lsyt gene product or LYST/Lyst-derived peptides, an appropriate polyadenylation site 5'-AATAAA-3') if one was not contained within the original cloned segment. Typically, the poly-A addition site is placed about 30 to 2000 nucleotides "downstream" of the termination site of the protein at a position prior to transcription termination.

It is contemplated that virtually any of the commonly employed host cells can be used in connection with the expression of the LYST1, Lystl, LYST2, or Lyst2 proteins and epitopes derived therefrom in accordance herewith. Examples include cell lines typically employed for eukaryotic expression such as 239, AtT-20, HepG2, VERO, HeLa, CHO, WI 38, BHK, COS-7, RIN and MDCK cell lines.

WO 97/28262 PCT/US97/01748 It C It is further contemplated that the proteins, peptides, or epitopic peptides derived from native or recombinant LYST1, Lystl, LYST2, or Lyst2 proteins may be "overexpressed", i.e., expressed in increased levels relative to its natural expression in human cells, or even relative to the expression of other proteins in a recombinant host cell containing LYSTI-, Lystl-, LYST2-, or Lyst2-encoding DNA segments. Such overexpression may be assessed by a variety of methods, lincluding radiolabeling and/or protein purification. However, facile and direct methods are preferred, for example, those involving SDS/PAGE and protein staining or Western blotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot. A specific increase in the level of the recombinant protein or peptide in comparison to the level in natural LYST1-, Lystl-, LYST2-, or Lyst2-producing cells is indicative of overexpression, as is a relative abundance of the specific protein in relation to the other proteins produced by the host cell and, visible on a gel.

As used herein, the term "engineered" or "recombinant" cell is intended to refer to a cell into which a recombinant gene, such as a gene encoding LYST1, Lystl, LYST2, or Lyst2 has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man. Recombinantly introduced genes will either be in the form of a single structural gene, an entire genomic clone comprising a structural gene and flanking DNA, or an operon or other functional nucleic acid segment which may also include genes positioned either upstream and/or downstream of the promotor, regulatory elements, with or without introns, or a cDNA clone comprising the structural gene itself, or even genes not naturally associated with the particular gene of interest.

Where the introduction of a recombinant version of one or more of the foregoing genes is required, it will be important to introduce the gene such that it is under the control of a promoter that effectively directs the expression of the gene in the cell type chosen for engineering. In general, one will desire to employ a promoter that allows constitutive (constant) expression of the gene of interest. Commonly used constitutive eukaryotic promoters include viral promotors such as the cytomegalovirus (CMV) promoter, the Rous sarcoma long-terminal repeat (LTR) sequence, or the SV40 early gene promoter. The use of these constitutive promoters will ensure a high, constant level of expression of the introduced genes. The inventors have noticed that the level of expression from the introduced genes of interest can vary in different clones, or genes isolated from different strains or bacteria. Thus, the level of expression of a particular WO 97/28262 PCTIUS97/01748 recombinant gene can be chosen by evaluating different clones derived from each transfection study; once that line is chosen, the constitutive promoter ensures that the desired level of expression is permanently maintained. It may also be possible to use promoters that are specific for cell type used for engineering, such as the insulin promoter in insulinoma cell lines, or the prolactin or growth hormone promoters in anterior pituitary cell lines.

2.4 Detection of LYST/Lyst Gene Products A further aspect of the invention is the preparation of immunological compositions, and in particular anti- LYST/Lyst antibodies for diagnostic and therapeutic methods relating to the detection and diagnosis of CHS. Methods for diagnosing CHS and the detection of LYST/Lyst encoding nucleic acid segments in clinical samples using nucleic acid compositions are also obtained from the invention. The nucleic acid sequences encoding LYST/Lyst are useful as diagnostic probes using conventional techniques such as in Southern hybridization analyses or Northern hybridization analyses to detect the presence of LYST/Lyst nucleic acid segments within a clinical sample from a patient suspected of having such a condition. In a preferred embodiment, nucleic acid sequences as disclosed in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11 and SEQ ID NO:13 are preferable as probes for such hybridization analyses.

Methods for Producing an Immune Response Also disclosed in a method of generating an immune response in an animal. The method generally involves administering to an animal a pharmaceutical composition comprising an immunologically effective amount of a peptide composition disclosed herein. Preferred peptide compositions include the peptide disclosed in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14.

The invention also encompasses LYST/Lyst and LYST/Lyst -derived peptide antigen compositions together with pharmaceutically-acceptable excipients, carriers, diluents, adjuvants, and other components, such as additional peptides, antigens, or outer membrane preparations, as may be employed in the formulation of particular vaccines.

Antibodies may be of several types including those raised in heterologous donor animals or human volunteers immunized with the LYST/Lyst gene product, monoclonal antibodies (mAbs) resulting from hybridomas derived from fusions of B cells from immunized animals or WO 97/28262 PCT/US97/01748 humans with compatible myeloma cell lines, so-called "humanized" mAbs resulting from expression of gene fusions of combinatorial determining regions of mAb-encoding genes from heterologous species with genes encoding human antibodies, or LYST/Lyst -reactive antibody-containing fractions of plasma from human donors suspected of having CHS. It is contemplated that any of the techniques described above might be used for the vaccination of subjects for the purpose of antibody production. Optimal dosing of such antibodies is highly dependent upon the pharmacokinetics of the specific antibody population in the particular species to be treated.

Using the peptide antigens described herein, the present invention also provides methods of generating an immune response, which methods generally comprise administering to an animal, a pharmaceutically-acceptable composition comprising an immunologically effective amount of a LYST/Lyst peptide composition. Preferred animals include mammals, and particularly humans.

Other preferred animals include murines, bovines, equines, porcines, canines, and felines. The composition may include partially or significantly purified LYST/Lyst peptide epitopes, obtained from natural or recombinant sources, which proteins or peptides may be obtainable naturally or either chemically synthesized, or alternatively produced in vitro from recombinant host cells expressing DNA segments encoding such epitopes. Smaller peptides that include reactive epitopes, such as those between about 10 and about 50, or even between about 50 and about 100 amino acids in length will often be preferred. The antigenic proteins or peptides may also be combined with other agents, such as other LYST/Lyst -related peptides or nucleic acid compositions, if desired.

By "immunologically effective amount" is meant an amount of a peptide composition that is capable of generating an immune response in the recipient animal. This includes both the generation of an antibody response (B cell response), and/or the stimulation of a cytotoxic immune response (T cell response). The generation of such an immune response will have utility in both the production of useful bioreagents, CTLs and, more particularly, reactive antibodies, for use in diagnostic embodiments, and will also have utility in various prophylactic or therapeutic embodiments. Therefore, although these methods for the stimulation of an immune response include vaccination regimens and treatment regimens, it will be understood that achieving either of these end results is not necessary for practicing these aspects of the invention.

WO 97/28262 PCTIUS97/01748 2Z Further means contemplated by the inventors for generating an immune response in an animal includes administering to the animal, or human subject, a pharmaceutically-acceptable composition comprising an immunologically effective amount of a nucleic acid composition encoding a LYST/Lyst epitope, or an immunologically effective amount of an attenuated live organism that includes and expresses such a nucleic acid composition. The "immunologically effective amounts" are those amounts capable of stimulating a B cell and/or T cell response.

Immunoformulations of this invention, whether intended for vaccination, treatment, or for the generation of antibodies useful in the detection of CHS, may comprise native, or syntheticallyderived antigenic peptide fragments from these proteins. As such, antigenic functional equivalents of the proteins and peptides described herein also fall within the scope of the present invention.

An "antigenically functional equivalent" protein or peptide is one that incorporates an epitope that is immunologically cross-reactive with one or more epitopes derived from any of the particular proteins disclosed. Antigenically functional equivalents, or epitopic sequences, may be first designed or predicted and then tested, or may simply be directly tested for cross-reactivity.

The identification or design of suitable epitopes, and/or their functional equivalents, suitable for use in immunoformulations, vaccines, or simply as antigens for use in detection protocols), is a relatively straightforward matter. For example, one may employ the methods of Hopp, as enabled in U.S. Patent 4,554,101, incorporated herein by reference, that teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences, for example, Chou and Fasman (1974a,b; 1978a,b; 1979); Jameson and Wolf (1988); Wolf et al., (1988); and Kyte and Doolittle (1982) address this subject. The amino acid sequence of these "epitopic core sequences" may then be readily incorporated into peptides, either through the application of peptide synthesis or recombinant technology.

It is proposed that the use of shorter antigenic peptides, about 25 to about 50, or even about 15 to 25 amino acids in length, that incorporate epitopes of the LYST/Lyst protein will provide advantages in certain circumstances, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution.

WO 97/28262 PCTUS97/01748 In still further embodiments, the present invention concerns immunodetection methods and associated kits. It is contemplated that the proteins or peptides of the invention may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention, may be employed to detect LYST/Lyst proteins or peptides. Either type of kit may be used in the immunodetection of compounds, present within clinical samples, that are indicative of CHS. The kits may also be used in antigen or antibody purification, as appropriate.

In general, the preferred immunodetection methods will include first obtaining a sample suspected of containing a LYST/Lyst -reactive antibody, such as a biological sample from a patient, and contacting the sample with a first LYST/Lyst protein or peptide under conditions effective to allow the formation of an immunocomplex (primary immune complex). One then detects the presence of any primary immunocomplexes that are formed. Preferable

LYST/LYST

proteins include LYSTI and LYST2 from human origins, and Lystl and Lyst2 proteins derived from murine origins.

Contacting the chosen sample with the LYST/Lyst protein or peptide under conditions effective to allow the formation of (primary) immune complexes is generally a matter of simply adding the protein or peptide composition to the sample. One then incubates the mixture for a period of time sufficient to allow the added antigens to form immune complexes with, to bind to, any antibodies present within the sample. After this time, the sample composition, such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any nonspecifically bound antigen species, allowing only those specifically bound species within the immune complexes to be detected.

The detection ofimmunocomplex formation is well known in the art and may be achieved through the application of numerous approaches known to the skilled artisan and described in various publications, such as, Nakamura et al., (1987), incorporated herein by reference.

Detection of primary immune complexes is generally based upon the detection of a label or marker, such as a radioactive, fluorescent, biological or enzymatic label, with enzyme tags such as alkaline phosphatase, urease, horseradish peroxidase and glucose oxidase being suitable. The particular antigen employed may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of bound antigen present in the composition to be determined.

WO 97/28262 PCT/US97/01748 Z4 Alternatively, the primary immune.complexes may be detected by means of a second binding ligand that is linked to a detectable label and that has binding affinity for the first protein or peptide. The second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies and the remaining bound label is then detected.

For diagnostic purposes, it is proposed that virtually any sample suspected of containing the antibodies of interest may be employed. Exemplary samples include clinical samples obtained from a patient such as blood or serum samples, bronchoalveolar fluid, ear swabs, sputum samples, middle ear fluid or even perhaps urine samples may be employed. This allows for the diagnosis of CHS and related disorders. Furthermore, it is contemplated that such embodiments may have application to non-clinical samples, such as in the titering of antibody samples, in the selection of hybridomas, and the like. Alternatively, the clinical samples may be from veterinary sources and may include such domestic animals as cattle, sheep, and goats. Samples from feline, canine, and equine sources may also be used in accordance with the methods described herein.

In related embodiments, the present invention contemplates the preparation of kits that may be employed to detect the presence ofLYST/Lyst -specific antibodies in a sample. Generally speaking, kits in accordance with the present invention will include a suitable protein or peptide together with an immunodetection reagent, and a means for containing the protein or peptide and reagent.

The immunodetection reagent will typically comprise a label associated with a LYST/Lyst protein or peptide, or associated with a secondary binding ligand. Exemplary ligands might include a secondary antibody directed against the first LYST/Lyst or peptide or antibody, or a biotin or avidin (or streptavidin) ligand having an associated label. Detectable labels linked to antibodies that have binding affinity for a human antibody are also contemplated, for protocols where the first reagent is a LYST/Lyst peptide that is used to bind to a reactive antibody from a human sample. Of course, as noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention.

WO 97/28262 PCTUS97/01748 The kits may contain antigen or antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.

The container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antigen may be placed, and preferably suitably allocated.

Where a second binding ligand is provided, the kit will also generally contain a second vial or other container into which this ligand or antibody may be placed. The kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, injection or blow-molded plastic containers into which the desired vials are retained.

2.6 Formulation as Vaccines It is expected that to achieve an "immunologically effective formulation" it may be desirable to administer LYST- or Lyst-encoding proteins to the human or animal subject in a pharmaceutically acceptable composition comprising an immunologically effective amount of LYST or Lyst proteins or peptides mixed with other excipients, carriers, or diluents which may improve or otherwise alter stimulation of B cell and/or T cell responses, or immunologically inert salts, organic acids and bases, carbohydrates, and the like, which promote stability of such mixtures. Immunostimulatory excipients, often referred to as adjuvants, may include salts of aluminum (often referred to as Alums), simple or complex fatty acids and sterol compounds, physiologically acceptable oils, polymeric carbohydrates, chemically or genetically modified protein toxins, and various particulate or emulsified combinations thereof LYST or Lyst proteins or peptides within these mixtures, or each variant if more than one are present, would be expected to comprise about 0.0001 to 1.0 milligrams, or more preferably about 0.001 to 0.1 milligrams, or even more preferably less than 0.1 milligrams per dose.

It is also contemplated that attenuated organisms may be engineered to express recombinant LYST or Lyst proteins or peptides, and the organisms themselves be delivery vehicles for the invention. Pox-, polio-, adeno-, or other viruses, and bacteria such as Salmonella, Shigella, Listeria, Streptococcus species may also be used in conjunction with the methods and compositions disclosed herein.

The naked DNA technology, often referred to as genetic immunization, has been shown to be suitable for protection against infectious organisms. Such DNA segments could be used in a WO 97/28262 PCTIUS97/01748 variety of forms including naked DNA and plasmid DNA, and may administered to the subject in a variety of ways including parenteral, mucosal, and so-called microprojectile-based "gene-gun" inoculations. The use of LYSTor Lyst nucleic acid compositions of the present invention in such immunization techniques is thus proposed to be useful as a vaccination strategy against Lyme disease.

It is recognized by those skilled in the art that an optimal dosing schedule of a vaccination regimen may include as many as five to six, but preferably three to five, or even more preferably one to three administrations of the immunizing entity given at intervals of as few as two to four weeks, to as long as five to ten years, or occasionally at even longer intervals.

2.7 USE OF LYST1 PEPTIDES/APTAMERS As PHARMACEUTICALS THAT BLOCK OR MIMIC LYST1 FUNCTION Lyst regulates degranulation oflysosomes, late endosomes and acidic secretory granules primarily in leukocytes. Blockade of such degranulation using dominant-negatively acting truncated Lyst peptides may reasonably be expected to be efficacious in inflammatory and autoimmune diseases such as asthma, urticaria, inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, systemic vasculitis, glomerulonephritis, multiple sclerosis, post-angioplasty restenosis. Proof of this principal is documented in Clark et al., 1982, who demonstrated that bg mice are protected from lupus nephritis.

2.8 USE OF PHARMACEUTICAL COMPOUNDS THAT BLOCK OR MIMIC LYST1 FUNCTION Lyst regulates degranulation oflysosomes, late endosomes and acidic secretory granules primarily in leukocytes. Blockade of such degranulation using dominant-negatively acting truncated Lyst peptides may reasonably be expected to be efficacious in inflammatory and autoimmune diseases such as asthma, urticaria, inflammatory bowel disease systemic lupus erythematosus, rheumatoid arthritis, psoriasis, systemic vasculitis, glomerulonephritis, multiple sclerosis, post-angioplasty restenosis. Proof of this principal is documented in Clark et al., (1982) who demonstrated that bg mice are protected from lupus nephritis.

Lyst peptides that mimic or augment Lyst function may reasonably be expected to be efficacious in the treatment ofneoplasia. Proof of this principle is documented in Aboud et al.

(1993) and Hayakawa et al. (1986), who demonstrate that bg mice and CHS patients are WO 97/28262 PCT[US97/01748 21 susceptible to development offneoplasia, and have more aggressive neoplasms with accelerated metastases.

2.9 USE OF LYST2 PEPTIDES/APTAMERS AS PHARMACEUTICAL AGENTS THAT BLOCK LYST2 FUNCTION OR REPRODUCE LYST2 FUNCTIONS Lyst2 is thought to act to regulate degranulation of vesicles within cells in the brain and kidney. Bblockade of such degranulation using dominant-negatively acting truncated Lyst2 peptides may reasonably be expected to be efficacious for the treatment ofneurologic and renal degenerative diseases such as Alzheimer's disease, motor neuron disease, Parkinson's disease, acute tubular necrosis, glomerulonephritis and glomerulosclerosis.

2.10 USE OF PHARMACEUTICAL COMPOUNDS THAT BLOCK OR MIMIC LYST2 FUNCTIONS Drugs that mimic the action of dominant-negatively acting truncated Lyst2 peptides.

Lyst2 is thought to act to regulate degranulation of vesicles within cells in the brain and kidney.

Blockade of such degranulation using dominant-negatively acting truncated Lyst2 peptides may reasonably be expected to be efficacious for the treatment of neurologic and renal degenerative diseases such as Alzheimer's disease, motor neuron disease, Parkinson's disease, acute tubular necrosis, glomerulonephritis and glomerulosclerosis.

3. BRIEF DESCRIPTION OF THE DRAWINGS The drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1A. Ethidium bromide-stained pulsed field gels of DNA from clones derived from a mouse YAC library. YAC clone numbers are shown above each panel and molecular size standards (in kilobases) to the left of each panel. 1380 is the host S. cerevisiae strain and does not contain a YAC. Sizes of YAC clones are: 151H1 950-kb, 195A8 650-kb, 64F5 580-kb, 93E4 370-kb, 68E12 500-kb, 55F3 550-kb, 135G3 750-kb, 148H8 1000-kb, 84A8 370-kb, 148E11 650-kb; 165F7 500-kb.

WO 97/28262 PCTIUS97/01748 FIG. 1B. Autoradiographs of corresponding Southern blots from the gels shown in FIG. 1A hybridized with pBR322 (which cross-hybridizes to pYAC4. YAC clone numbers are shown above each panel and molecular size standards (in kilobases) to the left of each panel. 1380 is the host S. cerevisiae strain and does not contain a YAC. Sizes of YAC clones are: 151H1 950-kb, 195A8 650-kb, 64F5 580-kb, 93E4 370-kb, 68E12 500-kb, 55F3 550-kb, 135G3 750-kb, 148H8 1000-kb, 84A8 370-kb, 148E11 650-kb; 165F7 500-kb.

FIG. 2. STS content mapping of bg critical region YAC and P1 clones. The presence of an STS (y-axis) in a YAC/P1 clone (x-axis) is indicated by a filled box. Each contig is identified by the degree of shading of the box. The bg critical region extends from proximal to D13Mit134 to the interval between D13MiU207 and D13Mit162/D13Mit305 (crossover location indicated by a double line). STS used for isolation of YAC clones were Nid5' for 151H1, 195A8, 64F5, 93E4, 68E12, and 55F3, Estm9 for 148E11, D13Mit134 for 165F7, D13Sfk13 for 84A8, and D13Mit207 for 135G3 and 148H8. P1 clones 8591 and 8592 were identified with DI3Sfk13. YAC clone 64F5 is chimeric; YAC clone 84A8 has acquired an internal deletion which includes D13Sfk6. The relative orientation with respect to the centromere of the contig composed of the 9 clones 195A8- 55F3 has not been established; The position of clones 165F7 and 148E11 with respect to this contig has not been established.

FIG. 3A. Genetic mapping of bg on mouse Chr 1. Haplotype analysis of proximal mouse Chr 1 genetic markers in 504 C57BL/6J-bgj X (C57BL/6J-bg x CAST/EiJ)FI backcross mice. Closed boxes represent the homozygous C3H pattern and open boxes the Fi pattern. Number of mice of each haplotype are indicated.

FIG. 3B. Genetic mapping of bg on mouse Chr 1. Haplotype analysis of proximal mouse Chr 1 genetic markers in 111 (C57BL/6J -Wh-bg x Mus domesticus PAC)Fi X C57BL/6J-bg' backcross mice. Closed boxes represent the homozygous C3H pattern and open boxes the FI pattern. Number of mice of each haplotype are indicated.

FIG. 3C. Genetic mapping of bg on mouse Chr 1. Haplotype analysis of proximal mouse Chr 1 genetic markers in 111 (C57BL/6J-Wh-bgJ x Mus musculus PWK)Fi X C57BL/6J- WO 97/28262 PCTIUS97/01748 2q bg J backcross mice. Closed boxes represent the homozygous C3H pattern and open boxes the F, pattern. Number of mice of each haplotype are indicated.

FIG. 3D. Genetic mapping of bg on mouse Chr 1. Composite linkage map of mouse Chr 13 in the vicinity of bg. Loci are positioned according to their Approximate relative positions of loci were ascertained by integration of data from the above three backcrosses and from Dietrich et al., (1994).

FIG. 4. Autoradiograph of a pulse field gel Southern blot of mouse DNA probed with Nid.

Restriction endonucleases are shown above the panel and molecular size standards (in kilobases) to the left. +/+=DBA/2J DNA; bg=SB-bg/bg DNA. Upon reprobing this blot with Gli3 or Estm9 all DBA/2J and SB-bg bands were of identical size.

FIG. 5A. DNA sequence of the CH gene (LYST1) from position 1 DNA sequence continues in FIG. to position 1400. The FIG. 5B. Continuation of the DNA sequence of the position 1401 and continuing to position 2800.

FIG. 5C. Continuation of the DNA sequence of the position 2801 and continuing to position 3514.

CH gene in FIG. 5A beginning at CH gene in FIG. 5B beginning at FIG. 6. Amino acid sequence of the CH protein.

FIG. 7A. Genetic mapping of the B1 gene.

FIG. 7B. Genetic mapping of the bg and B1 genes.

FIG. 7C. Detailed map showing the localiztion of the bg and Bl genes.

FIG. 8. Bl cDNA clones.

FIG. 9A. Deletion of part of B1 in bg 1 Probe used in Southern analysis is probe A from FIG. 8.

FIG. 9B. Deletion of part of B1 in Probe used in Southern analysis is probe B from FIG. 8.

WO 97/28262 PCT/US97/01748 FIG. 9C. Deletion of part of B1 in bg 1 Probe used in Southern analysis is probe C from FIG. 8. (In bp" J a deletion from bp 1250 to 2400 was observed.

FIG. 10. Physical mapping ofB1 gene within bg critical region.

FIG. 11. Genetic and physical map of the bg non-recombinant interval on mouse chromosome 13 showing the location of Lyst. Mouse chromosome 13 is shown by the horizontal line with the centromere on the left. The bg critical region is delineated by chromosome crossovers (denoted with an X) in animals 134 and 137 of an interspecific mouse backcross [C57BL/6-bg x (C57BL/6J-bg' x CAST/EiJ)Fi]. Microsatellite markers D13Mit172 and D13Mit239 flank bg proximally; D13Mit162 and D13Mit305 lie distal to bg (indicated by turquoise circles). YAC and P1 clones identified by PCR T M screening (Kusumi et al., 1993; Pierce et al., 1992) with oligonucleotides corresponding to Nid or D13Sfk13 are shown above the chromosome. Novel sequence-tagged sites (STS, indicated by dark blue circles), generated by inverse repetitive element PCR or direct or direct cDNA selection, were used to order clones within the contiguous array.

Novel mouse chromosome 13 STSs are numbered 1-18, corresponding to D13Sfkl to D13Sfk8, respectively. Lyst was isolated from YAC 195A8, a 650-kb clone, by using direct cDNA selection. The physical location of Lyst-associated STSs on YAC and P1 clones are shown in red (MGD accession number MGD-PMEX-13).

FIG. 12A. Intragenic deletion of Lyst in bg'" DNA. Southern blot identification of an intragenic Lyst deletion in bg"' DNA. A Southern blot was sequentially hybridized (Barbosa et al., 1995) with 3 Lyst probes; This panel shows the probe (nucleotides 1,262- 3,433 of Lyst cDNA) which extends upstream from the bg" deletion. Restriction endonucleases are indicated at the bottom of the panel, and molecular size standards (in kb) are shown to the left. Similar results were obtained with 3 additional restriction endonucleases. The bg"' mutation was discovered in 1992 at The Jackson Laboratory in a C57BL/6J-jb mouse at generation N4 after transfer from B6C3Fe-a/a. The mutationjb had, in turn, been discovered 14 generations earlier in B6C3Fe-a/a-hyh mice at generation N3 after transfer from C57BL/10J. The hyh mutation arose in C57BL/10J mice, and was maintained in that strain until transfer at F15. Thus the possible contributors of genetic information to bg

J

include C57BL/6J, C3HeB/FeJ and C57BL/10J. Southern blots were WO 97/28262 PCT/US97/01748 prepared from genomic DNA of all potential progenitor mouse strains, but only C57BL/10J, C57BL/6J and C57BL/6J-bg 1 are shown.

FIG. 12B. Intragenic deletion of Lyst in bg"' DNA. Southern blot identification of an intragenic Lyst deletion in bg"j DNA. A Southern blot was sequentially hybridized (Barbosa et al., 1995) with 3 Lyst probes. This panel shows the probe (nucleotides 2,835- 3,433 ofLyst cDNA) is completely deleted. Restriction endonucleases are indicated at the bottom of each panel, and molecular size standards (in kb) are shown to the left.

FIG. 12C. Intragenic deletion of Lyst in bg" J DNA. Southern blot identification of an intragenic Lyst deletion in bg" DNA. A Southern blot was sequentially hybridized (Barbosa et al., 1995) with 3 Lyst probes; Shown in this panel are results when the probe (nucleotides 3,594-4,237 of Lyst cDNA) extends downstream from the big bg'" deletion.

Restriction endonucleases are indicated at the bottom of each panel, and molecular size standards (in kb) are shown to the left. Similar results were obtained with 3 additional restriction endonucleases.

FIG. 12D. Intragenic deletion of Lyst in bg 1 DNA. PCR TM analysis of the bg'" deletion.

C57BL/10J, C3HeB/FeJ, C57BL/6J and C57BL/6J-bg" genomic DNA and Lyst cDNA were used as templates in the PCRTM reactions. Amplicons illustrated correspond to: Lyst cDNA nucleotides 1,337-1,837, which represent exon P and are upstream from the deletion. No amplicon was observed in control PCRTM reactions performed without template. More than 30 other STSs that had been localized within the bg nonrecombinant interval PCRTM amplified normally from bg" DNA FIG. 12E. Intragenic deletion of Lyst in bg 1 DNA. PCRTM analysis of the bg" deletion.

C57BL/10J, C3HeB/FeJ, C57BL/6J and C57BL/6J-bg" genomic DNA and Lyst cDNA were used as templates in the PCRTM reactions. Amplicons illustrated correspond to nucleotides 2,670-3,210, which represent exon y, which is deleted in bg"' DNA. No amplicon was observed in control PCRTM reactions performed without template. More than 30 other STSs that had been localized within the bg non-recombinant interval PCR

T

amplified normally from bg"' DNA.

WO 97/28262 PCTIUS97/01748 2 FIG. 12F. Intragenic deletion of Lyst in bg"j DNA. PCR T M analysis of the bg" deletion.

C57BL/10J, C3HeB/FeJ, C57BL/6J and C57BL/6J-bg" genomic DNA and Lyst cDNA were used as templates in the PCRTM reactions. Amplicons illustrated correspond to nucleotides 4,913-5,433, which represents an exon downstream from the deletion. No amplicon was observed in control PCRTM reactions performed without template.

FIG. 12G. Intragenic deletion of Lyst in bg"' DNA. Genomic structure of Lyst in the vicinity of the bg'" deletion. Lyst exons 0, y, 6, s, and 4) are depicted by black boxes, and intervening introns by a solid line. Nucleotides of the mouse Lyst cDNA that correspond to exonic boundaries are indicated above the boxes. The 3' end of exon 0, and all of exons y and 6, are deleted in bg" J DNA. The region of Lyst protein that is deleted in bg'" contains a pair of helices with N-terminal phosphorylation sites. Genomic structure and intronic sequences were ascertained by sequence analysis of nested PCRTM products, performed with exonic primers and P1 clone DNA as template (Kingsmore et al., 1994).

Boundaries of the bg'" deletion were determined by PCRTM of genomic DNA.

FIG. 13A. Northern blot analysis of mouse and human Lyst. Northern blots of 2 pg poly(A)' RNA from various mouse tissues (Clontech) hybridized with probes that correspond to nucleotides 4,423-4,621 of mouse Lyst cDNA.

FIG. 13B. Northern blot analysis of mouse and human Lyst. Northern blots of 2 pg poly(A)' RNA from various mouse tissues (Clontech) hybridized with probes that correspond to nucleotides 1,430-2,457 (exon p)of mouse Lyst cDNA. Molecular size standards (in kb) are shown to the left. Hybridization of mouse mRNA with probes from mouse Lyst exons a and y gave identical results to those shown with exon 0, whereas probes from exons 6, F, and 4 gave results identical to those shown in FIG. 13A.

FIG. 13C. Northern blot analysis of mouse and human Lyst. Northern blot of 2 ig poly(A)' RNA from various human lymphoid tissues, hybridized with a probe that corresponds to nucleotides 357-800 of human LYST cDNA. Molecular size standards (in kb) are shown to the left.

FIG. 13D. Northern blot analysis of mouse and human Lyst. Northern blot of 2 pg poly(A) RNA from human cancer cell lines, hybridized with a probe that corresponds to WO 97/28262 PCT/US9701748 *33 nucleotides 357-800 of human LYST cDNA. Molecular size standards (in kb) are shown to the left.

FIG. 14A. Mutation analysis of LYST cDNA from CHS patients. A Northern blot of 2 cg aliquots of lymphoblastoid poly(A) RNA from CHS patients and a control. The probe used for hybridization corresponds to nucleotides 490 to 817 of LYST.

FIG. 14B. SSCP analysis of cDNA corresponding to LYSTnucleotides 439 to 806. Each lane contains samples from individual patients as indicated. Note the appearance of an extra band in lanes corresponding to patients 371 and 373.

FIG. 14C. Sequence chromatograms showing mutations in LYST cDNA clones from patients 371 and 373. The upper part is normal human LYST cDNA sequence. The arrows indicate the positions of a G insertion (patient 371) and C to T substitution (patient 373). The antisense strand of LYSTis shown.

FIG. 15A. Physical mapping of LYST Monochromosomal somatic cell hybrid blot (BIOS Laboratories, New Haven, Connecticut) containing DNA from 24 somatic cell hybrid cell lines and three control DNAs (human, hamster, or mouse) digested with EcoRI. The cell line and chromosome number are indicated at the top of the figure. *Mix lane consists of mg human DNA and 4.5 mg mouse DNA. **Human/hamster somatic cell hybrid. All others are human/mouse hybrids. Molecular size standards (in kb) are shown to the right.

The blot was hybridized with a probe corresponding to nucleotides 2923-4865 of human LYSTcDNA.

FIG. 15B. Southern blot of CHS critical region YACs digested with TaqI. The YAC coordinates are indicated at the top and molecular size standards (in kb) are shown to the left. The probe used for the hybridization corresponds to LYST nucleotides 490 to 817.

FIG. 15C. The same Southern blot shown in panel B, rehybridized with a probe corresponding to LYST nucleotides 4551 to 4977. A third LSYT probe (corresponding to nucleotides 3032-4722) also hybridized to the same YAC clones.

FIG. 15D. Physical map of human chromosome 1 showing the location of LYST within a YAC contig of the CHS critical region (Barrat et al. 1996). The upper part represents chromosome 1. The microsatellite markers DIS179 (centromeric) and WI-12396 WO 97/28262 PCT/US97/01748 3q (telomeric) flank the CHS locus.. YAC clones are shown below the chromosome. The figure is not drawn to scale.

FIG. 16A. Genomic organization of LYST. Schematic representation of PCRTM clones corresponding to the human LYST cDNA (Genbank accession number U70064). The solid and open bars represent the LYST coding region and the 5' UTR, respectively.

Nucleotide 5095 corresponds to the transition between sequences conserved with Lyst (Barbosa et al., 1996) and BG (Perou etal., 1996a). The three human ESTs identified by database searches with the mouse sequence #2 and Genbank accession numbers: L77889; W26957; H51623) are shown at the top. Clones #6 and #8 are RT-PCRTM products. Clone #7 is a 2 kb 5'RACE product.

FIG. 16B. Alternative splicing of mouse Lyst. Solid boxes represent Lyst exons o and T.

Splicing of exon a to exon t occurs in the Lyst-I mRNA (12 kb). The hatched box represents the intronic region that forms the 3' end of the Lyst-II ORF (5.9 kb). An asterisk indicates a stop codon and an indicates a polyadenylation signal within the intron. Nucleotide positions indicated are from Genbank accession number L77884 (Lyst-II) and U70015 (Lyst-I).

FIG. 16C. Detection of Lyst-I and Lyst-II by RT-PCR T M and genomic PCR T M DNAse-treated mouse melanocyte RNA was reverse transcribed and amplified with primers Fl/RI (expected amplicon size 273 bp) or F1/R2 (expected amplicon size 560 bp). RNAse-treated C57BL/6J DNA was amplified with primers Fl/R1. The primer sequences are: Fl, 5'-TGTGGAATACATCCAATGAATCCGAGAGTGC-3'; F2, 5'-GAGCCAAGAAAGAGGCTGAT-3'; R1, 5'-GGTTTCGGACTCAAAAGTTTGTCGGAACTT-3'; R2, 5'-GAGACCCATATGGAGATTTC-3'.

WO 97/28262 PCT/US97/01748 4. DESCRIPTION OF ILLUSTRATIVE

EMBODIMENTS

4.1 LYST-ENCODING NUCLEIC ACID SEGMENTS As used herein, the term "LYSTI gene" is used to refer to a gene or DNA coding region that encodes a Chediak-Higashi protein, polypeptide or peptide.

The definition of a "LYST1 gene", as used herein, is a gene that hybridizes, under relatively stringent hybridization conditions (see, Maniatis et al., 1982), to DNA sequences presently known to include LYST1 gene sequences. It will, of course, be understood that one or more than one genes encoding LYST1 proteins or peptides may be used in the methods and compositions of the invention. The nucleic acid compositions and methods disclosed herein may entail the administration of one, two, three, or more, genes or gene segments. The maximum number of genes that may be used is limited only by practical considerations, such as the effort involved in simultaneously preparing a large number of gene constructs or even the possibility of eliciting a significant adverse cytotoxic effect.

As used herein, the term "LYST2 gene" is used to refer to a gene or DNA coding region that encodes a LYST2 protein, polypeptide or peptide.

The definition of a "LYST2 gene", as used herein, is a gene that hybridizes, under relatively stringent hybridization conditions (see, Maniatis et al., 1982), to DNA sequences presently known to include LYST2 gene sequences. It will, of course, be understood that one or more than one genes encoding LYST2 proteins or peptides may be used in the methods and compositions of the invention. The nucleic acid compositions and methods disclosed herein may entail the administration of one, two, three, or more, genes or gene segments. The maximum number of genes that may be used is limited only by practical considerations, such as the effort involved in simultaneously preparing a large number of gene constructs or even the possibility of eliciting a significant adverse cytotoxic effect.

In those embodiments involving multiple genes of the present invention, the LYST and Lyst genes disclosed herein may be combined on a single genetic construct under control of one or more promoters, or they may be prepared as separate constructs of the same of different types.

Thus, an almost endless combination of different genes and genetic constructs may be employed.

Certain gene combinations may be designed to, or their use may otherwise result in, achieving synergistic effects on formation of an immune response, or the development of antibodies to gene WO 97/28262 PCTIUS97/01748 3(.

products encoded by such nucleic acid segments, or in the production of diagnostic and treatment protocols for, among other things, Chediak-Higashi Syndrome. Any and all such combinations are intended to fall within the scope of the present invention. Indeed, many synergistic effects have been described in the scientific literature, so that one of ordinary skill in the art would readily be able to identify likely synergistic gene combinations, or even gene-protein combinations.

It will also be understood that, if desired, the nucleic segment or gene could be administered in combination with further agents, such as, proteins or polypeptides or various pharmaceutically active agents. So long as genetic material forms part of the composition, there is virtually no limit to other components which may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or tissues.

4.2 THERAPEUTIC AND DIAGNOSTIC KITS Therapeutic kits comprising, in suitable container means, a LYST or Lyst composition of the present invention in a pharmaceutically acceptable formulation represent another aspect of the invention. The LYST or Lyst composition may be native LYST or Lyst protein, truncated LYST or Lyst protein, site-specifically mutated LYST or Lyst-encoding DNAs, or LYST- or Lystderived peptide epitopes, or alternatively antibodies which bind the native LYST or Lyst gene product, truncated LYST or Lyst protein, site-specifically mutated LYST or Lyst protein, or LYST- or Lyst-encoded peptide epitopes. In other embodiments, the LYST or Lyst composition may be nucleic acid segments encoding one or more native LYST or Lyst proteins, truncated LYST or Lyst proteins, site-specifically mutated LYST or Lyst proteins, or peptide epitope derivatives of LYST or Lyst. Such nucleic acid segments may be DNA or RNA, and may be either native, recombinant, or mutagenized nucleic acid segments.

The kits may comprise a single container means that contains the LYST or Lyst composition. The container means may, if desired, contain a pharmaceutically acceptable sterile excipient, having associated with it, the LYST or Lyst composition and, optionally, a detectable label or imaging agent. The formulation may be in the form of a gelatinous composition, a collagenous- LYST or Lyst composition, or may even be in a more fluid form. The container means may itself be a syringe, pipette, or other such like apparatus, from which the LYST or Lyst composition may be applied to a tissue site, injected into an animal, or otherwise administered as WO 97/28262 PCTfUS97/01748 3"7 needed. However, the single container means may contain a dry, or lyophilized, mixture of a LYST or Lyst composition, which may or may not require pre-wetting before use.

Alternatively, the kits of the invention may comprise distinct container means for each component. In such cases, one container would contain the LYST or Lyst composition, either as a sterile DNA solution or in a lyophilized form, and the other container would include the matrix, which may or may not itself be pre-wetted with a sterile solution, or be in a gelatinous, liquid or other syringeable form.

The kits may also comprise a second or third container means for containing a sterile, pharmaceutically acceptable buffer, diluent or solvent. Such a solution may be required to formulate the LYST or Lyst component into a more suitable form for application to the body, as a topical preparation, or alternatively, in oral, parenteral, or intravenous forms. It should be noted, however, that all components of a kit could be supplied in a dry form (lyophilized), which would allow for "wetting" upon contact with body fluids. Thus, the presence of any type of pharmaceutically acceptable buffer or solvent is not a requirement for the kits of the invention.

The kits may also comprise a second or third container means for containing a pharmaceutically acceptable detectable imaging agent or composition.

The container means will generally be a container such as a vial, test tube, flask, bottle, syringe or other container means, into which the components of the kit may placed. The matrix and gene components may also be aliquoted into smaller containers, should this be desired. The kits of the present invention may also include a means for containing the individual containers in close confinement for commercial sale, such as, injection or blow-molded plastic containers into which the desired vials or syringes are retained.

Irrespective of the number of containers, the kits of the invention may also comprise, or be packaged with, an instrument for assisting with the placement of the ultimate LYST or Lyst composition within the body of an animal. Such an instrument may be a syringe, pipette, forceps, or any such medically approved delivery vehicle.

4.3 METHODS OF NUCLEIC ACID DELIVERY AND DNA TRANSFECTION In certain embodiments, it is contemplated that the nucleic acid segments disclosed herein will be used to transfect appropriate host cells. Technology for introduction of DNA into cells is WO 97/28262 PCT/US97/01748 well-known to those of skill in the art. Four general methods for delivering a nucleic segment into cells have been described: chemical methods (Graham and VanDerEb, 1973); physical methods such as microinjection (Capecchi, 1980), electroporation (Wong and Neumann, 1982; Fromm et al., 1985) and the gene gun (Yang et al., 1990); viral vectors (Clapp, 1993; Eglitis and Anderson, 1988); and receptor-mediated mechanisms (Curiel et al., 1991; Wagner et al., 1992).

4.4 LIPOSOMES AND NANOCAPSULES In certain embodiments, the inventors contemplate the use of liposomes and/or nanocapsules for the introduction of particular peptides or nucleic acid segments into host cells.

Such formulations may be preferred for the introduction of pharmaceutically-acceptable formulations of the nucleic acids, peptides, and/or antibodies disclosed herein. The formation and use of liposomes is generally known to those of skill in the art (see for example, Couvreur et al., 1977 which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy of intracellular bacterial infections and diseases). Recently, liposomes were developed with improved serum stability and circulation half-times (Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987).

Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry- Michelland et al., 1987). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 tpm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made, as described (Couvreur et al., 1977; 1988).

Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 lpm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 C, containing an aqueous solution in the core.

WO 97/28262 PCT/US97/01748 39 In addition to the teachings of Couvreur et al. (1988), the following information may be utilized in generating liposomal formulations. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs.

Liposomes interact with cells via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. It often is difficult to determine which mechanism is operative and more than one may operate at the same time.

METHODS FOR PREPARING ANTIBODY COMPOSITIONS In another aspect, the present invention contemplates an antibody that is immunoreactive with a polypeptide of the invention. As stated above, one of the uses for LYST- or Lyst-derived epitopic peptides according to the present invention is to generate antibodies. Reference to antibodies throughout the specification includes whole polyclonal and monoclonal antibodies (mAbs), and parts thereof, either alone or conjugated with other moieties. Antibody parts include Fab and F(ab) 2 fragments and single chain antibodies. The antibodies may be made in vivo in suitable laboratory animals or in vitro using recombinant DNA techniques. In a preferred embodiment, an antibody is a polyclonal antibody. Means for preparing and characterizing antibodies are well known in the art (See, Harlow and Lane, 1988).

Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized WO 97/28262 PCTIUS97/01748 animal. A wide range of animal species.can be used for the production of antisera. Typically an animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster or a guinea pig.

Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies.

Antibodies, both polyclonal and monoclonal, specific LYST- or Lyst-derived epitopes may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art. A composition containing antigenic epitopes of particular proteins can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against LYST- or Lyst-derived peptides. Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.

The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen, as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also may be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs (below).

One of the important features obtained from the present invention is a polyclonal sera that is relatively homogenous with respect to the specificity of the antibodies therein. Typically, polyclonal antisera is derived from a variety of different "clones," B-cells of different lineage.

mAbs, by contrast, are defined as coming from antibody-producing cells with a common B-cell ancestor, hence their "mono" clonality.

When peptides are used as antigens to raise polyclonal sera, one would expect considerably less variation in the clonal nature of the sera than if a whole antigen were employed.

Unfortunately, if incomplete fragments of an epitope are presented, the peptide may very well assume multiple (and probably non-native) conformations. As a result, even short peptides can WO 97/28262 PCT/US97/01748 produce polyclonal antisera with relatively plural specificities and, unfortunately, an antisera that does not react or reacts poorly with the native molecule.

Polyclonal antisera according to present invention is produced against peptides that are predicted to comprise whole, intact epitopes. It is believed that these epitopes are, therefore, more stable in an immunologic sense and thus express a more consistent immunologic target for the immune system. Under this model, the number of potential B-cell clones that will respond to this peptide is considerably smaller and, hence, the homogeneity of the resulting sera will be higher. In various embodiments, the present invention provides for polyclonal antisera where the clonality, the percentage of clone reacting with the same molecular determinant, is at least 80%. Even higher clonality 90%, 95% or greater is contemplated.

To obtain mAbs, one would also initially immunize an experimental animal, often preferably a mouse, with a LYST- or Lyst-containing composition. One would then, after a period of time sufficient to allow antibody generation, obtain a population of spleen or lymph cells from the animal. The spleen or lymph cells can then be fused with cell lines, such as human or mouse myeloma strains, to produce antibody-secreting hybridomas. These hybridomas may be isolated to obtain individual clones which can then be screened for production of antibody to the desired peptide.

Following immunization, spleen cells are removed and fused, using a standard fusion protocol with plasmacytoma cells to produce hybridomas secreting mAbs against the LYST or Lyst protein. Hybridomas which produce mAbs to the selected antigens are identified using standard techniques, such as ELISA and Western blot methods. Hybridoma clones can then be cultured in liquid media and the culture supernatants purified to provide the LYST- or Lystspecific mAbs.

It is proposed that the mAbs of the present invention will also find useful application in immunochemical procedures, such as ELISA and Western blot methods, as well as other procedures such as immunoprecipitation, immunocytological methods, etc. which may utilize antibodies specific to the LYST or Lyst protein. In particular, anti-LYST/Lyst antibodies may be used in immunoabsorbent protocols to purify native or recombinant LYST/Lyst proteins or LYST/Lyst-derived peptide species or synthetic or natural variants thereof WO 97/28262 PCTfUS97/01748 12- The antibodies disclosed herein may be employed in antibody cloning protocols to obtain cDNAs or genes encoding LYST/Lyst proteins from other species or organisms, or to identify proteins having significant homology to the LYST/Lyst gene product. They may also be used in inhibition studies to analyze the effects of LYST/Lyst protein in cells, tissues, or whole animals.

Anti- LYST/Lyst antibodies will also be useful in immunolocalization studies to analyze the distribution of cells expressing LYST/Lyst protein during particular cellular activities, or for example, to determine the cellular or tissue-specific distribution of LYST/Lyst under different physiological conditions. A particularly useful application of such antibodies is in purifying native or recombinant LYST/Lyst proteins, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.

4.6 RECOMBINANT EXPRESSION OF "LYST FAMILY" PEPTIDES Recombinant clones expressing the "LYST family" nucleic acid segments may be used to prepare purified recombinant LYST protein (rLYST), purified rLYST-derived peptide antigens as well as mutant or variant recombinant protein species in significant quantities. The selected antigens, and variants thereof, are proposed to have significant utility in diagnosing and treating CHS. For example, it is proposed that rLYSTs, peptide variants thereof, and/or antibodies against such rLYSTs may also be used in immunoassays to detect the presence of LYST or as vaccines or immunotherapeutics to treat CHS and related disorders. Additionally, by application of techniques such as DNA mutagenesis, the present invention allows the ready preparation of socalled "second generation" molecules having modified or simplified protein structures. Second generation proteins will typically share one or more properties in common with the full-length antigen, such as a particular antigenic/immunogenic epitopic core sequence. Epitopic sequences can be obtained from relatively short molecules prepared from knowledge of the peptide, or encoding DNA sequence information. Such variant molecules may not only be derived from selected immunogenic/ antigenic regions of the protein structure, but may additionally, or alternatively, include one or more functionally equivalent amino acids selected on the basis of similarities or even differences with respect to the natural sequence.

WO 97/28262 PCT/US97/01748 -/3 4.7 ANTIBODY COMPOSITIONS AND FORMULATIONS

THEREOF

Means for preparing and characterizing antibodies are well known in the art (See, e.g., Harlow and Lane (1988); incorporated herein by reference). The methods for generating mAbs generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat.

Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.

As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a'carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.

mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, a purified or partially purified protein, polypeptide or peptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich WO 97/28262 PCT/US97/01748 source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately about 5 x 10 7 to about 2 x 108 lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XXO Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1- Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8 -azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% PEG, by Gefter et al. (1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10.

6 to about 1 x 10- 8 However, this does not pose a problem, as the viable, fused hybrids are WO 97/28262 PCT/US97/01748 differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.

This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs.

The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific mAb produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using WO 97/28262 PCTIS97/01748 filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

4.8 IMMUNOASSAYS As noted, it is proposed that native and synthetically-derived peptides and peptide epitopes of the invention will find utility as immunogens, in connection with vaccine development, or as antigens in immunoassays for the detection of reactive antibodies. Turning first to immunoassays, in their most simple and direct sense, preferred immunoassays of the invention include the various types of enzyme linked immunosorbent assays (ELISAs), as are known to those of skill in the art. However, it will be readily appreciated that the utility of LYST-derived proteins and peptides is not limited to such assays, and that other useful embodiments include RIAs and other non-enzyme linked antibody binding assays and procedures.

In preferred ELISA assays, proteins or peptides incorporating LYST, rLYST, or LYSTderived protein antigen sequences are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity, such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, one would then generally desire to bind or coat a nonspecific protein that is known to be antigenically neutral with regard to the test antisera, such as bovine serum albumin (BSA) or casein, onto the well. This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

After binding of antigenic material to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation. Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background. The layered antisera is then allowed to incubate for, from 2 to 4 hours, at temperatures preferably on the order of about 250 to about 270. Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween

M

or borate buffer.

WO 97/28262 PCT/US901748 Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and the amount of immunocomplex formation may be determined by subjecting the complex to a second antibody having specificity for the first.

Of course, in that the test sample will typically be of human origin, the second antibody will preferably be an antibody having specificity for human antibodies. To provide a detecting means, the second antibody will preferably have an associated detectable label, such as an enzyme label, that will generate a signal, such as color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the antiserabound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions that favor the development of immunocomplex formation incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-TweenM).

After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H 2 0 2 in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, using a visible spectrum spectrophotometer.

ELISAs may be used in conjunction with the invention. In one such ELISA assay, proteins or peptides incorporating antigenic sequences of the present invention are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, it is desirable to bind or coat the assay plate wells with a nonspecific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of powdered milk. This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

4.9 IMMUNOPRECIPITATION The anti-LYST protein antibodies of the present invention are particularly useful for the isolation of LYST protein antigens by immunoprecipitation. Immunoprecipitation involves the WO 97/28262 PCTIES9701748 separation of the target antigen componentfrom a complex mixture, and is used to discriminate or isolate minute amounts of protein.

In an alternative embodiment the antibodies of the present invention are useful for the close juxtaposition of two antigens. This is particularly useful for increasing the localized concentration of antigens, enzyme-substrate pairs.

4.10 WESTERN BLOTS The compositions of the present invention will find great use in immunoblot or western blot analysis. The anti-LYST antibodies may be used as high-affinity primary reagents for the identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof In conjunction with immunoprecipitation, followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background. This is especially useful when the antigens studied are immunoglobulins (precluding the use of immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with the detecting agent, or they migrate at the same relative molecular weight as a cross-reacting signal. Immunologicallybased detection methods in conjunction with Western blotting (including enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety) are considered to be of particular use in this regard.

4.11 PHARMACEUTICAL

COMPOSITIONS

The pharmaceutical compositions disclosed herein may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.

WO 97/28262 PCT/US97/01748 The tablets, troches, pills, capsules. and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.

Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

The active compounds may also be administered parenterally or intraperitoneally.

Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.

Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the WO 97/28262 PCTJS97/01748 injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

For oral prophylaxis the polypeptide may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate The active ingredient may also be dispersed in dentifrices, including gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.

The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid WO 97/28262 PCTIUS97/01748 solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.

The composition can be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaC1 solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated.

The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.

4.12. EPITOPIC CORE SEQUENCES The present invention is also directed to protein or peptide compositions, free from total cells and other peptides, which comprise a purified protein or peptide which incorporates an epitope that is immunologically cross-reactive with one or more of the antibodies of the present invention.

WO 97/28262 PCTfUS97/01748 As used herein, the term "incorporatmg an epitope(s) that is immunologically crossreactive with one or more anti-LYST protein antibodies" is intended to refer to a peptide or protein antigen which includes a primary, secondary or tertiary structure similar to an epitope located within a LYST polypeptide. The level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against the LYST polypeptide will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein antigen. Various immunoassay methods may be employed in conjunction with such antibodies, such as, for example, Western blotting, ELISA, RIA, and the like, all of which are known to those of skill in the art.

The identification of LYST-derived epitopes such as those derived from the LYST gene or LYST-like gene products and/or their functional equivalents, suitable for use in vaccines is a relatively straightforward matter. For example, one may employ the methods of Hopp, as taught in U.S. Patent 4,554,101, incorporated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences (see, for example, Jameson and Wolf, 1988; Wolf et al., 1988; U.S. Patent Number 4,554,101). The amino acid sequence of these "epitopic core sequences" may then be readily incorporated into peptides, either through the application of peptide synthesis or recombinant technology.

Preferred peptides for use in accordance with the present invention will generally be on the order of about 5 to about 25 amino acids in length, and more preferably about 8 to about amino acids in length. It is proposed that shorter antigenic peptide sequences will provide advantages in certain circumstances, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution.

It is proposed that particular advantages of the present invention may be realized through the preparation of synthetic peptides which include modified and/or extended epitopic/immunogenic core sequences which result in a "universal" epitopic peptide directed to the LYST gene product or LYST-related sequences. It is proposed that these regions represent WO 97/28262 PCT/US97/01748 63 those which are most likely to promote T-cell or B-cell stimulation in an animal, and, hence, elicit specific antibody production in such an animal.

An epitopic core sequence, as used herein, is a relatively short stretch of amino acids that is "complementary" to, and therefore will bind, antigen binding sites on LYST protein epitopespecific antibodies. Additionally or alternatively, an epitopic core sequence is one that will elicit antibodies that are cross-reactive with antibodies directed against the peptide compositions of the present invention. It will be understood that in the context of the present disclosure, the term "complementary" refers to amino acids or peptides that exhibit an attractive force towards each other. Thus, certain epitope core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the desired protein antigen with the corresponding protein-directed antisera.

In general, the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core sequence or sequences. The smallest useful core sequence expected by the present disclosure would generally be on the order of about 5 amino acids in length, with sequences on the order of 8 or 25 being more preferred. Thus, this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention. However, the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.

The identification of epitopic core sequences is known to those of skill in the art, for example, as described in U.S. Patent 4,554,101, incorporated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. Moreover, numerous computer programs are available for use in predicting antigenic portions of proteins (see Jameson and Wolf, 1988; Wolf et al., 1988).

Computerized peptide sequence analysis programs DNAStar® software, DNAStar, Inc., Madison, WI) may also be useful in designing synthetic LYST peptides and peptide analogs in accordance with the present disclosure.

To confirm that a protein or peptide is immunologically cross-reactive with, or a biological functional equivalent of, one or more epitopes of the disclosed peptides is also a straightforward matter. This can be readily determined using specific assays, of a single proposed epitopic sequence, or using more general screens, of a pool of randomly generated synthetic peptides WO 97/28262 PCT/US97/01748 or protein fragments. The screening assays.may be employed to identify either equivalent antigens or cross-reactive antibodies. In any event, the principle is the same, based upon competition for binding sites between antibodies and antigens.

Suitable competition assays that may be employed include protocols based upon immunohistochemical assays, ELISAs, RIAs, Western or dot blotting and the like. In any of the competitive assays, one of the binding components, generally the known element, such as the LYST gene product or LYST-derived peptides, or a known antibody, will be labeled with a detectable label and the test components, that generally remain unlabeled, will be tested for their ability to reduce the amount of label that is bound to the corresponding reactive antibody or antigen.

As an exemplary embodiment, to conduct a competition study between a LYST protein and any test antigen, one would first label LYST with a detectable label, such as, biotin or an enzymatic, radioactive or fluorogenic label, to enable subsequent identification. One would then incubate the labeled antigen with the other, test, antigen to be examined at various ratios 1:1, 1:10 and 1:100) and, after mixing, one would then add the mixture to an antibody of the present invention. Preferably, the known antibody would be immobilized, e.g, by attaching to an ELISA plate. The ability of the mixture to bind to the antibody would be determined by detecting the presence of the specifically bound label. This value would then be compared to a control value in which no potentially competing (test) antigen was included in the incubation.

The assay may be any one of a range of immunological assays based upon hybridization, and the reactive antigens would be detected by means of detecting their label, using streptavidin in the case ofbiotinylated antigens or by using a chromogenic substrate in connection with an enzymatic label or by simply detecting a radioactive or fluorescent label. An antigen that binds to the same antibody as LYST, for example, will be able to effectively compete for binding to and thus will significantly reduce LYST binding, as evidenced by a reduction in the amount of label detected.

The reactivity of the labeled antigen, a LYST composition, in the absence of any test antigen would be the control high value. The control low value would be obtained by incubating the labeled antigen with an excess of unlabeled LYST antigen, when competition would occur and reduce binding. A significant reduction in labeled antigen reactivity in the presence of a test WO 97/28262 PCT/US97/01748 antigen is indicative of a test antigen that is "cross-reactive", that has binding affinity for the same antibody. "A significant reduction", in terms of the present application, may be defined as a reproducible consistently observed) reduction in binding.

In addition to the peptidyl compounds described herein, the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key portions of the peptide structure. Such compounds, which may be termed peptidomimetics, may be used in the same manner as the peptides of the invention and hence are also functional equivalents. The generation of a structural functional equivalent may be achieved by the techniques of modelling and chemical design known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope of the present invention.

Syntheses of epitopic sequences, or peptides which include an antigenic epitope within their sequence, are readily achieved using conventional synthetic techniques such as the solid phase method through the use of a commercially-available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptide antigens synthesized in this manner may then be aliquoted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may be readily stored in aqueous solutions for fairly long periods of time if desired, up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity. However, where extended aqueous storage is contemplated it will generally be desirable to include agents including buffers such as Tris or phosphate buffers to maintain a pH of about 7.0 to about 7.5. Moreover, it may be desirable to include agents which will inhibit microbial growth, such as sodium azide or Merthiolate. For extended storage in an aqueous state it will be desirable to store the solutions at 4 0 C, or more preferably, frozen. Of course, where the peptides are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled) or buffer prior to use.

4.13 SITE-SPECIFIC MUTAGENESIS Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the WO 97/28262 PCT/US97/01748 underlying DNA. The technique, well-known to those of skill in the art, further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.

Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 14 to about 25 nucleotides in length is preferred, with about 5 to about 10 residues on both sides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications. As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage are readily commercially-available and their use is generally well-known to those skilled in the art.

Double-stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double-stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically.

This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain WO 97/28262 PCTIUS97/01748 57 sequence variants. Specific details regarding these methods and protocols are found in the teachings of Maloy et al, 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each incorporated herein by reference, for that purpose.

4.14 BIOLOGICAL FUNCTIONAL EQUIVALENTS Modification and changes may be made in the structure of the peptides of the present invention and DNA segments which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics. The following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, secondgeneration molecule. The amino acid changes may be achieved by changing the codons of the DNA sequence, according to the codon chart listed in TABLE 1.

WO 97/28262 PCTIUTS97/01748 .TABLE 1 Amino Acids Alanine Ala Cysteine Cys Aspartic acid Asp Glutamic acid Glu Phenylalanine Phe Glycine Gly Histidine His Isoleucine Ile Lysine Lys Leucine Leu Methionine Met Asparagine Asn Proline Pro Glutamine Gin Arginine Arg Serine Ser Threonine Thr Valine Val Tryptophan Trp Tyrosine Tyr .TABLE 1

GCA

UGC

GAC

GAA

UUC

GGA

CAC

AUA

AAA

UUA

AUG

AAC

CCA

CAA

AGA

AGC

ACA

GUA

UGG

UAC

GCC

UGU

GAU

GAG

UUU

GGC

CAU

AUC

AAG

UUG

AAU

CCC

CAG

AGG

AGU

ACC

GUC

UAU

Codons GCG GCU GGG GGU

AUU

CUA CUC CUG CCG CCU

CUU

CGA

UCA

ACG

GUG

CGC

UCC

ACU

GUU

CGG

UCG

CGU

UCU

For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.

WO 97/28262 PCT/US97/01748 In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine valine leucine phenylalanine cysteine/cystine methionine alanine glycine threonine serine tryptophan tyrosine proline histidine glutamate glutamine aspartate asparagine lysine and arginine It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine lysine aspartate glutamate serine asparagine glutamine glycine threonine proline alanine histidine cysteine methionine valine leucine isoleucine tyrosine phenylalanine tryptophan It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

60 As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid sidechain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

5.1 EXAMPLE 1 MAPPING OF THE BG CRITICAL REGION ON MOUSE CHR 13 Three mouse mutations whose molecular basis is unknown, beige crinkled and progressive motor neuronopathy are clustered within 2 cM onproximal muse Chr 13. As part of a regional positional cloning effort, a high resolution physical map has been established of a 0.24 cM interval of mouse Chr 13 which corresponds to the bg critical region. 11 Yeast-artificial chromosomes (YACs) Sand 2 P1 clones, isolated using bg critical region STS, were characterised by STS-content mapping. This was achieved using existing microsatellite markers and 20 novel sequence tagged sites 35 (STS) which were generated from critical region YAC clone DNA by inverse-repetitive element PCR™ and direct selection. 2400-kb of the bg-critical region was isolated in YAC and P1 clones. Expressed sequence tags were identified from a bg-critical region YAC clone by direct selection, and represent potential candidates for bg and cr.

Positional cloning represents and approach to disease gene identification based solely upon chromosomal location. In the years since its inception, positional cloning has become established as a general, relatively efficient mode of identification of genes Scausing mammalian H:\Enma\Keep\Specis\18562.97.doc 16/02/00 WO 97/28262 PCTIUS97/01748 Mendelian disorders (Collins, 1995). Recently developed techniques and resources have both disencumbered and codified positional cloning; precise genetic mapping of a locus is followed by physical mapping and cloning of the resultant nonrecombinant interval in overlapping genomic clones (contigs) constructed using vectors which accommodate large DNA inserts. Transcribed sequences are then systematically identified from contig genomic clones and screened for mutations in affected individuals. An additional advantage of positional cloning is that it represents a regional, rather than disease-specific, approach. Thus reagents and resources developed for the purpose of cloning a specific disease gene, such as novel sequence tagged sites (STS), precise genetic maps, and establishment of relationships among clones in a contig, are also useful in positionally cloning other loci mapping within the same genomic region.

The region of proximal mouse Chr 13 adjacent to the extra-toes (Xt) locus is rich in mutant phenotypes, and represents an interval where a regional approach to disease gene identification may be synergistic. Xt is homologous to the human disorder Greig cephalopolysyndactyly; using a positional candidate approach, mutations in a zinc-finger gene (Gli3) were shown to underlie Xt (Vortkamp et al., 1992; Hui and Joyner, 1993). Very close to Xt lies the recessive mutation progressive motor neuronopathy (pmn), a model for Werdnig- Hoffmann spinal muscular atrophy (0 recombinants in 246 meioses, Brunialti et al., 1995). The recessive mutation crinkled (cr) maps approximately 2 cM proximal to Xt (23 recombinants in 1197 meioses; Swank et al., 1991; Lyon et al., 1967). Finally, beige the homolog of human Chediak-Higashi syndrome, maps between cr and Xt (Lane, 1971; Lyon and Meredith, 1969). bg is particularly amenable to a positional cloning approach for 3 additional reasons: the existence of numerous bg alleles facilitates candidate gene mutation analysis; bg is associated with a characteristic cellular phenotype (giant, perinuclear, dysfunctional lysosomes) offering the possibility of screening candidate genes by genetic complementation; and direct selection can be utilized to identify transcribed sequences which are candidates for bg from YAC clones since all cell types are affected in bg homozygotes.

Positional cloning of bg has been performed as an antecedent to identification of the homologous human gene, which is probably defective in human Chediak-Higashi syndrome.

Using backcross mice, bg was previously located to a 0.24 cM interval on Chr 13. The example WO 97/28262 PCTIUS97/01748 illustrates the further characterization of the bg critical region with 20 novel sequence tagged sites (STS), and the isolation of overlapping YAC and PI clones which encompass most of this region of mouse Chr 13.

5.1.1 MATERIALS AND METHODS 5.1.1.1 YAC MANIPULATION A mouse genomic DNA library constructed in the vector pYAC4 (Kusumi et 1994; Research Genetics Inc.) was screened by PCR T with primers derived from STS flanking bg.

False positive PCR™ products were minimized by raising annealing temperatures, and addition of an enhancer of polymerase specificity as necessary (Perfect Match, Stratagene, La Jolla, CA).

Veracity of PCR T products was checked by product digestion with suitable restriction endonucleases, and by inclusion of control yeast DNA in all PCR T reactions. Individual colonies of yeast clones containing YACs of interest were isolated on plates and frozen in 50% glycerol to prevent occurrence of microdeletions. YAC clones were grown in liquid YPD medium, converted to spheroplasts at exponential growth using Zymolase (ICN Pharmaceuticals, Costa Mesa, CA), and chromosomal DNA purified in agarose. YAC DNA was separated from host yeast chromosomes using preparative pulsed field electrophoresis (PFGE) with low melting point agarose (SeaPlaque GTG, FMC Bioproducts, Rockland, ME), and excised with a sterile blade.

5.1.1.2 P1 CLONES A mouse genomic DNA library constructed in the vector P1 (Pierce et al., 1992; Genome Systems Inc., St. Louis, MO) was screened by PCRT with primers derived from STS flanking bg.

Stabs corresponding to positive clones were streaked on kanamycin plates, and DNA prepared from individual colonies as described (Pierce et al., 1992).

5.1.1.3 PULSED FIELD ELECTROPHORESIS Preparation of high molecular weight DNA in agarose blocks, restriction enzyme digestion, PFGE, and Southern transfer were performed as previously described (Kingsmore et al., 1989). In brief, mouse splenocytes, lymph node cells, or yeast spheroplasts, were suspended in 0.5% low-melting point agarose (InCert®, FMC BioProducts) at 1-2 x 10 7 cells per ml (mammalian cells) or 1-2 x 1010 cells per ml (yeast). DNA was prepared by incubation of agarose blocks in 500 mM EDTA (pH 1% sodium lauroyl sarcosinate, 2% proteinase K at WO 97/28262 PCTIUS97/01748 twice for 24 h. Blocks were then washed, treated with phenylmethylsulfonylfluoride, washed again, and digested with 2-10 units/pgDNA of restriction endonucleases (Boehringer-Mannheim Biochemicals, Indianapolis, IN), if necessary. PFGE was carried out in 1% agarose gels (Fastlane, FMC BioProducts) at 14°C in IX TBE using a Gene Navigator unit (Pharmacia, Piscataway, NJ). Separation of 50-1500 kb DNA molecules was achieved using pulses ramped from 70-145 sec at 145 V for 46 h. Gels were stained with ethidium bromide to visualize molecular size standards (oligomers of X phage, and chromosomes of Saccharomyces cerevisiae [FMC BioProducts]). Southern transfer of DNA onto Zeta-probe T membranes (Bio-Rad Laboratories), and filter hybridizations were performed as previously described (Kingsmore et al., 1989). Assignment of two probes to a common restriction fragment was based on sequential hybridization of a filter and exhibition of identity by double or partial digests.

5.1.1.4 MOLECULAR PROBES All probes were labeled by the hexanucleotide technique with 3 2 P]dCTP as previously described (Kingsmore et 1989). Restriction endonuclease fragments representing ends of YAC clones were identified by Southern blot hybridization with pBR322 (which hybridizes efficiently to pYAC4); YAC clone internal restriction endonuclease fragments were identified by hybridization with a mouse B 1 repetitive element probe.

5.1.1.5 INTERSPERSED REPETITIVE ELEMENT-POLYMERASE CHAIN REACTION IRE-PCR was performed essentially as described using mouse B1 repetitive element primers and PFGE-purified YAC DNA as template (Hunter et al., 1993; Simmler et al., 1991).

The BI repetitive element-specific primers used were 5'-CCAGGACACCAGGGCTACAGAG-3' (SEQ ID NO:75) (forward primer, derived from the 3'-end of Bl) and /or CCCGAGTGCTGGGATTAAAG-3' (SEQ ID NO:76) (reverse primer, derived from the of B1). Inter-B1 PCR T was performed with the forward primer alone, the reverse primer alone, or both primers together. PCR T amplification reactions were performed using 40 ng of YAC DNA, 1 M of each primer, and 200 gM of each dNTP in a 20 pl reaction. Cycling parameters were 95°C for 2 min, followed by 32 cycles of 94 0 C for 20 sec, 55°C for 30 sec, and 72 0 C for 2 min. IRE-PCR products were isolated either by band excision from low-melting agarose gels, or by TA subcloning (Invitrogen). IRE-PCR products were sequenced, screened for the WO 97/28262 PCT/US97/01748 presence of common mouse repetitive element sequences, and nonrepetitive regions of the sequence used to design oligonucleotides suitable for sequence tagged sites (STS).

5.1.1.6 DIRECT SELECTION Direct selection was performed as previously described (Lovett et al., 1991; Lovett, 1994). Briefly, cDNA was generated from mouse spleen by reverse transcription using randomand oligo(dT)-priming, ligated to amplification cassettes, and PCR T amplified. Preparative PFGE was used to purify YAC 195A8 DNA, which was biotin-labelled, denatured, and hybridized in solution to the denatured cDNA pool. Repetitive elements, cDNA corresponding to rRNA, and yeast genes were blocked to Cot=20. YAC DNA (with annealed cDNAs) was captured on streptavidin-coated beads, washed at high stringency, and encoded cDNAs eluted.

Eluted cDNAs were PCRm-amplified, and subjected to a further round of direct selection.

Selected cDNAs were reamplified by PCR

T

subcloned into Xgtl0, and individual clones picked into SM buffer in 96-well plates. Direct selection products were amplified from phage-containing supernatents by PCR T with the following primers: 5'-GTTGTAAAACGACGGCCAGTGGCAAGTTCAGCCTGGTTAAG-3' (SEQ ID NO:77); and '-CACAGGAAACAGCTATGACCAGAGTATTTCTTCCAGGGTA-3' (SEQ ID NO:78).

Direct selection amplicons were cycle sequenced with standard M13 forward and reverse primers. Oligonucleotides suitable for STS were designed using direct selection product sequences.

5.1.1.7 STS PCR T M

PCR

T amplification reactions were performed using 40 ng of template DNA (YAC clone, P1 clone, S. cerevisiae strain 1380, or C57BL/6J genomic DNA), 1 IM of each primer, and 200 gM of each dNTP in a 20 pl reaction as described (Barbosa et al., 1995). Cycling parameters were 95 0 C for 2 min, followed by 34 cycles of 94°C for 20 sec, 45-58 0 C for 30 sec, and 72 0 C for 20 sec. Amplification products were separated on 3% agarose gels, and visualized by ethidium bromide staining, or by end-labeling one of the primers using 32 P]ATP and T4 polynucleotide kinase, and separation of products on 6% denaturing polyacrylamide gels, with autoradiographic visualization. Simple sequence length polymorphism (SSLP) primers were as described (Dietrich WO 97/28262 PCTfUS9/0148 et al., 1994; Research Genetics Inc., Hunstsville, AL). Novel STS primer sequences, amplicon sizes, and annealing temperatures are summarized in Table 2.

5.1.2 RESULTS AND DISCUSSION 5.1.2.1 ISOLATION OF YACS AND P1i 11 YAC clones and 2 P1 clones were isolated from mouse YAC and P1 libraries by

PCR

T using markers genetically mapped within the bg critical region. YAC clone sizes, as determined by PFGE, Southern blotting and hybridization with pBR322, are illustrated in FIG. 1.

YAC clones were examined for chimerism, microdeletions, and overlaps by STS content mapping. Previously described SSLP were the first source of STS to be utilized. The genomic region encompassing bg is particularly rich in such SSLP (38 have been localized within a 2 cM interval containing bg; Dietrich et al., 1994). Additional proximal chromosome 13 STS were generated using IRE-PCRTM and direct selection.

5.1.2.2 NOVEL CHR 13 STS DERIVED BY IRE-PCR T M IRE-PCRTM represents a rapid and facile method with which to saturate a genomic region with novel STS for initial characterization of YAC clones and contig development (Hunter et al., 1993; Simmler et al., 1991). IRE-PCR was performed using YAC DNA as template and primers derived from ends of the mouse repetitive element B which were oriented in opposite directions. IRE-PCRT products were subcloned, sequenced, and nonrepetitive regions used to design oligonucleotides suitable for sequence tagged sites. 12 novel STS (D3Sfkl-D13Sfk12) were developed by this method (Table and physically assigned to Chr 13 YAC and P1 clones by PCR T (FIG. 2).

5.1.2.3 NOVEL CHR 13 STS DERIVED BY DIRECT SELECTION Direct selection was performed with YAC 195A8, a 650-kb YAC which was easily purified from preparative pulsed field gels since it did not comigrate with host yeast chromosomes. 192 candidate cDNA fragments were eluted from YAC195A8 following two rounds of direct selection with mouse splenocyte cDNA. 56 of these direct selection products were sequenced. Comparison with DNA sequence databases revealed 2 nidogen (Nid), 32 novel, 12 repetitive elements (Bl=2, B2=1, LINE1=4, IAP=2, XL30=1, MT=1, satellite=l), and 9 contaminants (rRNA=3, actin=l, Nip2=l, plasmid=4). The presence of WO 97/28262 PCT/US97/01748 Nid cDNA fragments among these products confirmed the efficacy of the selection procedure in enriching for YAC 195A8-encoded genes. Furthermore, of 8 STS corresponding to novel direct selection products, 7 mapped back to YAC195A8 by PCR T analysis (D13Sfk3-D13Sfk9; Table 2, FIG. D13Sfkl3 and D13Sfk8 also hybridized sufficiently well to Southern blots to permit physical mapping adjacent to Nid on a polymorphic NotI fragment (1100-kb in DBA/2J DNA and 1150-kb in SB/LeJ DNA). D13Sfk13 was also genetically mapped within the bg critical region in 504 backcross mice [C57BL/6J-bg' X (C57BL/6J-bg x CAST/Ei)Fi] using a TaqI polymorphism.

5.1.2.4 ARRANGEMENT OF PROXIMAL CHR 13 YAC AND P1 CLONES IN CONTIGS YAC and P1 clones were typed for the presence or absence of STS derived from SSLP,

IRE-PCR

M amplicons, and direct selection products. STS content mapping enabled examination of clones for chimerism and microdeletions. One YAC clone, 64F5, was chimeric. This YAC, while 580-kb in size (FIG. contained only D13Mit44, and not STS derived from the or 3'ends of Nid (FIG. Since the latter two STS are separated by less than 65-kb in mouse genomic DNA (Durkin et al., 1995), and since D13Mit44 is located within the Nid gene, the portion of YAC 64F5 derived from Chr 13 was concluded to be less than YAC clone (84A8) contained an internal deletion which included D13Sfk6 (FIG. 2).

Furthermore, the physical size of 84A8 (370-kb) was considerably smaller than expected: the distance between the other genetic markers it encompassed was approximately 600-kb, confirming a substantial genomic deletion within this YAC. Some YAC clones have been reported to be unstable in culture, and become progressively smaller with time (Nehls et al., 1995). YAC 84A8 may exhibit such instability.

STS content mapping also enabled ordering of YAC and P1 clones within the bg critical region and integration of clones into 2 contigs (FIG. Contig 1 comprised 7 YAC and 2 P1 clones, extended from D13Sfk19 to D13Sfk2, and was approximately 1150-kb in length. The orientation this contig with respect to centromere was not established. The second contig 2 consisted of 2 YAC clones. It extended from D13Mit207 (proximal) to D13SfklO (distal), and was approximately 1000-kb in length. Contig 2 spanned the crossover defining the distal border of the bg critical region (FIG. Despite STS content mapping, 2 additional critical region YAC clones remained unlinked with these contigs (165F7 and 148E11). Isolation of YAC end clones WO 97/28262 PCT/US97/01748 will be necessary to definitively evaluate whether overlaps exist between these YACs and contig 1 or 2.

Efforts to identify YAC clones corresponding to one critical region genetic marker (D13Mit114) and the two STS which define the proximal border of the bg critical region (D13Mit172 and D13Mit239) were unsuccessful; furthermore, these STS were not present in any of the Chr 13 YAC/P1 clones identified. These data suggest that a region of the nonrecombinant interval remains unrepresented in the present YAC and P1 clones, or, alternatively, that additional microdeletions exist in the YAC clones. Based upon evaluation of overlaps between YAC and P1 clones, the bg critical region was estimated to be at least 2400-kb in length.

Direct selection products identified from YAC 195A8 using splenocyte cDNA not only allowed STS content mapping of Chr 13 YACs, but also constitute candidate genes for bg and cr.

Both of these mouse mutations appear to result from defects in constitutively expressed genes by virtue of abnormal phenotypes in all organs examined. The large number of bg alleles available enables effective screening of candidate genes by a combination of Southern and northern hybridization and RT- PCR

T

using nucleic acid from multiple bg alleles and coisogenic controls.

While such studies are inefficient methods for detection of point mutations, they are highly effective in detection of intragenic deletions, retrotranspositions, and genomic rearrangements, which together account for a large enough proportion of spontaneous mouse mutations to make likely the detection of a mutation in one of the bg alleles. While only one allele of cr exists, it arose in offspring of a mouse treated with nitrogen mustard, and therefore is more likely to be associated with a genomic rearrangement detectable using the same screening techniques.

In summary, approximately 2400-kb of the bg critical region has been physically mapped and isolated in the form of YAC and P1 clones. These studies represent an necessary intermediate step in positional cloning of bg, and may also be of value in positional cloning of cr and pmn.

TABLE 2 Novel Sequence Tagged Sites (STS) Isolated from bg Critical Region YACs by

IRE-PCR

Tm (DJ3SjkJ-Dl3Sflcl2) or Direct Selection (Dl 3Sfk13-Dl3Sfkl 9) Locus Assay Forward Primer SEQ ID Reverse Primer SEQ ID Ann. Amplicon (5r-31) NO: NO: Temp. Size (bp) DJ3SJkJ Irel CATTCTTTATTGACAGTGTT 15 CACATTGGCACAGGAAAC 37 500C 132 bp DJ3SJk2 Ire2 GTGAACCCTACCATATCT 16 TTACGAATGTGCCTGGTG 38 50 0 C 153 bp DJ3S~k3 Ire3 ATGCCATTCTITATTGACAG 17 TCCAAACACACTAAACCTG 39 500C 181 bp "I3Sjk4 Ire4 CATTGGCACAGGAAACAAC 18 CATGCCATTCTTTATTGACA 40 501C 134 bp "DI3SjkS Ire6 AAACCCTGTCTCGAAACAA 19 CTCAGTAGACTATACGAG 41 50 0 C 155 bp DJ3Sjk6 IrelO GAATTCCCAAGGACAGGT 20 GAGTrCAAGGTCATCCTC 42 50 0 C 153 bp Dl3Slc7 Irel3 TAGGAGGTGTGGCC'TrG 21 GCCTAAGCCCATTATCG 43 50 0 C 132 bp DI3SJk8 Irelt7 AGAGACGGCGGACACTTA 22 TAAATGCTGCCATAAACTCC 44 50 0 C 143 bp Dl3Sflc9 Ire3t7 TAAATATGAGGCGGGCAG 23 AGAAGTGACTTCAGGTAATA 45 50 0 C 146 bp DJ 3SJkJO0 Ire6t7 ATACTCTAAGTAAGATACAC 24 AGTCTCTCACACTTACAC 46 50 0 C 153 bp DJ3SJJl Ire9 TGTTTTAACTGTTTGCTAA 25 GACCCAAGTCAGCTTTC 4750 0 C 157 bp DJ3SJkI2 Irel.1 ACGCAGTGGGCATGCTG 26 CCAGTGTGTCACTTAAGC 48 52 0 C 190 bp DI3SJkJ3 BI CACAGGCTGTGACTGGAA 27 AGGGAGATGTATCATCTGC 49 56 0 C 89 bp DJ3SflcJ4 B9 AGTAGCCACAGGCCCTA 28 AGGGATCACCATGCTTTG 50 52 0 C 78 bp DJ3SjkJS CI#2 GAGATTACCCCAATAGTA 29 ACTTGGTCTTGGGGTCC 51 50 0 C 60 bp DJ3Sfkl6 C7. AGTGGAAGGAGGCTGTC 30 GAAAGCAGTGTAATGAGG 52 57 0 C 76 bp 2 continued Locus Assay Forward Primer SEQ ID Reverse Primer SEQ ID -Ann. Amplicon Name NO: Tern Size (bp) DJ3S7kI7 E7 CCATGGCGATGAAGCGG 31 TGCCTCTACATGGGAGC 53 541C 75 bp D13SkJ8 F71 ATGATGCAAAGAACCCAG 32 GCAAGCATTTAGTTAAACG 54 52'C 120 bp DI13 SJkJ 9 H7 'TTCAACTACATAGTGAA-TT 3 CTTG'ITCTTGTATATCTG 5 5C 13b Nid Nid5' TCCCTAACACATCCCTAA 34 ACAATGAAATCCTCCACC 56 54'C 168 bp Nd Nid3' CCTCATGT]7AGGGTAGAG--. 35 GTGACTTGATCCAGACTG 57 54 0 C 150 bp Esm9 Rs25b3' CCGTITAGTGTGTAGTT 36CTGTTCACTGTTCTC 58 52C 142 bp WO 97/28262 PCT/US97/01748 5.2 EXAMPLE 2 MAPPING OF THE BEIGE LOCUS TO MOUSE LYST 13 This example illustrates the generation of a high resolution genetic map of proximal Chr 13 in the vicinity of bg, and the identification of two genes which are tightly linked to bg. These studies precisely localize bg on Chr 13, and provide a foundation for YAC contig development and efficient screening of candidate genes for bg.

5.2.1 MATERIALS AND METHODS 5.2.1.1 MICE C57BL/6J-bg' X (C57BL/6J-bg x CAST/EiJ)Fi backcross mice were bred and maintained as described (Barbosa et al., 1995). (C57BL/6J-bg x PWK)Fi X C57BL/6J-bg' backcross mice, and (C57BL/6J-bg x PAC)Fi X C57BL/6J-bg J backcross mice used have been described (Holcombe et al., 1991).

5.2.1.2 SOUTHERN HYBRIDIZATION DNA was isolated from mouse organs using standard techniques and digested with restriction endonucleases, and 10 utg samples were subjected to electrophoresis on 0.9% agarose gels. DNA was transferred to Zeta-probe membranes (Bio-Rad Laboratories, Hercules, CA), and filter hybridizations were performed as previously described (Barbosa et al., 1995).

5.2.1.3 NORTHERN BLOT ANALYSIS ig of total RNA prepared from liver, spleen and kidney of C57BL/6J-+/+, C57BL/6Jbg', SB/LeJ-bg, and C3H/HeJ-bg' mice using standard techniques, was separated on formaldehyde agarose gels, transferred to Zeta-probe membranes (Bio-Rad Laboratories), and hybridized as previously described (Kingsmore et al., 1994).

5.2.1.4 RT- PCR T M

ASSAYS

Total RNA was prepared from liver of C57BL/6J-+/+, C57BL/6J-bg, SB/LeJ-bg, and C3H/HeJbg" mice by extraction with phenol guanidine isothiocyanate (TRIzol7, Gibco BRL, Gaithersburg, MD). The template for quantitative RT- PCR" assays was 1-10 ng of first-strand WO 97/28262 PCT/US97/01748 7' cDNA, which had been synthesized from total RNA with an oligo(dT) primer and Moloney murine leukemia virus reverse transcriptase (Stratagene, La Jolla, CA). The nidogen (Nid) primers used for RT- PCR M correspond to bp 3805-3822, and bp 3938-3955 of the mouse Nid cDNA (Durkin et al., 1988). The Estm9 primers used were: 5'-CAGGTGGAGATGCTGTTC-3' (Fl) (SEQ ID NO:59) 5'-GAGATGCCTTCAGGCAGT-3' (RI) (SEQ ID 5'-CCGTTAGTGTGTAGTCTC-3' (F2) (SEQ ID NO:61) 5'-CTTGCTCTCACTGTTCTC-3' (R2) (SEQ ID NO:62).

These correspond to the 5' and 3' ends, respectively, of an Estm9 cDNA (Bettenhausen and Gossler, 1995). RT-PCR7 products were amplified from bg, bg, bg and RNA with Nid primers or Estm9 primers Fl-R1 or F2-R2. Quantitative RT-PCR7 of aldolase A, which is constitutively expressed, was also performed, to ensure that equal amounts of bg, bg', bg", and template were used (Aldolase A primer 1: 5'-TGGATGGGCTGTCTGAACGC-3', (SEQ ID NO:63); primer 2: 5'-TGCTGGCAGATGCTGGCATA-3', (SEQ ID NO:64).

PCR

M reactions were performed in a 50 p.1 volume containing 1-20 ng of cDNA, 1 M of each primer, 200 pM each dNTP, 10 mM Tris-HC1, pH 8.8, 50 mM KCI, 1.5 mM MgCI 2 and 1.25 U AmpliTaq7 DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT). Cycling profiles consisted of an initial denaturation (94EC for 2 min) followed by 25 cycles of 94EC for 30 sec, 55-58EC for 30 sec, and 72EC for 1 minute per kb of expected product length. PCR T products were separated by electrophoresis on agarose gels, and quantified by intensity of ethidium bromide staining.

5.2.1.5 SSLP PCR T M

PCR

M amplification reactions were performed using 40 ng of genomic DNA, 1 4M of each primer (Dietrich et 1994; Research Genetics, Inc., Huntsville, AL), and 200 pM of each dNTP in a 20 pl. reaction as described (Barbosa et al., 1995). Cycling parameters were 95EC for 2 min, followed by 36-38 cycles of 94EC for 20 sec, 58EC for 30 sec, 72EC for 10 sec. Where WO 97/28262 PCT/US97/01748 '72possible, amplification products (20 pl) were separated on 3% agarose gels, and visualized by ethidium bromide staining. SSLP with allele sizes differing among strains by less than 8 bp were typed by end-labeling one of the primers using [y 32 P]ATP and T4 polynucleotide kinase, separation of amplification products (4 pl) on 6% denaturing polyacrylamide gels, and visualization by autoradiography. SSLP allele sizes are summarized in FIG. 3A, FIG. 3B, FIG.

3C and FIG. 3D.

5.2.1.6 PULSED FIELD ELECTROPHORESIS Preparation of high molecular weight DNA in agarose blocks, restriction enzyme digestion, pulsed field electrophoresis (PFGE), and Southern transfer were performed as previously described (Kingsmore et al., 1989). In brief, mouse splenocytes or lymph node cells were suspended in 0.5% low-melting point agarose (InCert, FMC BioProducts, Rockland, ME) at 1-2 H 107 cells per ml. DNA was prepared by incubation of agarose blocks in 500 mM EDTA (pH 1% sodium lauroyl sarcosinate, 2% proteinase K at 50EC twice for 24 h. Blocks were then washed, treated with phenylmethylsulfonylfluoride, washed again, and digested with 2-10 units/4g DNA of restriction endonucleases (Boehringer Mannheim Biochemicals). PFGE was carried out in 1% agarose gels (Fastlane, FMC BioProducts) at 14EC in IX TBE using a Gene Navigator system (Pharmacia, Piscataway, NJ). Separation of 50-1500 kb DNA molecules was achieved using pulses ramped from 70-145 sec at 145 V for 46 hr; 1000-6000 kb DNA was resolved by pulses of 15-90 min at 50 V for 6 or 10 days. Gels were stained with ethidium bromide to visualize molecular size standards (oligomers of X phage, and chromosomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe [FMC BioProducts]). Southern transfer of DNA onto Zeta-probe membranes (Bio-Rad Laboratories), and filter hybridizations were performed as previously described (Kingsmore et al., 1989). Assignment of two probes to a common restriction fragment was based on sequential hybridization of a filter and exhibition of identity by double- or partial-digests.

5.2.1.7 MOLECULAR PROBES All probes were labeled by the hexanucleotide technique with 32 P]dCTP as previously described (Kingsmore et al., 1989). The nidogen (Nid) probe used was pN-5 (Jenkins et al., 1991). The glioblastoma oncogene homolog-3 (Gli3) probe was derived from pGli3a (Hui and Joyner, 1993). The probes used for the T cell receptor chain (Tcrg), and the mid-gestation WO 97/28262 PCT/US97/01748 73 embryo cDNA ESTM9, have been described previously (Holcombe et 1991). Informative CAST/EiJ RFLV sizes are summarized in FIG. 3D; informative PAC and PWK RFLV for Tcrg were as described (Holcombe et al., 1991).

5.2.2 RESULTS Previous mapping studies, using 3 separate backcrosses segregating for the bg locus (2 intraspecific backcrosses [(C3H/HeJ x C57BL/6J-bg)Fi X C57BL/6J-bg J and [(C57BL/6J Wd-bg x Mus domesticus PAC)FI X C57BL/6J-bg i and an intersubspecific backcross [(C57BL/6J-Wh-bg xMus musculus PWK)FI X C57BL/6J-bg]}, have shown bg to lie proximal to Tcrg on mouse Chr 13 (Holcombe et al., 1987, 1991). In order to assess candidate genes for linkage to bg and as a precedent to positional cloning, the inventors have now generated a highresolution linkage map of proximal mouse Chr 13 using the latter 2 backcrosses and a third, novel backcross.

5.2.2.1 PHENOTYPIC ANALYSIS OF BG BACKCROSS MICE Three backcrosses segregating for bg were utilized; Phenotypic analysis of 109 (C57BL/6J -Wh-bg x Mus domesticus PAC)FI X C57BL/6J-bg backcross mice, and 111 (C57BL/6Jh bg J x Mus musculus PWK)FI X C57BL/6J-bg J backcross mice has been reported previously (Holcombe et al., 1991). The third backcross was established between C57BL/6J-bg' mice and Mus castaneus (CAST/EiJ), and 504 [C57BL/6J-bg X (C57BL/6J-bg' x CAST/EiJ)Fi progeny were generated. Mus castaneus was chosen as the second parent in the latter intrasubspecific backcross due to the increased likelihood of detection of DNA polymorphism in comparison to intraspecific crosses. Mice were phenotyped for the presence or absence of a beige-colored coat; Penetrance of bg in all of the crosses was complete (359 of 726 backcross mice exhibited a beige-colored coat).

5.2.2.2 IDENTIFICATION OF INFORMATIVE RFLV AND SSLP Informative RFLV were ascertained by hybridizing gene probes to Southern blots containing genomic DNA from C57BL/6J-bg and CAST/EiJ, PAC, or PWK parental mice digested with various restriction endonucleases. Table 3 lists the sizes of unique CAST/EiJ RFLV for Gli3 and Nid. PWK and PAC RFLV for Tcrg have been described previously (Holcombe et al., 1991); CAST/EiJ RFLV for Estm9 have been described previously. Informative SSLP were WO 97/28262 PCTIUTS97/01748 ascertained by PCR T of genomic DNA .from C57BL/6J-bg' and CAST/EiJ, PAC, and PWK parental mice. Approximate sizes of SSLP- PCRT products are listed in Table 3.

5.2.2.3 PRECISE GENETIC MAPPING OF BG ON PROXIMAL MOUSE CHR 13 111 (C57BL/6J -Wh-bg x Mus domesticus PAC)FI X C57BL/6J-bg backcross mice, 111 (C57BL/6J-W^-bgJ x Mus musculus PWK)F 1 X C57BL/6J-bg J backcross mice, and 504 [C57BL/6J-bg X (C57BL/6J-bg J x CAST/EiJ)Fi backcross mice were genotyped for a total of 23 SSLPs and 3 RFLVs known to map to proximal mouse Chr 13. At each locus, backcross DNA displayed either the homozygous or heterozygous FI pattern. Linkage relationships were determined using segregation analysis (Green, 1981), and the best gene order decided by minimization of crossover events and elimination of double crossover events (Bishop, 1985).

Haplotype analysis for each cross is shown in FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D.

Upon retyping of previously published genotypes of the PAC and PWK backcrosses (Holcombe et al., 1991), 4 errors were detected. In each case, the coat-color had been incorrectly assigned, resulting in the generation of a double crossover within a genetic interval of less than 0.5 cM; since such events are predicated against by positive interference, these animals were excluded from subsequent analysis. Upon exclusion of these animals, no significant differences in gene order or recombination frequencies were found among the three crosses.

The best gene order and recombination frequency standard deviation) for the [C57BL/6J-bg' X (C57BL/6J-bg' x CAST/EiJ)F 1 backcioss was: centromere D13Mit158, D13Mit72, D13Mit205, D13Mit206, D13Mit239 0.20 0.20 cM bg, Nid, Estm9, D13Mit44, D13Mit114, D13Mit134, D13Mit207 0.20 0.20 cM Gli3, D13Mit56, D13Mit62, D13Mit174, D13Mit237, D13Mit240, D13Mit305 0.20 0.20 cM D3Mit218, D13Mit219, D13Mit271 0.40 0.28 cM D13Mit3, D13Mi1133 telomere.

The best gene order and recombination frequency standard deviation) for the [(C57BL/6J -Wh-bg x Mus domesticus PAC)FI X C57BL/6J-bg J] backcross was: centromere D13Mit79 5.4 2.1 cM D13Mit 0.9 0.9 cM bg, D13Mit44, D13Mit134, D13Mi174, D13Mit205 0.9 0.9 cM Tcrg, D13Mit218, D13Mit219 3.6 1.8 cM D13Mit3 telomere.

The best gene order and recombination frequency standard deviation) for the [(C57BL/6J-Wh-bg' x Mus musculus PWK)FI X C57BL/6J-bg backcross was: centromere WO 97/28262 PCTIUS97/01748 D13Mit79 5.4 2.1 cM D13Mitl 0.9 0.9 cM bg', D13Mi44, D13Mit134, D13Mit205, D13Mit237 0.9 0.9 cM D13Mit174 0.9 0.9 cM Tcrg, D13Mit218, D13Mit219 0.9 0.9 cM D13Mit3 telomere.

A composite linkage map of proximal mouse Chr 13, derived by integration of these 3 crosses, is shown in FIG. 3D. The combined results delimit the region containing bg to a 0.24 0.17 interval on Chr 13, flanked proximally by the genetic markers D13Mit172 and D13Mit239, and distally by Gli3, D13Mit56, D13Mitl62, D13Mit237, D13Mit240, and D13Mit305. bg cosegregated with 6 genetic markers (Nid, Estm9, D13Mit44, D13Mit114, D13Mit134 and D13Mit207). Backcross mice with recombination events which define the bg nonrecombinant interval were derived from the [C57BL/6J-bg' X (C57BL/6J-bg J x CAST/EiJ)F 1 backcross.

5.2.2.4 EVALUATION OF THE CANDIDACY OF NID AND ESTM9 FOR CAUSALITY IN BG Given the availability of numerous bg alleles, it was reasoned that northern, Southern, and RT- PCR T M analyses would be effective modalities for initial evaluation of the candidacy of Nid and Estm9 for causality in bg.

Southern blots were generated with DNA from 6 bg alleles: SB/LeJ-bg, C57BL/6J-bg

J

C3H/HeJ-bg

J

DBA/2J-bg C57BL/6J-bg

L

j, C57BL/6J-bg and from appropriate coisogenic controls using 5 restriction endonucleases (EcoRI, HindIII, BamHI, MspI, and TaqI).

No restriction fragment length differences were observed between bg alleles and coisogenic controls upon hybridization with Nid or Estm9, excluding a deletion or insertion in these genes from causality in these bg alleles.

Expression of Nid and Estm9 in bg mice was examined by northern blot analysis and quantitative RT-PCR7. Hybridization of northern blots of liver and kidney RNA from bg, bg', and bg with probes for Nid and Estm9, yielded signals of similar size and intensity in bg and RNA. Furthermore, no difference in amplicon size or amount was observed upon quantitative RT-PCR7 using liver or kidney RNA from bg, bg and bg 2 mice and oligonucleotides for Nid or Estm9, indicating expression of Nid and Estm9 to be grossly intact in bg.

WO 97/28262 PCT/US97/01748 5.2.2.5 PHYSICAL MAPPING OF PROXIMAL MOUSE CHR 13 IN THE VICINITY OF BG Cytogenetic and physical mapping studies have demonstrated mouse mutations induced by gonadal x-irradiation to be frequently associated with genomic rearrangements (typically deletions or translocations). The SB/LeJ-bg allele was discovered among the offspring of a male which had received such treatment. In order to examine SB/LeJ-bg DNA for a genomic rearrangement, physical mapping studies were undertaken by pulsed field gel electrophoresis using high molecular weight DNA and restriction endonucleases which cleave infrequently. PFGE-Southern blots were generated using DNA from DBA/2, C57BL/6J-bgJ, CAST/EiJ and SB/LeJ-bg splenocytes, and probed sequentially with the 3 genes which map in the vicinity of bg (Nid, Estm9, and Gli3).

Physical linkage of these genes was not possible, since hybridization with Estm9, Gli3 and Nid gene probes revealed no bands of identical size (Table 4).

No differences were observed in the sizes of bands identified in SB./LeJ-bg and control DNA upon hybridization with Gli3 or Estm9 (Table However, hybridization of the same blots with an Nid gene probe did reveal band size disparities. With 5 restriction endonucleases (NotI, MluI, NruI, and Srfi complete digests, Nael partial digest, and NotI/MluI double digest), differences were observed between DBA/2 and the other DNAs (C57BL/6J-bg, CAST/EiJ and SB/LeJ-bg). In each case, the DBA/2 fragment was 25-50kb smaller than the band identified in C57BL/6J-bg, SB/LeJ-bg, or CAST/EiJ DNA (FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D; Table No differences in Nid band sizes were evident among other mouse strains examined (C57BL/6J-bg, SB/LeJ-bg, and CAST/EiJ). Other restriction endonucleases, which identify smaller fragments when probed with Nid (BssHII, ClaI, NaeI, Smal, Xhol) were identical in all strains tested (FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, Table Nid fragment size differences were observed using both methylation-sensitive and -insensitive restriction endonucleases.

5.2.3 DISCUSSION Previous studies have localized bg to proximal Chr 13. Lyon et al.,(1969) demonstrated bg to be 0.5 cM proximal to the mutation Xt, which corresponds to the Gli3 gene. Several groups have demonstrated tight linkage between bg and Tcrg (Holcombe et al.,1987, 1991; Justice et 1990). Jenkins et al.,(1991) found bg to cosegregate with Nid in 123 meiotic events. Precise genetic mapping of bg has been undertaken with respect to these genes and recently identified SSLP markers (Dietrich et al., 1994) as an antecedent to generation of a YAC contig of the genomic region encompassing bg. These results are in agreement with previous WO 97/28262 PCT/US97/01748 77 studies of genetic marker order on chromosome 13, although the greater number of meioses utilized in the present study permitted separation of loci which cosegregated in previous studies, and enabled localization of bg to a 0.24 cM interval on proximal mouse Chr 13. No statistically significant differences in genetic distances between markers were observed among the present crosses or between them and previous studies. Cosegregation of bg and Nid was observed in 504 meiotic events, suggesting bg to map within a linkage group conserved between proximal mouse Chr 13 and the distal long arm of human Chr 1 (Jenkins et al, 1991). By implication, the homologous human locus, CHS, may be expected to lie on human Chr lq42.1-1q43, which represent the approximate limits of this conserved linkage group (Jenkins et al., 1991; Mattei et al., 1994). Localization of bg to a 0.24 cM interval will enable the generation of a YAC contig encompassing bg. Those genetic markers which cosegregate with bg will serve as nucleation points for rapid contig assembly.

If it is assumed that a haploid mouse genome is 1500cM in size and contains 60,000, randomly distributed genes, it would be expected that the 0.24 cM bg critical region should contain 10 genes. In the present report, two genes, Nid and Estm9, were localized within this interval, and thereby represent candidate genes for the bg locus Nidogen, however, can be excluded from candidacy for bg for functional reasons. While bg mice exhibit a constitutive intracellular defect in lysosomal trafficking, nidogen is a component of basement membranes, a specialized extracellular matrix structure limited to certain tissues (Durkin et al., 1988). The candidacy of Estm9 cannot yet be evaluated on functional grounds. Estm9 is a novel mouse expressed sequence which was recently identified from a day 10.5 p.c. mouse embryo cDNA library (Bettenhausen and Gossler, 1995). Comparison of partial Estm9 cDNA sequences with DNA and peptide databases demonstrate significant sequence similarity only with uncharacterized human ESTs. While the function of Estm9 is unknown, expression analysis reveals it to be constitutively expressed, temporally and spatially, in the mouse (Bettenhausen and Gossler, 1995).

Initial genetic evaluation of the candidacy of Nid and Estm9 for bg by northern and Southern blot hybridization or quantitative RT- PCR

M

revealed no differences between several bg alleles and coisogenic controls. These studies do not definitively exclude Nid or Estm9 from candidacy for bg. A more robust method of evaluation for bg candidate genes would be genetic 30 complementation. Cell lines derived from bg mice exhibit pathognomonic phenotypes (Burkhardt et al., 1993; Gow et al., 1993; Baetz et al., 1995), which can be abrogated by genetic complementation (Perou and Kaplan, 1993; Penner and Prieur, 1987; Gow et al., 1993). Studies WO 97/28262 PCTIUS97/01748 to examine the ability of Nid or Estm9 to complement bg-associated phenotypes in vitro are being pursued.

Physical mapping studies of the bg critical region were undertaken to evaluate the radiation-induced SB-bg allele for the presence of a gross genomic rearrangement. SB-bg specific restriction fragment length differences were not observed with Nid, Estm9, or Gli3 gene probes. Furthermore, all critical region SSLP amplicons (D13Mit44, D13Mit114, D13Mit134 and D13Mit207) were present in SB-bg DNA. Together, these data preclude the existence of a gross genomic rearrangement in SB-bg DNA. However, DBA/2-specific pulsed-field electrophoresis RFLPs were observed with Nid using 5 restriction endonucleases. In each case, the DBA/2 fragment identified with Nid was 25-50 kb smaller than the corresponding band identified in control DNA (FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D). No difference in band sizes were observed among other strains or upon reprobing of PFGE-Southern blots with Gli3 or Estm9. Since fragment size differences were observed with many rare-cutting restriction endonucleases, including several which are methylation-insensitive, it is unlikely that they are merely interstrain differences in DNA methylation or point mutations. Instead, it is suggested that a genomic rearrangement has occurred in the DBA/2 mouse at a distance of less than 900 kb from Nid (FIG. 3D). The rearrangement may represent a small (25-50 kb) genomic deletion in the DBA/2 mouse. The functional significance of such a putative rearrangement is uncertain.

Interestingly, a similar phenomenon was recently described in the vicinity of the human nidogen gene (Goodrich and Holcombe, 1995) upon hybridization to pulsed field gel electrophoresis Southern blots of human genomic DNA digested with Sail, nidogen identified polymorphic band sizes in Caucasian populations. In 2 CHS patients that have been examined to date, homozygosity for one NID allele was observed, suggesting the possibility of linkage of human CHS and NID (Goodrich and Holcombe, 1995). Definitive mapping of human CHS, however, must await identification of the mouse bg gene. On a practical note, the interstrain differences in pulsed field restriction fragment length provide a physical landmark within the bg nonrecombinant interval. Thus bg candidate genes can be easily screened for physical linkage of with Nid as a means of determining whether or not they lie within the bg nonrecombinant interval.

In summary, the bg locus has been localized, which is the mouse homolog of human CHS, to a genomic interval corresponding to approximately one four-hundredth of mouse Chr 13. This represents an important intermediate step in the positional cloning of bg, and thereby human CHS.

WO 97/28262 WO 9728262PCTIUS97/01748 TABLE 3 Informative Proximal Chr 13 SSLP Allele Sizes SSLPAIlele DJ3MWt DJ3Mit D3M144(Nid) DJ3Mit6 D]3MCt9 DJ3Mit 14 DJ3Mit 33 Dl3Mit34 DJ3MWtI8 DJ3MitJ62 DJ3Mit-172 DiSMiti74 DJ3MiIOS D13Mit2O6 DJ3M1O7 D.13Mit2l8 DJ3Mit2J9 DJ3Mit23 7 DJ3Mit239 D13MiI24O D13Mit27J D13Mit3O5 *=Estimated size Size (bp) 147 159 124 226 107 149 150 123 144 105 146* 123 120 146 136 178 266 114 99 166 127 118 NDnot done C57BLI6J-bgj 149 196 116 260

NULL

137 181 137 135 135 154 135 140 168 148 194 282 102 116 124 135 144 CAST/EiJPAC 137* 240* 116* 226 103

ND

150 137*

ND

ND

ND

129* 128

ND

ND

186* 270 118* 120* 124*

ND

ND

PWK

178 116* 228* 91 149* 181 153

ND

105 158* 131* 132*

ND

ND

186* 282* 92* 130*

ND

ND

WO 97/28262 PCT/US97/01748 TABLE 4 Restriction Fragment Length Polymorphisms Used To Genetically Map Nid And GOi In 504 [C57BL/6J-BgJ X (C57BL/6J-BgJ X CAST/Eij)FIJ Mice Gene Probe Strain Informative Band Sizes (kb) Rest. Endo.

NO C57BL/6J-bg' MspI 3.2, CAST/EiJ 4.4,0.8 GOz C57BL/6J-b&/ MspI 5.0, 2.4, 1.5, 1.2 CAST/EiJ 5.0 3.01 2.4, 0.9 WO 97/28262 PCT/US97/01748 STABLE PFGE restriction fragment sizes (in kb) of Nid and Estm9 in SB/LeJ-bg and DBA/2J DNA Restriction Nid Estm9 Endonuclease DBA/2 SB-bg/bg* DBA/2 SB-bg/bg NotI 1100 1150 670, 300 670, 300 MluI 1800, 975 1850, 1025 770, 670 770, 670 NotV/MIuI 975 1025 Not done Not done Srfl 1000 1050 Not done Not done NaeI 900, 400 950, 400 280, 250 280, 250 ClaI 925, 650 925, 650 630, 500 630, 500 Sma l 200, 150 200, 150 Not done Not done BssHII 250, 25 250, 25 Not done Not done *SB/LeJ-bg band sizes were also observed using CAST/EiJ and C57BL/6J genomic DNAs.

5.3 EXAMPLE 3 IDENTIFICATION OF THE HOMOLOGOUS BEIGE AND CHS GENES As described above, the inventors have localized the bg locus within a 0.24 centimorgan interval on mouse chromosome 13, and isolated contiguous arrays of YACs that cover 2,400 kb of this interval. Candidate cDNAs for bg were isolated from YAC 195A8, which contains 650 kb of the bg non-recombinant interval using direct cDNA selection with mouse spleen cDNA (FIG. 11). Of 56 candidate cDNA clones analyzed from a direct-selection study, evidence for causality in bg was found in one (see below), and this gene was designated Lyst (lysosomal trafficking regulator). As this clone was 132 nucleotides long, additional Lyst sequences were sought by screening three mouse cDNA libraries and performing polymerase chain reaction (PCRTM) amplification of cDNA ends (Kingsmore et al., 1994). Ten overlapping Lyst clones were identified, representing -7 kb (Genbank accession number, L77889). These were physically assigned to mouse chromosome 13 with pulsed field gel electrophoresis (PFGE) Southern blots, confirming that they were all derived from a single gene (mouse genome database accession number, MGD-PMEX-14). The Lyst probes identified the same polymorphic PFGE restriction fragments as nidogen (Nid), indicating that Lyst and Nid are clustered within 650 kb. Lyst was also mapped genetically in 504[C57BL/6-bg x (C57BL/6J-bg x CAST/EiJ)F 1 backcross mice by means of three TaqI restriction fragment length polymorphisms (RFLPs). The Lyst RFLPs WO 97/28262 PCTIUS97/01748 cosegregated with bg (and Nid), confirming their colocalization on proximal mouse chromosome 13 (MGD accession number, MGD-CREX-615).

Evidence for Lyst mutations was found in two bg alleles. A 5-kb genomic deletion that contained the 3' end ofLyst exon P, and exons y and 5, was identified in bg 1 DNA (FIG. 12).

The bg"' deletion corresponds to the loss of -400 internal amino acids of the predicted Lyst peptide. Furthermore, whereas the 5' end of the bg 1 deletion occurs within Lyst exon 0, the 3' end is intronic. Therefore the truncated Lyst mRNA in bg"J mice is also anticipated to splice incorrectly, terminate prematurely, and lack polyadenylation.

Quantitative reverse transcription (RT)-PCRTM demonstrated a moderate decrease in Lyst mRNA in bg and bg' liver, and a gross reduction in bg (Lyst AOD after normalization for P-actin mRNA; 1.00; bg 2 0.19; bg/bg, 0.28; bg'/bg', 0.40). A commensurate reduction in bg transcript abundance was noted by using several primer pairs derived from different regions of the Lyst cDNA. Aberrant Lyst RT-PCRTM products were not observed. The particularly striking (more than fivefold) reduction in Lyst expression evident in bg" homozygotes suggested the existence of a mutation in bg" Lyst that results in decreased transcription or mRNA instability. The molecular basis of the decrease in Lyst mRNA in bg" is not yet known, but it is reminiscent of the leaky ablation of mature message associated with an intronic retrotransposition event (Kingsmore et al., 1994).

The predicted open reading frame (ORF) of Lyst was 4,635 nucleotides, encoding a protein of 1,545 amino acids and relative molecular mass 172,500 (Mr 172.5K) (FIG. 13a).

Nucleotides 51-74 are rich in CG nucleotides, a common feature of the 5' region of housekeeping genes. Comparison with DNA databases indicated that Lyst is novel, and resembles only uncharacterized human-expressed sequence tags (ESTs). The sequence of a cDNA clone corresponding to one such human EST (Genbank accession number L77889) matched the region of mouse Lyst (nucleotide identity was 76% in the 5' untranslated region (UTR), 91% in the ORF, and amin-acid identity was 97%; FIG. 13c); another human EST matched the 3' region of the mouse Lyst coding domain (Genbank accession number W26957). On hybridization to PFGE Southern blots of mouse DNA, the human clones identified restriction fragments that were indistinguishable from mouse Lystl; physical mapping of the human clones to the same region of the mouse genome as Lyst indicates that they are indeed homologous to Lyst.

WO 97/28262 PCTIUS97/01748 It has been suggested that CHS and bg represent homologous disorders, as their clinical features(Blume and Wolff, 1972) and defects in lysosomal transport (Burkhardt et al., 1993) are identical. Homology of bg and CHS is supported by genetic complementation studies; fusion of fibroblasts from bg mice and CHS patients failed to reverse lysosomal abnormalities, in contrast to fusions with normal cells (Perou and Kaplan, 1993). Furthermore, recent genetic linkage studies have shown that CHS maps within a linkage group conserved between human Chromosome 1q43 and the bg region on mouse Chromosome 13. Therefore LYST mutations in CHS patients were sought by sequencing LYST lymphoblast and fibroblast cDNAs corresponding to these ESTs from 10 CHS patients. In one patient, a single-base insertional mutation was found at nucleotides 117-118 of the LYST coding domain, resulting in a frame shift and termination after amino acid 62 (FIG. 13c).

Previous studies showing spontaneous aggregation of membrane-bound concanavalin A (capping) suggest that there is a defect in microtubule dynamics in bg cells (Oliver, Zurier and Berlin, 1975; Oliver and Zurier, 1976). In a search of the SWISSPROT database, using Blitz and BLASTP, a similarity was found between a domain in Lyst and stathmin (oncoprotein 18), a phosphoprotein that may regulate polymeration of microtubules (Belmont and Mitchison, 1996) (27% identity from residues 463 to 536; best expected occurrence by chance, 4.36 x 10 The domain is stathmin that matches Lyst is helical and has heptad repeats that participate in coiledcoil interactions with other proteins (Sobel, 1991; Maucuer et al., 1995). The stathmin-like region of Lyst is also predicted to be helical and formed coiled coils. However, it is the charged residues, rather than the hydrophobic ones, that are conserved between Lyst and stathmin, suggesting that the sequence similarity is not primarily due to conserved secondary structure.

Thus this region of Lyst potentially encodes a coiled-coil protein-interaction domain that may regulate microtubule-mediated lysosome transport. Although Lyst is no predicted to have transmembrane helices, the C-terminal tetrapeptide (CYSP; amino acids 1,542-1,545) is strikingly similar to known prenylation sites, which could provide attachment to lysosomal/late endosomal membranes through thioester linkage with the cysteine.

Previous studies of bg leukocytes have shown correction of microtubule function (as assessed by Concavalin A capping) and natural killer activity when treated with inhibitors of protein kinase C (PKC) breakdown (Sato et al., 1990; Ito et al., 1989), suggesting that bg might be regulated by phosphorylation. Lyst contains 25 sites of potential phosphorylation by PKC, 36 by casein kinase II (CKII) (many of which overlap those of PKC), two by cAMP-dependent WO 97/28262 PCT/US97/01748 protein kinase, and one by tyrosine kinase (FIG. 13b). Almost half of the predicted helices outside the stathmin-like region (14 of 30) have a PKC- or CKII-phosphorylation signal at their amino terminus, and eight of them form consecutive helical pairs. Thus Lyst seems to contain helical bundles with clusters of phosphorylation sites at either end. Stathmin also has an Nterminal phosphorylation site and helix motif, and these Lyst domains may have a similar 'signal relaying' function to stathmin (Sobel, 1991; Maucuer et al., 1995). Furthermore, phosphorylation of these positions could provide a control mechanism by causing a conformational shift in the bundles, thereby affecting interactions with other molecules.

Northern analysis and RT-PCR T M indicated that Lyst is ubiquitously transcribed, both temporally and spatially, in mouse and human tissues (FIG. 14). Northern blot analysis also revealed complex alternative splicing of Lyst mRNA, with both constitutive and anatomically restricted Lyst mRNA isoforms. The largest Lyst transcript in human and mouse was 12-14 kb, but this transcript was not constitutively expressed. In mRNA from mouse spleen, human peripheral blood leukocytes, promyelocytic leukaemia HL-60, and several leukaemia lines, the 12-14 kb isoform was either undetectable or barely detectable, but smaller Lyst transcripts were abundant (FIG. 14). Given the significance for bg mice and CHS patients of defects in the lysosomal and late-endosomal compartments of granulocytes, NK cells and cytolytic T lymphocytes (Gallin et al., 1974; Roder and Duwe, 1979; Saxena et al., 1982; Baetz et al, 1995), it is likely that these Lyst mRNAs of-3 kb and 4 kb represent the transcripts of primary functional significance. Probes derived from the 5' or 3' ends of the Lyst 5.4 EXAMPLE 4 MUTATION ANALYSIS AND PHYSICAL AND GENETIC MAPPING ESTABLISH HUMAN L YST AS THE CHS GENE 5.4.1 MATERIALS AND METHODS 5.4.1.1 CLONING OF THE HUMAN LYST GENE Segments of the human LYST sequence were obtained by an anchored, nested PCRTM RACE-PCRTM) using liver cDNA as a template (Clontech Laboratories, Palo Alto, CA), by RT-PCRTM using total RNA and by sequencing of human ESTs similar in sequence to mouse Lyst.

For the 5' RACE-PCRTM two nested primers were used that were derived from a human EST (GenBank accession number W26957) and had the following nucleotide sequence: WO 97/28262 PCT/US97/01748 5'-CCAAGATGAAAGCAGCCGATGGGGAAAACT-3' (SEQ ID NO: 65) and 5'-TCAGCCTCTTTCTTGCTCCGTGAAACTGCT-3' (SEQ ID NO: 66).

For RT-PCRTM experiments, total RNA was prepared from the promyelocytic HL-60 cell line. Reverse transcription was performed with Expand (Boehringer Mannheim, Meylan France) with the following primer pairs: 5'-AGTTTATGAGTCCAAATGAT-3' (SEQ ID NO: 67) and 5'-GAATGATGAAGTTGCTCTGA-3' (bp 490-2034) (SEQ ID NO:68), 5'-CAGCAGTTCTTCAGATGGA-3' (SEQ ID NO:69) and 5'-ATCTTTCTGTTGTTCCCCTA-3' (bp 1,891-3050) (SEQ ID 5'-TAGGGGAGCAACAGAAAGAT-3' (SEQ ID NO:71) and 5'-GCTCATAGTAGTATCACTTT-3' (bp 3320-4722) (SEQ ID NO:72).

The primers used to amplify the cDNA between bp 1891 and 3050 were derived from the mouse Lyst sequence. Human primers were designed from the sequence of the PCRTM product (1159 bp) and used to amplify the flanking sequences.

5.4.1.2 DNA SEQUENCING AND SEQUENCE ANALYSIS PCRTM products were cloned using a TA cloning kit (Invitrogen Corporation, San Diego California) and both strands were cycle sequenced. The sequences were analyzed with the GCG Package (Devereux etal., 1984) and searches of the National Center for Biotechnology Information database were performed using the BLAST network server (Altschul et al., 1990) (National Library of Medicine, via INTERNET) and the Whitehead Institute Sequence Analysis Programs (MIT, Cambridge, Massachusetts).

5.4.1.3 SOUTHERN AND NORTHERN BLOT ANALYSIS Preparation of mouse, human and yeast DNA samples, digestion with restriction endonucleases, agarose gel electrophoresis and Southern transfers were performed using standard techniques (Maniatis et al., 1984). The EcoRI monochromosomal somatic cell hybrid blot was obtained from BIOS Laboratories (New Haven, Connecticut). Isolation of poly(A)+RNA from fibroblast and EBV-transformed B lymphoblast cell lines, formaldehyde agarose gel WO 97/28262 PCTIUS97/01748 electrophoresis and Northern blotting were perfo ed according to standard procedures (Maniatis et al., 1984). Membranes were hybridization with various LYST or actin probes labeled with a32P-dCTP. Mouse genetic mapping analyses were performed as described (Barbosa etal., 1995).

5.4.1.4 SSCP ANALYSIS Detection of nucleotide changes by SSCP was performed as described by Orita et al.

(1989). Briefly, each PCRTM product was mixed with an equal volume of denaturing buffer and heated to 95C for 3 min., after which the samples were loaded onto 0.8 mm thick, 10% native polyacrylamide gels. Gels were run at ambient temperature at 9 W for 6-10 hours, depending on the size of the PCR T M product. Bands were visualized by silver-staining (Beidler et al., 1982).

5.4.1.5 ALLELE-SPECIFIC OLIGONUCLEOTIDE ANALYSIS PCRTM products spanning the mutation site in patient 371 were transferred to nylon membranes using a slot blot apparatus. Approximately 5 ng of each PCRTM product was treated with a denaturing solution (0.5 M NaOH, 1.5 M NaCI), split in half and loaded in duplicate. Two 17 mer oligonucleotides were synthesized that span the region containing the mutation. One contained the sequence of the normal allele (5'-CGCACATGGCAACCCTT-3')(SEQ ID NO:73), while the other contained the sequence of the mutant allele (5'-GCACATGGGCAACCCTT-3') (SEQ ID NO:74). These were end-labeled with y 32 P-dATP using T4 polynucleotide kinase and hybridized to the membranes at 50°C. Hybridization and wash buffers were as described (Church and Gilbert, 1984). Membranes were sequentially washed at 45 0 C, 55°C and 65°C for 10 min each and exposed to X-ray film.

5.4.2 RESULTS 5.4.2.1 A QUESTION OF TWO BG GENES In order to resolve the dilemma created by the existence of two different bg candidate genes (Lyst and BG), the inventors isolated and sequenced additional mouse cDNA and genomic clones corresponding to the 3' end of Lyst. An anchored, nested PCRTM (3'RACE-PCRTM) from this region yielded two fragments (1.25 kb and 2 kb). The 1.25 kb clone contained the previously published 3' end of Lyst, while the 2 kb clone contained sequences derived from Lyst (at the 5' end) WO 97/28262 PCTiS9701748 and from BG (at the 3' end). Reverse- transcription and PCRTM (RT-PCR T M confirmed that nucleotides 1-4706 of Lyst also represent the previously undetermined 5' end of the BG open reading frame (FIG. 15c). A full length cDNA was assembled from nucleotides 1-4706 of Lyst, the 2 kb 3'RACE-PCRTM clone and 6824 nucleotides of BG cDNA. This 11,817 bp cDNA sequence (Lyst-I, Genbank accession number U70015) corresponds to the largest mRNA observed in Northern blots (-12 kb) (Goodrich and Holcombe, 1995).

Analysis of a P1 genomic clone (number 8592) containing Lyst and BG revealed that the 11,817bp Lyst-I cDNA results from splicing of Lyst exon a (containing nucleotide 4706) to downstream exon r (FIG. 15b). Incomplete splicing and reading through the intron o' interposed between exons o and t yields the 5893 bp cDNA described by Barbosa etal. (1996) (Lyst-II, FIG. 15b, Genbank accession number L77884). Intron o' encodes 37 in-frame amino acids followed by a stop codon and a polyadenylation signal. Lyst-II corresponds to a smaller (-4kb) mRNA observed on Northern blots. Lyst-I and Lyst-II are both present in poly(A) RNA from many mouse tissues (FIG. 15b). The putative Lyst-I protein is of relative molecular mass 425,287 (Mr 425K) while that of Lyst-II is predicted to be of Mr 172.5K.

5.4.2.2 SEQUENCE OF HUMAN L YST1 AND L YST2 cDNAs cDNAs corresponding to LYST], the human homolog ofLystl-isoform I (which is the largest mRNA isoform of the bg gene) were obtained by identification of human expressed sequence tags (ESTs) similar in sequence to mouse Lysti by database searches (Genbank accession numbers L77889, W26957 and H51623). Intervening cDNA sequences were isolated using RT-PCRTM with primers derived from mouse Lysti sequence and adjacent ESTs. The partial LYSTI cDNA sequence (Genbank Accession number U70064; 7.1 kb) was assembled by alignment of these clones with mouse Lystl cDNA. Human LYST1 has 82% predicted amino acid identity with mouse Lystl over 1,990 amino acids. The predicted human LYSTI amino acid sequence contains a 6 amino acid insertion relative to mouse Lystl at residue 1,039. Recently, another group has published the sequence of the human LYST1 cDNA (Nagle et al., 1996). The cDNA sequence of the present invention differs in at 4 nucleotides and 3 predicted amino acids from that of Nagle et al. (1996). This 13.5 kb cDNA sequence corresponds to the largest mRNA (LYSTJ-isoform I) observed on northern blots of human tissues (caption in FIG. These northern blots also demonstrated the existence of a smaller LYST isoform kb, designated LYST-isoform II) that was similar in size to the smaller mouse Lysti mRNA, and that appeared to WO 97/28262 PCTIUS97/01748 differ in distribution of expression in human tissues from LYST-isoform I. Assuming that the genomic derivation of human LYST-isoform II was the same as mouse Lystl-isoform II, the sequence of the 3' end of the human LYST1-II isoform was sought by cloning human LYST1 intron F' using PCR T M of human genomic DNA with primers derived from LYST1 exon F and mouse intron F' (caption in FIG. The sequence of the 5' end of human LYST1 intron F' contained 17 codons in frame with LYST1 exon F, followed by a stop codon. By amplification of a LYSTI-isoform II cDNA from human peripheral blood RNA by RT-PCR T M with primers from a LYST1 exon and LYST1 intron it was demonstrated that this intron was indeed retained in human LYSTJ-isoform II mRNA. Nucleotides 1-5905 of human LYST]-isoform II cDNA are identical to LYSTl-isoform I, and are followed by intron F' sequence (Genbank accession number U84744)(FIG. The predicted intron-encoded amino termini of the mouse Lystl-isoform II and human LYST1- isoform II peptides shared 65% identity.

The only significant sequence similarity ofLYST1- isoform II to known proteins was with the stathmin family. Identity with mouse Lystl-isoform II in this region (amino acids 376-540) was 92% (and similarity was 99%)(FIG. 5.4.2.3 GENETIC AND PHYSICAL MAPPING OF L YST A 2 kb human LYST probe was assigned to human chromosome 1 by hybridization to human-rodent somatic cell hybrid DNA (FIG. 16). All of the bands that segregated with human DNA hybridized only to somatic cell hybrids containing human chromosome 1 DNA.

In order to precisely map LYST on human chromosome 1, LYST probes were hybridized to YAC clones encompassing the CHS critical region (FIG. 16b and FIG. 16c) (Barrat et al. 1996).

Three probes, derived from different segments of the LYST cDNA each hybridized to five CHS critical region YACs (FIG. 16d), confirming localization to the correct interval.

Genetic mapping in 504 [C57BL/6J-bg' x (C57BL/6J-bg' x CAST/EiJ)Fi] backcross mice was used to determine whether LYST was the human homolog of the mouse bg gene. Using one XbaI and two TaqI RFLPs, LYST was shown to cosegregate with bg and Lyst on mouse Chromosome 13.

WO 97/28262 PCT/US97/01748 5.4.2.4 MUTATION ANALYSIS As an initial screen for LYST mutations in CHS patients, we analyzed northern blots of poly(A) RNA from CHS patients. The largest LYSTmRNA species (LYST-I, approximately 12 kb) was greatly reduced in abundance or absent in lymphoblastoid mRNA of patients P1 and P3, respectively (FIG. 4a), while the smaller LYSTtranscript (LYST-II, approximately 4.4 kb) was both present and undiminished in abundance. Rehybridization of this blot with an actin probe confirmed that absence of the larger transcript was not due to uneven gel loading or RNA degradation. Fibroblast poly(A)+ RNA from three other CHS patients (369, 371 and 373) showed a moderate reduction in LYST-I mRNA (51-60% of control by densitometry), while the LYST-II mRNA was essentially unaltered in abundance (103-147% of control).

Single-strand conformation polymorphism (SSCP) analysis was undertaken using cDNA samples derived from lymphoblastoid or fibroblast cells lines from CHS patients. Anomalous bands were detected in PCRTM products from the 5' end of the LYSTORF in two unrelated CHS patients different from those with aberrant northern blot patterns (371 and 373, FIG. 4b).

Subsequent sequence analysis identified a C to T transition at nucleotide 148 of the coding domain in patient 373 (FIG. 4c). Four of nine cDNA clones derived from patient 373 contained this mutation. Restriction enzyme digestion confirmed this mutation. TaqI digestion of LYST cDNA (nucleotide 520 to 808) showed loss of this restriction site in patient 373 to be heterozygous. The C to T substitution creates a stop codon at amino acid 50 Patient 371 had previously been shown to have a frame-shift mutation with a G insertion at nucleotide 118 of the coding domain (FIG. 4c)[Barbosa et al, 1996]. Each of five cDNA clones isolated from lymphoblasts of patient 371 were found to contain this mutation.

Allele-specific oligonucleotide hybridization of cDNA from this patient failed to detect a signal with an oligonucleotide corresponding to the normal allele, suggesting that the patient is either homozygous or hemizygous for this mutation.

Mutations were identified in three other CHS patients: cDNA isolated from EBV-transformed lymphoblasts from patient 372 (deposited at the Coriell Institute as GM03365) contained a homozygous C to T transition at nucleotide 3310 of the coding domain, that created a stop codon at amino acid 1104 (R1 104X)[Nagle et al., 1996]. Patient 370 contained a homozygous C to T transition at nucleotide 3085 of the coding domain, that created a stop codon at amino acid 1029 (Q1029X). Patient 369 had a heterozygous frame shift mutation. Nucleotides WO 97/28262 PCT/S97/01748 3073 and 3074 of the coding domain were.deleted in two of five cDNA clones isolated from this patient. The deletion results in a frame shift at codon 1026 and termination at codon 1030.

Lymphoblasts from all of these patients (369, 370, 371, 372, 373, P1 and P3) contain the giant perinuclear lysosomal vesicles that are the hallmark of CHS. Patients 369, 370, and 371 had typical clinical presentations of CHS, with recurrent childhood infections and oculocutaneous albinism. The parents of patients 369 and 370 are known not to have been cosanguinous. In contrast, the clinical course of patients 372 and 373 was milder: Lymphoblasts were immortalized from patient 372 at 27 years of age. He had oculocutaneous albinism, recurrent skin infections, and peripheral neuropathy. Patient 373 has not had systemic infections and is alive at age 37.

Patient 373 does, however, have hypopigmented hair and irides as well as peripheral neuropathy.

5.4.2.5 EXPRESSION OF L YST-I AND L YST-II IN HUMAN TISSUES Analysis of northern blots of mouse mRNA had suggested that the relative abundance of mouse Lyst-I and Lyst-II transcripts differed from tissue to tissue (Barbosa et al., 1996). The relative abundance ofLYSTmRNA isoforms in human tissues at different developmental stages was examined by sequential hybridization of a poly(A) RNA dot blot with several LYST cDNA probes. The quantity ofpoly(A) RNA loaded on the blot was normalized to eight housekeeping genes (phospholipase, ribosomal protein S9, tubulin, a highly basic 23 kD protein, glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine guanine phosphoribosil transferase, $-actin, and ubiquitin) to allow estimation of the relative abundance ofLYST mRNA isoforms in different tissues.

Using a probe that hybridized only to LYST-I transcripts (the largest LYST isoform) on northern blots (Barbosa et al., 1996), LYST-I mRNA was found to be most abundant in thymus (adult and fetal), peripheral blood leukocytes, bone marrow, and several regions of the adult brain. In contrast, no LYST-I mRNA was detected in fetal brain. Negligible LYST-I transcription was also apparent in heart, lung, kidney, or liver at any developmental stage.

A somewhat different pattern of expression was evident upon rehybridization of the blot with a probe derived from the 5' end of the coding domain of LYST, a region that hybridized to both LYST-I and LYST-II mRNAs on northern blots (Barbosa et al., 1996). Consonant with the pattern of LYST-I transcription was abundant expression detected with this probe in peripheral 3 0 blood leukocytes, thymus (adult and fetal), and bone marrow, and negligible expression detected WO 97/28262 PCT/US97/01748 in skeletal muscle. However, several tissues with abundant LYST-I transcripts, exhibited considerably less hybridization signal with the LYST-I LYST-II probe, including most regions of the adult brain, fetal and adult thymus, and spleen. Furthermore, several tissues with negligible LYST-I transcription exhibited intense hybridization with the LYST-I LYST-II probe, including adult and fetal heart, kidney, liver, and lung, and adult aorta, thyroid gland, salivary gland, appendix, and fetal brain.

5.4.3 DIscussIoN As described above, the novel mouse gene, Lyst (Lysosomal trafficking regulator), was identified from a bg critical region YAC and showed that it was mutated in two bg alleles. The inventors also identified two human ESTs similar in sequence to mouse Lyst and identified a mutation in one of these ESTs in a CHS patient. Simultaneously, another group published a partial cDNA sequence (BG) that had been isolated from the same YAC (Perou et al., 1996a).

This partial cDNA was mutated in two other bg alleles, but was different in sequence from Lyst.

The inventors have resolved this bg gene dilemma by demonstrating that Lyst and BG sequences are derived from a single gene with alternatively spliced mRNAs. The unrelated cDNA sequences that had been reported are derived from non-overlapping parts of two Lyst isoforms with different predicted C-terminal regions. The inventors described a 5893 bp cDNA (Lyst) whilePerou et al.

reported a partial cDNA sequence (BG) without a 5' end (Perou et al., 1996a). By sequencing additional RT-PCRTM products, the inventors have shown that nucleotides 1-4706 of Lyst also represent the previously undetermined 5' region of BG. Alternative splicing at nucleotide 4706, however, results in bg gene isoforms that contain the 3' region of BG orLyst. Splicing of Lyst exon o (containing nucleotide 4706) to exon results in an mRNA (Lyst-I) that corresponds to the largest band observed on Northern blots and that contains BG sequence at the 3' end.

Incomplete splicing at nucleotide 4706 results in the 5893 bp cDNA (Lyst-II) described by Barbosa et al. (1995) and contains intron-derived sequence at the 3' end. Lyst-II corresponds to a smaller mRNA observed on Northern blots. While several other genes generate an alternative C-terminus by incomplete splicing (Myers et al., 1995; Sugimoto et al., 1995; Sygiyama et al., 1996; Zhao and Manlley, 1996; Van De Wetering et al., 1996), the bg gene is unique in that the predicted structures of the two C-termini are quite different. The C-terminus of Lyst-I contains a 'WD'-repeat domain that is similar to the P-subunit ofheterotrimeric G proteins and which may assume a propeller-like secondary structure (Lambright et al., 1996). In contrast, Lyst-II has a WO 97/28262 PCT/US97/01748 C-terminal prenylation motif that could provide attachment to the lysosomal membrane.

Although the prenylation signal is absent from Lyst-I, it contains a hydrophobic region that is predicted to be membrane associated. The significance of these divergent features is increased by the fact that Lyst is not predicted to have transmembrane helices.

Identification of the human homolog of the bg gene, LYST, provided a second line of evidence that Lyst and BG are derived from a single gene, since the LYST sequence overlaps both Lyst and BG. The LYST cDNA identified corresponds to the mouse Lyst-I isoform. Northern blots of human tissues had suggested that a similar complexity exists in the transcription of LYST, the homologous human gene (Barbosa et al., 1996). We recently identified two human ESTs homologous to mouse Lyst and described a mutation in one of these ESTs in a CHS patient (Barbosa et al., 1996). Subsequently, another group published the cDNA sequence of the largest LYSTisoforms (LYST-I), and identified mutations in this gene in 2 additional patients with CHS (Nagle et al., 1996). Here we have described the identification of a second isoform of human LYST. This cDNA, designated LYST-II, encodes a protein of 1531 amino acids that is homologous to mouse Lyst-II. Like the latter, human LYST-II mRNA arises through incomplete splicing and retention of a transcribed intron that encodes the C-terminus of the predicted LYST-II protein. The mouse and human LYST-II -specific codons share 65 predicted amino acid identity. The stop codon, however, is not precisely conserved between human and mouse LYST-II. While mouse Lyst-II is predicted to contain a C-terminal prenylation motif (CYSP), translation of human LYST-II is predicted to terminate 22 codons earlier and to lack this motif Several of the predicted structural features of mouse Lyst were conserved in human. The most notable of these was a region similar in sequence to stathmin (amino acids 376-540). While mouse and human LYST had an overall amino acid identity of 81%, identity in the stathmin-like domain was 92% (and similarity was Stathmin is a coiled-coil phosphoprotein thought to regulate microtubule polymerization and to act as a relay for intracellular signal transduction (Sobel 1991; Belmont and Mitchison, 1996). This region of LYST may encode a coiled-coil protein interaction domain and may regulate microtubule-mediated lysosome trafficking.

Intriguingly, a defect in microtubule dynamics has previously been documented in CHS (Oliver et al., 1975) and intact microtubules are required for maintenance of lysosomal morphology and trafficking (Matteoni and Kreis, 1987; Swanson et al., 1987; Swanson et al., 1992; Oka and Weigel, 1983).

WO 97/28262 PCT/US97/01748 Other putative structural features ofLYST that are conserved between human and mouse are several pairs of predicted helices with a protein kinase C- or casein kinase II-phosphorylation signal at their N-terminus. These helical bundles have been hypothesized to have a signal transduction function similar to stathmin. The conserved phosphorylation sites have been hypothesized to affect interactions ofLYST with other molecules through phosphorylation dependent conformational shifts in the helical bundles. The conservation of these features between human and mouse lends credence to their biological relevance.

In order to evaluate the candidacy of LYST for CHS, segments of the LYST sequence were mapped in the human genome. The CHS locus was recently assigned to human chromosome 1q42-43 (Goodrich and Holcombe, 1995; Barrat et al. 1996; Fukai et al., 1996), a result that had been expected based on linkage conservation between the mouse chromosome 13 region containing the bg locus and human chromosome lq42-q43 (Beguez-Cesar, 1943). DIS2680 and DIS163 were previously shown to represent the telomeric and centromeric limits, respectively, of the CHS critical region (Barrat et al. 1996). Human LYST mapped within this CHS critical region. The localization of all LYSTPCRTM products to CHS critical region YACs also precluded the possibility that the LYST sequence had been assembled from segments of closely related genes.

Northern blots demonstrated a 12 kb mRNA (corresponding to LYST-I) to be severely reduced in abundance in two CHS patients. A 4.4 kb band (corresponding to LYST-II), however, was present in mRNA from these patients in normal abundance. These results suggest that, at least in some patients, CHS results from loss of the protein encoded by LYST-I rather than LYST-II. This result is surprising since previous Northern blots had suggested that the major LYST mRNA in granular cells was LYST-II, while LYST-I was either undetectable or barely detectable in these cells. Because lysosomal trafficking defects in granular cells account for the clinical features of CHS (Griffiths, 1996), it had been hypothesized that the 4.4 kb LYST-II mRNA represented the transcript of primary functional significance. In this context, it is interesting to note that the bg mutation results in the generation of a premature stop codon in Lyst-I that is unlikely to affect Lyst-II mRNA processing (Perou et al., 1996a). These results suggest that defects in LYST-I alone can elicit CHS and that LYST-II expression alone cannot compensate for loss of LYST-I.

Mutations were identified within the coding domain ofLYST in five CHS patients, two of which have been reported previously (Barbosa et al., 1996; Nagle et al., 1996). The genetic WO 97/28262 PCTIUS97/01748 lesions in three CHS (patients 370, 372 and 373) were C to T transitions that resulted in premature termination (Q1029X, RI 104X and R50X, respectively)[Nagle et al., 1996]. Two other patients had coding domain frame shift mutations that induced premature termination. One of these, patient 371, had a G insertion at nucleotide 118 of the coding domain, leading to premature termination at codon 63 (Barbosa et al., 1996). Allele-specific oligonucleotide analysis indicated that this mutation was either homozygous or that mRNA corresponding to this region is not produced from the other allele (hemizygosity). Patient 369 was heterozygous for a dinucleotide deletion that results in premature termination at codon 1030. Interestingly, all bg and CHS mutations identified to date are predicted to result in the production of either truncated or absent LYST proteins (Barbosa et al., 1996; Nagle et al., 1996). Unlike Fanconi anemia, type C, there does not appear to be a correlation between the length of the truncated LYST proteins (which may or may not be stable) with clinical features or disease severity in CHS patients.

However, until the other mutant allele in patients 369 and 373 are identified, and the exact effects of each mutation at the protein level are characterized, such correlation is imprecise.

Comparison of transcription of LYST-I and LYST-II in human tissues at different developmental stages revealed an overlapping but distinct pattern of expression. A quantitative estimate of the expression of the smaller LYST mRNA isoforms was obtained by subtraction of the relative hybridization intensity obtained with an LYST-I specific probe from that obtained with a probe that hybridizes to all LYST transcripts. LYST-I transcripts predominated in thymus, fetal thymus, spleen, and brain (with the exception of amygdala, occipital lobe, putamen, and pituitary gland). Both LYST-I and LYST-II transcripts were abundant in the latter brain tissues, peripheral blood leukocytes, and bone marrow. Only the smaller LYST isoforms were expressed in several tissues, including heart, fetal heart, aorta, thyroid gland, salivary gland, kidney, liver, fetal liver, appendix, lung, fetal lung, and fetal brain. The developmental pattern ofLYSTmRNA isoform expression in brain was particularly interesting, since only the smaller LYSTisoforms were expressed in fetal brain, whereas the largest isoform (LYST-I) predominated in many regions of the adult brain.

In summary, the inventors have shown that the same gene is mutated in human CHS and bg mice. Without bone marrow transplantation, CHS patients typically die in childhood of infection and malignancy. The existence of an animal model of CHS with a similar genetic lesion will assist efforts to develop novel therapies for this disease.

WO 97/28262 EXAMPLE 5 DNA SEQUENCES OF MOUSE LYSTI 5.5.1 cDNA SEQUENCE OF LONG ISOFORM (SEQ ID NO:3) PCT/US97/01748 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501

TGAGAGCTCI

CCGGCGGGCC

GCTGAGACAG-

TGTTACAGAP

CATTGGCAOG

GTCCAGAGGC

AACTOTTGG.

AACTAAATTC

AC T CTTCT TO

ACAGCAGGCA

CCAAAGAAAA

T TAGAAGGAA TO GAAATIAT C AT GCAAGAAA

GAAAAGAGTA

GT TCCTGACG CC TOTGCCAC T CCAGAGAAG GC CAGO TGC T

TATGTCATGT

GCTTTGAATC

TGAG TTCC TA

AGGGACAACT

CAACGGATGC

CACT GOAGAG T TAGGGCAGC T TOTCCCGA

ATTTTCCAAG

GAGTTCTACA

TTTTACTTCA

AGGATTTAAT

CAAGGGAOGG

AA.CAGTGTAA

AC T TCAT CAT

CCCACTTCAT

TTTAAAAATC

TGTGCAGTAT

GCTTGCGCTT

CTATCAGGTG

TGTGAT CGCC

TCCAGCAGCA

GGAGGAGCAG

TACTGTGGAO

COT TGTGTGG T TTCAGGGGA

GGATGCATTA

ATAACATTCA

GTAGTTGTTC

PAGAGGAGT T

OTCAGACTCA

C TTCAGATT T

CGCTGGCCT(

AGAGCGCGC(

TTTTATOTAC

LGTGCTGAAAC

TGAGTTTCTC.

CAGAAGCCAC

CAGTAOCTTC

TATCATTGAI

TGTCGCTCCI

*ACAGATTTTP

*GAACTCAAI

GTGCTCCATC

AG G CGACAG C

GACACAGCTC

CGGT TGTGAC CAGT CTCC TA

CAAAGAACAG

TCATTCTGAG

T TGT CTAT TO

TTTGTTATOT

ATAATTCTTC

GOAGGCTTTG

GGT TTO TGCA TOT TTCGAAC

GCAA.TGCCAG

TTTAAAAATT

GAOAAAAGAA

TATCGTOAC

GOTOOTOATO

GTCAAGOOAT

O TOT TTGGAI\ T GTTOOT TCA

TAAAAATAAT

TOOATGTGCA

GOAGOACOAC

AGCTT TOTAA

OCAGAGOGOT

GOTGOAGCAG

TAOACAGTGT

OCT TTAOTGC

TATACTGAAT

AGO TAT CAC C TOT GAO CAAC

TGCTGGTCOT

TOO TGOOCAG

GAGGCTTATO

GATTGOAAAT

AGTGGAAAT T GAAT TAGTAO CATG T GTAG C ATT TAAAAAC 3GCAGCCTTGG 3GOGGACOACA

;TTCATGAACO

;AOTGOTOTGT

;ATTGATGTCA

;GGAAGAAGAA

TOCATGGAOG

CAGGOOOTGA

TCOTTGGTG

LACOTGCCACT

TTGCAAAAAT

TGGTOAGGTT

GTAAAAGTAC

*TOCAOOTCTG

TAAZ\CACAGG

*AGGAAGGOCA

GTOCTTTCTG

ACAGGAT TOA

TCAGTAAOAT

CTATTGGAAA

COTAGOACTO

GGGAO TGOTG

GGTTGGACAG

OGTGOTGOAO

AAAGT CT TAG AGAGOT TGO T AACAC TACAG

GAGCOTTOT

TCTTGTOTTO

GGATTTAGTT

CAGOAG TTOT

GAPLGCTGOAG

GAGTACTGTG

CAAGGAAAAG

CGOGATOTTT

AAGCCCOTTT

GCTGCTGOAT

GTTTCCOTGA

TGGAATCTGT

ATGCTTTTAA

GTOCTGAGOA

GAGAATTAAA

TGGCTAAGTT

ACOTOOGGOT

CAGOGGOTOT

AGAGOTTTGT

OAOATTTGTA

GTATAATTAT

ATOATTGTOA

OAACTGAAAC

TOTAOOTGTC

TGAGTCGGGA

GAGCGGAGGT

CAAAT TATAT

OATGAGOAOG

ACOAGCTTTG

GAAGAGGAGA

AGGATTTOTG

OAT GOAGAGA

TGGAAGATAC

GTCATOTGAT

OAACTCAGGG

TOT GOAAAAG COAT CGT TAT AC TCCGAAGG

AGGCTCOACG

COTCGTAGOO

ACAOCATGTC

AATGGTGACA

GAATAAT TOT

AAGTTTGTAA

AG T GTAG TAO

TAACCAGAGT

AAGAGCCGGT

OTTATGTCAG

GATAAAAT TTG T GGAAAAGCA

GAGGTCOAGG

AOTAOOTGAG

AGAGT GOAGO CAAGAAT TTA

TCAGATGGAA

AACATTTGAA

AAAAAGGTGA

ACACCGGCGT

CAGGGOTCCT

GAAGAGACCG

CGCCGTGTGC

GCACCACGTG

TGTTGTATGG

GTTGOOAGOA

kACTTCTTGT

BAAGCAGCTT

kGGAGAGACA

TGCCCAGTCC

3

AAGAOTTGO

TTTCAAGAA

kTTTACTCCA kTCTTTAATCC \.CAGOTAAGC I kGTATTTGCCI :TACTTAAAT

TTCTOOTGOA

GAAGOCTTAT

ACAAGOTGAA

GACAGCAAC T

CAATGCAGTG

CAOACATGGC

TTACTTAOOA

AGAACTCOTG

OTGTOCAGGA

ATAATOCTGA

AAAAT TATAT

TAAAOCTTTT

TCTGTAAGAG

CAAC TCAGAT OGOTGO OACG AAAC T GACO

TGTGGAAAAC

TATTAAGTGA

CTT T TGAO T GT T TGACAT T C CACAO TGAC

GAOAOTTTGG

AGCTTTGGTT

TAGAOGTTAG

AOTGAATTGC

GCCTGAGOO T

TAGGGCTTTGT

CT TOTGGA2AG T TCAAAT COO

TOCAGOACCA

TGG T GO TTA

AGCTCTGATA

A'AT CAGAGOA T GTGAG TAT T

GGTTTCAGCT

CAGAGGGAGA

GCTCAOCAGT

r GTOOAGATO !kTCCTAAGTC

CTGAAAGCTT

3GATCAGTTA 3CAACATCTG

TGCAAGGCA

P TO TACOGA rGTGGAAGTG 3ACAGAT TAO 3AAAGGCAAT 'TGTGC TOCA

~TTCOTTCAG

'CAGGAAGTG

CAGGGTAAT

WO 97/28262 WO 9728262PCTIUS97/01748 2551 2601 2651 2701 2751 2801 2851 2901 2951 3001 3051 3101 3151 3201 3251 3301 3351 3401 3451 3501 3551 3601 3651 3701 3751 3801 3851 3901 3951 4001 4051 4101 4151 4201 4251 4301 4351 4401 4451 4501 4551 4601 4651 4701 4751 4801 4851 4901 4951 5001 5051 5101 5151 5201 5251 AAGAGATTTG TTTTTAAGTT ATTACTTAGA TGGGATTCGA ATTGTCAGCC TAGGGGAACA TGGGTTAGAC ATCCAACAGG TTCATAAGCA GCAAGCTTCT TATGCCAGCC TOAGAGAGC GGATGTTCAC ATAAACACCA GTGTCAGTAA AGAAGCAGAC GATAOTTOTG GCTATGACAG ACCATGTOTG TOTOTTGAGG ACCATGCAGO AGACATTTGG TOAGTOTTOC AGAAACAA\TT TGAAT TAATA TTTATGATAA ATCAAGG.AG AAGGCAGGGA ATOAGGATAT CTTAOCCCGA AACAGCACCA GAOCTGGGAT GATTCCAGTO ACAGCOTGTG AOTGAATCTG TTCCTGGGGA GAOTTOTOTC OAGAGCATAC TTOATAGTGO CAGAGOOTGT CAGGGCTTGT OTGTGGAAAA CCAGTCAAAG GTGGCAGAA TGCTTOGAGT AGCOOTGGGG GOTGTGACTG AGAAGAGTCA CGGAGATTTT TCAGAAGAAG TTOTGGGTGA GGAAkGAAGGC GATGTTGACA CCCAAGACGA TTTCAGTGGA TCGATTGTTT GGGAAATAAT ATACOTGAG GCTAGCAAAG CTAAACTTGA GAMAATTGTC AGGCAGAAGG GAACTGTGAA AATCCTTCTA

GTAATGGAGT

AGT CAT TCCC

ACAGAAAGAT

AG T T GCGTO TCAGAT TO T

TGATCCAAAA

TAAACC TCT T

TCTGATAGGG

C CCT CCCAGT ACGT TGTCT T TO CAT GT GT C TCACAGGOT T

TCCAGAAACT

GAAATGAGTA

GCTGACACTG

T TC TGAGAAA

CTTCCCACAA

ACGAAAGGCA

GACTTTTGGA

CAACAGAAGA

TATAT T GTOT CAGAAT TAGO

AATCATTCAG

TCO TC TGAG

CTGAGGATTC

TAT GAAGCGG

TGGAGTAGAA

CAAACAACT T

ATTTGCATGC

TGTGOTTGCT

AAAAGAACAT

GGCGGGTTCT

OOAGAGAGTA

GT TCAGAAGA

TGTACAGAAA

TACTATGAGC

GT TTAAGCAA

AAACCACATA

TGAAAGCATT

GOT GCAGT TO O TTAAGTGTG CT TGOAGTO T

AAAOGAAAGA

OOTOTGTGTG

AGT CTGCOAA GAGO OAT TAA ACO TTOCCOT

GTTGGATOTA

GGTGGTTTC

ATTOAGAAGT

GAAAT GAAAA

AAGGGAGATG

GAGTGCTGAC

GTGCAGAGCA

AT TGAAC TAA TGAAACTO TO

TAGACOTOGA

OGTCOTTOTC

OAGGAAGTT T COAT TCACCA GO TT TTO TAT

TGAGTOAGAA

GTOACATGOT

GAATGTTTGO

OATGTTGAAO

AAGTGTGOOA

CATAOAGAGG

OOAAOAGOTA

TATOATOTGC

AGOG TGC GTG GAT TGTG000T TTTATGAGTO AAOAAAGTGA OTOTOTOOTO GACATTTGTC TGGAATTGGA GCTAOOGTOT GAAOTGAGGG AAOACCTTTO AAAGOCTTTA TTTGATGOC CAGATTTGGG CCOTGGTGAT GAAGAGOTGT TGTCCCAGOO TOAGTGTTGT AGTTTGAAAO ATAGTGAAAG OAATOOTGAG TTAAATOCTG \AGOAGAAGG AOTTGAAAAO OTCACTOAOG TGGGATTAAA TTTGCTTTCT

AAOTCTGATT

TTTGATGAGO

TATTTCTGGA

.AAAATTGTTG

TOCTTTCOTG

C TOOTGGGAT ATT TCOGAG GOT TTOOTCT AC TGC TGGCC

ATGTGGAATA

AG TAAAGAAA

GAGOAGAAGG

AGAC TCATAG AT TGATGAT C

GTGTGTGOAT

GTGGAATCAC

ACTTACC TAT TO TOCATAT G GTGO TOOOAA CATOAT TGGT GAAAAT GOGA T OAOAAGAGG

CATGOCGTGT

TOOAAGOATG

TOAAGAAOGT

GAAATOTOOT

AAAGTGATT T

CATACCOGA

TOTTAAOAGT AA-AGOOTTAG GOAAGAAAGA GGC TGATAGA TGGCATATAG OOOCAATOOA AOACOTGTOA GAAGGATTTA OATOOAATGA ATOCGAGAGT AGAAAOAAAO OATCAGTTCT TGATAGAOOA GAAGT TACAG AAGAOGGOTG TATTOATTTG OAAGTGTGGG OTGATCOOCA GGACT OAAAT GATGACACGA AGGAGAATAT TTTCTTTOCA ATTCAGOATO OTCAAGGGAA GGTCTCTGGG OAGAGGAAGA GAAAAAOAAG OTTATOATCA OATTGCTTAA OATOOOAAGA OCTGGGGAAO TTGCTOOTCT OOTTTTTCCT GTATGCTTGT AAATATGGAO AGOCAGTCAT

OATGTGTTTG

TTCTCTCOTC

TGAATATTTT

CTGGTGGATC

CT TAA TOT T

TTOTTCTCCT

CCTTCAOAGT

T GGTGT T TA GO TTAT TGAG GAGAGT T TTO

CCTGCOGTTG

GTGTTTOTCT

GOT GOAGAAA

GGAAGAOAGO

AATOOAT OAA AT T TCAC TGG

CAGTGGOACT

A GO TGTC TO

AGOAAATGGO

AAAGAATGTO

CTGATGTCAT

GACAGCAATA

1GAG TOTOTG

TOAATGGAGO

GGACCCAACT

r GAO TAOTOO AGAGC T TTOT

ATACAAOAGG

AACACAAAOT

TOT TGTAT C OTT TGGAGAA

TGGTATTOAC

GTOTGAOOT T

TOACAGAAAO

AAGAGOAOGG

COTATAGGOT

CTGGGACAGA

TGTGGGTTTA

GGGGAAAAAG

AGT TT TGAAG TOO TGGT GAO GG T OAAAGO T TTAT OTT TO AC TAG CACAG

AAOAOTTAGT

OATGGGGAAA

CT TGGAT TTT AAAOAT TT TO

OAATTAGOTG

TAAAATTGGO

ACACAT CCAT AAATAOAT TA WO 97/28262 PCTIUS97/01748 9 r) TTTGAGATGT GATGAAATCA GAGACCTTTT TATGACCAAG ATGTTGGTCT CTTAATTGAA AGTCTTTCAG TTGTTTATAC 5301 5351 5401 5451 5501 5551 5601 5651 5701 5751 5801 5851 5901 5951 6001 6051 6101 6151 6201 6251 6301 6351 6401 6451 6501 6551 6601 6651 6701 6751 6801 6851 6901 6951 7001 7051 7101 7151 7201 7251 7301 7351 7401 7451 7501 7551 7601 7651 7701 7751 7801 7851 7901 7951 8001

ATAAGACAT

AAAGAAGTGG

AACTTGCTGT

AGGGCCAAGT

GCCCAGAGCA

GGAAT GTAAA CAT TTGT TT T ACT CAAG CAT

ACAGAGAACA

AAG TGTTGGT CT TC TTGAAG

AGAGTTCAAG

GCCATCCAGG

TCTCTTTGCT

TAGCACTGCG

CAGGAACTGG

CAAACAGAAA

GTTGCTGCGG

CTGGATGTTG

TGCTGCAGGA ACTGTTACTT GGTGTGTGGG AGACTCTGCT GCACCACCAG CAGCAGTTTA TTCACCACTT CCTACTGACC CAGCTTACAT CTATGCCCCG TGCAGAAGTC CTTGGTTCTC TCAATTTCCT GTTAGCTGTA AATCCCACAA ACTTCTACTT GGAGAAAGTG CAGTCACTCG AAGCCTTTCC CAGCCCTGGA GCAGCTCCTC CTGAAGGAAC GGCTGCTCAG ATGGTTCGAT TACCAC TAAT AC GAG CACAA GACAAGTTAC ?AAAATATTGC AGGAAGACCC TACGCACTGA GCTGTGAGTC TGCAAAGACT CACGCCTCTG CCAATGGGGT CAGAG TAGAT CACAAA.GATG GTCCTGGGGA TGAGTCTTAC GCCTCATTCC AGCGA7XGCCA TCCCTCTCAG AATGGATCTG ATAGGAATGC TGATGACTGG GCAAGCTACA ACCGAGCCAC

GCATTCTGCA

AGGGT TG TT G

GGTTATACTG

AAAAT TGTAA T GCAT TGT TG TGAA.GAAGT T AGT CT CATGC GAC TGGAAGA

AGCAGCTTTG

ATAT TAAGCA T GTCAGGTT T AGAAGTT T GT C TCCAGACT T

CACCCTCCTA

CTCTTTGCAC

CGTACC TGAG

TTCCTGGTAA

CAG TTC TTC C

CTAGAAGTCT

AAACTGGCT G

AGATGCCAAC

AAACAAGCAA

GTT TGT GAA T TCCAGAGGA T GGGAACAGA

CCACGTCGGC

AAGCAC TGT C

CAGTTGCTAG

GAGAACTTTA

AAGCACCCAC

TATATGAAC T

GAAGATGTGA

TAACCACCCA

CATACAT TAA

CGTGGCTTTT

GGAG TTGT TG T GGAT GAAGA AAGT GGT CTG

CAATGTCCTC

CTT GT TCCAA

TTATGTAA:TA

GAACGAATAC

T TACAAT TCA

GAAGACCTTA

GACACAAAAT

TGGAATTTAT

TCGCCTTCTG

TGGTTCTCGC

T GC GCTCAGT AGC CAAGAGC CCTGCTCAGT ACACCATCTA GAAAACTCAG CCCTCTCAAA TCTTGCTAGA ACCTTCTCAA

TGAACCAGTG

GAC CCT TCAG

CTCAZ\AGGCC

TGAGATTGGT

AACTTAGTAG

TCTTTAATTA

TGGAC TCT CT

GCTTTCACAT

ATCCACGTCA

TATAATCCAA

TAT GGAATAA GAAGTCCT CA GT TGCTGAAC

TACAGGAACA

AGATCATTTG

GGAATTATTG

C TAATAC TTA

ATAGATGGCA

GCATTCTAGC

TAAGCCCATC

AATATTGTTC

ACCAGCATTT

CAAGT T TGGG

CCAGAGAAAC

AGAGGAAGCA

TGGAGGC TC T

TCACCGAGGT

GCCCAGATCA

CTGACAACC T GCAAGCC TT G

CAGGTGGCCA

CCTTTTCTCC

AGT GT CATT G

CTTAA.GTGGG

TGGACAGGAT

TCACCTGCTA

TAGAGCATCA

CC TTAT TAGC

GAGTGCTTTG

ATTTGATCTG

TCATTCCCGT

TTGCACAATG

GGTAGCAGAC

CAG TAGCAGC AAAT TGCT CG TGC TTGCAGT

TTGTACTTCT

ATGGC TT TGG

AAGGAGCACA

CCCACCGCCA

A7AATTCCCTC

GGCCAGCGAT

AC C CCAGC CA

ATTCGACTCA

CTCAAAGGAA

TCCAACCTAC

GGGGC TGGCA

CTGTGAAGAA

AG TACAGCCA ATGAT TCACC T TTGAAGACC

GTGAGCATGG

GATGT TAAGC

GGCAGAGCAA

TCCGGG TAGA

GCCCACGTGG

CAGAGAGGGG

TGAAAATCAT

ACAGTTATTT

TGT TTG TCAC

AGATCTTTCA

AG C GGAG GGC TGCC T TTACT CACAGC GGAT CCTACT TATT T T TTAG TGTT

AGAATCTTTT

TTCATCAGCA

T CT TGGAGCC

TCCCCAGGGC

GATGAT GACA

CAAGGGACTG

GGCTGGCGTT

AGTC T TGTT G TGCT TATGAG AAGACT GTCT

GTTCTTCTTG

TAT TCAAGCA

TCCAGCAAGG

AAG GAG CAAA CAACCAGT TG TTGAAAT GT T

GAGGAAGTGA

TCTCGGACTA

CTCTTTTACT

ATGC TAC TGG

CCTGAATGGA

CATGTGATAT

T CC TCAGGCA

TGGATATCTT

CCC TGCAGC T

GCCAATCATG

T TCAGT GCCT

TGGCTCAGAC

GAGCTACACT

GGC CTCGGAG

GATACCTATC

TCCT GC CTGA

GATATTCTTC

AGTAAT TAAA

AGGACAAGTT

TATCTTCATA

CTTTGGTCGA

AGCACATGGA

ATAGAGACC T

TCTTCTGCAA

ACAATGGTCT

T TAGAAAAGA ACAGCAGCT T CACAG TATT T

CATAATAGCA

TAGAGT TCTC ACTC TGAAAG

TGCTGTGGAT

T GCTATGCTT

TAGTCCTTGT

CTGTTACATG

T CTGAAGAAC

GGGGAACTCA

CCGAT TGGCC ACT GT TCCAG

CTCTCTATGA

GT T TTAAAC T

ACTCTATGTA

ACAT TCC TGT

TTCATAGCAG

TAGAGTGATT

AAAACAAGAG

CAGGCTGCTT

TCCAGTGCAC

CCGAAGCGGA GAAGCATTGC AGAGTCTCTG CTGATGAAGA CTATGATGCA GAGGAGGATG WO 97/28262 WO 9728262PCT/US97/01748 8051 8101 8151 8201 8251 8301 8351 8401 8451 8501 8551 8601 8651 8701 8751 8801 8851 8901 8951 9001 9051 9101 9151 9201 9251 9301 9351 9401 9451 9501 9551 9601 9651 9701 9751 9801 9851 9901 9951 10001 10051 10101 10151 10201 10251 10301 10351 10401 10451 10501 10551 10601 10651 10701 10751 GCAGAGC TOG GAT TAT TTAC

CAGAAAATAC

GAAGAAGACA

AGAAATGTTA

GTAAAACTAG

AAAGAAACC C

TCOAACCCAO

AACCAGCTCA

CATGGAGCCA

GGATGAGTTA

CT CTAAAGTT

CCAGAGCTCA

AGAGAGTGTG

AAAGTC TCT T

GCTGCAGACA

GAAGGTGATC

GOAGGCACTG

TGGTATGAC

AGGGCCAAAC

O OAATAAGTA COCOCAOTC T TAO TGT CATA

GTATCAGTGT

A7\ATGTGGGA O TOGAGCCTO AGGAAAT TAA

GTAGAAATOT

TAAOAAGGT T

OAAATOTTOT

TOT GGACAA

GGGOOGGTOO

T TT OTGACTA AGAAAOO TAT T TACGT TGAC

GAGAGGATGA

TAO TOOAAOA

OAOTAAAATG

GAAOATT TOA AT GAOTGATG CT TAGT GAAO

GGGTTAAOOA

T TCATCCTTA

OATOTGTCAO

CGT T GTTOA GATGTC TOTG

GATAAAAACC

O CAG C CGAOCC

GTTGGOTTGT

GGAAGTCAOT

GGGAGTACGT

OAGCCOATG

OATCTGTGGT

AGOAAGGTGT

CTAAGCTGGG

CTOAGAGGCT

CAAGAGTTGA

ATCOAAAGC

TGOATOATGA

ACO TATO T C OGT TTCTAAG TOO GAGAC CA

ACTGTACAAG

O CAGGATATA AGT TGGT TT T

AGTGAAGAAG

ATGTGGOOAO

T TAAGAT CAT

AGCAAAGGOT

OCAGAGGOTO

TOACCOAGGO

CAGOACAT CA

GCAGGAATGC

CAATCTAOTA

OGAGAGAGGA

OCT CCTGAGG

OTTAOOTTTT

GACAAAGCTG

TGCACCATCT

TGTAO TTT GT

CAAGGGGAGT

AGAAGT TCAC

TTTTAACAAA

CGTGATGACG

GGAGTACGGO

TTACOAATTT

TTCAATGATO

TGTTAGTGAG

CTAPAGCCTAT

ACATACAAGT

OOOOATGOCT

GOGGOACOGT

T T TTAGCCT T TOTAOAAAO TGAAGGAGC T OG TGAAGGCT OGTCAATCT T

TTCACCGGCA

TGGATOGAOT

AGCOATCAAT

OAGT TGAAGA

TAOGGGOAGA

TGGAGOOAkG TAGTC OAGT T CAT CC GAGOCC AG G TTCOC CC0 GAGAAAGAT T

TTATCACGA

GAGAAG CAT C

GATATGOTGA

913 GOAGAGGOTO ACCATOTTAG ATTCAGATAT TATTGAOATT AAGAOOTOAG TTTOTOAGAO GCAAOCTTCT GTATATAATC TGGATGGOTT CAAAGTGTGT OAGOAGTGGA CTATAATOCT GCTTGGAAGA TTGOTAGOGO AACGGAAGOA GATAOTTGAG CTTOGTGAOT GTCTTAGCOO GTATTTGTOA GAGTTGATAO AAkAT GGACAO AGOAGAAC TG AAGTGCATOO OGOOCAGTGO CAGAGAG GAG CAA\AAGAAGT OATGGOAGAA AAOGGTGAAO GAT TTOATAAT OCAAGGATAT TGTATOAOTO TOOOAAGGOA GAGGGATGTA CAAAGTTGAO AT COAGOAGO TGAOAOATGA TOOAAOTTCA TGGCAGTTGG GACGTTTGOA GAGATGCTAT GACAGACAGA AGC AGAAGG TGAAGATAAA AOTOATTOTT STOT GAA.OAG T TGAGAAC TO TGAAAT TTOT

CATTTCAAAA

ATTGGTTOAA

GGGGTOTTCT

ATATTTTGTO

ATAGTTOATG

OTCOOCACAA

ATAATOATCA

OTTATGAATG

CCT TCOAAA ATGA1AAGTGA

AAOAACOAGC

ATCTAAAATC

TTGAAAGGAA

C TGAG TGO CA

CAGAGOAGC

AT OCAAOAGA C TAAO TAT TO T CTGO TOAGG CCT TOTOTO AATCGAAkGAT GT TGT TAGGT CAG TTGAAAG

TGGAOATATG

AGATAATGOT

CATTTGACAA

AATAACCTOC

CAAGTGAATC

AGAGACACAG

GGAAGAOAAT

TLAGAGOOGGO

AGCGOTGGT

T GGOAGAAOA

TGTACOAGAG

AAOATCACOG

TGAATATTTG

TCATGOAGTA

AC TOTTGAOO

AGOTGTGCAG

AOTTGGAGGA

OCTGTGCAAO

GO TOCAC TTO AT OAAGAT CA

ACAAOTTGGO

GATTOCAGAG

T TGAOT T GC

OOTOOCTGGG

AGOAC TAGAG

TAGTGTTTGG

GTOTTOOACO

TOCAGTGCAC

OOOOAOG TOA

OTTAACATOG

OGOTTTOCA

OTTTGTCATG

AGTGOTOCAG

TGGTTOCCTG

ACT TOTG TOT

AAOAAOAOOA

CAACATCTTA

OATOAGACTG

CTGGGGAATT

GCT CTGAC G

ATOATTTTOT

GGOAACTAAG

CT CC TAT TAG

CATOCTOACA

OTOTGACAAA OOTGTGCTAT AOTCATTTAA AOAAGOATGC COGGTGTTC COOTTOATOC TOAATGATCC ATOTATOTAC TATAAAGAAA .AAGAAGACOG GGAGTATOGO AAGGGAGOTO COTACOACTA TGGCTOOCAO OTGCTOAGGA TGOOGOOTTT GAGTTTCGAC ATTOCAGACO GOOTOTOCTO OTTTGAGTOO TTTTTOTATC TTCCTGAGTT TGTTOGTCAG AATGGAGAGO CAOGOAAOGA TOOTOGGOTG TOTGACOATG TGTCCOAGAA O TAOAAGOAA AAGGGGAAGG OTGOTACATA TTTTGGAATG AGAOGGGOT T TAGAAAO CAT GTTGTTOCAO ACAGOOCATG AAGGAGAGCT TOCAGOAGOT GAGACOOGAG AAOOAGTC.AA GATAAAAGGO TTGAAGTGGG TACCTGTGGT OTGCTTCAGC OAGGCAOTGO COACCAGAGO TOTGATGACO TAOAAOAkGG ATATTOAGTG GTCTGOTATO CGGTTGAAAA GTAAGOAGAG WO 97/28262 WO 9728262PCTIUS97/01748 10801 10851 10901 10951 11001 11051 11101 11151 11201 11251 11301 11351 11401 11451 11501 11551 11601 11651 11701 11751 11801

TGAGCCACCA

GTTGTGCCTG

GGTGTCATCA

AATTGAAATG

CCGGCTTATG

GACGGGACCT

TTTGGCTGGA

CAGGTGACAT

AGACTCTGGA

GATCATTTGT

ACGTCATTGC

TGGGAC TTGA

CATCATAAGC

ACAGTGAGGG

AAGCTGCCCA

AGAAGGAGT G ACCAAC TGCA

CGTGCCTCTG

C TGCCC TCAT AAT TAAAGT C

AAGATCAGGC

ATCAACTTCA

GGTGCC TGAO CAGCC TATAC

GAGAGTCAGA

TGTC TGCAAG

GCATAGTATG

CACAAAAGC C TGCTAO TGT G

CCGTGAATGG

TO TGTAGCT T

TGGGGGATTA

AGCC TGTGAG C TGACATTC T

GACAGTGATC

T GT TCTACT C

TOCCCCACAGG

GAAGCAGAT G

TGCGCGCTTC

TCCATAAkTGG

AGAATCTTGA

TCGTGCC

ql'

TTCAGAGTTC

AGT TGTCAGC CAACAGGC TC

TGCATCTCTA

CCGTACAGCG

GGACC TGAAC

CTGTGACGGC

TGTGACTCAG

GGACCTCGTT

TCTC CAACCA

GAAAATGGCA

AGAGAT TACA

CCTGTGATGG

GCATGGTGCC

TT TCC TCAGC

ACATAAGCAC

AC TGAGCAGA CCCAGC CTCC

CGTGGAAGGC

TGCTTTTTCC

ACAGCAGCAC

TC TTCAC TGG AC CAGCAGOA

TGGACACACA

TGATGATAAG

AGGC TGTGOT

TGTCTCTGCC

CTGGCGGGGG

GGACATGTCC

GCC TGAGGGA

TTGTAAGGCT

TTTCCOAAAT

CCAC CATTTG

GGAAGGACCA

AGO TACGCAG CGC TCT GOGA

TATCCAGGAA

GT GGGCC TGA

TGGGTCTGCA

CAAAAGGT TA CAGG TAACCA

GAGCAAGTGT

OGOOCTOAGA

GAG GAGAT CA CGT GAG CAGA

ATGTACAAAG

AG TGAAACG T

OAGTGAOOTG

AC TGOAGAGA

GTCTCCATOA

ATGGAGOAOA

OAAA.TAAGC C TACAC TGOCA GCAGCGTG TG O TGGATGAAG

GCOTGGOTCO

AGACAACACA

GAG TAAAG CO CACAO TAGCC

GGCTGAATCA

5.5.2 CDNA SEQUENCE OF SHORT IsoFoRm (SEQ ED 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351

TGAGAGCTOA

CCGGCGGGCG

GCTGAGACAG

T GT TAOAGAA

CATTGGCAOG

GTCCAGAGGG

ACXTOT TGGA

AACTAAATTC

AC TOTTOT TO

ACAGCAGGCA

COAAAGAAAA

TTAGAAGGAA

TCGAAAAATC

ATGOAAGAAA

GAAAAGAGTA

GT TCCTGACG CO TOTGOOAO T CCAGAGAAG GOCAGO TGC T

TATGTOATGT

GOTTTGAATC

TGAGT TOOTA

AGGGACAACT

CAACGGATGO

CAOTGCAGAG

TTAGGGOAGO

T TCT CCOGA

ATTTTCCAAG

CGCTGGCCTG

AGAG CGO G CG

TTTTATCTAG

GTGC TGAAAG TGAGT TTO TG

CAGAAGOOAG

CAG TACO T TG TAT OAT TGAT

TGTCGCTCCT

AOAGATTTTA

GAAO TOAAGT GTGC TOCAT C

AGGOGACAGO

GACACAGOTO

OGGTTGTGAG

CAGCT TOTA

OAAAGAAOAG

TOAT TCTGAG

TTGTOTATTC

T TT GTTATO T ATAATTOT TO

GCAGGOTTTG

GGTT TOTGOA TOT TTCGAAO

GCAATGCCAG

TTTAAAAATT

GAOAAAAGAA

TATCGTCAoC GOAGO CT TGG

GOGGACCACA

TTOATGAACC

AC TGC TOTGT

ATTGATGTCA

GGAAGAAGAA

TOOATGGACG

CAGG C CC TGA

TOOCTTGGTG

ACOTGCOACT

T TGCAAAAAT TGGTOAGGT T

GTAAAAGTAO

T COACO TCTG

TAAAOAAGG

AGGAAGGO CA

GTOCTTTCTG

AOAGGATTOA

T CAGTAAOAT

OTATTGGAAA

CC TAG CAC TO

GGGAOTGCTG

GGTTGGAOAG

CGTGCTGCAC

AAAGTOTTAG

AGAG T TGCT AACAC TACAG

GAGCOTTCT

TGAGTOGGGA

GAG CG GAG GT

CAAATTATAT

OATGAGOACG

ACOAGO TTTG

GAAGAGGAGA

AGGAT T TTG

OATGCAGAGA

TGGAA.GATAC

GTCATCTGAT

OAAC TOAGGG

TOTGCAAAAG

CCATCGTTAT

ACT C CGAAG G

AGGCTCOACG

OCT CGTAGOO

AOAOOATGTC

AArrGGTGAOA GAATAAT TOT

AAGTTTGTAA

AGTG TAG TAO

TAACOAGAGT

AAGAGCOGGT

OTTATGTCAG

GAAAAATTTG

TGGAAAAGCA

GAGGTCCAGG

ACTACCTGAG

TTCTCTGCA

GAAGOCTTAT

ACAAGC TGAA GAOAGOAAC T

CAATGCAGTG

OAOAOATGGC

T TACT TAO CA AGAAO TOOTG

CTGTCOAGGA

ATAATCCTGA

AAAAT TATAT TAAACO T TTT TOT GTAAGAG

OAAOTOAGAT

O GOTGCCAOG AAAOO TGAC

TGTGGAAAAO

TAT TAAGTGA OCT T TTGACT

GTTTGACATT

OOAOACTGAC

GACACTTTGG

AGO TT TGGTT

TAGACGTTAG

ACT GAATTGO GCCTGAGCC T

AGGGCTTTGT

CT TOTGGAAG WO 97/28262 WO 9728262PCT/US97/01748 1401 1451 1501 1551 1601 1651 1701 1751 180]1 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651 2701 2751 2801 2851 2901 2951 3001 3051 3101 3151 3201 3251 3301 3351 3401 3451 3501 3551 3601 3651 3701 3751 3801 3851 3901 3951 4001 4051 4101 GAGTTCTAO2 T T TTACTT 02 AGGAT TTAA'.

CAAGGGACG(

AACAGTGTAI

ACTTCATOA'j

CCCACTTCA'

T TTAAAAATC

TGTGCAGTAI

GCTTGOGCT'I

CTATCAGGTC

TGTGATCGCC

TCCAGCAGCT

GGAGGAGCAG.

TAC TGT GGAC

COTTGTGTGC

TTTCAGGGGP

GGATGCATTP

ATAAOAT TC2A G TAGT TGT TO AAGAGGAGT T CT CAGAC TCA

CTTCAGATTT

AAGAGAT TTG

ATTACTTAGA

ATTGTCAGCC

TGGGTTAGAC

T TCATAAGCA

TATGCCAGCO

GGATGTTCAO

GTGTCAGTAA

GATACTTCTG

ACCATGTCTG

AC CAT GCAGC

TCAGTCTTCC

TGAAT TAATA AT CAAGGAAG

ATCAGGATAT

AACAGCACCA

GATTCOAGT C ACT GAATCTG GACT TC T CTC

TTCATAGTGC

CAGGGCTTGT

CCAGTCAAAG

TGCTTCGAGT

GC TGTGAOTG CGGAGAT TTT

TTCTGGGTGA

GATGTTGACA

TT TCAGTGGA

GGGAAATAAT

GCTAGCAAAG

GAAAATTGTC

GAACTGTGAA

kGCTCCTCATC I GTCAAGCCAT P? CTCTTTGGAA 3 TGTTCCTTCA k TAAMAATAAT

PTCCATGTGCA

GOAGCACCAC

AGCTTTCTAA

CCAGAGCGCT

GCTGCAGCAG

TACACAGTGT

COTTTACTGO

TATACTGAAT

AGOTATCACC

TCTGACCAAC

TGCTGGTCCT

LTCCTGOCCAG

GAGGCTTATC

GATTGCAAAT

AGTGGAAATT

GAAT TAG TAC

OATGTGTAGC

AT TTAAAAAC TT T TTAAGT T TGGGAT TOGA

TAGGGGAACA

ATCCAACAGG

GCAAGCTTCT

TCAGAGAGCC

ATAAACACCA

AGAAGCAGAC

GCTATGACAG

TOTCTTGAGG

AGACATTTGG

AGAAACAAT T

TTTATGATAA

AAGGCAGGGA

OTTACCOGA

GAOCTGGGAT

AOAGOOTGTG

TTOCTGGGGA2

CAGAGOATAC

CAGAGCCTGT

CTGTGGAAAA

GTGGCAGAAA

AGCCCTGGGG2

AGAAGAGTCAI

TCAGAAGAAGC

GGAAGAAGGC

CCCAAGACGA I1 TOGATTGTTT C ATACCOTGAG IT CTAAACTTGA T1 AGGCAGAAGG ATCTTCTA C l0o

TCTTGTCTTO

GGATTTAGTT

CAGCAGTTOT

GAAGCTGCAG

GAGTACTGTG

CAAGGAAAAG

O GOGATC TTT AAGCCCCTT T

GOTGOTGOAT

G TT TCCC TGA T GGAATCT GT ATG T T TTAA

GTOOTGAGOA

GAGAAT TAAA TGGC TAAGT T AC T COGGO T CAGOGGO TOT AGAGO T TTGT

OACATTTGTA

GTATAATTAT

ATOATTGTCA

CAA T GAAAO TO TACO TGT C

GTAATGGAGT

AGTOATTCO

AOAGAAAGAT

AGT TGCCGTC

TOAGATTCTO

TGATCCAAAA

TAAAOCTOTT

TCTGATAGGG

COOTCCCAGT

.AOGTTGTCTT

I'OCATGTGTO

I'OACAGGCTT

TOCAGAAACT2

GAAATGAGTA

GCTGACACTG2

TTCTGAGAAA

"TTCOCAOAAL

kCGAAAGGCA 3ACTTTTGGAC

'AACAGAAGAI

E'ATATTGTGTC

'AGAA.TTAGO kATOATTCAG C L'OCTOTGAG C ~TGAGGATTO Ij 'ATGAAGCGG .T 'GGAGTAGAA Ij

~AAACAACTT

TTTGOATGO TI 'GTGCTTGCT C ~AAAGAAOAT TI ~GCGGGTTCT T

AGAGTGCAGC

OAAGAAT TTA

TOAGATGGAA

AAOATT TG.AA AAAAAGGT GA

AOAOOGGOGT

CAGGGOTCCT

GAAGAGACCG

CGOOGTGTGC

GOACOACGTG

TGT TGTAT GG

GTTGOOAGCA

AAOT TOTTGT

AAAGOAGOTT

AGGAGAGACA

TGCOOAGTC

GAAGAOTTGC

OTT TCAAGAA

ATTTACTOOA

ATOTTTAATO

ACAG T.AAGC

AGTATTTGOO

OTAC TTAAAT

AAA.OOACATA

TGAAAGOATT

GO TGCAGT TO

CTTAAGTGTG

OT TGOAGTCT AkAACGAAAGA

COTOTGTGTG

AGTCTGCCAA

GAGCCATTAA

.CCTTCCCCT

GTTGGATOTA

GGTGGTTTC

kTTCAGAAGT 3AAATGAAA

XLAGGGAGATG

3AGTGCTGAO 3TGOAGAGOA CTTATGAGTO 2 3TOTOTOCTG

EGGAATTGGA

3AAOTGAGGG

\AAGCOTTTA

~AGATTTGGGC

;AAGAGOTGT IJ ~CAGTGTTGT I

TAGTGAAAGC

~TAAATCCTG

LCTTGA7\AAC

C

~GGGATTAAA

TI

:ATGTGTTTG

T

TOTOTOOTC P GAATATTTT 2

TTCAAATOC

TCOAGOACCA

TGGC TGOTTA

AGCTCTGATA

AATCAGAGOA

TGTGAGTATT

GGT T TAGO T

CAGAGGGAGA

GOT OACCAGT TGTCOAGAT C

ATOCTAAGTC

CTGAAAG T T

GGATCAGTTA

GCAAOATOTG

O TGOAAGGCA TTCTAO OGA T GT GGAAGTG GACAGAT TAO

GAAAGGOAAT

C TGTGO T CA AT TOOT TAG

TOAGGAAGTG

CCAGGGTAA.T

ATTGAACTAA

TGAAA TOT G

TAGACGTCGA

GGTOCTTOTO

CAGGAAGTTT

OCATTOACCA

GCTTTTOTAT

TGAGTCAGAA

GT OAOATGO T

GAATGTTTGC

CATGT TGAAO 4~AGTGTGCCA

CATAOAGAGG

CCAAGAGC TA

TATOATCTGC

kGOG T GGTG 3ATTGTGGCT

ACAAAGTGA

3ACATTTGTC 3CTACCOTO T k.ACACCTTTC ST TGATGCCC

CCOTGGTGAT

'GTOOOAGOO

~GTT TGAAAC

'AATCCTGAG

.AGOAGAAGG

TOAC TOAOG

TTGCTTTOT

~GAGCTT TOT

LTAC.AAOAGG

LAOACAAACT

WO 97/28262 PTU9/14 PCT/US97/oI748 loI 4151 4201 4251 4301 4351 4401 4451 4501 4551 4601 4651 4701 4751 4801 4851 4901 4951 5001 5051 5101 5151 5201 5251 5301 5351 5401 5451 5501 5551 5601 5651 5701 5751 5801 5851 AAC TC TGAT T

TTTGATGAGC

TAT TTO TGGA

AAAATTGTTG

TCCTTTOOTG

OTCCTGGGAT

ATTTOCOGAG

GCT TTOO TOT

AOTGCTGGC

ATGTGGAATA

AGTAAAGAAA

GAG CAGG TAT

GCTGCTTGCC

TAAATGTTAT

O TGAG TTT TO

GGGAAGTGGG

OAT GAC TGTG O TATGGCCAT COOT TOTAGT

AAOAGAGTTC

GAAT T TTGT T

TOATATAGCA

TAACCTGTTA

TGTTCOTCTT

AGTAOATCTT

OTTATOACAC

AGAGGCAAC T GO TAGO TOAC

AGGGTTTGGO

AATAAT GATO

ATTATAGATO

ATGGOATCTC

CAACAAAAGO

OAOOO TOAGG

AGCTTTTACT

TOOAAGOATG

TCAAGAACGT

GAAATC0TOOT A7AAGTGATTT

OATAOOOOGA

TOTTAAOAGT

GOAACAAAGA

TGCATATAG

AOAOOTGTOA

CATOOAATGA

AGAAAOAAAO

GATGCAGGG

TGAGTTTTAA

TOAOOTTAAA

AAOAGCCTT

CAT GOT TGT

TTAGTCTGGO

TTTTATTTTA

TACTOTCoT

ATOTGTAAOT

TTTGTTOTOT

GOTAGAGGAA

AAAATGGTGG

TTTTAAAAGT

AGO TTAG TT T

ATATTOOAOO

OAAGAT TTAA AGO OATAAAC ATOA T TAC

ATTTGACAG

GTGOOTTTGT

OTOOOTGGC

AGAACCT TGG OCT T TAGT T GAG GAO 0TC OCAGAGAGTA OTGGTGCATO TOTTGGTATC GTTOAGAAGA OTTAAOTOTT OTTTGGAGAA TGTAOAGAAA TTOTTOTOOT TGGTATTOAO TAOTATGAGO OOTTOAOAGT GTOTGAOOTT GTTTAAGOAA TGGTGTOTTA TOAOAGAAAO AAAGCCTTAG GOTTATTGAG AAGAGCAOGG GGOTGATAGA GAGAGTTTTO COTATAGGOT OOOOAATOOA OOTCOGTTG

OTCGGAOAGA

GAAGGATTTA GTCTTTCTOT TGTGGGTTTA ATOOGAGACT GOTGOAGAAA GGGGAAAAAG OATCACTTOT GCAAGAOAGO AGTTTTGAAG TOTGATOTAT ATAOTAACAT TOTTCAAATA GOATATOTGG OAGTATTTtA ATCTATTOTT GATOOTAOTT OACTAOTCAA TTAOOAAAGO GAAATOTTOA TTGTOTOTAA ACTTTAGATA TTOTCAAOA GOTGTTGAAG TTAGOAGCO TTOTGATAOT ACATACT TAT AAATAAAAC ACTTOTOOTT OTGTGTOTTA OAOOAATGGO GATOATTTAT ATGTAACAGT OOAAAGT TAG GAAG.AAOTGC TGTTAGGATG TAOTGAAATT TOTTTTTTAA GOAATCAAOA GTTTOTTAAG GTAGTOTTAA AAAOTGGCTG TGTATTTTTT OTAATATTTT TATACCOTAA TAATTGATAA OTGAGOTTTT GGAOATCAO TGTTTATGTT AAOATAAAGT CACATOATAG TAAOAAATAG TCATTGOT GTOAOAGATA ATGGGAATAT GTAGTAAGGT GOCATTCCGA GGGGTAAGOA AOTTOTOTOA GOGGAGACAA AOTGTGATTO ATGCTTATTT CAAGGTTGTT OAOTAOOTTA TGOAGOTTTT OTAAGAAGAG TATTAATAAT AAOAATTTTT TTAGTGOAG GOATCTGTTG CATGGTOAGT TGTGTTAGAG TGACOOAATO TATGGAGTGT GGOTGACCAT GGTOOTTTAG TAAAGCATTT AATAAOTTTT AAAAOAOTGG COGGGCGOOC GCOAOOGCGG TGG 5.6 EXAMPLE 6 DEDUCED AmiNo ACID SEQUENCES OF MOUSE LYSTI PROTEINS 5.6.1 PEPTIDE SEQUENCE OF LONG ISOFORM (SEQ ID NO:4) 1 51 101 151 201 251 301 351 401 451 501 551 MSTDSNSLAR EFLTDVNQLO NAVVQRAEAR CFLLLTKLNS IIDQALTORE ELLTLLLSLL SSDIILTKEK NSSLQKSTQC KLYLECSAPS HRYSVRDARK TQLSTSDSEG NSDEKSTVVS LVAKPDPSAT KEQVLSDTMS VENSREVILR NNSPFDLOHV LLSLLEKVOK FDIALNHNSS NQSDTLEGQL VSAGWTEEPV ALVQRM~LFRT KNLTELLRAA LKIRAOLEKQ PEPFSPRQKK LPELLEGVLQ LLISOLQSAA SNPFYFSQAA QMEWLLTRDG VPSEAAEHLK ALTNSVIKIM HRRCEYSHFM QHHRDLSGLL VSAFKNQLSK AVCAI{QOLRL LQQVSLSTTo VQILSCVHSV

EEEEEETHMA

PLVWKI PVQE GQVSAKVNL F

KHRRLI{ALPR

QDSNGDI LSE

LALSVVPTLT

VLHLMSVDVS

TLQEVQEGFV

DLVQE FlQHQ S TVKKVKSEQ S PFEE TAEGD

GCCOMDPKS

TLGQYLVHGR

QQATD FNLPL

RKIRRQRKST

FLTQSPKEGH

PAALSILSN4

EFLACFGDOO

TAEAMPE SLR

FSKYRHRALL

GFNLFGTAVL

LHHSMOTRKP.

VQYPEROOOI

VIAPILLHAFK

WO 97/28262 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651 2701 2751 2801 2851 2901 2951 3001 3051 3101 3151 3201 3251 3301 LPALKAFQQH ILNVLSKLLV GETLQGTLCG AGPTSGLPSP FQEDRLHNIQ IANHICNLLQ QLSIPSAQTH MCSQLKQYLP NHIIELNYLD GIRSHSLKAF LSVGPSLHKQ QASSDSPCSL LCVAFLCVSK FADS DRE SAN PSPECLHI{AA DIWSMCRWIY FRSHTEDQGR RQGEMSRNEN SADSVRGFQS QPVLPTSAEQ SLLDICLHSA RACQQJQ4ELE KELFDALLRV ALGNHSADLG QCCSLKLLGE EEGYEADSES LENLTIIGEII YPETCMLGLN SLLIQQGTVK ILLGGFLNIL LTLLWRIFLE KSPCTEILLL GVLSQKPPGI LNSKALGLLR LPLLGQNCWP HLSEGFSVSL EDSSFEGAEG DRPEVTESIN SGTFTFRVCM DSNDDTKAVS KNVHGEISTW VSGQRKTDVI ESLQLAGKWD LGNLLLFNGA DYSKYINKDI LRCDEIRDLF EPVIRLKGQV KTQPSQRPFS EIGGAGTFVF LFARVVELSS GLSMIHQVLV KQKCTVGFHI I TQDVKLLQE LLLDWKIWNK LLNAHVVH-F LLTCQVLQEH ELLTVIFNFL. LAVHPPTNTY HSSSGGQAFP SPGFLVISPS PAFPTYLPLT RAQKLAASLG EEAFISSCES AKTVCEMEAL PRSDDDSPGD ESYPRRPDNL RWPSLVDRNA DDWENFTFS P LSGVLLVLPD AMLEDVMDRI RASKEQKDKF LKNRGFSLLA FDLEEVKHME LFQKWSVI PV VADMLLDNGL LYVLCNTVAA ACSSSGTQYF RVIEDLIVLL RSTANHDSES PVHSPSAIRH ASDELHSNNQ RRMSQEHPSQ IDILRTPENT SQSKTSVSQT KVCIGSSKTS VSKQQWTKIL ILEIVH-EPAH QDILRDCLSP AELLMNALKL CGHKCIPPSA TVNNNQQSLF QRLDFKSKDI KVDLSASRHW QECIQQLTHD RCYLTIPNKY LLRDRQKSEG IRVNRRCTSV APSRETAGEL S FS WTYEE 1K EVHRRWWQLR I LTNNLPNLL FYGNI TALTN PVFPFILSDY VSETLDLNDP EYRKGAREDD PMPPVQPYHY SFDIPDRTFH STNTTWRLSS VRQNGERVNH VNLPPWARND PCTIUS97/01748 DQLGGAELSP RIKKAACNIC TVDSDQLAKL SYRFQGILPs SGSEDLLWKW DALEAYQSFV KGNVVVQWKL YNYIFNPVLQ RGVELVHHCQ QEVLQIYLKT LPVLLKSRVI RDLFLSCNGV ETLIVSLGEQ QKDAAVLDVD GLDIQQELPS RKFYASLREP DPKKRKTIHQ DVHINTINLF ESEDTSGYDS PPSEPLSHML PCLSLEDWVL MLNSVFQKQF HRLGGFQVCH ELIFM'IIQKL QELIRISYPE LTLKGDVSSA TAPDLGFLRK IVATESVPGE RKAFMSQQSE TSLQSIRLLE LPSQGIJSVEN ILCELREHLS QSKV.AETELA PGDAVTEKSH PSEEELLSQP GDFSEEAEDS NPEDVDTQDD GVELNPEAEG FSGSIVSNNL LLSASKAKLD VTJAHVFESFL KIVRQKEKNI TQTNSDFQAC QRVLVDLLVS LMSSRTCSED GIHKIVESDF TMSPSQCLTF PFLHTPSLSN RA.RISRGKKE ADRESFPYRL LSSWHIAPIH VGLMWNTSNE SESAAERGKR XTKKRNKPSVL PGDRLIEDGC IHLISLGSKA LMIQVWADPH LAQVESQENI FFPSKWQHLV LTYIQHPQGK LDFVLPRKTS LSSDSNKTFC MIGHCLTSQE KIGSQEAFFL YACGPNYTSI MPCKYGQPVI MTKKEVDVGL LIESLSVVYT TCOPAQYTIY SKEAQSILLE PSQLKGLQPT ECKAIQGILH CEETQALALR VILSLIKYSQ QRTQELENCN LKTLLEGCCG EEVIHVSEHG EFKLDVESHA AEQGVWETLL AALEVLIRVE HHQQQFNIKQ REGQLTSMPR EVCRSFVKII AEVLGSPPDL VCHNPTNFYF SLHTDGKIFQ EKVQSLAYLR AFTAAPPEGT SSSNIVPQRM AAQMVRSRSL FSVDKLQNIA DANPEKQNLL GRPYALKTSK LGAHASANGV SRGSPRFRA RVDHKDVGTE KGLASFQRSQ STVASLGLAF PSQNGSAVAS AYEASYNPAT STHSVIEDCL IPICCGLYEL IQADILLVLV NHPSPATQQG VIKLLHAYIN NQLYLUHRGTQ ELLECFVEMF FGRPIGLDEE LGLIETSLYD NVLLHiNALLL LLQVLNSCSK LNGLEKNIPV NEYKLLACDT QQLFIAVTIH GYLHNSKNKR TQNM~ALALQL RVLQAALEFI SVPPKRRSIA GSRKFPLAQT ESLLMKMRSV ASEAELAQRL QRLTIIJAVNR IIYQELNSDT ETSEEDMHHE QPSVYNPFQK EMLTYLLDGF GSCKETLRDQ LGRLLAILIS PTHTVQERKQ SPQHGAKLVL YLSELIHNHQ DELSEEEMDT PSKPELIKII REEQIKKYESE ESVSKGSWQK SKIAADTTQA VSLSQGIERK KVIQHIRGMY RAVWYDPIYY PTSWQLDPTE GPNRERRRLQ VLRPPLSYLF EDKTHSSFSS TVKDKAASES LLGKCGMYFV EDNASDAVES SSLQGELEPA DNAVEIFLTN GRTLLLAFDN NKVRDDVYQS LWYSGQITNF EYLTHLNKH-A GRSFNDLMQY SIYRNLSKPT AVQYKEKEDR YVDTYKYLEE GSHYSNSGTV LHFLVPMPPF TKMFLAYQDQ FESMTDVKEL IPEFFYLPEF LVNREGFDFG PRLFILIHRQ ALESDHVSQN ICHWIDLVFG WO 97/28262 PTU9/14 PCT/US97/01748 3351 3401 3451 3501 3551 3601 3651 3701 3751 YKQKGKASVQ ATNVFHPATY LFHTAHASRP

GAKLNIEGEL

TKGLKWGEYV GSPSAPVPVV LMTYNKEQGV RSMNNTNT QW QQHQVTScAW VPDSCQLFTG GHTEEITGLC VCKPYSVMIS vSASETSGDT ATVCDSAGGG PEGVS INVIA GGLENGIVRL HHLYTANSEG TVIAWCRKDQ FGMDVSAVED PVQRRALETM IKTYGQTPRQ PAA.VGLLVQF AFRETREPVK EVTHPSPLSW CFSQPHGERF GSLQALPTRA ICGLSRNFCL SAILSWGYAD NILRLKSKQS EPPINFIQSS SKCGVTTAYT NRLTSSTPSE IEMESQM-LY VSRDGTCIVW DLNRLCYVQS LAGHKSPVTA SDLRLWTVNG DLVGHVHCRE IICSVAFSNQ WSTWDLKPV. EITFPKSNKP IISLTFSCDG QRVKLPMFYs FLSSYAAG 5.6.2 PEPTIDE SEQUENCE OF SHORT ISOFORM (SEQ ID NO:6) 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501

MSTDSNSLAR

GFLLLTKLNS

SSDT TLTKEK

HRYSVRDARK

LVAKPDPSAT

NNS PFDLCHV

NQSDTLEGQL

KNLTE LLRAA

LPELLEGVLQ

QMEWLLTRDG

HRRCEYSHFM

AVCAHQCLRL

LPALKAFQQH

GETLQGTLCG

FQEDRLHNI Q QLS IPSAQTH

NIIELNYLD

LSVGPSLHKQ

LCVAFLCVSK

PS PECLHHAA

FRSHTEDQGR

SADSVRG FQS

SLLDICLHSA

KPLFDALLRV

QCC SLKLLGE LENLTHGET T

SLLIQQGTVK

LTLLWRT FLE

GVLSQKPPGT

LPLLGQNCWP

EDSSFEGAGM

EFLIDVNQLC

I TDQALTCRE

NSSLQKSTQG

TQLSTSDSEG

KEQVLS DTMS LLS LLEKVCK

VSAGWTEEPV

LKTRACLEKQ

LL ISCLQSAA

VPSEAAEHLK

QHHRDLSGLL

LQQVSLSTTC

ILNVLSKLLV

AGPTSGLPSP

TANH ICNLLQ

MCSQLKQYLP

GTRSHSLKAF

QASSDSPCSL

EADSDRESAN

DIWSMCRWIY

RQGEMSRNEN

QPVLPTSAEQ

RACQQKMELE

ALGNHSADLG

EEGYEADSES

YPE TCMLGLN

TLLGGFLNIL

KSPCTEILLL

LNSKALGLLR

HLSEGFSVSL

MAGS DLYTK I

NAVVQPAEAR

ELLTLLLSLL

KLYLEGSAPS

NSDEKSTVVS

VENS REVI LR

FDTALNHNSS

ALVQR4L FRT PEPFS PRQKK

SNPFYFSQAN

AL INS VI KIM

VSAFKNQLSK

VQILSGVHSV

DQLGGAELS P

SYRFQGILPS

KGNVVVQWKL

QEVLQ IYLKT

ETLTVSLGEQ

RKFYAS LRE P

ESEDTSGYDS

MLNSVFQKQ F

QELTRISYPE

IVATESVPGE

LPS QGLSVEN PGDAVTEKS H

NPEDVDTQDD

LLSASKAKLD

TQTNSDFQAC

G IHKI VESD F RART SRGKKE VGLMWNT SNE LQIAACLS FK EEEEEETH4A PLVWKT PVQE GQVSAKVNL F

KHRRLHALPR

QDSNGDT LSE LALSVVP TL T

VLHLMSVDVS

TLQEVQEGFV

DLVQE FTQHQ S TVKKVKSEQ S PFEE TAE GD GI CCCMDPKS RI KKAACN IC

SGSEDLLWKW

YNYI FNPVLQ

LPVLLKSRVI

QKDAAVL DVD

DPKKRKTTHQ

P PS EPLS HML I-RLGG FQVCH LTLKGDVS SA

RKAFMSQQSE

TLCELREHLS

PSEEELLSQP

GVELNPEAEG

VLAHVFE SFL

QRVLVDLLVS

TMSPSQCLTF

ADRE SFPYRL

SESAAERGKR

HTWQYFNVFF

TLGQYLVHGR

QQATDFNLPL

RKIRRQRKS T FLTQS PKEGH

PAALSILSNM

E FLAGFGDCC

TAEAMPESLR

FSKYRHRALL

GFNLFGTAVL

LHHSMC TRKR

VQYPERCCCI

VIAPLLHAFK

TVDS DQLAKL DALEAYQS FV RGVE LVHHC Q

RDLFLSCNGV

GLDIQQELPS

DVHINTTNLF

PCLSLEDVVL

ELI FMIIQKL

TAPDLGFLRK

TSLQS TRLLE QS KVAE TELA GDFS EEAEDS

FSGSTVSNNL

KTVRQKEKNI

LMSSRTCSED

PFLHTPSLSN

LSSWHIAPIH

VKKRNKP SVL

KCYSP

5.7 EXAMPLE 7 DNA SEQUENCES OF HUMAN LYST1 GENE 5.7.1 cDNA SEQUENCE OF LONG ISOFORM (SEQ ID NO:7) 1 CGCAAGGGCT TCTAAGAAGC CATCCCAATG ACCTTTTGGC TTTGAGAAGA 51 GCAGTCCTCA TACCAGAGTG TTTGGGGTTT TGGCCTCTTT CAGTGTTTAT WO 97/28262 Ar 101 TCATTCTTAC

GTGOGAAAGT

151 CTTTTGCCTT

CACCATCTGT

201 ACAGCAGTAT

TTTCCCTTGC

251 ACCGTGTGTG

TTCCTCAATG

301 GAACAGAAGC

CAGGATCTAA

351 GCATGGAAGA GAACCACTTT 401 ATTAGCCTGC AACAAAGAGT 451 TTCAGGTGAA

GCTTTGTTCT

501 CCAAATGATA

TAGACTGTAA

551 TCATGAGCAC

CGACAGTAAC

601 AACCGGCTTT

GCAATGCAGT

651 AGAGGAGGAG

ACGCACATGG

701 GAGGATTTCT

ATTACTTACC

751 ACATOTAGAG

AAGAA.CTCCT

801 ATGGAAGATA

CCTGTCCAAG

851 TCTCAGCAGA

TATAATCCTG

901 TCCACTCAGG

AAAAATTACA

951 TTCTGCAAAA

GTAAATGTTT

1001 CCCATCGCTA

TTCTGTAAGA

1051 GATTCAGAAG

CCAATTCAGA

1101 AAGGCCCCAT

CTGCTGCATC

1151 ACCCCAAAGC

TAAACTTGAC

1201 GATGCTATGG

CTTTGGAAAA

1251 AAACACTGAC

ATTTTAAGTG

1301 TGAACAATTC TCCATTTGAC 1351 AAAGTTTGTA AGTTTGACGT 1401 C-AGTGTAGTG CCCACACTAA 1451 GCAGTCTGAC

CGACAACTTG

1501 GAAGAACCGG

TGGCTTTGAT

1551 TCTTCTGTCA

GTAGATGTTA

1601 GGAAAAATTT

AACTGAATTG

1651 CTAGAAAAGC AGCCTGACCC 1701 GGAGGTTCAG

GAAGATTTTG

1751 TTTTACCTGA GCTTTTGGAA 1801 CAGAGTGCAG

CTTCAAATCC

1851 TCAAGAATTC ATTCAGCATC 1901 TTCAAATGGA ATGGCTGGTT 1951 GAGCATTTGA

AAGCCCTAAT

PCTIUS97/01748 IDot TGTATTCCGA GGTTTCTGTG GTGCATGAAC TCCCGTGTCT TCCTCGGGTG

ACATCAGAGT

CATCTAATGG GGTTTGGGCT

GTTTGACTCA

CCAGGGGAAT AATCCTACCC TAA.GTCAGCT CTGCAAACAA GAGACCCAGC TTGCTTAACA CCTTGCAGCT ACCTGGGAAG ACGGTTGCTA TCCTTGCTCA TCTAAAAGAG

GCAATCACCG

AAGAATATTT GTTTCATCTA

GTTTATGAGT

ATGTCACAGC AGTGGTGA,A

GACTGCTCGG

TCACTGGCAC GTGAATTTCT

GACCGATGTC

GGTCCAGAGG GTGGAGGCCA GGGAGGAAGA CAACCCTTGG ACAGTACCTT

GTCOATGGTC

AAGCTAAATT CTATAATTGA

TCAGGCATTG

GACTCTTCTT CTGTCTCTCC

TTCCACTGGT

AAGAAAAGGC AACAGATTTT

AACCTACCGC

ACCAAAGAAA AGAACTCAAG TTCACAAAGA TTTAGAAGGA AGTGCCCTGT CTAGTCAGGT TTCGAAAAAG CAGAGGACAG CGTAAAATTA GATGCAAGAA AGACACAGCT CTCCACCTCA TGAAAAAGGC ATAGCAATGA

ATAAGCATAG

ATTTTTTAAC ATCGTTTCCT AAA.CAAGACC CGCTTAGCAA CCAAAGAACA GACTCCTCCA TTCCAGAGAG ATTATTCCAA GACAGGGGTC AGCCAGCTGC CTTGTCTGTT ATCAGTAACA TTATGTCATG TTTTGTTATC TTTATTAGAA TACCTTGAAT CAT-AATTCTC CTTTAGCAGC CTGAATTCCT AGCAGGCTTT GGGGACTGCT GAGAGTCGAG TAGTTTCTGC AGGTTGGACC TCAAAGGATG CTCTTTCGAA CAGTGTTGCA GTACTGCAGA GATGATGCCA GAAAATCTTA CTTAGAGCAG CTTTAAAAAT TAGAATATGC TTTTGCACCA AGAC.AAAAGA AAACACTGCA TGTTTTCAAA GTATCGTCAT AGAGCCCTTC GGAGTTCTTC AGATTCTGAT CTGTTGTCTT CTTCTACTTC AGTCAAGCCA TGGATTTGGT ATGGATTTAA TTTATTTGAA ACAGCAGTTC TTAAGAGATG GAGTTCCTCC CGAGGCCTCA AAATAGTGTG ATGAAAATAA TGAGCACTGT AACTTCATCA TTCGATGTGT ACAAGAAJAAA TCTCATTTTA TGCATCATCA CCGAGATCTC TTTTAAAAAC CAGGTTTCCA AAAACCCATT ATGTTTATTA TCCTGAGCGG TGCTGTTGCA TGCTTGCGCT TACTGCAGCA GGCTTCCTTG CCTATCGGGT GTTCATAACA TTGGAATATG CTGTAATCAT TCCTTTGCTC CATGCTTTTA TTTCAGCAGC ATATATTGAA TATCCTTAAC AGGAGGAGCA GAGATATCAC CAAAAATTAA GTACTGTTGA CTCTGACCAA CTAGCCCAAT AACTTATGTG ATGCTGAACT CTCCTCAAGT TATTTCAAGGG ATCCTGCCCA GCAGTGGATC GGGATGCTTT AAAGGCTTAT CAGAACTTTG CATAGTATAC AGATTGCAAA TCACATTTGC TATAGTTGTT CAGTGGAAAT TATATAATTA M.AGAGGAGT TGAATTAGCA CATCATTGTC GCTCAAAGTC ATGTATGTAG CCATCATAAC 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651 2701 2751 2801 CAAAAAAG TG

GGCACAGACG

TCAGGT CT TC TGAAGAGAC T T TGCAGT GTG

AGCAGCACTT

CT GT TGTATG

AATTGCCAGC

AAACTTATTT

AAAAGCAGC T

TAGAAGAGAC

TTATCCAGTC

TGAAGATTTG

T T TTTGGAGA

AATTTAATCC

CATATTTAAT

AACACCTAAG

AAAT CAGAGC AT GTGAATAT

TGGTTTCGGC

GCAGATGGAG

TGCCCATCAG

GTGTCCAGAT

GATCCCAAAT

AC TGAAAAAT

TGGATCAGTT

TGTAATATTT

AC TGCAGGGA CT TCT TACAG

TTGTGGAAAT

AGACAGAT TA

AGAAAGGCAA

CCTGTGCTCC

CG TTACT TCA WO 97/28262 2851 2901 2951 3001 3051 3101 3151 3201 3251 3301 3351 3401 3451 3501 3551 3601 3651 3701 3751 3801 3851 3901 3951 4001 4051 4101 4151 4201 4251 4301 4351 4401 4451 4501 4551 4601 4651 4701 4751 4801 4851 4901 4951 5001 5051 5101 5151 5201 5251 5301 5351 5401 5451 5501 5551 OAGTGCTTGO

OTOAGGAOGT

OOTGCTTAAA

TOCAGGGTAA

TAAGTCAAAT AATO GAAT TA CTAAAAGCAT

TTGAAAOTOT

TGOCTCAGTT

OOAGATATTG

OOTCTGTAOA

TGTGGGTACT

TOTOOTOAGA

GTOTCAGCAA

AAAGAGACGG AAGACTGTTA TATTCOTOTG

TGTGGOTTTT

AGGGAGTOGG

CCAATGACTC

CAGOGAGCoT

TTAAGTOATA

TOTTGOOTTO

TCCTGAAOAT

TGTCGTTGGA

TCTACATGTT

GCTTGGTGGT

TTOOGAGTAT

AACTGTTCAG AAGTOACAAA AGTGTAAATG

AAAAOCAGGA

!K

GCTTCAGATT

TAAGAGATT

T

AATTGCTTAA

GATAATCAGC

ATGGGATAGA

TOTTTTOATC

ATTTTATGCT

ACCAAGATGT

TTATGCGTAA

AGATAGATAC

T

T GOTGC CATG AT GCACCAAG GAG TTOAGT G GCCATAAGT T

GAGGAGCAAG

TTTAAACAGA

OTT TGGCTAT

AAGAGTGOTG

AAATG TGGAA TAT TTAOAAG GAAGCO OTTO

GATGGAATTG

TTGAAATGAG

GOAAAGC OTT

AGOAGATTTT

OAGAAGAATT

TOTOAGTGTT

~GATAGTGAA

ACTTAAAGTO

TAT GAAGGAA

OAGAACTAGG

T TAOAGOATA OGO TCOGAG T TCAAAG TAT GO OAGAAO TA GT T G TGGAA

AGGTGATTGA

GTTGOOOTCG

TGAGAAGAGT

TTTOAGAAGA

GAAGAAGAAG

AACOOOAGGAT

OATOAAGOAG

AT TTATC00TG

AGOOAAAOTT

T TAGGOAGAA

AAAAATCTTT

TTTTOAAGOA

GTTOAAGAAC

GAGAAAT CT C

TGAAAGTGAT

TGOAOGOT~C

ATTTTAAACA

GAG OAAGAAA

OTTGGOACAT

COACACCTAT

TATOCATGAA

GAAAOAAAT

C

GACAGACOAG

AGAA~GGATGT

AAGTGTGGGO

GAT T AAATG GGAGAATAT 7

TACAGOAGOCC

GTOTOTGGAC

AA-AAAOAAG T

ATTGTTTATC

CTGGGAAATT

*GATTTATTAT

TAGTOTAAAA

TTTOTTCOAT

GAAGCAAAGC

AC GAOT T TTG

GTOAAOAGAA

AGTATATTAT

AAOAOAACTA

GGAATTATTO

CATOAATOTG

AGO TGAGGAT

GTTACGAAGC

GATGGGGTAG

TOQAAATGAC

AGATTTGTAT

GATGTGOTTG

AGATAAAGAAT

TAGGAGGGTT

TGOOAGAGAG

ATGTTOAGAA

O TTG TACAAA

ACTACTATGA

AAAT TTAAG.O

GTAAGGOOA:T

GAGGOTGATA

AG C CCCAG T

OAGAAGGTTT

GOTGAGAGTA

ATTAATTTTA

AAGG T GAGA AT TOATATAA TGAT COOCAC

ATGAOATGAA

7TOO TOOCAA O OAAGGGAAA

AGAGGAAGCC

TTGTCATOTG

AT 0CAAGA

TGCTTOTOTT

PCT/US97/01748 TATGTAAAAA

CTOTGCOTAT

GTTTTTGAGT

TGTAATGGAG

ATGGTATTOG

AAGTOATTCT

OTAGGGGAGC

AACAGAAAGA

OATTGAACAG

AAGGAGTTGT

ATCAGOAAGC

TTATTOAGAT

GGOOTOAAAG

AAGOTTATOO

TOATATCAAC

AOAATAAAC

GTAAAGAAGO

AGAGTOTGAC

TOTGGOTATG

AOAGOAOAGO

TATATOTOTO

GAGAGCCTTG

OAGOAGACAT

TTGGTCTATG

TTOOAGAAAC

AGTTTTATAG

AATATTTATG

ATAATACAGA

GAAAAAAGGA

GGGAGATACA

ATTTOTOAAC

CTAAGAGAAC

AAAAAGTGAC

COCATACOAT

AOAGTT7TAGG

TAM.TTAGAG

GAAGTTTOAG

CTAOTGAAGC

TCAAGAAAGT

GAGACOTOAC

TGGOOATTTG

TOTTOATGGT

GAGTTAOOTA

ATCAGAACTT

GGACOATOTT

TOOCAGTCAA

TATTTGATGC

OOTGOTTCGA

GAAOATAATG ATGO TAT GAO GTCATCOCAG

OOTGGTGATT

GTAGTTTTAA AOTTTTAGTr AGOAATOCTG

AAGATGGCGA

TGAAACAGAA

GGTTTCAGTG

ACOTOACTCA

AGGGGAAATA

AATTTGOTTT

CTGCTAGTAA

TGAGAGTTTT

TTGAAAATTA

TOATGOAACA

GGGAAOTGTG

TTAAOAOAGG

ATGATTCTGA

TOTTTTGGTA

TCTTTGATGA

TTOTTTTGAG

AATATTTOTG

OTGGGTATTC

TGAAAATTAT

GTATOTAAOC

TTOOOTTTAO

OATOAOAAAA GTATOOTGGG

TTACTOGAAA

GCTGGAATTA

COCATGTAT T

GTTTTTCTGC

OTTGAGTATT

TAT TGGTGGA

GAGCTAACOO

AATTCTTOTT

GOOC TTOACA AAOGGTG T TT

GGGTTTATTG

GAGAGAGTT T CACO TGOCGT CAGTGT T TOC O TAOAGAAAA

COAGATAGCA

GTAOATAAAT

TTTCACTGGG

AATGCCACTO

AGO TG T TTTA

AGAAGAGOAC

TOOOOATOGG

TGOTGGGGOA

OTGTGGTTTA

AGGAAAGAAG

G T TTTGATGG

COTGGTGAAA

AT CCAAAGO G

TTATOTTTOG

OTAGCAOAGG

GAGT T TACG

OTGCTTTOAT

AAACTGOTGG

ATGTGGAGTG

ATAAAGAUA

TACAGAGAGO

GACTCATAGA

TTGATGAT~C

TGTGTGCATG

TTGAATOAOA

OTOACOTACT

CTOCATATGG

TGCTTOOAAG

ATGATTGGOC

AAAATGGGAC

CACAAGAGGC

GCAAATGGOA

AOATTTAGTA

AGGAGGATTO

ATGGGAAAAT

TGATGTTAOT

TTGGATTTTA

ATAGOAATJAA

AACATTTTGC

GAGTTTTTGC AGTTGGOTGG CAAOGGAGOT AAGGTTGGTT WO 97/28262 PCTIUS97/01748 5601 5651 5701 5751 5801 5851 5901 5951 6001 6051 6101 6151 6201 6251 6301 6351 6401 6451 6501 6551 6601 6651 6701 6751 6801 6851 I OvE CTTTTATCTG TATGOTTGTG GACCCAACCA

I

AGTATGGCAA GCCAGTCAAT

GACTACTOCA

TTGCGATGTG AACAAATCAG AGAATTTTTT 7 TATTGGTCTC TTAATTGGAG TCTTTCAGTT

C

TGCTCCAGTA TACCATOTAT GAACCAGTGA

T

AAAACOCAAC TCTCTCAAAG ACOCTTCAGO

TI

CTTATTAGAA CCTCATCATC TAAAGAATCT

C

CTATTCAAGG CATTCTGCAC

GAAATTGGTG

CTCTTTGCCA GGGTTGTTGA ACTCAGTAGO

T

AGCACTGCGA OTTATACTOT CATTAATTAA

P~

ATGAATTAGA AAATTGTAAT GGACTTTCTA

T

AAACAAAAAT GCATTGTTGG GTTTTACATT

T

ATGCTGTGGT GAAGATATTA TTTATATGAA

T

TGGATGTAGA CTCTAATGCT ATAATCCAAG

A

OTATTGCTTG ACTGGAAGAT ATGGAGTAAA

G

AACTTTGCTA GOAGOTOTAG AAGTCCTCAT

C

AGATGTTTAA TATTAAGCAG TTATTGAAAG

C

CTACTGAOTT GTCAGGTTTT

GCAGGAATAO

CATGCCCCGA GAGATGGCAA GATCTTTCAG

G

GTACCTGAGG CATTCCAGCA GTGGAGGAAG

G

TTATGGTAAT AAGCCCATOT GGTTTTACTG

C

AATTCCTCTA ATATTATTOC ACAAOAGATG

G

TAGAAGCCTA OCAGOATTCC CTACTTCTTC

A

AACTGACTGG AAGTTTGGGT TGTAGTATCG

A

GATACTTATG TTGCCACCCA ATCAAAGAAA

C

CGACACACTG AAAAAAGGCA AAGAGGACGC

A

CTGCAAAAALC TGTTTGTGAA ATGGAAGCTG

T

GTCAGTGATG TCCCAAAGGG AGTGCTGGGA

T

TCATAAACAG TTGGGAGCAG AACCCAGGTC

A

ATGAGTCCTG CCCANOGCCG AGCCCTATGO

A

~ACATCTGTA

ATGCCATGTA

ATATAT TAA TAAAGAAAT

T

TGACCAAGA

AAGATGTGGA

TTTATACAA OTTAOTGTCC ~TAGACTTAA AGGTCAAATG

'CAAAAGAAG

:OAACO TACT

;AACTGGOAT

'GTGAAGAAA

~TACAACCAA

GATTCATCA

TGAAGACCC

GAGAATGGA

~TGTTAAGCT

CAGAGCAAG

AGAGCAGAT

T CAAG TGGT

AAGAGGGGC

AGAAAGTGO

TCCOTTATG

TTCACCATA

CCGCCATA

CTACTAACG

CAGG TTACA

AAAATTCTT

TTCATCAGT

CC TOTCAGC

TTCCAGTGG

GAAGAT

GAO

TTCAGAGCAT

GAATATAAAA

ATTTGTTTTT

CTCAAGCATT

CAAAGAG TAO GG TG TTGAT C T TCT TGAAGG GAGT TTAAG T GT TAGAGGAA

GTGTTTGGGA

CACCACCAGC

T CAT CAC TT T AA T CACAOCC GG TCAAT CAT

AGOCCTGGAT

TGAAGGAGAG

TGOTGCGTTO

CAAT CACAAA

AAATATTGCA

TGGGGAGT TO AG T G TGAGT

OCAGGTCTCT

TC.AAAGOAGA

AGT OCT GGGG 6901 6951 7001 7051 5.7.2 cDNA SEQUENCE OF SHORT ISOFORM (SEQ ID NO:9) 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 CGOAAGGGCT

TCTIAAGAAGC

GOAGTCCTCA

TACOAGAGTG

TOATTOTTAC

GTGGGAAAGT

CTTTTGOOTT

CACCATCTGT

ACAGCAGTAT

TTTCCCTTGC

ACCGTGTGTG

TTCCTCAATG

GAAkCAGAAGC CAGGATO TAA GCATGGAAGA

GAACCAGTTT

ATTAGCCTGC

AACAAAGAGT

TTOAGGTGAk

GCTTTGTTCT

CCAATGATA

TAGACTGTAA,

TCATGAGCAC

CGACAGTAAC

AAOCGGOTTT

GOAATGCAGT

AGAGGAGGAG ACGCACAT GG GAGGATTTCT

ATTACTTACC

ACATGTAGAG

AAGAACTCOT

ATGGAAGATA

CCTGTOCAAG

TOTOAGCAGA

TATAATCCTG

TCCACTCAGG

AAAAATTACA

TTCTGCAAA&

GTAAATGTTT

CATOCCAATG

ACOTTTTGGC

TTTGGGGTTT

TGGCCTOTTT

TGTATTCCGA

GGTTTCTGTG

TCOGTGTCT

TCOTOGGGTG

CATCTAATGG

GGTTTGGGCT

CCAGGGGAAT

AATCCTACCC

CTGCAAACAA

GAGACCCAGC

CCTTGOAGCT

ACCTGGGAAG

TCCTTGCTOA

TCTAAAAGAG

AAGAATATTT

GTTTCATCTA

ATGTCACAGC

AGTGGTGAAA

TCACTGGCAO

GTGATTTCT

GGTOCAGAGG

GTGGAGGCOA

CAACOCTTGG

ACAGTACCTT

AAGOTAAATT

CTATAATTGA

GACTCTTCTT CTGTCTOTCC AAGAAAAGGC AACAGAT TT T ACCAAAGAAA AGAAC TCAAG TTTAGAAGGA

AGTGCCOTGT

TTCGAAAAAG CAGACGAOAG

TTTGAGAAGA

CAGTGTT TAT

GTGCATGAAG

ACATCAGAGT

G T TTGAC TOA

TAAGTCAGOT

T TGOT TAACA

ACGGTTGCTA

GCAATCACCG

GTTTATGAGT

GAO TGC TOGG

GAOOGATGTC

GGGAGGAAGA

GTCCATGGTC

T CAGG CAT TG

TTOOACTGGT

AAOCCTACOGO

TTCAOAAAGA

CTAGTCAGGT

CGTAAAkATTA WO 97/28262 WO 9728262PCTIUS97/ifJ74s 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651 2701 2751 2801 2851 2901 2951 3001 3051 3101 3151 3201 3251 3301 3351 3401 3451 3501 3551 3601 3651 3701 OCCATOGCTA

TTCTGTAAGA

GATTOAGAAG CCA-AT TCAGA AAGGCCCCAT

CTGCTGCATC

ACCCOAAAGC

TAAACTTGAO

GATGOTATGG CTTTGGAAAA AAACACTGAC

ATTTTAAGTG

TGAACAATTC

TOOATTTGAC

AAAGTTTGTA

AGTTTGACGT

CAGTGTAGTG

COCACAOTAA

GCAGTCTGAG

CGAOAACTTG

GAAGAACOGG TGGOTTTGAT TCTTCTGTCA

GTAGATGTTA

GGAMAAATTT

AACTGAATTG

CTAGAAAAGC

AGOCTGACCO

GGAGGTTCAG

GAAGATTTTG

TTTTACCTGA GCTTTTGGAA OAGAGTGCAG

CTTCAAATCC

TCAAGAATTC

ATTCAGCATC

TTCAAATGGA

ATGGCTGGTT

GAGCATTTGA

AAGCOTAAT

CAAAAAAGTG

AAATCAGAGC

GGOACAGACG

ATGTGAATAT

TCAGGTCTTC

TGGTTTCGGC

TGAAGAGAO T GCAGAT GGAG TTGCAGTGTG

TGCOCATOAG

AGCAGCACTT

GTGTCCAGAT

OTGTTGTATG

GATCCCAAAT

AATTGCCAGC

ACTGAMAAAT

AAACTTATTT

TGGATOAGTT

AT\AAGOAGCT TGTAATATTT TAGAZ-GAGAC ACTGCAGGGA TTATOCAGTC

CTTCTTACAG

TGAAGATTTG

TTGTGGAAAT

TTTTTGGA GA AGACAGATTA AAT TTAATOC AGAAAGGOAA CATATTTAAT

CCTGTGCTCC

AACACCTAAG CGTTACTTCA CAGTGCTTGC CTCAGGACGT CCTGCTTAAA TCCAGGGTAA TAAGT OAAAT AATOGAAT TA CTAAAAGCAT

TTGAATCT

TGCCTCAGTT

OOAGATATTG

CCTCTGTACA TGTGGGTACT TCTCCTCAGA GTCTOAGCAA AAAGAGACGG AAGAOTGTTA TATTCCTCTG TGTGGCTTTT AGGGAGTCGG COATGAOTO CAGOGAGOOT TTAAGTCATA TCTTGCCTTC TOCTGAACAT TGTCGTTGGA TCTACATGTT GOTTGGTGGT TTCCGAGTAT AACTGTTCAG AAGTCACAAA AGTGTAAATG AAAAOOAGGA TAT GAAGGAA GAT TTAT TAT CAGAACTAGG TAGTCTAAAA Itol GATGCAAGAA AGACACAGC T TGAAAAAGGO ATAGCAATGA ATTTTTTAAC

ATCGTTTCCT

CGCTTAGOAA OCAAAGAACA TTCCAGAGAG ATTATTCOAA AGCCAGCTGC

CTTGTCTGTT

TTATGTCATG TTTTGTTATo TACCTTGAAT

CATAA.TTCTC

CTGAATTOCT

AGCAGGCTTT

GAGAGTCGAG TAGTTTCTGC TOAAAGGATG

CTCTTTCGAA

GTACTGCAGA GATGATGCCA CTTAGAGCAG

CTTTAAAAAT

TTT TGCACOA AGACAAAAGA TGTTTTCAAA

GTATCGTOAT

GGAGTTCTTO AGATTCTGAT CTTCTACTTC

AGTCAAGCCA

ATGGATTTAAk TTTATTTGAA T TAAGAGAT G

AAATAGTGTG

AACTTCATCA

TCTCATTTTA

TTTTAAAAAC

ATOTT TAT TA

TGOTTGCGCT

CCTATCGGGT

CTGTAATCAT

TTTOAGCAGC

AGGAGGAGCA

C TACT GT TGA

AACTTATGTG

ATTTCAAGGG

GGGATGCTTT

CATAGTATAC

TATAG TT GT T

AAAGAGGAGT

GCTCAAAGTC

GCTTCAGATT

TAAGAGATT T

.AATTGCTTAA

GATAATCAGC

AT GGGATAGA TOT TTT CAT C

ATTTTATGCT

ACCAAGATGT

TTATGCGTAA

AGAAGATAO T

TGCTGCCATG

ATGCACCAAG

GAGTTCAGTG

GCCATAAGT T

GAGGAGCAAG

GAG T TCTCO AT GAAAAT.AA T TOCAT GT GT

TGOATCATCA

CAGCTTTOCA

TCCTOAGOGG

TACTGCACCA

GTTCATAACA

TCCTTTGCTC

ATATATTGAA

GAGATATOAC

CT CTGAC CAA

ATGCTGAACT

ATCCTGCCCA

AAAGGCT TAT

AGATTGCAAA

CAGTGGAAAT

TGAAT TAG CA

ATGTATGTAG

TATGTAAAAA

OTT TT TGAGT

ATGGTATTCG

OTAGGGGAGC

OAT TGAACAG AT CAGCAAG C

GGCCTCAAAG

TCATATCAAC

GTAAAGAAGC

TOT GGCTATG

TATATOTOTO

CAGOAGACAT

TT CCAGAAAO

AATATTTATG

GAAAAAAGGA

CTOOAOCTCA

ATAAGCATAG

AAACAAGAC

GAO TOO TOCA

GACAGGTO

ATCAGTAAOA

TTTATTAGAA

OTT TACAGO GOGGAC TOOT AGGT TGGACC

OAGTGTTGCA

GAAAAT T TA

TAGAATATGC

AAACA T OCA

AGAGCCOTTO

CTGTTGTOTT

TGGATT TGGT

AOAGCAGTTC

CGAGGOCTOA

T GAG CAC TG T

ACAAGAAA

COGAGATO TO AAAAOO CAT T

TGOTGTTGOA

GOCT TOOTTG

TTGGAATATG

CATGCTTTTA

TATOCTTAAC

CAAAAATTAA

O TACCCAAT

OTOOTCAAGT

GOAGTGGATO

OAGAAOTTTG

TOAOATTTGO

TATATAAT TA

OATOATTGTO

COAT OATAAC CT CTG000TAT

TGTAATGGAG

AAGT OAT TOT

.AACAGAAAGA

.AAGGAGTTGT

TTATTCAGAT

AAGCTTATCC

AOAATAAAC 0 AGAGTOT GAO

ACAGOACAGO

GAGAGOOTTG

TTGGTCTATG

AGTTTTATAG

ATAATACAGA

GGGAGATACA

CTAAGAGAAC

CCCATAOOAT

TAAATTAGAG

TTTAACAGA ATTTCTOAAC CTTTGGOTAT AAAAAGTGAO AAGAGTGCTG AOAGTTTAGG WO 97/28262 3751 3801 3851 3901 3951 4001 4051 4101 4151 4201 4251 4301 4351 4401 4451 4501 4551 4601 4651 4701 4751 4801 4851 4901 4951 5001 5051 5101 151 5201 T TACAGCATA

CGCTCCCGAG

T TCAAAGTAT CCCAGAAC TA G TC TGTGGAA

AGGTGATTGA

GTTGCCCTCG

T GAGAAGAGT

TTTCAGAAGA

GAAGAAGAAG

AAC CCAGGAT

CATCAAGCAG

AT TTAT CC TG AGCCAAACT T T TAGGCAGAA AAAAT CTT T

TTTTCAAGCA

GT T CAAAC

GAGAAATCTC

TGAAAGTGAT

TGCACGCTCC

ATTTTAAACA

GAGCAAGAAA

CTTGGCACAT

CCACACCTAT

TATCCATGAA

GAAACAAATC

ATGACAGGAT

TTCCTTGAGC

ATTCACCTTA

TTTCTTCCAI

GAAGCAAAGC

ACGAC T TTTC

GTCAACAGAT

AGTATATTAI

AACACAAC T.

GGAATTATTC

CATCAATCTG

AGCTGAGGAI

G TTACGAAGC

GATGGGGTAG

TCCAAATGAC

AGATTTGTAIr

GATGTGCTTG

AGAAAAGAAT

TAGGAGGGTT

TGCCAGAGAG

ATGTTCAGA-A

CTTGTACAAA

ACTACTATGA

AAAT TTAAGC

GTAAGGCCAT

GACGCTGATA

AGCCCCAGTC

CAGAAGGTTT

GCTGAGAGTA

ATTAATTTTA

TATCTGATTT

CGTAAAAATG

AAGATCCTAC

PCT/US97/01748 AAATGTGGAA GAAGTTTCAG CTACTGAAGC TATTTACAAG TCAAGAAAGT GAGACCTCAC GAAGCCCTTC TGGCCATTTG TCTTCATGGT GATGGAATTG GAGTTACCTA ATCAGAACTT TTGAAATGAG GGACCATCTT TCCCAGTCAA GCAAAGCCTT TATTTGATGC CCTGCTTCGA AGCAGATTTT GAACATAATG ATGCTATGAC CAGAAGAATT GTCATCCCAG CCTGGTGATT TCTCAGTGTT GTAGTTTTAA ACTTTTAGTT AGATAGTGAA AGCAATCCTG AAGATGGCGA ACTTAAAGTC TGAAACAGAA

GGTTTCAGTG

TTACTCGAAA ACCTCACTCA AGGGGAAATA GCTGGAATTA AATTTGCTTT CTGCTAGTAA CCCATGTATT TGAGAGTTTT TTGAAAATTA GTTTTTCTGC TCATGCAACA GGGAACTGTG CTTGAGTATT TTAACACAGG ATGATTCTGA TATTGGTGGA TCTTTTGGTA TCTTTGATGA GAGCTAACCC TTCTTTTGAG AATATTTCTG AATTCTTCTT CTGGGTATTC TGAAAATTAT *GCCCTTCACA GTATCTAACC TTCCCTTTAC AACGGTGTTT CATCACAAAA GTATCCTGGG GGGTTTATTG AGAAGAGCAC GAGTTTCACG GAGAGAGTTT TCCCCATCGG CTGCTTTCAT CACCTGCCGT TGCTGGGGCA AAACTGCTGG CAGTGTTTCC CTGTGGTTTA ATGTGGAGTG CTACAGAAAA AGGAAAGAAG ATAAAGAAAA CCAGATAGCA GTTTTGATGG TACAGCTATG GTACACAAAG ATTGTTTTCA GACTATAATT TGGTAGTGTT CTTAACACTC TTAACATGTT 5.8 EXAMPLE 8 DEDUCED AMINO ACID SEQUENCES OF llumAN LYSTi PROTEIN 5.8.1 PEPTIDE SEQUENCE OF LONG ISOFORM (SEQ ID NO:8) 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 MS TDSNS LAR

GFLLLTKLNS

SADI ILTKEK

HRYSVRDARK

PKAKLDRLAT

NNSPFDLCHV

SLSDNLESRV

KNLTELLRAA

LPELLEGVLQ

QMEWLVLRDG

HRRCEYSHFM

AVCAHQCLRL

LPALKNFQQH

EE TLQGNLCD FGEDRLHS IQ HL SVT SAQS H

SQIIELNCLN

E FL TDVNRLC NAVVQRVEA.

IIDQALTCRE ELLTLLLSLL NSSSQRSTQE KLHLEGSALS TQLSTSDSEA NSDEKGIAMN KEQTPPDAMA LENSREIIPR LLSLLEKVCK FDVTLNHNSP VSAGWTEE PV ALT QFML FRT LKTRICLEKQ PDPFAPRQKK ILICCLQSAA SNPFYFSQAM VPPEASEHLK ALINSVMKIM HHHRDLS GLL VSAFKNQVSK LQQASLSSTC VQILSGVHNI ILNILNKLIL DQLGGAEISP AELSSSLSSP SYRFQGILPS IANHICNLIQ KGNIVVQWKL VCSHHNQCLP QDVLQIYVKT GIRSHSLKAF ETLIISLGEQ EEEEEETEI4A TLGQYLVHCR PLVWKIPVQE EKATDFNLPL SQVSAKVNVF RKSRRQRKIT KHRRPHLLHH FLTSFPKQDH QGSNTDILSE PAALSVTSNM

L?

4 ASVVPTLT EFLAGFGDCC VLHLLSVDVS TAENMPENLR TLQEVQEDFV FSKYRHRALL DLVQEFI QHH GFNLFETAVL STVKKVKSEQ LHHSMCTRKR NPFEETADGD VYYPERCCCI GICCCMDPKS VI IPLLHAFK KIKKAACNIC TVDSDQLAQL SGSEDLLWKW DALKAYQNFV YNYI FNPVLQ RGVELAHHCQ LPILLKSRVI RDLFLSCNGV QKDASVPDID GIDIEQKELS WO 97/28262 WO 9728262PCT[US97/O 1748 851 SVT-VGTSFHH 901 FLCVAFLCVS 951 LPSPEHMHQA 1001 LFRSHKEEQG 1051 ELGSLKKSAD 1101 QSIRLLEALL 1151 VIETQLAKPL 1201 SEEAEDSQCC 1251 SSSPNDLLEN 1301 RQKEKNVFLL 1351 SRTCSEELTL 1401 HAPNLSNGVS 1451 WHIAPVHLPL 1501 NKSLILPDSS 1551 VWADPHNATL 1601 QQPQGKRRIH 1651 CLSSQEEFLQ 1701 YGKPVNDYSK 1751 LQYTIYEPVI 1801 IQGTLHEIGG 1851 ELENCNGLSM 1901 DVDSNAIIQD 1951 MFNIKQLLKA 2001 T I og QQAYSDSPQS LSKFYAGLKE AYPKRRKTVN KEAESDRESA NDSEDTSGYD STASEPLSHM ADIWSMCRWI YMLSSVFQKQ FYRLGGFRVC KKEGDTSVNE NQDLNRISQP KRTMKEDLLS SLGKLELQHI SSINVEEVSA TEAAPEEAKL AICLHGARTS QQKMELELPN QNLSVESILF FDALLRVALG NYSADFEHND AMTEKSHQSA SFKLLVEEEG YEADSESNPE DGETQDDGVD LTQGEIIYPE ICMLELNLLS ASKAKLDVLA MQQGTVKNLL GGFLSILTQD DSDFQACQRV LLRIFLEKSP CTKILLLGIL KIIESDTTMS SQKYPGILNS KAMGLLRRAR VSRSKKEAD.

LGQNCWPNLS EGFSVSLWFN VECIHEAEST FDGTESDRPE GAEYINPGER LTEEGCIHII IFRVCMDSND DMKAVLLAQV ESQENTFLPS GKISIWVSGQ RKPDVTLDFM LPRKTSLSSD LAGKWDLGNL LLFNGAKVGS QEAFYLYACG YINKEILRCE QIREFFMTKK DVDIGLLIGV RLKGQMKTQL SQRPFSSKEV QSILLEPHHL TGIFVFLFAR VVELSSCEET QALALRVILS IHQVLIKQKC IVGFYILKTL LEGCCGEDII VKLLEELLLD WKTWSKAEQG VWETT 4

LAALE

OVVHHFLLTC OVLOEYKEGO LTPMPREMAR

QDVHINTINL

LPCISLESLV

HKLIFMI IQK

LAIKSDPIPS

FTSQESETSL

EMRDHLSQSK

EELSSQPGDF

LKSETEGFSA

HVFESFLKI I LVDLLVS LMS

PSQYLTFPLL

ES FPHRLLS S

TEKGKKIKKR

SLGSKALMTQ

KWQHLVLTYL

SNKTFCMI GH~

PNI-TSVMPCK

FQLFI QLTVL KNLQP TEYKT L IKYNQQRVH YMNENGE FKL VL I PADHHQQ

SFRRKCGQSC

5.8.2 PEPTIDE SEQUENCE OF SHORT ISOFORM (SEQ ID NO:1O) 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 MS TDSNSLAR

GFLLLTKLNS

SADI ILTKEK

HRYSVRD.ARK

PKAKLDRLAT

NNS PFDLCHV

SLSDNLESRV

KNLTELLRAA

LPELLEGVLQ

QMEWLVLRDG

HRRCEYSHFM

AVCAIIQCLRL

LPALKNFQQH

EETLQGNLCD

FGEDRLHSIQ

HLSVTSAQSH

SQl IELNCLN

SVHVGTSFHH

FLCVAFLCVS

LPSPEHIKHQA

LFRSHKEEQG

ELGSLKKSAD

QS IRLLEALL VIE TQLAKPL

SEEAEDSQCC

SSSPNDLLEN

E FLTDVNRLC NAVVQRVEAR TIDQALTCRE ELLTLLLSLL NSSSQRSTQE KLHLEGSALS TQLSTSDSEA NSDEKGIAMN KEQTPPDAMA LENSREIIPR LLSLLEKVCK FDVTLNHNSP VSAGWTEEPV ALIQR 4LFRT LKIRICLEKQ PDPFAPRQKK ILICCLQSAA SNPFYFSQAM VPPEASEHLK ALINSVMKIM HH-HRDLS GLL VSAFKNQVSK LQQASLSSTC VQILSGVHNI ILNTLNKLIL DQLGGAEISP AELSSSLSSP SYRFQGILPS IANHICNLIQ KGNIVVQWKL VCSHHNQCLP QDVLQIYVKT GTRSHSLKAF ETLIISLGEQ QQAYSDSPQS LSKFYAGLKE KEAESDRESA NDSEDTSGYD ADIWSMCRWI YMLSSVFQKQ KKEGDTSVNE NQDLNRISQP SLGKLELQHI SSINVEEVSA AICLHGARTS QQKMELELPN FDALLRVALG NYSADFEHND SFKLLVEEEG YEADSESNPE LTQGEI IYPE ICMLELNLLS EEEEEETHlA PLVWKI PVQE S QVSAKVNVF

KHRRPHLLHH

QGSNTDI LSE

LAASVVPTLT

VLHLLSVDVS

TLQEVQEDFV

DLVQE FlQHH ST VKKVKS EQ

NPFEETADGD

GI CCCMDPKS

KIKKAACNIC

SGSEDLLWKW

YNYI FNPVLQ

LPILLKSRVT

QKDASVPDID

AYPKRRKTVN

STASEPLSHM

FYRLGG FRVC

KRTMKEDLLS

TEAAPEEAKL

QNLSVESILF

AM'TEKSHQSA

DGETQDDGVD

ASKAKLDVLA

TLGQYLVHGR

EKAT DFNLP L RKSRRQRKI T

FLTSFPKQDH

PAALSVI SNV

EFLAGFGDCC

TAEMNPENLR

FSKYRHRALL

GFNLFETAVL

LHHSMCTRKR

VYYPERCCCI

VIIPLLHAFK

TVDS DQLAQL

DALKAYQNFV

RGVELAHHCQ

RDLFLSCNGV

GIDIEQKELS

QDVHINT INL LPC015LE SLV HKLIFMI IQK

LAIKSDPIPS

FTSQESETSL

EMRDHLSQSK

EELS SQPGDF

LKSETEGFSA

HVFESFLKI I WO 97/28262 WO 9728262PCTIUS97/01748 1301 1351 1401 1451 1501 11O RQKEKNVFLL MQQGTVKNLL GGFLSILTQD DSDFQACQRV LVDLLVSLMS SRTCSEELTL IJLRIFLEKSP CTKILLLGIL KIIESDTTMS PSQYLTFPLL HAPNLSNGVS SQKYPGILNS KAI4GLLRPAR VSRSKKEADR ESFPHRLLSS WHIAPVHLPL LGQNCWPHLS EGFSVSLWFN VECIHEAEST TEKGKKIKKR NKSLILPDSS FDGTGMMTGL SDLYTKTVFR L 5.9 EXAMPLE 9 IDENTIFICATION OF A DNA SEGMENT ENCODING LYST2 Lyst2 was identified in a search for human genes similar in sequence to Lyst 1 (the CH gene). Mouse Lysti cDNA sequence was compared with Genbank sequences, and significant similarity was noted between residues 3275 to 3413 of Lysti (Genbank Accession number U70015) and R17955, R17955 is an uncharacterized human expressed sequence tag 292 bp in length. The corresponding partial length cDNA clone (#32273) was obtained from Image consortium. This cDNA clone was derived from a cDNA library of human infant brain, and is 1979-bp in length. The clone was designated human LYST2.

10 EXAMPLE 10 DNA SEQUENCE OF THE HUMAN L YsT2 GENE The LYST2 clone was sequenced using standard methodologies. The DNA sequence is given below (SEQ ID NO: 11): 1 ATACTTCTGA 51 ATGTTTGTCA 101 AGTGGTAAAT 151 TTGTGCGGAT 201 CTTCATCAGT 251 AGCAGTTCGT 301 TGAACCTGGA 351 CAGATACAGA 401 TCCGCCTCGG 451 TOATGTTTAA 501 CCTTCAAATT 551 GACCATCC 601 ATAGATGGCA 651 GATCAAGCCC 701 TTCAGGTGTA 751 AAATCAATGC 801 ATCTGTGGAT 851 GAAATTGACT T GTAAAGGAA

ACAGTALTGG

GAT GT TGAT C CAAkCAGGATG

GGATCGACCT

GO TC TGAATG

TAGTATCACT

ACT T TGGACA AAO TOTGCCA AGAT CAGAT G

CTCCAGTAAC

GCAGTGGTGA

CTAATTCCAG

ATATAATCTT

TTOCCCCTTG

GOCCCTAGAA-\

TATATTTGGC

T TTT TCACTA GAT OCT GTGC

GACGCCATCT

T GOACOT GTG

CAACAGGATG

CCATGTGGOA

OAGTGACTTG

AGTTOTACTA

GGAGTCAGAG

GGCAAAAAAA

GTGAATTTGT

TATAAGCAGO

CT TGAO TTAT

TCAGGGAGGC

CAGT TGO TTA T TTOOT T CA

TGATAATGGT

GCCAAOACTO

CAGOOGACTO

GAGCTCCAGG

OCTACCAGAG

AAGAT GAAG T

CTGAAGACT

T TOT TGCCAA GAG GAO CAGA G.AAGGCTOT G CAT GGAGGCA T TGAGOCACA

CAGAGTCCGO

GOT GAAGT TT T GOCCOCACT T

TTTGCAGTGA

ATAOTCCTTG CAACAOAGTA GGOO TOAGAG ACCATOT TOO

AACAAACGGC

ACAT TG TTT T

TCTGGGATAA

CAGAT TGTAT CAT TGAAATG

AGATOACAGA

GTGGTAACAG

GAGOTTOAGA

TTGGCCATTG

GATOCAT TAA TAGCCAATAA OCT CGT TGAO

CAGATAATOG

GTTTATACTA

GGATGTGGT C

OAGAGTATAO

O TATATTOT T

CAGAAAOAGG

ACT TGOT TGG WO, 97/28262 WO 9728262PCTIUS97/01748 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951

CCAGGTCCGA

CGAGATGCCA

AGGAGACAAC

CAGGCCATGA

CTTGTTATCA

TGGAGATTTG

GOT TGATATC GGGCGAT TCA

GATCAATGAT

TGGTCACCGG

T TCAAGCAAC T GGAC TTGTC AGCAT TGTAG

GAACAGATAC

GGCACAAGTG

TAOATCGACC

T TGTAGT CAG

AGCTGCTATC

GTTTTGGTAA

ATGTGTCACA

TTACAiAAGTT

TTCAAGCTGA

GTCATACATT

CCC TGO TGC T CC TAACAGCA

COATGAAGTT

G TGGT GOTAA C TGAGAGCC C TG TC TCAGC

GT.AATTTCAG

TCAACAOGGG

AGGGGAOAAT

TGTACATTTA

CCATGACCAG

OTT TTAATAT

TGAAGATAAA

CTGCATGGAA

TOO GT TTGTA CAAC CAT TTT AAGCAAGOT T T TAT T TCATA

AGAGACTTTT

TAAGCT TTGA

GAAAAA

9JTGGGGACT

CTGGTACTGG

GTGACTATCC

GTCTGTGTTT

AGAGGGOCCT

TTGAAGGAC

GAAGGCCACT

CAT TAATGGG C OATTOT OCT

GGGGTAGTAG

ACCCTGGATG

AGGAC TOTGA

AGATTTTAAT

GGAAGAACCA

AGGCAATATC

CATTCOATCA

ACT TTGTGTG

ATATCATGTA

TAT TGT TGTT

GACAATTCTG

ACCTAACOTG

AAAAAAAAA

GOTACATCGT

AGTGGGOGGC

GGCACCAAGA

OTGTCTGTGC

TGOCTTGTCC

AGAAAO TGO GTAT CATATA

AAACTTTTGG

GAGCAGTGAC

AGGTCTGGCA

T GAT GOTGGC

TCAOTGGCAT

CGGTGGCATT

AAAGC CAAGT TOT GGTGGAA

CACCCAGCAA

T T TT TTCACG

AATTATATGA

TAT TGAGAAA

AGGAACOTTG

CATCCCATTT

GTCO GGAT CT

ACOATATOAT

GOC GTOO TOA

AGAACTTGGG

ACACOATOAC

TTATTCCCAC

C TAT G7ACGA

OTOAAATGGA

GGCCAGAACC

GGCO TG TGAO AT TAGAGCAA

GGCTTCTGGT

ATGAGCATOA

TAAAGC TGAG AAAAT TOGT C TAG T GTACA ACT GAACAC C AT TAGGAGAT AGGT TG TAGG TG TCOAG T TG

CCAGCCTOTT

(SEQ ID NO:11) This DNA sequence corresponds to the 3' end of the coding domain of human LYST2 and the 3' untranslated region.

5.11 EXAMPLE 11 AMINO ACID SEQUENCE OF THE HUMAN LYST2 PROTEIN Translation of the DNA of SEQ ID NO: I1I provided the deduced amino acid sequence of the LYST2 protein (SEQ ID NO: 12) which is shown below: 1 TSDVKELIPE FYYLPEMFVN SNGYNLGVRE 51 VRINRMALES EFVSCQLHQW IDLIFGYKQR 101 NLDSITDPVL REA1MEAQIQN FGQTPSQLLI 151 MFKDQMQQDV IMVLKFPSNS PVTHVAANTL 201 RWHNTVGLRG APGYSLDQAH HLPIEMDPLT 251 INAHCFVVTA DNRYILICGF WDKSFRVYTT 301 RSESYIGGDC YIVSGSRDAT LLLWYWSGRH

DEVWVNDVDL

GPEAVRALNV

EPHPPRNSA4 PHLT IPAVVT ANNS GVNKRQ

ETGKLTQIVF

HIIGDNPNSS

P PWAKKPED F

FHYLTYEGSV

HLOFLPQSPL

VTCSRLFAVN

ITDLVDQSIQ

GHWDVVTCLA

DYPAPRAVLT

WO 97/28262 PCT/US97/01748 351 GHDHEVVCVS VCAELGLVIS GAKEGPCLVH TITGDLLRAL

EGPENCLFPR

401 LISVSSEGHC IIYYERGRFS NFSINGKLLA QMEINDSTRA

ILLSSDGQNL

451 VTGGDNGVVE VWQACDFKQL YI (SEQ ID NO:2) Amino acids 2 to 140 of the predicted human LYST2 protein share only a 51.8% amino acid identity with amino acids 3275 to 3413 of mouse and human Lystl. The C-terminal residues of LYST2 are not similar to LYST1, but do have a similar predicted secondary structure: This region of LYSTI contains WD repeats and is predicted to assume a propellor-like secondary structure, similar to the beta subunit of heterotrimeric G proteins. The corresponding region of LYST2 also contains WD repeats and is also similar in sequence to the beta subunit of heterotrimeric G proteins (30.4% identity from LYST2 amino acid 285 to 418 to the guanine nucleotide-binding protein beta subunit-like protein P49027). Furthermore, the stop codons of mouse Lyst] and human LYST2 occur approximately the same distance from the matching region.

5.12 EXAMPLE 12 GENETIC MAPPING OF THE LYST2 GENE By hybridization to Southern blots of human-rodent somatic cell hybrids, LYST2 was shown to map on human Chromosome 13. This is in contrast to LYSTI, which maps on human Chromosome 1. Using an Mspl restriction fragment length polymorphism, Lyst2 was mapped by cros-hybridization in the mouse. Linkage analysis using DNA from 93 intersubspecific backcross [C57BL/6J-bg J X (C57BL/6J-bg J x CAST/EiJ)F mice revealed Lyst2 to map to mouse Chromosome3 between D3Mit21 and D3Mit22. This contrasts with Lyst, which maps on mouse Chromosome 13. Pulsed field gel electrophoresis blots of mouse DNA hybridized with a Lyst2 probe showed a single band, indicating that Lyst2 is a single genetic locus.

5.13 EXAMPLE 13 EXPRESSION ANALYSIS OF THE LYST2 GENE Hybridization of northern blots of human and mouse tissues with LYST2 revealed the following pattern of expression: Lyst2 is abundantly expressed in mouse brain, and moderately expressed in mouse kidney, and weakly expressed in mouse heart, lung, skeletal muscle, and testis. Lyst2 is not expressed in mouse spleen or liver. The largest (and most prominent) band observed on northern blots was 13kb in size (very similar to the largest Lyst mRNA). Additional transcripts on 6kb and 5kb were evident in mouse brain RNA.

WO 97/28262 PCT/US97/01748 i In selected human tissues, LYST2 was expressed as follows: Moderate expression was observed in melanoma cells, weak expression in HeLa cells, colorectal carcinoma cells, and in spleen, lymph node, thymus, and appendix. No expression was detected in peripheral blood leucocyte, bone marrow, fetal liver, lung carcinoma, or leukemia cell lines (K562, MOLT4, Raji, The major transcript was 13-kb in size in human RNA.

In summary, LYST2 appears to be similar in size to the largest LYST1 mRNA, but has a very different tissue distribution of expression, being abundantly expressed only in brain. LYST2 appears to be a brain-specific homologue of LYST1, and may function to regulate protein trafficking to the lysosome and late endosome within the brain.

The relative abundance of LYST2 mRNA isoforms in human tissues at different developmental stages was examined by sequential hybridization of a poly(A) RNA dot blot with a LYST2 cDNA probe. The quantity of poly(A) RNA loaded on the blot was normalized to eight housekeeping genes (phospholipase, ribosomal protein S9, tubulin, a highly basic 23-kDa protein, glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine guanine phosphoribosil transferase, P-actin, and ubiquitin) to allow estimation of the relative abundance ofLYST2 mRNA isoforms in different tissues.

Abundant LYST2 transcripts were detected in all brain regions and in kidney. LYST2 transcripts were detected in those regions at all developmental stages.

5.14 EXAMPLE 14 IDENTIFICATION OF MOUSE LYST2 cDNA CLONES A mouse embryo (day 14.5 post-coitum) cDNA library was hybridized with a probe corresponding to human LYST2. Two clones were isolated and sequenced. They contained overlapping sequences that were assembled by alignment with human LYST2 and represent 2543 bp of cDNA sequence.

5.15 EXAMPLE 15 DNA SEQUENCE OF THE MOUSE LYST2 GENE 1 GCAGCAGGGC GAACCGGACC TCTGTGATGT TTAATTTTCC

TGACCAAGCA

51 ACAGTTAAAA AAGTTGTCTA CAGCTTGCCT CGGGTTGGAG

TGGGGACCAG

101 CTATGGTTTG CCACAAGCCA GGAGGATATC ACTGGCCACT

CCTCGACAGC

151 TGTATAAGTC TTCCAATATG ACTCAGCGCT GGCAAAGAAG GGAAATCTCC WO 97/28262 PCT/US97/01748 201 AAOTTTGAGT

ATTTGATGTT

251 TGATCTGAAC

OAGTATECTG

301 CAGAGGAGTT

GGACCTGACT

351 CCAAAAGGTG

CTTTGAACCC

401 TGAGACATGG

GAGGAGGATC

451 ACTCAACGGO

GAOTTCC

501 ACAACCTTCT

TCCTCAATGC

551 AACCTTCTCA

TCCATTGCAA

601 CCGATGTCAA

GGAACTAATT

651 GTCAA.OAGCA

ATGGGTACOA

701 TAATGATGTG

GACOTGCCCO

751 GGATCAACAG

GATGGOCCTG

801 CAATGGATTG

ACCTTATATT

851 CCGTGOTOTC

AATGTTTTOC

901 TGGACAGCAT

CACAGACOCT

951 CAGAACTTTG

CCCAGACGCC

1001 TAGGACTTCA

GCCATGCATC

1051 TCAAAGATCA

GATGOAGCAG

1101 AATTCTCCTG

TGACTCATGT

1151 CCCTGCAGTG

GTGACAGTGA

1201 GGCACAACAC

AGTOGGOCTC

1251 GCACACCATC

TTOOCATTGA

1301 TGTGAACAAG

CGGOAGATOA

1351 ATGCCCACTG

CTTCGTGGTC

1401 GGGTTTTGGG

ATAAAAGTTT

1451 GACACAGATT

GTATTTGGOC

1501 CGGAGTCCTA

OATTGGTGGA

1551 GCCACOTTGC

TTCTCTGGTA

1601 CAACCCCAAT

AGCAGTGACT

1651 ATGACCATGA AGTTGTCTGT 1701 ATCAGTGGTG

CTAAAGAGGG

1751 TCTGOTG.AAG

GCCCTGGAAG

1801 TTTCGGTATC

CAGTGAAGGC

1851 TTTAGOAAOT

TCAGCATCAA

1901 TGATTCOACT

AGGGCTATTO

1951 CTGGAGGGGA

CAATGGTGTG

2001 CAGCTGTACA

TTTAOCCAGG

2051 ATCCCATGAC

CAAAGGACTO

2101 TAOTTTTAAT

ATAGATTTTA

2151 CTGAAGAGAA

GCAGOAGAAG

2201 GTGGAAAGGC

AGTATOTOTG

2251 TGTACATCCA

TOCCACOCAG

2301 TTOACCCTGT

TTTTCCAGGA

2351 TATCATGTAA

ATTATCTGAA

2401 ATCGCCGTTT ATTGAGAAAA 2451 AOAATTCTGA

GGAAOCTTGT

2501 CCTAACTTGC

ATOOOATTTO

(SEQ ID NO:13) 114

TCTCAACACG

TGTTTCCATG

CTCOCAGGAA

GAAGAGAGCA

AAAGCCCACC

CTTTCATGGC

AAATGATGGG

GGT OAT GGAG CCAGAGT TOT

TCTTGGAGTG

CO TGGGOCAA

GAAAGTGAAT

TGGCTACAA

ACTACTTGAC

GTGCTCCGGG

ATCTCAGTTG

TGTGTTCCCT

GATGTGATCA

GGCTGCCAAC

CC TGCAGCCG

AGAGGAGCO

GATGGACCCA

CAGAC OTTG T ACAGO TGATA

CAGAGTTTAC

ACTGGGATGT

GACTGCTACA

C TGGAG TGGG

ATCCTGCGCC

GTCTOCGTCT

COOT TGCCTC

GACCAGAAAA

CAOTGOATCA

TGGGAAACTT

TCOTGAGCAG

GTGGAGGTCT

ATGTGATGOT

T GAT CAC TGG

ATOGGTGGOA

COAOATTOAA

GT GGGAO GOT

OAGTOGCOGA

O TGAACACOA

TTAGGAGCOG

GGTTGTAGGA

GCOCAGTTGT

CAGCCT GGG

ATAGOAGGTC

GGTGTTAACA

AOTTOAGGCA

GTGT TT TACG OTTOCAC TAO T TGT TOGGAT A7AATTTGACC

AACCAGTOAG

AT TAO GTACO AGGGAG GAO G

GAAGOOAGAA

TTGTTTOTTG

CAGOGAGGGC

OTACGAAGGO

AGGCCATGGT

OTOATTGAGC

TO CACAGAG C T GG TGOTGA ACCO TGCCCC ACT GT TTGCA

COGGATACTO

TTAATOGCAA

AGACCAGAGO

ATOGO TACAT T CGACAGAAA

TGTOAOATGC

TAGTGTOTGG

CGTCAOCACA

CAGAG T GTC

GTGCAGAAOT

GTTCATAOCA

CT GO TTAT TT TATAT TAT GA T TGGO TCAA

OGATGGACAG

GGOAGGCOTG

GGOATTAGAG

OATGGCTTCC

TTATGAGOAT

G TGAGAGCAO GG T CACAT C ACAT OATAGT

GOTGOTGTOA

TTTTGGTAAT

AGCCTCACAA

TAOAAAGTTT

CT TOACTOG T

GGACGTATAA

AAC TAT GAAT TO TG TCAAAG

CAGAGOGOTA

AAOACACATT

TGAGCCATTC

ATOCAGACCG

AGAGATACAT

AGAGATG T TT

AAGTGGTGGT

GAOTTTGTGC

COAACTCCAT

CAGAGGCAGT

TOTGTAAACO

TGCAOAGATA

OGOATOOGOC

OOAOTCATGT

GTTT COAT CC

AOCTGACOAT

G TGAACAGAT

CTTGGATCAA

ATAAOTCTGG

ATOOAGATOA

OOTCATOTGT

OAGGGAAAOT

OTGGOCAGGT

AT T CGGGAC

TOATOGGAGA

O TOACAGGC OGGACTOGT T

TCACTGGAAA

COACGCCTAA

GCGAGGAOGG

TGGAGATCAA

AAOCTGGTGA

TGAOTTTAAG

CGATGGATTT

GGOAGCATTG

CAG.AACAGTA

AAGTGCTTCT

GGOOTOTGCT

OGGGAGOOAT

AGOAAGOTTA

TATTTOATAT

GAGAC TT TT G

AAGCTTTGAA

GC

5.16 EXAMPLE 16 DEDUCED AMINO AmI SEQUENCE OF MOUSE LYST2 PROTEIN 1 SRANRTSVMF 51 YKSSNNTQRW 101 EELDLTLPGN

NFPDQATVKK

QRREISNFEY

FRHLSKPKGA

VVYSLPRVGV

LMFLNTIAGR

LNPKPRAVFYA

GTSYGLPQAR

TYNDLNQYPV

ERYETWEEDQ

RISLATPRQL

FPWVLTNYES

S PP FHYNTHY WO 97/28262 PCT/US97/01748 151 201 251 301 351 401 451 501 551 601 651 S TAT SPLS WL

DVKELIPEFY

I NRMALE SE F

DSITDPVLRE

KDQMQQDVIM

HNTVGLRGAP

AH-C FVVTADN

ESYIGGDCYI

DHEVVCVSVC

SVSSEGHCI I

GGDNGVVEVW

VRIEPFTTFF LNANDGKFDH YVPEMFVNSN GYHLGVREDE VSCQLHQWID LIFGYKQRGP AM~VAQIQNFA QTPSQLLIEP VLKFPSNSPV THVAANTLPH GYSLDQAHHL PIEMDPLIAN RYILICGFWD KSFRVYSTET VSGSRDATLL LWYWSGRHHI AELGLVISGA KEGPCLVHTI YYERGRFSNF SINGKLLAQN QACDFKQLYI YPGCDAGIRA PDRTFSS TAR

VVVNDVDLPP

EAVPALNVFH

HPPRTSAMHL

LT IPAVVTVT

NSGVNKRQIT

GKLTQIVFGH

IGDNPNSSDY

TGNLLKALEG

EINDSTRAIL

MDLSHDQRTL

SWRTSQRDTs

WAKKPEDF'VR

YLTYEGSVNL

CSLPQSPLMF

CSRLFAVNRW

DLVDQS IQIN

WDVVTCLARS

PAPRAVL TGH PENCLFPRL I

LSSDGQNLVT

ITGMASGS

IV

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WO 97/28262 PCT/US97/01748 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: University of Florida STREET: 223 Grinter Hall CITY: Gainesville STATE: Florida COUNTRY: USA POSTAL CODE (ZIP): 32611 (ii) TITLE OF INVENTION: Lystl and Lyst2 Gene Compositions and Methods of Use (iii) NUMBER OF SEQUENCES: 78 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 60/011,146 FILING DATE: 01-FEB-1996 (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: US UNKNOWN FILING DATE: 23-DEC-1996 (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 60/033,599 FILING DATE: 20-DEC-1996 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 3514 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TTTAAAAATT AGAGCTTGCT TGGAAAAGCA GCCTGAGCCT TTCTCCCCGA AACACTACAG GAGGTCCAGG AGGGCTTTGT ATTTTCCAAG TATCGTCACC ACTACCTGAG CTTCTGGAAG GAGTTCTACA GCTCCTCATC TCTTGTCTTC TTCAAATCCC TTTTACTTCA GTCAAGCCAT GGATTTAGTT CAAGAATTTA AGGATTTAAT CTCTTTGGAA CAGCAGTTCT TCAGATGGAA TGGCTGCTTA TGTTCCTTCA GAAGCTGCAG AACATTTGAA AGCTCTGATA AACAGTGTAA GAGTACTGTG AAAAAGGTGA AATCAGAGCA ACTTCATCAT TCCATGTGCA ACACCGGCGT TGTGAGTATT CCCACTTCAT GCAGCACCAC CGCGATCTTT GGTTTCAGCT TTTAAAAATC AGCTTTCTAA AAGCCCCTTT GAAGAGACCG

GACAAAAGAA

GAGCCCTTCT

AGAGTGCAGC

TCCAGCACCA

CAAGGGACGG

TAAAAATAAT

CAAGGAAAAG

CAGGGCTCCT

CAGAGGGAGA

120 180 240 300 360 420 480 540 WO 97/28262 PTU9/14 PCTIUS97/01748

TGTGCAGTAT

GCTGCAGCAG

TGGAATCTGT

GTTGCCAGCA

GGATCAGTTA

TACTGTGGAC

TGCTGGTCCT

CAGCGGCTCT

CTTTCAAGAA

GAAAGGCAAT

AAGAGGAGTT

CATGTGTAGC

TCTACCTGTC

AAACCACATA

TGAAACTCTG

TGGGTTAGAC

GCAAGCTTCT

TGATCCATAA

CCTCTGTGTG

TGAGTCAGAA

ACCATGTCTG

AGACATTTGG

TCACAGGCTT

ATTCAGAAGT

CCAAGAGCTA

AACAGCACCA

ACAGCCTGTG

ACGAAAGGCA

GTCTCTCCTG

GCTACCGTCT

CCAGTCAAAG

AGCCCTGGGG

TCCCTCTGAG

CCAGAGCGCT

GTTTCCCTGA

TGTTGTATGG

CTGAAAGCTT

GGAGGAGCAG

TCTGACCAAC

ACCTCCGGCT

GAAGACTTGC

GACAGATTAC

GTAGTTGTT C

GAATTAGTAC

CAACTGAAAC

CTACTTAAAT

ATTGAACTAA

ATTGTCAGCC

ATCCAACAGG

TCAGATTCTC

AAACGAAAGA

GCTTTTCTAT

GATACTTCTG

TCTCTTGAGG

TCCATGTGTC

GGTGGTTTCC

CATACAGAGG

ATCAGGATAT

GACCTGGGAT

CTTCCCACAA

TTTAT GAGTC

GACATTTGTC

CAGGGCTTGT

GTGGCAGAAA

AAT CATT GAG

GAAGAGCTGT

GCTGCTGCAT

GCACCACGTG

ATCCTAAGTC

TCCAGCAGCA

AGCTATCACc

TGGCTAAGTT

TGCCCAGTCC

TGTGGAAGTC

ATAACATTCA

AGTGGAAATT

ATCATTGTCA

AGTATTTGCC

CCAGGGTAAT

ATTACTTAGA

TAGGGGAACA

AGTTGCCGT C

CTTGCAGTCT

CCATTCACCA

GTGTCAGTAA

GCTATGACAG

ACGTTGTCTT

GTTGGATCTA

AAGTGTGCCA

ATCAAGGAAG

CTTAC CC CGA

TTCTGAGAAA

GTGCAGAGCA

AACAAAGTGA

TTCATAGTGC

CTGTGGAAAA

CAGAATTAGC

CAGATTTGGG

TGTCCCAGCC

I IN~

CGCCGTGTGC

TGTCCAGATC

TGTGATCGCC

TATACTGAAT

GAGAATTAAA

AGGAGAGACA

TTCCTACCGA

GGATGCATTA

GATTGCAAAT

GTATAATTAT

ACAGCTAAGC

TCAGGAAGTG

AAGAGATTTG

TGGGATTCGA

ACAGAAAGAT

CTTAAGTGTG

CAGGAAGTTT

GGATGTTCAC

AGAAGCAGAC

CCCTCCCAGT

ACC T TCC CCT

CATGTTGAAC

TGAATTAATA

AAGGCAGGGA

GCTGACACTG

GAGTGCTGAC

GATTGTGGCT

GACTTCTCTC

CAGAGCCTGT

TATATTGTGT

AAAG CCT TTA

CCCTGGTGAT

CGGAGATTTT

GCTCACCAGT

CTATCAGGTG

CCTTTACTGC

GTCCTGAGCA

AAAGCAGCTT

CTGCAAGGCA

TTTCAGGGGA

GAG GCTTAT C

CACATTTGTA

AT CTTTAAT C

ATTCCTTCAG

CTTCAGATTT

TTTTTAAGTT

AGTCATTCCC

GCTGCAGTTC

GGTCCTTCTC

TAT GC CAGC C

ATAAACACCA

TCTGATAGGG

GAGCCATTAA

GAATGTTTGC

TCAGTCTTCC

TTTATGATAA

GAAATGAGTA

AAGGGAGATG

AGCGTGCGTG

ACTGAAT CTG

CAGAGCATAC

CAACAGAAGA

GAACTGAGGG

TTTGATGCCC

GCTGTGACTG

TCAGAAGAAG

GCTTGCGCTT

TACACAGTGT

ATGCTTTTAA

AACTTCTTGT

GCAACATCTG

CCTTGTGTGG

TCCTGCCCAG

AGAGCTTTGT

ATTTACTCCA

CT GT GCT CCA CT CAGACT CA

ATTTAAAAAC

GTAATGGAGT

TGAAAGCATT

TAGACGTCGA

TTCATAAGCA

TCAGAGAGCC

TAAACCTCTT

AGTCTGCCAA

GTCACATGCT

ACCATGCAGC

AGAAACAATT

TCCAGAAACT

GAAATGAAAA

TATCATCTGCj

GATTCCAGTC

TTCCTGGGGA

GACTTTTGGA

TGGAATTGGA

AACACCTTTC

TGCTTCGAGT

AGAAGAGTCA

CTGAGGATT C 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 WO 97/28262 PCTIUS97/01748

TCAGTGTTGT

CAATCCTGAG

TTTCAGTGGA

ATACCCTGAG

TGTGCTTGCT

TTCTCTCCTC

AACACAAACT

TTTGATGAGC

GAAATCTCCT

TACTATGAGC

TGGTGTCTTA

AAGAGCACGG

GCTTTCCTCT

ACACCTGTCA

AT C CGAGAGT

GAAGACAGCA

CCTGGTGACA

AGTTTGAAAC

GATGTTGACA

TCGATTGTTT

ATTTGCATGC

CATGTGTTTG

ATACAACAGG

AACTCTGATT

TCAAGAACGT

T GTACAGAAA

CCTTCACAGT

TCACAGAAAC

.ATTTCCCGAG

TGGCATATAG

GAAGGATTTA

GCTGCAGAAA

GTTTTGAAGG

GCTCGTGCCG

TT CTGGGTGA

CCCAAGACGA

CAAACAACTT

TGGGATTAAA

AGAGCTTTCT

GAACTGTGAA

TCCAAGCATG

GTTCAGAAGA

TTCTTCTCCT

GTCTGACCTT

CTCCTGGGAT

GCAAGAAAGA

C CC CAAT CCA GTGTTTCT CT

GGGGAAAAGG

AGCAGAAGGT

AATTCGGCAA.

rQGAAGAAGGC

TGGAGTAGAA

ACTTGAAAAC

TTTGCTTTCT

GAAAATTGTC

AATCCTTCTA

CCAGAGAGTA

CTTAACTCTT

TGGTATTCAC

TCCTTTCCTG

TCTTAACAGT

GGCTGATAGA

CCTGCCGTTG

TGTGGGTTTA

AGTAAGGAAA

GATAG1ACCAG

CGAG

TATGAAGCGG

TTAAAT CCTG

CTCACTCACG

GCTAGCAAAG

AGGCAGAAGG

GGCGGGTTCT

CTGGTGGAT C

CTTTGGAGAA

AAAATTGTTG

CATACCCCGA

AAAGCCTTAG

GAGAGTTTTC

CTGGGACAGA

ATGTGGAATA

AGAACAAACC

ATAGTGAAAG

AAGCAGAAGG

GGGAAATAAT

CTAAACTTGA

AAAAGAACAT

TGAATATTTT

TCTTGGTATC

TATTTCT GGA

AAAGTGATTT

GTTTAAGCAA

GCTTATT GAG

CCTATAGGCT

ACTGCTGGCC

CAT CCAAT GA

ATCAGTTCTG

2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3514 AAGTTACAGA ATCCATCAAT INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 1185 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Leu Lys Ile Arg Ala Cys Leu Glu Lys Gin Pro Glu Pro Phe 1 Arg Lys Ser Pro Gin Lys Lys Tyr Arg His Leu Gin Glu Val 25 Leu Giu Gly Phe Val Phe Ser Glu Gly Val Arg Ala Leu Leu Gin Leu Leu 40 Leu Pro Glu Leu Leu Ser Leu Ile Ser Tyr Phe Cys 55 Asp Gin Ser Ala Al a Phe Asn Pro Phe Ser Gin Ala Gly Met Gly Leu Val Gin Ile Gin His Phe Asn Leu Thr Ala Val Met Glu Trp Leu Leu Ala Leu Thr Arg Asp Gly Thr Ag As Gly Pro Ser Giu Ala GlHi LeLy Glu His Leu Lys WO 97/28262 PCT/US97/01748 110 Ile Asn Ser Val Ile Lys Ile Met Glu Glu 145 Val Ala Cys Thr Cys 225 Leu Lys Lys Lys Ser 305 Ser Gin Asn Lys Leu 385 Met Tyr Leu Gin 130 Tyr Ser Glu Ala Cys 210 Met Pro Leu Lys Leu 290 Gly Gly Ser His Leu 370 Val Cys Leu Phe 115 Leu Ser Ala Gly His 195 Val Asp Ala Leu Ala 275 Gly Leu Ser Phe lie 355 Tyr His Ser Lys Leu 435 His His SPhe Asp 180 Gin Gin Pro Leu Val 260 Ala Glu Pro Glu Val 340 Cys Asn His Gin Thr 420 Ser His Phe Lys 165 Val Cys Ile Lys Lys 245 Asp Cys Thr Ser Asp 325 Phe Asn Tyr Cys Leu 405 Leu Cys Ser Met 150 Asn Gin Leu Leu Ser 230 Ala Gin Asn Leu Pro 310 Leu Gin Leu Ile Gin 390 Lys Pro Asn Met 135 Gin Gin Tyr Arg Ser 215 Val Phe Leu Ile Gin 295 Ser Leu Glu Leu Phe 375 Gin Gin Val Gly 120 Cys His Leu Pro Leu 200 Gly Ile Gin Gly Cys 280 Gly Tyr Trp Asp Gin 360 Asn Leu Tyr Leu Val 440 Ser Thr His Ser Glu 185 Leu Val Ala Gin Gly 265 Thr Thr Arg Lys Arg 345 Lys Pro Ser Leu Leu 425 Asn Thr Arg Arg Lys 170 Arg Gin His Pro His 250 Ala Val Leu Phe Trp 330 Leu Gly Val Ile Pro 410 Lys His SVal Lys Asp 155 Ser Cys Gin Ser Leu 235 Ile Glu Asp Cys Gin 315 Asp His Asn Leu Pro 395 Gin Ser Ile Lys Arg 140 Leu Pro Cys Val Val 220 Leu Leu Leu Ser Gly 300 Gly Ala Asn Val Gin 380 Ser Glu Arg Ile Lys 125 His Ser Phe Cys Ser 205 Gly His Asn Ser Asp 285 Ala Ile Leu Ile Val 365 Arg Ala Val Val Glu 445 Val Arg Gly Glu Ile 190 Leu Ile Ala Val Pro 270 Gin Gly Leu Glu Gin 350 Val Gly Gin Leu Ile 430 Leu Lys SArg Leu Glu 175 Ala Ser Cys Phe Leu 255 Arg Leu Pro Pro Ala 335 Ile Gin Val Thr Gin 415 Arg Asn Ser Cys Leu 160 Thr Val Thr Cys Lys 240 Ser Ile Ala Thr Ser 320 Tyr Ala Trp Glu His 400 Ile Asp Tyr WO 97/28262 Leu Asp 450 PCT/US97/01748 l)I Gly Ile Arg Ser His Ser Leu Lys Ala Phe Giu Thr Leu Ile 455 460 Val Ser Le 1 465 Gly Leu Phe His Phe 545 Glu Ser Pro Ile Gly 625 Phe Arg Leu Arq I Pro 705 Arg I Arg I Cys C Glu P 7 Le Hi Tyi Glr 530 Leu Ser His Glu Tyr 610 Phe Arg sn Lys Lys 690 hr .ys Jeu In ~sn '70 u Asp s Lys Ala 515 1 Asp I Cys Glu Met Cys 595 Met Gin Ser Glu Gly 675 Ser 2 Ser 2 Ala I Leu C Gin I 755 Ile I Gi Tl Gli Sei Val Val Asp Leu 580 Leu Leu Va1 His ksn 560 Nsp la -1a ?he ;lu ~ys leu y Glu e Gin 485 -i Gin Leu His Ser Thr 565 Pro His Asn Cys Thr 645 Gin Val Asp Glu Met C 725 Ser I Met C Cys G G1 47 Gi1 Al.

Arc Ilc Lys 550 Ser Cys His Ser His 630 Glu Glu Ser Ser ;In 710 er .eu 1 u ;lu n Gin 0 n Glu a Ser j Glu Asn 535 Glu Gly Leu Ala Val 615 Glu Asp Leu Ser Val I 695 Ile Gin Leu I Leu C Leu I 775 Ly Lei.

Ser Pro 520 Thr Ala Tyr Ser Ala 600 Phe Leu Gin Ile 4k a 580 !rg Val 3In \sp ;1u 160 ~rg As 1 1 Prc AsT 505 Asp Ile Asp Asp Leu 585 Asp Gin Ile Gly Arg 665 Thr Gly Ala Ser Ile 745 Leu Glu 3 Ala :>Ser 490 Ser Pro Asn Ser Ser 570 Glu Ile Lys Phe Arg 650 Ile Ala I Phe Thr C Glu 1) 730 Cys I Pro S His L Al 47* Lei Prc Lye Lei.

Asp 555 Pro Asp Trp Gin Met 635 krg Ser ?ro 3In lu 15 'hr .eu er jeu a Val 5 Ser Cys Lys Phe 540 Arg Pro Val Ser Phe 620 Ile Gin Tyr Asp Ser 700 Ser Ser I His Gin C Ser C 780 Lei Val Sel Arg 525 Leu Glu Ser Val Met 605 His Ile Gly Pro Leu 685 Gin Val ,eu 3er ;ly 165 In 1 Asp Gly 7 Leu 510 Lys Cys Ser Glu Leu 590 Cys Arg Gin Glu Glu 670 Gly Pro Pro Gin Ala I 750 Leu S Ser I Va.

Pr 49 Ar Th Val Ala Pro 575 Pro Arg Leu Lys Met 655 Leu Phe Val 3er 735 ~rg ier ~ys 1 Asp 480 0 Ser g Lys Ile Ala Asn 560 Leu Ser Trp Gly Leu 640 Ser Thr Leu Leu Glu 720 Ile Ala Vai Val Ala Giu Thr Glu Leu Ala Lys Pro Leu Phe Asp 795 Ala Leu Leu Arg WO 97/28262 PCTIUS97/01748 Ala Leu Gly Asn His Ser Ala Asp Leu Gly Pro Gly Asp Ala Val Thr 805 810 815 Glu Lys Ser His Pro Ser Glu Glu Glu Leu Leu Ser Gin Pro Gly Asp 820 825 830 Phe Ser Glu Glu Ala Glu Asp Ser Gin Cys Cys Ser Leu Lys Leu Leu 835 840 845 Gly Glu Glu Glu Gly Tyr Glu Ala Asp Ser Glu Ser Asn Pro Glu Asp 850 855 860 Val Asp Thr Gin Asp Asp Gly Val Glu Leu Asn Pro Glu Ala Glu Gly 865 870 875 880 Phe Ser Gly Ser Ile Val Ser Asn Asn Leu Leu Glu Asn Leu Thr His 885 890 895 Gly Glu Ile Ile Tyr Pro Glu Ile Cys Met Leu Gly Leu Asn Leu Leu 900 905 910 Ser Ala Ser Lys Ala Lys Leu Asp Val Leu Ala His Val Phe Glu Ser 915 920 925 Phe Leu Lys Ile Val Arg Gin Lys Glu Lys Asn Ile Ser Leu Leu Ile 930 935 940 Gin Gin Gly Thr Val Lys Ile Leu Leu Gly Gly Phe Leu Asn Ile Leu 945 950 955 960 Thr Gin Thr Asn Ser Asp Phe Gin Ala Cys Gin Arg Val Leu Val Asp 965 970 975 Leu Leu Val Ser Leu Met Ser Ser Arg Thr Cys Ser Glu Asp Leu Thr 980 985 990 Leu Leu Trp Arg Ile Phe Leu Glu Lys Ser Pro Cys Thr Glu Ile Leu 995 1000 1005 Leu Leu Gly Ile His Lys Ile Val Glu Ser Asp Phe Thr Met Ser Pro 1010 1015 1020 Ser Gin Cys Leu Thr Phe Pro Phe Leu His Thr Pro Ser Leu Ser Asn 1025 1030 1035 1040 Gly Val Leu Set Gin Lys Pro Pro Gly Ile Leu Asn Ser Lys Ala Leu 1045 1050 1055 Gly Leu Leu Arg Arg Ala Arg Ile Ser Arg Gly Lys Lys Glu Ala Asp 1060 1065 1070 Arg Glu Ser Phe Pro Tyr Arg Leu Leu Ser Ser Trp His Ile Ala Pro 1075 1080 1085 Ile His Leu Pro Leu Leu Gly Gin Asn Cys Trp Pro His Leu Ser Glu 1090 1095 1100 Gly Phe Ser Val Ser Leu Val Gly Leu Met Trp Asn Thr Ser Asn Glu 1105 1110 1115 1120 Ser Glu Ser Ala Ala Glu Arg Gly Lys Arg Val Lys Lys Arg Asn Lys 1125 1130 1135 Pro Ser Val Leu Glu Asp Ser Ser Phe Glu Gly Ala Gly Met Met Ala WO 97/28262 PCTIUS97/01748 1140 1145 1150 Gly Ser Asp Leu Tyr Thr Lys Ile Leu Gin Ile Ala Ala Cys Leu Ser 1155 1160 1165 Phe Lys His Ile Trp Gin Tyr Phe Asn Val Phe Phe Lys Cys Tyr Ser 1170 1175 1180 Pro 1185 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 11817 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TGAGAC-CTCA CGCTGGCCTG GCAGCCTTGG TGAGTCGGGA TTCTCCTGCA CCGGCGGGCG

AGAGCGCGCG

TTCATGAACC

CATGAGCACG

CAATGCAGT G

AACTCTTGGA

TATCATTGAT

TCCCTTGGTC

GTCATCTGAT

AAAATTATAT

TCGAAAAATC

GACACAGCTC

TAAACACAGG

CCTCGTAGCC

TGTGGAAAAC

GCCAGCTGCT

TTTGTTATCT

CCTAGCACTC

TAACCAGAGT

AGCTTTGGTT

CACTGCAGAG

TTTAAAAATT

GCGGACCACA

CAAATTATAT

GACAGCAACT

GTCCAGAGGG

CAGTACCTTG

CAGGCCCTGA

TGGAAGATAC

ATAAT CCT GA

TTAGAAGGAA

AGGCGACAGC

TCCACCTCTG

AGGCTCCACG

AAACCTGACC

TCCAGAGAAG

TTGTCTATTC

CTATTGGAAA

AGTGTAGTAC

GACACTTTGG

CAACGGATGC

GCAATGCCAG

AGAGCTTGCT

GAGCGGAGGT

ACAAGCTGAA

CATTGGCACG

CAGAAGCCAG

TCCATGGAcG

CATGCAGAGA

CTGTCCAGGA

CCAAAGAAAA

GTGCTCCATC

GTAAAAGTAC

ACTCCGAAGG

CGCTGCCACG

CCT CTGCCAC

TCATTCTGAG

TCAGTAACAT

AAGTTTGTAA

CCACACTGAC

AGGGACAACT

TCTTTCGAAC

AAAGT CT TAG

TGGAAAAGCA

GAAGCCTTAT

TGTTACAGAA

TGAGTTTCTG

GGAAGAAGAA

AGGA!TTTCTG

AGAACTCCTG

ACAGCAGGCA

GAACTCAAGT

TGGTCAGGTT

CCATCGTTAT

CAACTCAGAT

GTTCCTGACG

CAAAGAACAG

ACAGGATTCA

GAATAATTCT

GTTTGACATT

TGAGTTCCTA

GGTTTCTGCA

CGTGCTGCAC

GAAAAATTTG

GCCTGAGCCT

GCTGAGACAG

GTGCTGAAAG

ATTGAT GTCA

GAAGAGGAGA

TTACTTACCA

ACT CTTCTTC

ACAGATTTTA

TTGCAAAAAT

TCTGCAAAAG

TCTGTAAGAG

GAAAAGAGTA

CAGTCTCCTA

GTCCTTTCTG

AATGGTGACA

CCTTTTGACT

GCTTTGAATC

GCAGGCTTTG

GGTTGGACAG

CTTATGTCAG

ACTGAATTGC

TT CT CC C CGA

TTTTATCTAG

ACTGCTCTGT

ACCAGCTTTG

CACACATGGC

AACTAAATTC

TGTCGCTCCT

AC CTCCACT

CAACTCAGGG

TAAACCTTTT

ATGCAAGAAA

CGGTTGTGAG

AGGAAGGCCA

ACACCATGTC

TATTAAGTGA

TATGTCATGT

ATAATTCTTC

GGGACTGCTG

AAGAGCCGGT

TAGACGTTAG

TTAGGGCAGC

GACAAAAGAA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 WO 97/28262 PCTUS97/O 1748

AACACTACAG

ACTACCTGAG

TT CAAAT CC C

AGGATTTAAT

TGTTCCTTCA

GAGTACTGTG

ACACCGGCGT

GGTTTCAGCT

TGTGCAGTAT

GCTGCAGCAG

GAGGTCCAGG

CTTCTGGAAG

TTTTACTTCA

CTCTTTGGAA

GAAGCTGCAG

AAAAAGGTGA

TGTGAGTATT

TTTAAAAATC

CCAGAGCGCT

GTTTCCCTGA

TGGAATCTGT TGTTGTATGG

GTTGCCAGCA

GGATCAGTTA

TACTGTGGAC

TGCTGGTCCT

CAGCGGCT CT

CTTTCAAGAA

GAAAGGCAAT

AAGAGGAGTT

CATGTGTAGC

TCTACCTGTC

AAACCACATA

TGAAACTCTG

TGGGTTAGAC

GCAAGCTTCT

TGATCCAAAA

CCTCTGTGTG

TGAGTCAGAA

AC CATCT CT G

AGACATTTGG

TCACAGGCTT

ATTCAGAAGT

CTGAAAGCTT

GGAGGAGCAG

T CTGACCAAC

ACCTCCGGCT

GAAGACTTGC

CACAGATTAC

GTAGTTGTTC

GAATTAGTAC

CAACTGAAAC

CTACTTAAAT

ATTGAACTAA

ATTGTCAGCC

ATCCAACAGG

TCAGATTCTC

AAACGAAAGA

GCTTTTCTAT

GATACTTCTG

TCTCTTGAGG

TCCATGTGT C

GGTGGTTTCC

CATACAGAGG

AGGGCTTTGT

GAGTTCTACA

GTCAAGCCAT

CAGCAGTTCT

AACATTTGAA

AATCAGAGCA

CCCACTTCAT

AGCTTTCTAA

GCTGCTGCAT

GCACCACGTG

ATCCTAAGTC

TCCAGCAGCA

AGCTATCACC

TGGCTAAGTT

TGCCCAGTCC

TGTGGAAGTG

ATAACATTCA

AGTGGAAATT

ATCATTGTCA

AGTATTTGCC

CCAGGGTAAT

ATTACTTAGA

TAGGGGAACA

AGTTGCCGTC

CTTGCAGTCT

CCATTCACCA

STGTCAGTAA

3CTATGACAG kCGTTGTCTT :3TTGGATCTA

AGTGTGCCA

%TCAAGGAAG

N~

ATTTTCCAAG

GCTCCTCATC

GGATTTAGTT

TCAGATGGAA

AGCTCTGATA

ACTTCATCAT

GCAGCACCAC

AAGCCCCTTT

CGCCGTGTGC

TGTCCAGATC

TGTGATCGCC

TATACTGAAT

GAGAATTAAA

AGGAGAGACA

TT CCTAC CGA

GGATGCATTA

GATTGCAAAT

GTATAATTAT

ACAGCTAAGC

TCAGGAAGTG

AAGAGATTT G

TGGGATTCGA

ACAGAAAGAT

CTTAAGTGTG

CAGGAAGTTT

GGATGTTCAC

PAGAAGCAGAC

CCCTCCCAGT

ACCTTCCCCT

CATGTTGAAC

TGAATTAATA

AAGGCAGGGA

TATCGTCACC

TCTTGTCTTC

CAAGAATTTA

TGGCTGCTTA

AACAGTGTAA

TCCATGTGCA

CC AT CTTT

GAAGAGACCG

GCTCACCAGT

CTATCAGGTG

CCTTTACTGC

GTCCTGAGCA

AAAGCAGCTT

CTGCAAGGCA

TTTCAGGGGA

GAGGCTTATC

CAC.ATTTGTA

AT CTTTAAT C

ATTCCTTCAG

CTTCAGATTT

TTTTTAAGTT

AGTCATTCCC

GCTGCAGTTC

GGTCCTTCTC

TATGCCAGCC

1TAAACACCA

TCTGATAGGG

GAGCCATTAA

GAATCTTTC

rCAGTCTTCC rTTATGATAA

GAAATGAGTA

GAG C CCTT CT

AGAGTGCAGC

TCCAGCACCA

CAAGGGACGG

TAAAAATAAT

CAAGGAAAAG

CAGGGCTCCT

CAGAGGGAGA

GCTTGCGCTT

TACACAGTGT

ATGCTTTTAA

AACTTCTTGT

GCAACATCTG

CCTTGTGTCG

TCCT GCCCAG

AGAGCTTTGT

ATTTACTCCA

CTCT GCTC CA

CTCAGACTCA

ATTTAAAAAC

GTAATGGAGT

rGAAAGCATT

TAGACGTCGA

rTCATAAGCA

TCAGAGAGCC

TAAACCTCTT

%GTCTGCCAA

3TCACATGCT

)CATGCCACC

kGAAACAATT

PCCAGAAACT

3AAATGAXAAA 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 WO 97/28262 PCT/US97/01748

CCAAGAGCT

AACAGCACC.

ACACCCTGT

ACCAAAGGC,

GTCTCTCCT(

GCTACCGTC'r

CCAGTCAA(

AGCCCTGGG(

TCCCTCTGAC

TCAGTGTTGI

CAAT CCTGAC-

TTTCAGTGGI

ATACCCTGAG

TGTGCTTGCT

TTCTCTCCTC

AACACAAACT

TTTGATGAGC

GAAATCTCCT

TACTATGAGC

TGGTGT CTTA

AAGAGCACGG

GCTTTCCTCT

ACACCTGTCA

AT C CGAGAGT

GGAAGACAGC

T CCTGGTGAC

ATTGATGATC

GGACTCAAAT

TTTCTTTCCA

AAAGAATGTC

CTTGGATTTT

CATGATTGGT

A ATCAGGATP

GACCTGGGA

G CTTCCCACA k TTTATGAGT

:GACATTTGT

r' CAGGGCTTG 3GTGGCAGAPk ;AATCATTCAi 3GAAGAGCTG'

AGTTTGAAA(

GATGTTGAC)

LTCGATTGT'

ATTTGCATGC

CATGTGTTTC

ATACAACAGG

AACTCTGAT:l

TCAAGAACGT

TGTACAGAAA

CCTTCACAGT

TCACAGAAAC

AT TT CCC GAG

TGGCATATAG

GAAGGATTTA

GCTGCAGAAA

AGTTTTGAAG

AGACTCATAG

CAAGTGTGGG

GATGACACGA

AGCAA.ATGGC

CATGGGGAAA

GTGCTCCCAA

CATTGCTTAA

,T CTTACCCCG ,T TTCTGAGAA A GTGCAGAGC.

C AACAAAGTG.

C TTCATAGTGI T CTCTGGAAA k CAGAATTAG( 77 CAGATTTGG( r TGTCCCAGC(

TTCTGGGTGI

k CCCAAGAcG; CAAACAACTq TGGGATTA~z ;AGAGCTT'l

GAACTGTGA

TCCAAGCATG

*GTTCAGAAGA

*TTCTTCTCCT

GTCTGACCTT

CTCCTGGGAT

GCAAGAAAGA

CCCCAAT CCA

GTGTTTCTCT

GGGGAAAAAG

GAGCAGAAGG

AAGACGGCTG

CT GAT CC CCA

AAGCTGTCTC

AACACTTAGT

TCTCCATATG

GAAAAACAAG

CAT C CCAAGA A GCTGACACTG A GAGTGCTGAC A GATTGTGGCT k GACTTCTCTC

CCAGAGCCTGT

k TATATTGTGT

-AAAGCCTTTA

3CCCTGGTGAT

CGGAGATTTT

SGGAAGAAGGC

STGGAGTAGAA

ACTTGAAAAC

LTTTGCTTTCT

GAAAATTGTC

AATCCTTCTA

CCAGAGAGTA

CTTAACTCTT

TGGTATTCAC

TCCTTTCCTG

TCTTAACAGT

GGCTGATAGA

CCTGCCGTTG

C

TGTGGGTTTA

AGTAAAGAAA

TGATAGACCA

TATTCATTTGA

CAGTGGCACT T ACTAGCACAG

G

ACTTACCTAT A GGTCTCTGGG

C

CTTATCATCA G, AGAGTCTCTG C

AAGGGAGATG

AGCGTGCGTG

ACTGAATCTG

CAGAGCATAC

CAACAGAAGA

GAACTGAGGG

TTTGATGCCC

GCTGTGACTG

TCAGAAGAAG

TATGAAGCGG

TTAAATCCTG

CTCACTCACG

GCTAGCAAAG

A\GGCAGAAGG

GGCGGGTTCT

CTGGTGGATC

CTTTGGAGAA

kAAATTGTTG 'ATACCCCGA C AAGCCTTAG C ;AGAGTTTTC C TGGGACAGA P

LTGTGGAATAC

LGAAACAAACC

,AAGTTACAGA

-TTTCACTGG

G

TTATCTTTC

G

TGGAATCAC

A

TTCAGCATC c AGAGGAAGA C

ACAGCAATA

1kATTAGCTG G

TATCATCTGC-

GATTCCAGTC

TTCCTGGGGA

GACTTTTGGA

TGGAATTGGA

AACACCTTTC

TGCTTCGAGT

AGAAGAGTCA

CT GAGGATT C

ATAGTGAAAG

AAGCAGAAGG

GGGAAATAT

CTAAACTTGA

k.AAAGAACAT rGAATATTTT

ECTTGGTATC

[ATTTCTGGA

,AAGTGATTT

TTTAAGCAA

;CTTATTGAG

CTATAGGCT

~CTGCTGGCC

'AT CCAAT GA 'AT CAGTT CT AT CCATCAA

GTCCAAAGC

TGTGTGCAT

GGAGAATAT

TCAAGGGAA

TGATGTCAT

kACATTTTG 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 CCTGGGGAAC TTGCTCCTCT TCAATGGAGC TAAAATTGGC TCACAAGAGG

CCTTTTTCCT

5220 WO 97/28262 PCT/US97/01748

GTATGCTTGT

TGACTACTCC

TATGACCAAG

AACTTGCTGT

GAAAACTCAG

AC CTTC T CAA

TGAGATTGGT

CTGTGAAGAA

ACAGAGAACA

CAAACAGAAA

TGAAGAAGTT

TATAATCCAA

GGCAGAGCAA

GCACCACCAG

CCTACTGACC

AGAAGTTTGT

GGAATTATTG

TGTTTGTCAC

GGAGAAAGTG

CAGCCCTGGA

CAGTTCTTCC

ACCAGCATTT

TTTTAGTGTT

AGGAAGACCC I TGCAAAGACT

C

TTCCAGAGGA

TI

GCCCAGATCA c CAAGGGACTG

G

TCCCTCTCAG

TGATGACTGG

AAGCACCCACA

GGACCCAAC

AAATACATT.

AAAGAAGTG

CCTGCTCAG'

CC CTC T CAA

CTCAAAGGC(

GGGGCTGGC)

ACT CAAG CA'

CAGGAACTGC

TGCATTGTTC

ATCCACGTCZ

GATGTTAAGC

GCTGTGTGGG

CAGCAGTTTP

TGTCAGGTTT

IkGATCATTTG

ACAGTTATTT

AATCCCACAA

.AGTCACTCG

rTCCTGGTAA kATATTGTTC

'CTACTTATT

3ACAAGTTAC

~ACGCACTGA

~TTTGTGAAA

CACCGAGGT

,ATGATGACA

~CCTCATTCC

ATGGATCTG

AGAACTTTA

-GTGTCATTG

T ACACATC CA' A ATAAAGAcA! G ATGTTGGTC'.

r ACACCAT CT) k. GACCCTTCAC

"TCCAACCTAC

k CATTTGTTT9 P' TAGCACTGCC.

3AAAATTTAZV GCTTTCACA7

GTGAGCATGCG

TGCTGCAGGA

AGACTCTGCT

ATATTAAGCA

TACAGGAACA

TGAAAATCAT

TCAATTTCCT

ACTTCTACTT

CGTACCTGAG

TAAGCCCATC

CACAGCGGAT

TACCACTAAT

AAAATATTGC

AAACAAGCAA

TGGAGGCTCT

TCCCCAGGGC

GTCCTGGGGA

AGCCAAGCCA

CAGTTGCTAG

CCTTTTCTCC

AAGACTGTCT

r CATGCCGTG r TTTGAGATG r CTTAATTGA k. TGAACCAGT, 3CTCAAAGGAK

GGAATGTAAJ

TCTCTTTGC'

GGTTATACT(

TGGACTCC'

TTTGAAGAC(

AGAGTTCAAC

ACTGTTACTq

AGCAGCTTTG

GTTGCTGAAC

CAGAGAGGGG

TGCAGAAGTC

GTTAGCTGTA

CTCTTTGCAC

GCATTCTAGC

TGCCTTTACT

GGCTGCTCAG

ACGAGCACAA

AGATGCCAAC

AGAGGAAGCA

TCTTGGAGCC

CAGAGTAGAT

TGAGTCTTAC

AAGCACTGTC

CAGGTGGCCA

TGCTTATGAG

GATACCTATC

T AAATATGGAC T GAT GAAAT CA A\ AGTCTTTCAG S ATTCGACTCA zk GCCCAGAGCA

SGCCATCCAGG

P AGGGTTGTTC 3 TCTTTAATTA

*ATGATTCACC

CTTCTTGAAG

;CTGGATGTTG

GACTGGAAGA

GATCCTCA

GCCCACGTGG

CAGCTTACAT

CTTGGTT CT C CACCCT CCTA

ATAGATGGCA

AGCGGAGGGC

GCAGCTCCTC

ATGGTTCGAT

AAACTGGCTG

CCAGAGAAAC I TTCATCAGCA

C

CACGCCTCTG

C

CACAAAGATG

T

CCACGTCGGC C GCPAGCCTTG

G

AGTCTTGTTGP

GCAAGCTACA A

TGCTGTGGATT

AGCCAGTCAT

GAGACCTTTT

TTGTTTATAC

AGGGCCAAGT

TCTTGCTAGA

GCATTCTGCA

AACTTAGTAG

AGTACAGCCA

AAGTGTTGGT

GTTGCTGCGG

AGT CT CATC TAT GGAATAA

TCCGGGTAGA

TTCACCACTT

CTATGCCCCG

CT CCAGACTT

CTAATACTTA

.GATCTTTCA

kAGCCTTTCC

CTGAAGGAAC

CTAGAAGTCT

"AAGTTTGGG

~GAATCTTTT

;CTGTGAGTC

]CAATGGGGT

'GGGAACAGA

:TGACAACCT

;GCTGGCGTT

,TAGGAATGC

~CCGAGCCAC

ATATGAACT

5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 CTTAAGTGGG GTTCTTCTTG TCCTGCCTGA TGCTATGCTT GAAGATGTGA TGGACAGGAT 7140 WO 97/28262 PCTIUS97,01748 TATTCAAGC;z AGTAATTA.Az

TCTGAAGAAC

GGAGTTGTTG

ATTTGATCTC

TCTCGGACTA

TCTTCTGCAA

ACTCTATGTA

GAACGAATAC

TGCTTGCAGT

TGGATATCTT

TAGAGTTCTC

TCCAGTGCAC

TGGTTCTCGC

GCCAGCGAT

GGCCTCGGAG

GATTATTTAC

AT C CCAAAG C

GCAACCTTCT

CAAAGTGTGT

GGGGTCTTGT

TCCAACCCAC

CCAGGATATA

GTATTTGTCA

AGCAGAACTG

CCCTTCCAAA

AGAGAGTGTG

CCAGAGGCTC

TGTATCACTC

CAAAGTTGAC

CAGAGCAGTC

AGGGCCAAAC

CCTCCTGAGG

SGATATTCTTC

SCTGTTACATCG

*CGTGGCTTTT

*GAGTGCTTTG

GAGGAAGTGA

ATAGAGACCT

*GTTTTAAACT

TTATGTAATA

AAATTGCTCG

TCCTCAGGCA

CATAATAGCA

CAGGCTGCTT

TCGCCTTCTG

AAATTCCCTC

GAGCTACACT

GCAGAGCTCG

CAAGAGTTGA

AAGACCTCAG

GTATATAATC

ATTGGTTCAA

AAAGAAACC C

ACTGTACAAG

CTTCGTGACT

GAGTTGATAC

CTTATGAATG

CCAGACCTCA

AGCAAAGGCT

GATTTCAAAT

TCCCAAGGCA

CTGAGTGCCA

TGGTATGACC

CGAGAGAGGA

GACAGACAGA

TAGTCCTTGT

CATACATTAA

CCTTATTAGC

TTGAAATGTT

AGCACATGGA

CTCTCTATGA

CTTGTTCCAA

CAGTAGCAGC

CATGTCATAT

CACAGTATTT

AAAACAAGAG

TGGAATTTAT

CCCACCGCCA

TGGCT CAGAC

CTATGATGCA

CTCAGAGGCT

ATTCAGATAT

TTTCTCAGAC

CATTTCAAAA

GTAAAACTAG

TCCGAGACCA

AACGGAAGCA

GTCTTAGCCC

ATAAT CAT CA

CTCTAAAGTT

TTAAGATCAT

CATGGCAGAA

CCAAGGATAT

TTGAAAGGAA

GCAGGCACTG

CAATCTACTA

GACGTTTGCA

AGTCAGAAGG

3W

TAACCACCCA

TAGAGCATCA

CAACCACTTG

CTTTGGTCGA

ACTGTTCCAG

CAATGTCCTC

GGTAGCAGAC

CCTGAATGGA

ACAGCAGCTT

TAGAGTGATT

GACACAAAAT

AAGGAGCACA

TTCAGTGCCT

AGAGTCTCTG

GAGGAGGATG

GCAGAGGCTC

TATTGACATT

TGAAATTTCT

AGAAATGTTA

CGTTTCTAAG

GCTTGGAAGA

GATACTTGAG

CT CCC CACAA

GGATGAGTTA

ATGTGGCCAC

CAGAGAGGAG

AACGGTGAAC

AT CTAAAAT C

GAAGGTGATC

GCAGGAATGC

TCCAACTTCA

GAGATGCTAT

TGTGCTCAGG

TCACCTGCTA

AAGGAGCAAA

TATCTTCATA

CCGATTGGCC

AAGTGGTCTG

TTGCACAATG

ATGCTACTGG

TTAG.AAAAGA

TTCATAGCAG

GAAGACCTTA

ATGGCTTTGG

GCCAATCATG

CCGAAGCGGA

CT CAT GAAGA

AGCCAAGAGC

ACCATCTTAC

TTGAC4AACTC

GAAGAAGACA

AC CTAT CT CT

CAGCAGTGGA

TTGCTAGCGC

ATAGTTCATG

CATGGAGCCA

AGTGAAGAAG

AAGTCCATCC

CAAAAGAAGT

AACAACCAC

GCTGCAGACA

CAGCACATCA

PTCCAGCAC

TGGCAGTTGG

CTAACTATTC

CCCCCACTCT

TCCAGCAAGG

AGCACAAGTT

GGGGAACTCA

TGGATGAAGA

TCATTCCCGT

CTCTTTTACT

ACAATGGTCT

ACATTCCTGT

TTACAATTCA

TTGTACTTCT

CCCTGCAGCT

ACTCTGAAAG

GAAGCATTGC

TGCGCTCAGT

ACCCCAGCCA

CTGTGAACAG

CAGAAAATAC

TGCATCATGA

TGGATGGCTT

CTAAAATCCT

ATATTTTCT C

AACCAGCTCA

1GTTGGTTTT

AAATGGACAC

CGCCCAGTGC

kTGAAAGTGA kAAGTCTCTT

TCACCCAGGC

GAGGGATGTA

rGACACATGA

%TCCAACAGA

,CAATAAGTA

TTACCTTTT

7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 9000 9060 9120 WO 97/28262 PCT/US97/01748 TGAAGATA7A7

CATCAGAGTC

GTTGTTAGGI

CT CGAGC CT C

AGAAGTTCAC

TGGCAGAACP.

CAT CCT CACA

CCTGTGGTAT

GGGCCGGTCC

TGTTAGTGAG

AGCTGTGCAG

GGAGTATCGC

TGGCTCCCAC

CACTAAAAT G

TTCTACAAAC

GATTCCAGAG

TGTTCGTCAG

TCCTCGGCTG

CATCTGTCAC

AGC CAT CAAT

TCCAGTGCAG

GTTGTTCCAC

TCCAGCAGCT

GGAAGTCACT

AGGTTCCCCC

TGGTTCCCTG

TCTGATGACC

GTCTGCTATC

TGAGCCACCA

GGTGCCTGAc

CAACAGGCTC

TGGACACACA

ACTCATTCTr

AATCGAAGAJ

AAATGTGGG;

*CAAGGGGAGI

*AGGCGCTGGI

*CTCCTATTAC

*AATAACCTCC

TCTGGACAAP

TTCAATGATC

ACTCTTGACC

TATAAAGAAA

AAGGGAGCTC

TACTCCAACA

TTTCTAGCCT

ACAACTTGGC

TTTTTCTATC

AATGGAGAGC

TTCATCCTTA

TGGATCGACT

GTCTTCCACC

AGACGGGCTT

ACAG CC CAT G

GTTGGCTTGT

CAT CC GAG CC

AGTGCTCCAG

CAGGCACTGC

TACAACAAGG

CTAAGCTGGG

ATCAACTTCA

AGTTGT CAGC

ACCAGCAGCA

GAGGAGATCA

C CCTTCTCCTC 7 GTATCAGTGT

STGTACTTTGT

STAGAGCCGGC

GGCAACTAAG

;CATTTGACAA

CAAATCTTCT

TTACCAATTT

TCATGCAGTA

TCAATGATCC

AAGAAGACCG

GAGAGGATGA

GCGGCACCGT

AT CAAGAT CA

GCCTCTCCTC

TTCCTGAGTT

GGGTTAACCA

TTCACCGGCA

TAGTGTTTGG

CTGCTACATA

TAGAAACCAT

CCAGCCGACC

TAGTCCAGTT

CTTTGTCATG

TACCTGTGGT

CCACCAGAGC

AGCA?\GGTGT

GATATGCTGA

TTCAGAGTTC

TCTTCACTGG

CGCCCTCAGA

CCGGCTTATG

.TACTGTCAAA

TGCACCATCT

GGAAGACAAT

AT CAT TT TCT

AGATAATGCT

TAACAAGGTT

GGAGTACGC

TGAATATTTG

CCCGGTGTTC

ATCTATCTAC

TTACGTTGAC

CCC CAT GC CT

GCTCCACTTC

GACTTTCGAC

CTTTGAGTCC

CTTAGTGAAC

CGTCAATCTT

AGCACTAGAG

CTACAAGCAA

TTTTGGAATG

GATAAAAACC

TGGAGCCAAG

CGCTTTCAGA

GATAAAAGGC

CTGCTTCAGC

CATCTGTGGT

GAGAAGCATG

CAACATCTTA

kCAGCAGCAC GAGCAAGTGT C kATTGAAATG ['GTCTGCAAG C GACAAAGCT G

AGAGAGACAG

GCCTCTGACG

TGGACATATG

GTAGAAATCT

CGTGATGACG

AACATCACCG

ACT CAT TTAA CC CTT CAT CC

AGAAACCTAT

ACATACAAGT

CCTGTGCAAC

CTGGTCAGGA

AT TC CAGAC C

ATGACTGATG

CGTGAAGGCT

CCTCCCTGGG

TCTGACCATG

AAGGGGAAGG

GATGTCTCTG

TACGGGCAGA

CTTAACATCG

GAGACCCGAG

TTGAAGTGGGC

'AGCCCCATGC

PTATCACGAA i kACAACACCA 'GGTTGAAAA C 'AGGTAACCA C

;GTGTCATCAC

'AGAGTCAGA T ~CGTACAGCG

T

CAAGTGAATC

CTGGGGAATT

CAGTTGAAAG

AGGAAATTAA

TTTTAACAAA

TGTACCAGAG

CTCTGACA

ACAAGCATGC

TTTCTGACTA

CTAAGCCTAT

ACTTGGAGGA

CCTACCACTA

TGCCGCCTTT

GAACATTTCA

TGAAGGAGCT

rTGACTTCGG

CACGCAACGA

rGTCCCAGAA

CGTCTGTTCA

CAGTTGAAGA

CCCCACGTCA

kAGGAGAGCT k.ACCAGTCAA 3GGAGTACGT

;AGAAAGATT

\CTTCTGTCT

TATTCAGTG

TAAGCAGAG

TTGTGCCTG

'AGCCTATAC

'GCATCTCTA

'GATGATAAG

9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 9960 10020 10080 10140 10200 10260 10320 10380 10440 10500 10560 10620 10680 10740 10800 10860 10920 10980 11040 WO 97/28262 PCTIEJS97/01748 CGTGAGCAGA GACGGGACCT

GCATAGTATG

GGACCTGAAC

AGGCTGTGCT ATGTACAAAG

TTTGGCTGGA

TGCTACTGTG

GGACCTCGTT

GCCTGAGGGA

ATGGAGCACA

CAT CATAAG C

GACAGTGATC

TTTCCTCAGC

CGCTCTGCGA

AGACAACACA

CT GC CCT CAT CACAAAAGC C

TGTGACTCAG

GGACATGTCC

GTCTCCATCA

TGGGACTTGA

CTGACATTCT

C CAT GGT GC C

AGCTACGCAG

GCCTGGCTCC

CGTGCCTCTG

TCCATAATGG

CTGTGACGGC

CTGGCGGGGG

ACTGCAGAGA

ACGTCATTGC

AGCCTGTGAG

CCTGTGATGG

GGAAGGACCA

CTGGATGAAG

ACCAACTGCA

TGCGCGCTTC

CGTGGAAGGC

TGTCTCTGCC

CAGTGACCTG

GATCATTTGT

TGGGGGATTA

AGAGATTACA

CCACCATTTG

GCAGCGTGTG

AGAAGGAGTG

GAAGCAGATG

CCCAGCCTCC

TGGGTCTGCA

AGTGAAACGT

AGACTCTGGA

TCTGTAGCTT

GAAAATGGCA

TTTCCCAAAT

TACACTGCCA

AAGCTGCCCA

TCCCCACAGG

ACTGAGCAGA

GTGGGCCTGA

CACACTAGCC

CAGGTGACAT

CCGTGAATGG

TCTCCAACCA

TTGTAAGGCT

CAAATAAGCC

ACAGTGAGGG

TGTTCTACTC

ACATAAGCAC

TATCCAGGAA

GAGTAAAGCC

AATTAAAGTC

11100 11160 11220 11280 11340 11400 11460 11520 11580 11640 11700 11760 11817 AGAATCTTGA TGCTTTTTCC CAAAAGGTTA GGCTGAATCA AAGATCAGGC

TCGTGCC

INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 3788 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Ser Thr Asp Ser Asn Ser Leu Ala Arg Giu Phe Leu Ile Asp Val Gin Leu Cys Asn Ala Val Val Arg Ala Glu Ala Glu Giu Giu Gly Arg Gly Gl u Thr His Met Al a 40 Thr Leu Giy Gin Tyr Ile Arg Giu Giu Leu Vai His Ile Asp Gin Phe Leu Leu Ala Leu Leu 55 Giu Lys Leu Asn' Ser Leu Thr Cys Arg Leu Leu Thr Leu Gin Leu Ser Leu Leu Leu Val Trp Lys Pro Val Gin Gin Ala Thr Asp Phe Asn Leu Pro Ser Leu Gin 115 Ser Ser Asp Ile Sex Thr Gin Ile Leu Thr Lys Giu 105 Lys Leu Tyr Leu Glu 125 Val Asn Leu Phe Arg 140 Lys Asn Ser 110 Gly Ser Ala Lys Ile Arg Pro Ser 130 Gly Gin Val Ser Ala 135 Arg Gin Arg Lys Sex Thr His Arg Tyr Sex Val Arg Asp Ala Axg Lys WO 97/28262 145 Thr Thr Thr Al a Arg 225 Pro Leu Ile Leu Thr 305 Al a Val Leu Lys Val 385 Leu Gin Val Val Ala.

465 Ser Asp Ser Glu Gin Leu Ser Thr Val1 Gin Th r 210 Gi u Aila Cys Al a Thr 290 Leu Leu Asp Th r Gin 370 Gin Pro Ser Gin Leu 450 Al a Th r Val Ser 195 Lys Val Al a His Leu 275 Giu Gi u Val Val1 Gi u 355 Pro Gi u Glu Al a Gi u 435 Gin Gi u Val Ser 180 Pro Glu Ile Leu Val 260 Asn Phe Gi y Gin Ser 340 Leu Glu Gi y Leu Al a 420 Phe Met His Lys 165 Lys His Lys Giu Gin Val Leu Arg 230 Ser Ile 245 Leu Leu His Asn Leu Ala Gin Leu 310 Arg Met 325 Thr Ala Leu Arg Pro Phe Phe Val 390 Leu Giu 405 Ser Asn Ile Gin Giu Trp Leu Lys 470 Lys Val 485 Arg Gi y Leu 215 Gin Leu Ser Ser Gi y 295 Val1 Leu Glu Al a Ser 375 Phe Gi y Pro His Leu 455 Ala Lys Arg His 200 Ser Asp Ser Leu Ser 280 Phe Ser Phe Al a Al a 360 Pro Ser Val Phe Gin 440 Leu Leu Ser Leu Leu Asp Ser As n Leu 265 Leu Gly 'Al a Arg Met 345 Leu Arg Lys Leu Tyr 425 Gi y Thr Ile Glu 1Gly 170 His Val Th r As n Met 250 Glu Al a Asp Gi y Thr 330 Pro Lys Gin Tyr Gin 410 Phe Phe Arg Asn Gin 490 155 As r Al a Al a Met Gi y 235 As n Lys Leu Cys T rp 315 Val Gi u Ile Lys Arg 395 Leu Ser \s n 1 ks p Ser 475 Leu S er *Leu *Lys Ser 220 Asp As n Val1 Ser Cys 300 Thr Leu Ser Arg.

Lys 380 His Leu- Gin Leu Gly 460 Val His Asp P ro Pro 205 Val Ile Ser Cys Val 285 As n Glu His Leu Al a 365 Thr Arg Ile Al a Phe 445 1 Ile H~is Giu Arg 190 Asp Giu Leu Pro Lys 270 Val1 Gin Gi u Leu Arg 350 Cys Leu Al a Ser Met 430 Gly Pro Lys Ser PCTIUS97/01748 160 Lys Ser 175 Phe Leu Pro Ser Asn Ser Ser Glu 240 Phe Asp 255 Phe Asp Pro Thr Ser Asp Pro Vai 320 Met Ser 335 Lys Asn Leu Giu Gin Giu Leu Leu 400 Cys Leu 415 Asp Leu Thr Ala Ser Glu Ile Met 480 Met Cys 495 WO 97/28262 Thr Arg Ly PCTIUS97/01748 HySM His Arg Arg Cys Glu Tyr Ser His Phe Met Gln His s His Ser Glu 545 Leu Vai Ala Gin Gly 625 Thr Thr Arg Lys Arg 705 Lys Pro Ser Leu Leu 785 Asn I Leu I Ar Ly 53( Arc Glr His Prc His 610 Ala Vai Leu Phe Trp 690 Leu Gly ial lie ?ro 770 -ys iis -ys g Asp 515 s Ser 0 3 Cys 1 Gin Ser Leu 595 Ile Glu Asp Cys Gin 675 Asp His Asn Leu Pro 755 Gin Ser 2 Ile I Ala I 8 Arc 500 Let Pro Cys Val Val 580 Leu Leu Leu Ser Gly 660 Gly Ala Asn Val Gin 740 Ser lu krg Ele ?he ~20 Ser Phe Cys Ser 565 Gly His Asn Ser Asp 645 Ala Ile Leu Ile Vai 725 Arg Ala Val Val I Glu I 805 Glu 'I Gi Glu Ile 550 Leu Ile Ala Val Pro 630 Gin Gly Leu Glu Gin 710 al ly 31n eu [le 790 .eu 'hr Lei Gli 531 Ser Cys Phe Leu 615 Arg Leu Pro Pro Ala 695 Ile Gin Vai Thr Gin 775 Arg Asn Leu i Leu 520 i Thr Val Thr Cys Lys 600 Ser Ile Ala Thr Ser 680 Tyr Ala Trp Glu His 760 Ile 'I Asp I Tyr I Ile 8 505 Val Ala Cys Thr Cys 585 Leu Lys Lys Lys Ser 665 Ser Sin zksn Lys Leu 745 4et [yr eu .eu Tal 125 510 Ser Glu Ala Cys 570 Met Pro Leu Lys Leu 650 Gly Gly Ser His Leu 730 Val I Cys Leu I Phe I Asp C 810 Ser I Ala Gly His 555 Val Asp Ala Leu Ala 635 Sly Leu Ser Phe Tie 715 Tyr iis 3er -ys eu 195 ;ly ~eu i Pht As! 54( Glr Glr Pro Leu Vai 620 Ala Glu Pro Glu Val 700 Cys Asn His Gin Thr 780 Ser Ile Giy e Lys 525 D Val 0 Cys Ile Lys Lys 605 Asp Cys Thr Ser Asp 685 Phe Asn Tyr Cys Leu 765 Leu Cys Arg Giu As Glr Let Leu Ser 590 Ala Gin Asn Leu Pro 670 Leu Gin Leu Ile Gin 750 Lys Pro Asn Ser Sin 830 Gin i Tyr a Arg I Ser 575 Val Phe Leu Ile Gin 655 Ser Leu Glu Leu Phe 2 735 Gin Gin 'I Val I Gly His S 815 Gin L Leu Pro Leu 560 Gly Ile Gin Gly Cys 640 Sly Tyr Trp ksp Gin 720 ksn ,eu 'yr eu

T

ai 100 er 'ys Asp Ala Ala Vai 835 Leu Asp Val Asp 840 Gly Leu Asp Ile Gin Gin Giu Leu WO 97/28262 PCT/US97/01748 liol Pro Ser Leu Ser Val Gly Pro Ser Leu His Lys Gin Gin Ala Ser Ser 850 855 860 Asp Ser Pro Cys Ser Leu Arg Lys Phe Tyr Ala Ser Leu Arg Glu Pro 865 870 875 880 Asp Pro Lys Lys Arg Lys Thr Ile His Gin Asp Val His Ile Asn Thr 885 890 895 Ile Asn Leu Phe Leu Cys Val Ala Phe Leu Cys Val Ser Lys Glu Ala 900 905 910 Asp Ser Asp Arg Glu Ser Ala Asn Glu Ser Glu Asp Thr Ser Gly Tyr 915 920 925 Asp Ser Pro Pro Ser Glu Pro Leu Ser His Met Leu Pro Cys Leu Ser 930 935 940 Leu Glu Asp Val Val Leu Pro Ser Pro Glu Cys Leu His His Ala Ala 945 950 955 960 Asp Ile Trp Ser Met Cys Arg Trp Ile Tyr Met Leu Asn Ser Val Phe 965 970 975 Gin Lys Gin Phe His Arg Leu Gly Gly Phe Gin Val Cys His Glu Leu 980 985 990 Ile Phe Met Ile Ile Gin Lys Leu Phe Arg Ser His Thr Glu Asp Gin 995 1000 1005 Gly Arg Arg Gin Gly Glu Met Ser Arg Asn Glu Asn Gin Glu Leu Ile 1010 1015 1020 Arg Ile Ser Tyr Pro Glu Leu Thr Leu Lys Gly Asp Val Ser Ser Ala 1025 1030 1035 1040 Thr Ala Pro Asp Leu Gly Phe Leu Arg Lys Ser Ala Asp Ser Val Arg 1045 1050 1055 Gly Phe Gin Ser Gin Pro Val Leu Pro Thr Ser Ala Glu Gin Ile Val 1060 1065 1070 Ala Thr Glu Ser Val Pro Gly Glu Arg Lys Ala Phe Met Ser Gin Gin 1075 1080 1085 Ser Glu Thr Ser Leu Gin Ser Ile Arg Leu Leu Glu Ser Leu Leu Asp 1090 1095 1100 Ile Cys Leu His Ser Ala Arg Ala Cys Gin Gin Lys Met Glu Leu Glu 1105 1110 1115 1120 Leu Pro Ser Gin Gly Leu Ser Val Glu Asn Ile Leu Cys Glu Leu Arg 1125 1130 1135 Glu His Leu Ser Gin Ser Lys Val Ala Glu Thr Glu Leu Ala Lys Pro 1140 1145 1150 Leu Phe Asp Ala Leu Leu Arg Val Ala Leu Gly Asn His Ser Ala Asp 1155 1160 1165 Leu Gly Pro Gly Asp Ala Val Thr Glu Lys Ser His Pro Ser Glu Glu 1170 1175 1180 Glu Leu Leu Ser Gin Pro Gly Asp Phe Ser Glu Glu Ala Glu Asp Ser WO 97/28262 PCT/US97/01748 1185 1190 1195 1200 Gin Cys Cys Ser Leu Lys Leu Leu Gly Glu Glu Glu Gly Tyr Glu Ala 1205 1210 1215 Asp Ser Glu Ser Asn Pro Glu Asp Val Asp Thr Gin Asp Asp Gly Val 1220 1225 1230 Glu Leu Asn Pro Glu Ala Glu Gly Phe Ser Gly Ser Ile Val Ser Asn 1235 1240 1245 Asn Leu Leu Glu Asn Leu Thr His Gly Glu Ile Ile Tyr Pro Glu Ile 1250 1255 1260 Cys Met Leu Gly Leu Asn Leu Leu Ser Ala Ser Lys Ala Lys Leu Asp 1265 1270 1275 1280 Val Leu Ala His Val Phe Glu Ser Phe Leu Lys Ile Val Arg Gin Lys 1285 1290 1295 Glu Lys Asn Ile Ser Leu Leu Ile Gin Gin Gly Thr Val Lys Ile Leu 1300 1305 1310 Leu Gly Gly Phe Leu Asn Ile Leu Thr Gin Thr Asn Ser Asp Phe Gin 1315 1320 1325 Ala Cys Gin Arg Val Leu Val Asp Leu Leu Val Ser Leu Met Ser Ser 1330 1335 1340 Arg Thr Cys Ser Glu Asp Leu Thr Leu Leu Trp Arg Ile Phe Leu Glu 1345 1350 1355 1360 Lys Ser Pro Cys Thr Glu Ile Leu Leu Leu Gly Ile His Lys Ile Val 1365 1370 1375 Glu Ser Asp Phe Thr Met Ser Pro Ser Gin Cys Leu Thr Phe Pro Phe 1380 1385 1390 Leu His Thr Pro Ser Leu Ser Asn Gly Val Leu Ser Gin Lys Pro Pro 1395 1400 1405 Gly Ile Leu Asn Ser Lys Ala Leu Gly Leu Leu Arg Arg Ala Arg Ile 1410 1415 1420 Ser Arg Gly Lys Lys Glu Ala Asp Arg Glu Ser Phe Pro Tyr Arg Leu 1425 1430 1435 1440 Leu Ser Ser Trp His Ile Ala Pro Ile His Leu Pro Leu Leu Gly Gin 1445 1450 1455 Asn Cys Trp Pro His Leu Ser Glu Gly Phe Ser Val Ser Leu Val Gly 1460 1465 1470 Leu Met Trp Asn Thr Ser Asn Glu Ser Glu Ser Ala Ala Glu Arg Gly 1475 1480 1485 Lys Arg Val Lys Lys Arg Asn Lys Pro Ser Val Leu Glu Asp Ser Ser 1490 1495 1500 Phe Glu Gly Ala Glu Gly Asp Arg Pro Glu Val Thr Glu Ser Ile Asn 1505 1510 1515 1520 Pro Gly Asp Arg Leu Ile Glu Asp Gly Cys Ile His Leu Ile Ser Leu 1525 1530 1535 WO 97/28262 PCT/US97/01748 Gly Ser Lys Ala Leu Met Ile Gin Val Trp Ala Asp Pro His Ser Gly 1540 1545 1550 Thr Phe Ile Phe Arg Val Cys Met Asp Ser Asn Asp Asp Thr Lys Ala 1555 1560 1565 Val Ser Leu Ala Gin Val Glu Ser Gin Glu Asn Ile Phe Phe Pro Ser 1570 1575 1580 Lys Trp Gin His Leu Val Leu Thr Tyr Ile Gin His Pro Gin Gly Lys 1585 1590 1595 1600 Lys Asn Val His Gly Glu Ile Ser Ile Trp Val Ser Gly Gin Arg Lys 1605 1610 1615 Thr Asp Val Ile Leu Asp Phe Val Leu Pro Arg Lys Thr Ser Leu Ser 1620 1625 1630 Ser Asp Ser Asn Lys Thr Phe Cys Met Ile Gly His Cys Leu Thr Ser 1635 1640 1645 Gin Glu Glu Ser Leu Gin Leu Ala Gly Lys Trp Asp Leu Gly Asn Leu 1650 1655 1660 Leu Leu Phe Asn Gly Ala Lys Ile Gly Ser Gin Glu Ala Phe Phe Leu 1665 1670 1675 1680 Tyr Ala Cys Gly Pro Asn Tyr Thr Ser Ile Met Pro Cys Lys Tyr Gly 1685 1690 1695 Gin Pro Val Ile Asp Tyr Ser Lys Tyr Ile Asn Lys Asp Ile Leu Arg 1700 1705 1710 Cys Asp Glu Ile Arg Asp Leu Phe Met Thr Lys Lys Glu Val Asp Val 1715 1720 1725 Gly Leu Leu Ile Glu Ser Leu Ser Val Val Tyr Thr Thr Cys Cys Pro 1730 1735 1740 Ala Gin Tyr Thr Ile Tyr Glu Pro Val Ile Arg Leu Lys Gly Gin Val 1745 1750 1755 1760 Lys Thr Gin Pro Ser Gin Arg Pro Phe Ser Ser Lys Glu Ala Gin Ser 1765 1770 1775 Ile Leu Leu Glu Pro Ser Gin Leu Lys Gly Leu Gin Pro Thr Glu Cys 1780 1785 1790 Lys Ala Ile Gin Gly Ile Leu His Glu Ile Gly Gly Ala Gly Thr Phe 1795 1800 1805 Val Phe Leu Phe Ala Arg Val Val Glu Leu Ser Ser Cys Glu Glu Thr 1810 1815 1820 Gin Ala Leu Ala Leu Arg Val Ile Leu Ser Leu Ile Lys Tyr Ser Gin 1825 1830 1835 1840 Gin Arg Thr Gin Glu Leu Glu Asn Cys Asn Gly Leu Ser Met Ile His 1845 1850 1855 Gin Val Leu Val Lys Gin Lys Cys Ile Val Gly Phe His Ile Leu Lys 1860 1865 1870 Thr Leu Leu Glu Gly Cys Cys Gly Glu Glu Val Ile His Val Ser Glu 1875 1880 1885 WO 97/28262 PCT/US97/01748 His Gly G l u Phe Lys Leu Asp Val Glu Ser His Ala Ile Ile Gin Asp 1890 1895 1900 Val Lys Leu Leu Gin Glu Leu Leu Leu Asp Trp Lys Ile Trp Asn Lys 1905 1910 1915 1920 Ala Glu Gin Gly Val Trp Glu Thr Leu Leu Ala Ala Leu Glu Val Leu 1925 1930 1935 Ile Arg Val Glu His His Gin Gin Gin Phe Asn Ile Lys Gin Leu Leu 1940 1945 1950 Asn Ala His Val Val His His Phe Leu Leu Thr Cys Gin Val Leu Gin 1955 1960 1965 Glu His Arg Glu Gly Gin Leu Thr Ser Met Pro Arg Glu Val Cys Arg 1970 1975 1980 Ser Phe Val Lys Ile Ile Ala Glu Val Leu Gly Ser Pro Pro Asp Leu 1985 1990 1995 2000 Glu Leu Leu Thr Val Ile Phe Asn Phe Leu Leu Ala Val His Pro Pro 2005 2010 2015 Thr Asn Thr Tyr Val Cys His Asn Pro Thr Asn Phe Tyr Phe Ser Leu 2020 2025 2030 His Ile Asp Gly Lys Ile Phe Gin Glu Lys Val Gin Ser Leu Ala Tyr 2035 2040 2045 Leu Arg His Ser Ser Ser Gly Gly Gin Ala Phe Pro Ser Pro Gly Phe 2050 2055 2060 Leu Val Ile Ser Pro Ser Ala Phe Thr Ala Ala Pro Pro Glu Gly Thr 2065 2070 2075 2080 Ser Ser Ser Asn Ile Val Pro Gin Arg Met Ala Ala Gin Met Val Arg 2085 2090 2095 Ser Arg Ser Leu Pro Ala Phe Pro Thr Tyr Leu Pro Leu Ile Arg Ala 2100 2105 2110 Gin Lys Leu Ala Ala Ser Leu Gly Phe Ser Val Asp Lys Leu Gin Asn 2115 2120 2125 Ile Ala Asp Ala Asn Pro Glu Lys Gin Asn Leu Leu Gly Arg Pro Tyr 2130 2135 2140 Ala Leu Lys Thr Ser Lys Glu Glu Ala Phe Ile Ser Ser Cys Glu Ser 2145 2150 2155 2160 Ala Lys Thr Val Cys Glu Met Glu Ala Leu Leu Gly Ala His Ala Ser 2165 2170 2175 Ala Asn Gly Val Ser Arg Gly Ser Pro Arg Phe Pro Arg Ala Arg Val 2180 2185 2190 Asp His Lys Asp Val Gly Thr Glu Pro Arg Ser Asp Asp Asp Ser Pro 2195 2200 2205 Gly Asp Glu Ser Tyr Pro Arg Arg Pro Asp Asn Leu Lys Gly Leu Ala 2210 2215 2220 Ser Phe Gin Arg Ser Gin Ser Thr Val Ala Ser Leu Gly Leu Ala Phe WO 97/28262 PCT/US97/01748 2225 2230 2235 2240 Pro Ser Gin Asn Gly Ser Ala Val Ala Ser Arg Trp Pro Ser Leu Val 2245 2250 2255 Asp Arg Asn Ala Asp Asp Trp Glu Asn Phe Thr Phe Ser Pro Ala Tyr 2260 2265 2270 Glu Ala Ser Tyr Asn Arg Ala Thr Ser Thr His Ser Val Ile Glu Asp 2275 2280 2285 Cys Leu Ile Pro Ile Cys Cys Gly Leu Tyr Glu Leu Leu Ser Gly Val 2290 2295 2300 Leu Leu Val Leu Pro Asp Ala Met Leu Glu Asp Val Met Asp Arg Ile 2305 2310 2315 2320 Ile Gin Ala Asp Ile Leu Leu Val Leu Val Asn His Pro Ser Pro Ala 2325 2330 2335 Ile Gin Gin Gly Val Ile Lys Leu Leu His Ala Tyr Ile Asn Arg Ala 2340 2345 2350 Ser Lys Glu Gin Lys Asp Lys Phe Leu Lys Asn Arg Gly Phe Ser Leu 2355 2360 2365 Leu Ala Asn Gin Leu Tyr Leu His Arg Gly Thr Gin Glu Leu Leu Glu 2370 2375 2380 Cys Phe Val Glu Met Phe Phe Gly Arg Pro Ile Gly Leu Asp Glu Glu 2385 2390 2395 2400 Phe Asp Leu Glu Glu Val Lys His Met Glu Leu Phe Gln Lys Trp Ser 2405 2410 2415 Val Ile Pro Val Leu Gly Leu Ile Glu Thr Ser Leu Tyr Asp Asn Val 2420 2425 2430 Leu Leu His Asn Ala Leu Leu Leu Leu Leu Gin Val Leu Asn Ser Cys 2435 2440 2445 Ser Lys Val Ala Asp Met Leu Leu Asp Asn Gly Leu Leu Tyr Val Leu 2450 2455 2460 Cys Asn Thr Val Ala Ala Leu Asn Gly Leu Glu Lys Asn Ile Pro Val 2465 2470 2475 2480 Asn Glu Tyr Lys Leu Leu Ala Cys Asp Ile Gin Gin Leu Phe Ile Ala 2485 2490 2495 Val Thr Ile His Ala Cys Ser Ser Ser Gly Thr Gin Tyr Phe Arg Val 2500 2505 2510 Ile Glu Asp Leu Ile Val Leu Leu Gly Tyr Leu His Asn Ser Lys Asn 2515 2520 2525 Lys Arg Thr Gin Asn Met Ala Leu Ala Leu Gin Leu Arg Val Leu Gin 2530 2535 2540 Ala Ala Leu Glu Phe Ile Arg Ser Thr Ala Asn His Asp Ser Glu Ser 2545 2550 2555 2560 Pro Val His Ser Pro Ser Ala His Arg His Ser Val Pro Pro Lvs Ara 2565 2570 2575 WO 97/28262 PCT/US97/01748 Arg Ser Ile Ala Gly Ser Arg Lys Phe Pro Leu Ala Gin Thr Glu Ser 2580 2585 2590 Leu Leu Met Lys Met Arg Ser Val Ala Ser Asp Glu Leu His Ser Met 2595 2600 2605 Met Gin Arg Arg Met Ser Gin Glu His Pro Ser Gin Ala Ser Glu Ala 2610 2615 2620 Glu Leu Ala Gin Arg Leu Gin Arg Leu Thr Ile Leu Ala Val Asn Arg 2625 2630 2635 2640 Ile Ile Tyr Gin Glu Leu Asn Ser Asp Ile Ile Asp Ile Leu Arg Thr 2645 2650 2655 Pro Glu Asn Thr Ser Gin Ser Lys Thr Ser Val Ser Gin Thr Glu Ile 2660 2665 2670 Ser Glu Glu Asp Met His His Glu Gin Pro Ser Val Tyr Asn Pro Phe 2675 2680 2685 Gin Lys Glu Met Leu Thr Tyr Leu Leu Asp Gly Phe Lys Val Cys Ile 2690 2695 2700 Gly Ser Ser Lys Thr Ser Val Ser Lys Gin Gin Trp Thr Lys Ile Leu 2705 2710 2715 2720 Gly Ser Cys Lys Glu Thr Leu Arg Asp Gin Leu Gly Arg Leu Leu Ala 2725 2730 2735 His Ile Leu Ser Pro Thr His Thr Val Gin Glu Arg Lys Gin Ile Leu 2740 2745 2750 Glu Ile Val His Glu Pro Ala His Gin Asp Ile Leu Arg Asp Cys Leu 2755 2760 2765 Ser Pro Ser Pro Gin His Gly Ala Lys Leu Val Leu Tyr Leu Ser Glu 2770 2775 2780 Leu Ile His Asn His Gin Asp Glu Leu Ser Glu Glu Glu Met Asp Thr 2785 2790 2795 2800 Ala Glu Leu Leu Met Asn Ala Leu Lys Leu Cys Gly His Lys Cys Ile 2805 2810 2815 Pro Pro Ser Ala Pro Ser Lys Pro Glu Leu Ile Lys Ile Ile Arg Glu 2820 2825 2830 Glu Gin Lys Lys Tyr Glu Ser Glu Glu Ser Val Ser Lys Gly Ser Trp 2835 2840 2845 Gin Lys Thr Val Asn Asn Asn Gin Gin Ser Leu Phe Gin Arg Leu Asp 2850 2855 2860 Phe Lys Ser Lys Asp Ile Ser Lys Ile Ala Ala Asp Ile Thr Gin Ala 2865 2870 2875 2880 Val Ser Leu Ser Gin Gly Ile Glu Arg Lys Lys Val Ile Gin His Ile 2885 2890 2895 Arg Gly Met Tyr Lys Val Asp Leu Ser Ala Ser Arg His Trp Gin Glu 2900 2905 2910 Cys Ile Gin Gin Leu Thr His Asp Arg Ala Val Trp Tyr Asp Pro Ile 2915 2920 2925 WO 97/28262 PCT/US97/01748 Tyr Tyr Pro Thr Ser Trp Gln.Leu Asp Pro Thr Glu Gly Pro Asn Arg 2930 2935 2940 Glu Arg Arg Arg Leu Gin Arg Cys Tyr Leu Thr Ile Pro Asn Lys Tyr 2945 2950 2955 2960 Leu Leu Arg Asp Arg Gin Lys Ser Glu Gly Val Leu Arg Pro Pro Leu 2965 2970 2975 Ser Tyr Leu Phe Glu Asp Lys Thr His Ser Ser Phe Ser Ser Thr Val 2980 2985 2990 Lys Asp Lys Ala Ala Ser Glu Ser Ile Arg Val Asn Arg Arg Cys Ile 2995 3000 3005 Ser Val Ala Pro Ser Arg Glu Thr Ala Gly Glu Leu Leu Leu Gly Lys 3010 3015 3020 Cys Gly Met Tyr Phe Val Glu Asp Asn Ala Ser Asp Ala Val Glu Ser 3025 3030 3035 3040 Ser Ser Leu Gin Gly Glu Leu Glu Pro Ala Ser Phe Ser Trp Thr Tyr 3045 3050 3055 Glu Glu Ile Lys Glu Val His Arg Arg Trp Trp Gin Leu Arg Asp Asn 3060 3065 3070 Ala Val Glu Ile Phe Leu Thr Asn Gly Arg Thr Leu Leu Leu Ala Phe 3075 3080 3085 Asp Asn Asn Lys Val Arg Asp Asp Val Tyr Gin Ser Ile Leu Thr Asn 3090 3095 3100 Asn Leu Pro Asn Leu Leu Glu Tyr Gly Asn Ile Thr Ala Leu Thr Asn 3105 3110 3115 3120 Leu Trp Tyr Ser Gly Gin Ile Thr Asn Phe Glu Tyr Leu Thr His Leu 3125 3130 3135 Asn Lys His Ala Gly Arg Ser Phe Asn Asp Leu Met Gin Tyr Pro Val 3140 3145 3150 Phe Pro Phe Ile Leu Ser Asp Tyr Val Ser Glu Thr Leu Asp Leu Asn 3155 3160 3165 Asp Pro Ser Ile Tyr Arg Asn Leu Ser Lys Pro Ile Ala Val Gin Tyr 3170 3175 3180 Lys Glu Lys Glu Asp Arg Tyr Val Asp Thr Tyr Lys Tyr Leu Glu Glu 3185 3190 3195 3200 Glu Tyr Arg Lys Gly Ala Arg Glu Asp Asp Pro Met Pro Pro Val Gin 3205 3210 3215 Pro Tyr His Tyr Gly Ser His Tyr Ser Asn Ser Gly Thr Val Leu His 3220 3225 3230 Phe Leu Val Arg Met Pro Pro Phe Thr Lys Met Phe Leu Ala Tyr Gin 3235 3240 3245 Asp Gln Ser Phe Asp Ile Pro Asp Arg Thr Phe His Ser Thr Asn Thr 3250 3255 3260 Thr Trp Arg Leu Ser Ser Phe Glu Ser Met Thr Asp Val Lys Glu Leu WO 97/28262 PCT/US97/01748 3265 3270 3275 3280 Ile Pro Glu Phe Phe Tyr Leu Pro Glu Phe Leu Val Asn Arg Glu Gly 3285 3290 3295 Phe Asp Phe Gly Val Arg Gin Asn Gly Glu Arg Val Asn His Val Asn 3300 3305 3310 Leu Pro Pro Trp Ala Arg Asn Asp Pro Arg Leu Phe Ile Leu Ile His 3315 3320 3325 Arg Gin Ala Leu Glu Ser Asp His Val Ser Gin Asn Ile Cys His Trp 3330 3335 3340 Ile Asp Leu Val Phe Gly Tyr Lys Gin Lys Gly Lys Ala Ser Val Gin 3345 3350 3355 3360 Ala Ile Asn Val Phe His Pro Ala Thr Tyr Phe Gly Met Asp Val Ser 3365 3370 3375 Ala Val Glu Asp Pro Val Gin Arg Arg Ala Leu Glu Thr Met Ile Lys 3380 3385 3390 Thr Tyr Gly Gin Thr Pro Arg Gin Leu Phe His Thr Ala His Ala Ser 3395 3400 3405 Arg Pro Gly Ala Lys Leu Asn Ile Glu Gly Glu Leu Pro Ala Ala Val 3410 3415 3420 Gly Leu Leu Val Gin Phe Ala Phe Arg Glu Thr Arg Glu Pro Val Lys 3425 3430 3435 3440 Glu Val Thr His Pro Ser Pro Leu Ser Trp Ile Lys Gly Leu Lys Trp 3445 3450 3455 Gly Glu Tyr Val Gly Ser Pro Ser Ala Pro Val Pro Val Val Cys Phe 3460 3465 3470 Ser Gin Pro His Gly Glu Arg Phe Gly Ser Leu Gin Ala Leu Pro Thr 3475 3480 3485 Arg Ala Ile Cys Gly Leu Ser Arg Asn Phe Cys Leu Leu Met Thr Tyr 3490 3495 3500 Asn Lys Glu Gin Gly Val Arg Ser Met Asn Asn Thr Asn Ile Gin Trp 3505 3510 3515 3520 Ser Ala Ile Leu Ser Trp Gly Tyr Ala Asp Asn Ile Leu Arg Leu Lys 3525 3530 3535 Ser Lys Gin Ser Glu Pro Pro Ile Asn Phe Ile Gin Ser Ser Gin Gin 3540 3545 3550 His Gin Val Thr Ser Cys Ala Trp Val Pro Asp Ser Cys Gin Leu Phe 3555 3560 3565 Thr Gly Ser Lys Cys Gly Val Ile Thr Ala Tyr Thr Asn Arg Leu Thr 3570 3575 3580 Ser Ser Thr Pro Ser Glu Ile Glu Met Glu Ser Gin Met His Leu Tyr 3585 3590 3595 3600 Gly His Thr Glu Glu Ile Thr Gly Leu Cys Val Cys Lys Pro Tyr Ser 3605 3610 3615 WO 97/28262 PCTJUS97/01748 Val Met Ile Ser Val ser Arg Asp Gly Thr Cys Ile Val Trp Asp Leu 3620 3625 3630 Asn Arg Leu Cys Tyr Vai Gin Ser Leu Ala Gly His Lys Ser Pro Val 3635 3640 3645 Thr Ala Val Ser Ala Ser Giu Thr Ser Gly Asp Ile Ala Thr Val Cys 3650 3655 3660 Asp Ser Ala Gly Gly Gly Ser Asp Leu Arg Leu Trp Thr Val Asn Gly 3665 3670 3675 3680 Asp Leu Val Gly His Vai His Cys Arg Giu Ile Ile Cys Ser Val Ala 3685 3690 3695 Phe Ser Asn Gin Pro Giu Giy Val Ser Ile Asn Vai Ile Ala Giy Gly 3700 3705 3710 Leu Glu Asn Gly Ile Val Arg Leu Trp Ser Thr Trp Asp Leu Lys Pro 3715 3720 3725 Vai Arg Giu Ile Thr Phe Pro Lys Ser Asn Lys Pro Ile Ile Ser Leu 3730 3735 3740 Thr Phe Ser Cys Asp Gly His His Leu Tyr Thr Ala Asn Ser Giu Gly 3745 3750 3755 3760 Thr Val Ile Ala Trp Cys Arg Lys Asp Gin Gin Arg Vai Lys Leu Pro 3765 3770 3775 Met Phe Tyr Ser Phe Leu Ser Ser Tyr Ala Ala Gly 3780 3785 INF'ORMATION FOR SEQ ID NO: Ci) SEQUENCE CHARACTERISTICS: CA) LENGTH: 5893 base pairs TYPE: nucleic acid STRAN~DEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TGAGAGCTCA CGCTGGCCTG GCAGCCTTGG TGAGTCGGGA TTCTCCTGCA CCGGCGGGCG

AGAGCGCGCG

TTCATGAACC

CATGAGCACG

CAATGCAGTG

AACTCTTGGA

TAT CATTGAT

TCCCTTGGTG

GTCATCTGAT

AAAATTATAT

TCGAAAAATC

GCGGACCACA

CAAATTATAT

GACAGCAACT

GTCCAGAGGG

CAGTACCTTG

CAGGCCCTGA

TGGAAGATAC

ATAATCCTGA

TTAGAAGGAA

AGGCGACAGC

GAGCGGAGGT

ACAAGCTGAA

CATTGGCACG

CAGAAGCCAG

TCCATGGACG

CAT GCAGAGA

CTGTCCAGGA

CCAAAGAAAA

GTGCTCCATC

GTAAAAGTAC

GAAGCCTTAT

TGTTACAGAA

TGAGTTTCTG

GGAAGAAGAA

AGGATTT CTG

AGAACTCCTG

ACAGCAGGCA

GAACTCAAGT

TGGTCAGGTT

CCATCGTTAT

GCTGAGACAG

GTGCTGAAAG

ATTGATGTCA

GAAGAGGAGA

TTACTTACCA

ACTCTTCTTC

ACAGATTTTA

TTGCAAAAAT

TCTGCAAAAG

TCTGTAAGAG

TTTTATCTAG

ACTGCTCTGT

ACCAGCTTTG

CACACATGGC

AACTAAATTC

TGTCGCTCCT

ACCTGCCACT

CAACTCAGGG

TAAACCTTTT

ATGCAAGAAA

120 180 240 300 360 420 480 540 600 660 WO 97/28262 PCT/US97/01748

GACACAGCTC

TAAACACAGG

CCTCGTAGCC

TGTGGAAAAC

GCCAGCTGCT

TTTGTTATCT

CCTAGCACTC

TAACCAGAGT

AGCTTTGGTT

CACTGCAGAG

TTTAAAAATT

AACACTACAG

ACTACCTGAG

TTCAAATCCC

AGGATTTAAT

TGTTCCTTCA

GAGTACTGTG

ACACCGGCGT

GGTTTCAGCT

TGTGCAGTAT

GtTGCAGCAG

TGGAATCTGT

GTTGCCAGCA

GGATCAGTTA

TACTGTGGAC

TGCTGGTCCT

CAGCGG CT CT

CTTTCAAGAA

GAAAGGCAAT

AAGAGGAGTT

CATGTGTAGC

TCTACCTGTC

*TCCACCTCTC

AGGCTCCACC

AAACCTGACC

*TCCAGAGAA(.

TTGTCTATTC

CTATT GGAAP

AGTGTAGTAC

GACACTTTGC

CAACGGATGC

GCAATGCCAG

AGAGCTTGCT

GAGGTCCAGG

CTTCTGGAAG

TTTTACTTCA

CTCTTTGGAA

GAAGCTGCAG

AAAAAGGTGA

TGTGAGTATT

TTTAAAAATC

CCAGAGCGCT

GTTTCCCTGA

TGTTGTATGG

CTGAAACTT

GGAGGAGCAG

TCTGACCAAC

ACCTCCGGCT

GAAGACTTGC

GACAGATTAC

GTAGTTGTTC

GAATTAGTAC

CAACTGAAAC

CTACTTAAAT

3ACTCCGAAGE

;CGCTGCCACC

*CCTCTGCCAC

TCATTCTGAC

TCAGTAACAT

AAGTTTGTAA

CCACACTGAC

AGGGACAACT

TCTTTCGAAC

AAAGTCTTAG

TGGAAAAGCA

AGGGCTTTGT

GAGTTCTACA

GTCAAGCCAT

CAGCAGTTCT

AACATTTGAA

AAT CAGAGCA

CCCACTTCAT

AGCTTTCTAA

GCTGCTGCAT

GCACCACGTG

ATCCTAAGTC

TCCAGCAGCA

AGCTATCACC

TGGCTAAGTT

TGCCCAGTCC

T GTGGAAGTG

ATAACATTCA

AGTGGAAATT

ATCATTGTCA

AGTATTTGCC

CCAGGGTAAT

ZAACTCAGAI

GTTCCTGACC

CAAAGAACAG

ACAGGATTCA

GAAkTAATTCT

GTTTGACATT

TGAGTTCCTA

GGTTTCTGCA

CGTGCTGCAC

GAAAAATTTG

GCCTGAGCCT

ATTTTCCAAG

GCTC CT CAT C

GGATTTAGTT

TCAGATGGAA

AGCTCTGATA

ACTTCATCAT

GCAGCACCAC

AAGCCCCTTT

CGCCGTGTGC

TGTCCAGATC

TGTGATCGCC

TATACTGAAT

GAGAATTAAA

AGGAGAGACA

TTCCTACCGA

GGATGCATTA

GATTGCAAAT

GTATAATTAT

ACAGCTAAGC

TCAGGAAGTG

AAGAGATTTG

GAAAAGAGT.7

CAGTCTCCTI

GTCCTTTCTC

AATGGTGACP

CCTTTTGAC7 GCTTT GAATc

GCAGGCTTTG

GGTTGGACAG

CTTATGTCAG

ACTGAATTGC

TTCTC CCC GA TAT CGT CAC C

TCTTGTCTTC

CAAGAATTTA

TGGCTGCTTA

AACAGTGTAA

TCCATGTGCA

CGCGATCTTT

GAAGAGACCG

GCTCACCAGT

CTATCAGGTG

CCTTTACTGC

GTCCTGAGCA

AAAGCAGCTT

CT GCAAGG CA

TTTCAGGGGA

GAGGCTTATC

CACATTTGTA

ATCTTTAATC

ATTCCTTCAG

CTTCAGATTT

TTTTTAAGTT

SCGGTTGTGAG

SAGGAAGGCCA

ACACCATGTC

TATTAAGTGA

TATGTCATGT

ATAATTCTTC

GGGACTGCTG

AAGAGCCGGT

TAGACGTTAG

TTAGGGCAGC

GACAAAAGAA

GAGCCCTTCT

AGAGTGCAGC

TCCAGCACCA

CAAGGGACGG

TAAAAATAAT

CAAGGAAAAG

CAGGGCTCCT

CAGAGGGAGA

GCTTGCGCTT

TACACAGTGT

ATGCTTTTAA

AACTTCTTGT

GCAACATCTG

CCTTGTGTGG

TCCTGCCCAG

AGAGCTTTGT

ATTTACTCCA

CTGTGCTCCA

CTCAGACTCA

ATTTAAAAAc

GTAATGGAGT

720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 WO 97/28262 PCTIUS97/01748

AAACCACATA

TGAAACTCTG

TGGGTTAGAC

GCAAGCTTCT

TGATCCAAAA

CCTCTGTGTG

TGAGTCAGAA

ACCATGTCTG

AGACATTTGG

TCACAGGCTT

ATTCAGAAGT

CCAAGAGCTA

AACAGCACCA

ACAGCCTGT G

ACGAAAGGCA

GTCTCTCCTG

GCTACCGTCT

CCAGTCAAAG

AGCCCTGGGG

TCCCTCTGAG

TCAGTGTTGT

CAATCCTGAG

TTTCAGTGGA

ATACCCTGAG

TGTGCTTGCT

TTCTCTCCTC

AACACAAACT

TTTGATGAGC

GAAATCTCCT

TACTATGAGC

TGGTGTCTTA

AAGAGCACGG

ATTGAACTAA ATTACTTAGA

ATTGTCAGCC

AT C CAACAGG

TCAGATTCTC

AAACGAAAGA

GCTTTTCTAT

GATACTTCTG

TCTCTTGAGG

TCCATGTGTC

GGTGGTTTCC

CATACAGAGG

AT CAGGATAT

GACCTGGGAT

CTTCCCACAA

TTTATGAGTC

GACATTTGTC

CAGGGCTTGT

GTGGCAGAAA

AATCATTCAG

GAAGAGCTGT

AGTTTGAAAC

GATGTTGACA

TCGATTGTTT

ATTTGCATGC

CATGTGTTTG

ATACAACAGG

AACTCTGATT

TCAAGAACGT

T GTACAGAAA

CCTTCACAGT

TCACAGAAAC

ATTT CC CGAG

TAGGGGAACA

AGTTGCCGTC

CTTGCAGTCT

CCATTCACCA

GTGTCAGTAA

GCTATGACAG

ACGTTGTCTT

GTTGGAT CTA

AAGTGTGCCA

ATCAAGGAAG

CTTACCCCGA

TTCTGAGAAA

GTGCAGAGCA

AACAAAGTGA

TTCATAGTGC

CTGTGGAAAA

CAGAAT TAG C

CAGATTTGGG

TGTCCCAGCC

TTCTGGGTGA

CCCAAGACGA

CAAACAACTT

TGGGATTAAA

AGAGCTTTCT

GAACTGTGAA

TCCAAGCAT C

GTTCAGAAGA

TTCTTCTCCT

GTCTGACCTT

CTCCTGGGAT

GCAAGAAAGA

TGGGATTCGA

ACAGAAAGAT

CTTAAGTGTG

CAGGAAGTTT

GGATGTTCAC

AGAAGCAGAC

C CCT CCCAGT

ACCTTCCCCT

CATGTTGAAC

TGAATTAATA

AAGGCAGGGA

GCT GACACTG

GAGTGCTGAC

GATTGT GGCT

GACTTCTCTC

CAGAGCCTGT

TATATTGTGT

AAAGCCTTTA

CCCTGGTGAT

CGGAGATTTT

GGAAGAAGGC

TGGAGTAGAA

ACTTGAAAAC

TTTGCTTTCT

GAAAATTGTC

AATCCTTCTA

CCAGAGAGTA

CTTAACTCTT

TGGTATTCAC

TCCTTTCCTG

TCTTAACAGT

GGCTGATAGA

AGTCATTCCC TGAAAGCATT

GCTGCAGTTC

GGTCCTTCTC

TAT G CCAGC C

ATAAACACCA

TCTGATAGGG

GAGCCATTAA

GAATGTTTGC

TCAGTCTTCC

TTTATGATAA

GAAATGAGTA

AAGGGAGATG

AGCGTGCGTG

ACT GAATCTG

CAGAGCATAC

CAACAGAAGA

GAACTGAGGG

TTTGATGCCC

GCTGTGACTG

T CAGAAGAAG

TATGAAGCGG

TTAAATCCTG

CTCACTCACG

GCTAGCAAAG

AGGCAGAAGG

GGCGGGTTCT

CTGGTGGATC

CTTTGGAGAA

AAAATTGTTG

CATACCCCGA

AAAGCCTTAG

GAGAGTTTTC

TAGACGTCGA

TTCATAAGCA

TCAGAGAGCC

TAAACCTCTT

ACTCTGCCAA

GTCACATGCT

ACCATGCAGC

AGAAACAATT

TCCAGAAACT

GAAATGAAAA

TAT CAT CT GC

GATTCCAGTC

TTCCTGGGGA

GACTTTTGGA

TGGAATTGGA

AACACCTTTC

TGCTTCGAGT

AGAAGAGTCA

CTGAGGATTC

ATAGTGAAAG

AAGCAGAAGG

GGGAAATAAT

CTAAACTTGA

AAAAGAACAT

TGAATATTTT

TCTTGGTATC

TATTTCTGGA

AAAGTGATTT

GTTTAAGCAA

GCTTATTGAG

CCTATAGGCT

2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 GCTTTCCTCT TGGCATATAG CCCCAATCCA CCTGCCGTTG CTGGGACAGA ACTGCTGGCC WO 97/28262 PCTIUS97/01748 ACACCTGTCA GAAGGATTTA GTGTTTCTCT ZGTGGGTTTA

ATCCGAGAGT

GGAAGACAGC

TCTTCAAATA

TAAATGTTAT

AAACAGCCTT

TTCTGCAACA

AGATAGTTAT

CACCAATGGC

AACAGAGTTC

TTTGTTCTCT

GTAGTCTTAA

TATACCCTAA

TGTTTATGTT

CTTATCACAC

CAAGATTTAA

ACTT CT CTCA

CAAGGTTGTT

TATTAATAAT

ATGGCATGTG

AGAACCTTGG

TAAAGCATTT

GCCACCGCGG

GCTGCAGAAA

AGTTTTGAAG

GCTGCTTGCC

TCACCTTAAA

GAAATCTTCA

GCTGTTGAAG

AAATAAAACC

CCCTTCTAGT

AT CTGTAACT

TCTTTTTTAA

AAACTGGCTG

TAATTGATAA

AGTACATCTT

ATATTCCACC

GTAGTAAGGT

GCGGAGACAA

CACTACCTTA

ATTATAGATC

CTCCCTGGGC

TATGGAGTGT

AATAACTTTT

TGG

GGGGAAAAAG

GAGCAGGTAT

TGAGTTTTA.A

GATCCTACTT

TTGTCTCTAA

TTAGCAGTCC

CTATGGCCAT

TACTGTCCCT

GAAGAACTGC

GCAATCAACA

TGTATTTTTT

TGTTCCTCTT

AGCTTAGTTT

TGCCATTGCT

GCCATTGGGA

ACTGTGATTC

AATAAT CAT C

GTGCCTTTGT

CATGGTCAGT

GGCTGACGAT

AAAACACTGG

AGTAAAGAAA

GAT GGCAGGG

GCATATCTGG

CACTACTGAA

ACTTTAGATA

CATGACTGTG

TTTTATTTTA

GATCATTTAT

TGTTAGGATG

GTTTCTTAAG

TAACCTGTTA

TTTTAAAAGT

AACATAAAGT

GTCACAGATA

GGGGTAAGCA

AGGGTTTGGC

ATTTGAGCAG

AACAATTTTT

TGTGTTAGAG

GGTCCTTTAG

AGTCTTTAGT

ATGTGG.AATA.

AGAAACAAAC

TCTGATCTAT

CAGTATTTTA

TTACCAAAGC

GGGAAGTGGG

TTAGTGTGGC

AGTTCTCCTT

ATGTAACAGT

TACTGAAATT

TCATATAGCA

AAAATGGTGG

CTGAGCTTTT

CACATCATAG

ATGGGAATAT

GCTAGCTCAC

ATCACTTAGC

TGCAGCTTTT

TTAGTGCAAG

TGACCCAATC

CACCCTCAGG

GAGGACCTGC

CAT CCAAT GA

CATCAGTTCT

ATACTAAGAT

ATGTATTCTT

CTGAGTTTTC

GATGCTCTGT

TTCTGATACT

CTGTGTCTTA

CCAAAGTTAG

GAATTTTGTT

GCTAGAGGAA

CTAATATTTT

GGACATGCAC

TAACAAATAG

AGAGGCAACT

AGCCATAAAC

ATGGTTATTT

CTAAGAAGAG

GCATCTGTTG

CAACAAAAGC

CCTTGTAGTT

CCGGGCGGCC

4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5893 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 1545 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Met Ser Thr Asp Ser Asn Ser Leu Ala Arg Glu Phe Leu Ile Asp Val 1 5 10 Asn Gln Leu Cys Asn Ala Val Val Gin Arg Ala Glu Ala Arg Glu Glu 25 Glu Glu Glu Glu Thr His Met Ala Thr Leu Gly Gin Tyr Leu Val His WO 97/28262 PCT/US9701748 154 Gi Ala Pro Asn Ser Pro Arg 145 Thr Thr Thr Ala Arg 225 Pro Leu Ile Leu Thr 305 Ala Val Leu Lys Arg Leu Leu Leu Leu Ser 130 Gin Gin Val Gin Thr 210 Glu Ala Cys Ala Thr 290 Leu Leu Asp Thr Gin 370 Gly Thr Va1 Pro Gin 115 Gly Arg Leu Va1 Ser 195 Lys Val Ala His Leu 275 Glu Glu Val Val G1u 355 Pro Phe Cys Trp Leu 100 Lys Gin Lys Ser Ser 180 Pro Glu Ile Leu Val 260 Asn Phe Gly Gin Ser 340 Leu Glu Leu Arg Lys Ser Ser Val Ser Thr 165 Lys Lys Gin Leu Ser 245 Leu His Leu Gin Arg I 325 Thr Leu I Pro I Let Glu 70 Ile Ser Thr Ser Thr 150 Ser His Glu Val Arg 230 Ile Leu Asn a Leu 310 4et kla %rg Phe Leu 55 Glu Pro Asp Gin Ala 135 His Asp Arg Giy Leu 215 Gin Leu Ser Ser Gly 295 Val Leu Glu Ala Ser 375 Thr Leu Val Ile Gly 120 Lys Arg Ser Arg His 200 Ser Asp Ser Leu Ser 280 Phe Ser Phe kla kia 360 Pro Lys Leu Asn Ser Ile Ile Asp Gin Leu Gin Ile 105 Lys Val Tyr Glu Leu 185 Leu Asp Ser Asn Leu 265 Leu Gly Ala Arg Met 345 Leu Arg Thr Glu 90 Leu Leu Asn Ser Gly 170 His Val Thr Asn Met 250 Glu Ala Asp ly rhr 330 Pro Lys Gin Leu 75 Gin Thr Tyr Leu Vai 155 Asn Ala Ala Met Giy 235 Asn Lys Leu Cys Trp 315 Val Glu Ile Lys Leu Gin Lys Leu Phe 140 Arg Ser Leu Lys Ser 220 Asp Asn Va1 Ser Cys 300 Thr Leu Ser Arq Lys 380 Leu Ala Glu Glu 125 Arg Asp Asp Pro Pro 205 Val Ile Ser Cys Val 285 Asn Glu His Leu Ala 365 Thr Ser Thr Lys 110 Gly Lys Ala Glu Arg 190 Asp Glu Leu Pro Lys 270 Val Gin Glu Leu krg 350 -ys Leu Leu Asp Asn Ser Ile Arg Lys 175 Phe Pro Asn Ser Phe 255 Phe Pro Ser Pro Met 335 Lys Leu Gin Leu Phe Ser Ala Arg Lys 160 Ser Leu Ser Ser Glu 240 Asp Asp Thr Asp Vai 320 Ser ksn Glu Glu WO 97/28262 Val Gin Glu Gly Phe Val Phe Ser 1:S: PCT/US97/01748 His Arg Ala Leu Leu 385 Leu Pro Gin Ser Val Gin Val Leu 450 Ala Ala 465 Ser Thr Thr Arg His Arg Ser Lys 530 Glu Arg 545 Leu Gin Val His Ala Pro Gin His 610 Gly Ala 625 Thr Val Thr Leu Arg Phe Lys Trp 690 Arg Leu 705 390 Glu Ala Glu 435 Gin Glu Val Lys Asp 515 Ser Cys Gin Ser Leu 595 Ile Glu Asp Cys Gin 675 Asp His Leu Ala 420 Phe Met His Lys Arg 500 Leu Pro Cys Val Val 580 Leu Leu Leu Ser Gly 660 Gly Ala Asn Leu 405 Ser Ile Glu Leu Lys 485 His Ser Phe Cys Ser 565 Gly His Asn Ser Asp 645 Ala Ile Leu Ile Glu Asn Gin Trp Lys 470 Val Arg Gly Glu Ile 550 Leu Ile Ala Val Pro 630 Gin Gly Leu Glu Gin 710 Gly Pro His Leu 455 Ala Lys Arg Leu Glu 535 Ala Ser Cys Phe Leu 615 Arg Leu Pro Pro Ala 695 Ile Val Phe Gin 440 Leu Leu Ser Cys Leu 520 Thr Val Thr Cys Lys 600 Ser Ile Ala Thr Ser 680 Tyr Ala Lys Leu Tyr 425 Gly Thr Ile Glu Glu 505 Val Ala Cys Thr Cys 585 Leu Lys Lys Lys Ser 665 Ser Gin Asn Tyr Arg 395 Gin Leu 410 Phe Ser Phe Asn Arg Asp Asn Ser 475 Gin Leu 490 Tyr Ser Ser Ala Glu Gly Ala His 555 Cys Val 570 Met Asp Pro Ala Leu Leu Lys Ala 635 Leu Gly 650 Gly Leu Gly Ser Ser Phe His Ile 715 Leu Tyr 730 Leu Gin Leu Gly 460 Val His His Phe Asp 540 Gin Gin Pro Leu Val 620 Ala Glu Pro Glu Val 700 Cys Ile Ala Phe 445 Val Ile His Phe Lys 525 Val Cys Ile Lys Lys 605 Asp Cys Thr Ser Asp 685 Phe Asn Ser Met 430 Gly Pro Lys Ser Met 510 Asn Gin Leu Leu Ser 590 Ala Gin Asn Leu Pro 670 Leu Gin Leu Cys 415 Asp Thr Ser Ile Met 495 Gin Gin Tyr Arg Ser 575 Val Phe Leu Ile Gin 655 Ser Leu Glu Leu 400 Leu Leu Ala Glu Met 480 Cys His Leu Pro Leu 560 Gly Ile Gin Gly Cys 640 Gly Tyr Trp Asp Gin 720 Lys Gly Asn Val Val Val Gin Trp Lys Asn Tyr Ile Phe Asn 735 WO 97/28262 PCT/EJS97/01748 Pro Val Leu Gin 740 Ser Ile Pro 755 Leu Pro Gin 770 Leu Lys Ser 785 Asn His Ile Leu Lys Ala Asp Ala Ala 835 Pro Ser Leu 850 Asp Ser Pro 865 Asp Pro Lys Ile Asn Leu Asp Ser Asp 915 Asp Ser Pro 930 Leu Glu Asp 945 Asp Ile Trp Gin Lys Gin Ile Phe Met 995 Gly Arg Arg 1010 Arg Ile Ser 1025 Thr Ala Pro Gly Phe Gin Ser Glu Arg Ile Phe 820 Val Ser Cys Lys Phe 900 Arg Pro Val Ser Phe 980 Ile Gin Tyr Asp Ser Arg Gly Val. Glu Leu Val 745 Ala Gin Thr His Met Cys 760 Val Leu Gin Ile Tyr Leu 775 Val Ile Arg Asp Leu Phe 790 Glu Leu Asn Tyr Leu Asp 805 810 Glu Thr Leu Ile Val Ser 825 Leu Asp Val Asp Gly Leu 840 Val Gly Pro Ser Leu His 855 Ser Leu Arg Lys Phe Tyr 870 Arg Lys Thr Ile His Gin 885 890 Leu Cys Val Ala Phe Leu 905 Glu Ser Ala Asn Glu Ser 920 Ser Glu Pro Leu Ser His 935 Val Leu Pro Ser Pro Glu 950 Met Cys Arg Trp Ile Tyr 965 970 His Arg Leu Gly Gly Phe 985 Ile Gin Lys Leu Phe Arg 1000 Gly Glu Met Ser Arg Asn 1015 Pro Glu Leu Thr Leu Lys 1030 Leu Gly Phe Leu Arg Lys 1045 105C Gin Pro Val Leu Pro Thr His His Cys Gin 750 Ser Gin Leu Lys 765 Lys Thr Leu Pro 780 Leu Ser Cys Asn 795 Gly Ile Arg Ser Leu Gly Glu Gin 830 Asp Ile Gin Gin 845 Lys Gin Gin Ala 860 Ala Ser Leu Arg 875 Asp Val His Ile Cys Val Ser Lys 910 Glu Asp Thr Ser 925 Met Leu Pro Cys 940 Cys Leu His His 955 Met Leu Asn Ser Gin Val Cys His 990 Ser His Thr Glu 1005 Glu Asn Gin Glu 1020 Gly Asp Val Ser 1035 Ser Ala Asp Set Ser Ala Gu G Ser Ala Glu Gln Gin Leu Gin Tyr Val Leu Gly Val 800 His Ser 815 Gin Lys Glu Leu Ser Ser Glu Pro 880 Asn Thr 895 Glu Ala Gly Tyr Leu Ser Ala Ala 960 Val Phe 975 Glu Leu Asp Gin Leu Ile Ser Ala 1040 Val Arg 1055 Ile Val 1060 Ala Thr Glu Ser Val Pro 1065 Gly Glu Arg Lys Ala 1070 Phe Met Ser Gin Gin WO 97/28262 PCT/US97/01748 159 1075 1080 1085 Ser Glu Thr Ser Leu Gin Ser Ile Arg Leu Leu Glu Ser Leu Leu Asp 1090 1095 1100 Ile Cys Leu His Ser Ala Arg Ala Cys Gin Gin Lys Met Glu Leu Glu 1105 1110 1115 1120 Leu Pro Ser Gin Gly Leu Ser Val Glu Asn Ile Leu Cys Glu Leu Arg 1125 1130 1135 Glu His Leu Ser Gin Ser Lys Val Ala Glu Thr Glu Leu Ala Lys Pro 1140 1145 1150 Leu Phe Asp Ala Leu Leu Arg Val Ala Leu Gly Asn His Ser Ala Asp 1155 1160 1165 Leu Gly Pro Gly Asp Ala Val Thr Glu Lys Ser His Pro Ser Glu Glu 1170 1175 1180 Glu Leu Leu Ser Gin Pro Gly Asp Phe Ser Glu Glu Ala Glu Asp Ser 1185 1190 1195 1200 Gin Cys Cys Ser Leu Lys Leu Leu Gly Glu Glu Glu Gly Tyr Glu Ala 1205 1210 1215 Asp Ser Glu Ser Asn Pro Glu Asp Val Asp Thr Gin Asp Asp Gly Val 1220 1225 1230 Glu Leu Asn Pro Glu Ala Glu Gly Phe Ser Gly Ser Ile Val Ser Asn 1235 1240 1245 Asn Leu Leu Glu Asn Leu Thr His Gly Glu Ile Ile Tyr Pro Glu Ile 1250 1255 1260 Cys Met Leu Gly Leu Asn Leu Leu Ser Ala Ser Lys Ala Lys Leu Asp 1265 1270 1275 1280 Val Leu Ala His Val Phe Glu Ser Phe Leu Lys Ile Val Arg Gin Lys 1285 1290 1295 Glu Lys Asn Ile Ser Leu Leu Ile Gin Gin Gly Thr Val Lys Ile Leu 1300 1305 1310 Leu Gly Gly Phe Leu Asn Ile Leu Thr Gin Thr Asn Ser Asp Phe Gin 1315 1320 1325 Ala Cys Gin Arg Val Leu Val Asp Leu Leu Val Ser Leu Met Ser Ser 1330 1335 1340 Arg Thr Cys Ser Glu Asp Leu Thr Leu Leu Trp Arg Ile Phe Leu Glu 1345 1350 1355 1360 Lys Ser Pro Cys Thr Glu Ile Leu Leu Leu Gly Ile His Lys Ile Val 1365 1370 1375 Glu Ser Asp Phe Thr Met Ser Pro Ser Gin Cys Leu Thr Phe Pro Phe 1380 1385 1390 Leu His Thr Pro Ser Leu Ser Asn Gly Val Leu Ser Gin Lys Pro Pro 1395 1400 1405 Gly Ile Leu Asn Ser Lys Ala Leu Gly Leu Leu Arg Arg Ala Arg Ile 1410 1415 1420 WO 97/28262 PCT/US97/01748 I scl Ser Arg Gly Lys Lys Giu Ala Asp Arg Glu Ser Phe Pro Tyr Arg Leu 1425 1430 -1435 1440 Leu Ser Ser Trp His Ile Ala Pro Ile His Leu Pro Leu Leu Gly Gin 1445 1450 1455 Asn Cys Trp Pro His Leu Ser Glu Gly Phe Ser Val Ser Leu Val Gly 1460 1465 1470 Leu Met Trp Asn Thr Ser Asn Giu Ser Giu Ser Ala Ala Glu Arg Gly 1475 1480 1485 Lys Arg Val Lys Lys Arg Asn Lys Pro Ser Val Leu Glu Asp Ser Ser 1490 1495 1500 Phe Glu Gly Ala Gly Met Met Ala Gly Ser Asp Leu Tyr Thr Lys Ile 1505 1510 1515 1520 Leu Gin Ile Ala Ala Cys Leu Ser Phe Lys His Ile Trp Gin Tyr Phe 1525 1530 1535 Asn Val Phe Phe Lys Cys Tyr Ser Pro 1540 1545 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 7080 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CGCAAGGGCT TCTAAGAAGC CATCCCAATG ACCTTTTGGC TTTGAGAAGA GCAGTCCTCA

TACCAGAGTG

TGTATTCCGA

TCCTCGGGTG

GTTTGACTCA

GAACAGAAGC

GAACCAGTTT

TCCTTGCTCA

GTTTCATCTA

GACTGCTCGG

AACCGGCTTT

ACGCACATGG

AAGCTAAATT

CTGTCTCTCC

TTTGGGGTTT

GGTTTCTGTG

ACATCAGAGT

ACCGTGTGTG

CAGGATCTAA

CCTTGCAGCT

TCTAAAAGAG

GTTTATGAGT

TCAT GAG CAC

GCAATGCAGT

CAACCCTTGG

CTATAATTGA

TTCCACTGGT

TGGCCTCTTT

GTGCATGAAG

ACAGCAGTAT

TTCCTCAATG

CTGCAAACAA

AC CT GGGAAG

GCAATCACCG

CCAAATGATA

CGACAGTAAC

GGTCCAGAGG

ACAGTACCTT

TCAGGCATT G AT GGAAGATA

CAGTGTTTAT

CTTTTGCCTT

TTTCCCTTGC

CCAGGGGAAT

GAGACCCAGC

ACGGTTGCTA

TTCAGGTGAA

TAGACTGTAA

TCACTGGCAC

GTGGAGGCCA

GTCCATGGTC

ACATGTAGAG

CCTGTCCAAG

TCATTCTTAC GTGGGAAAGT

CACCATCTGT

CATCTAATGG

AATCCTACCC

TTGCTTAACA

ATTAGCCTGC

GCTTTGTTCT

ATGTCACAGC

GTGAATTTCT

GGGAGGAAGA

GAGGATTTCT

AAGAACTCCT

AAGAAAAGGC

TCCCGTGTCT

GGTTTGGGCT

TAAGTCAGCT

GCATGGAAGA

AACAAAGAGT

AAGAATATTT

AGTGGT GAAA

GACCGATGTC

AGAGGAGGAG

ATTACTTACC

GACTCTTCTT

AACAGATTTT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 AACCTACCGC TCTCAGCAGA TATAATCCTG ACCAAAGAAA AGAACTCAAG TTCACAAAGA WO 97/28262 PCTUJS97/O 1748

TCCACTCAGG

GTAAATGTTT

GATGCAAGAA

ATAGCAATGA

AAACAAGACC

GATGCTATGG

ATTTTAAGTG

TTATGTCATG

CATAATT CT C

GGGGACTGCT

GAAGAACCGG

GTAGATGTTA

CTTAGAGCAG

AGACAAAAGA

AGAGCCCTTC

CAGAGTGCAG

ATTCAGCATC

TTAAGAGATG

ATGAAAATAA

ACAAGAAAAA

TCAGGTCTTC

GCAGATGGAG

TGCTTGCGCT

GTTCATAACA

CAT GCTTTTA

AAACTTATTT

TGTAATATTT

AACTTATGTG

ATCCTGCCCA

CAGAACTTTG

AATTTAATCC

CCTGTGCTCC

GCTCAAAGTC

AAAAATTACA

TTCGAAAAAG

AGACACAGCT

ATAAGCATAG

ACCCCAAAGC

CTTTGGAAAA

AGCCAGCTGC

TTTTGTTATC

CTTTAGCAGC

GCAGT CT GAG

TGGCTTTGAT

GTACTGCAGA

CTTTAAAAAT

AAACACTGCA

TTTTACCTGA

CTTCAAATCC

ATGGATTTAA

GAGTTCCTCC

TGAGCACTGT

GGCACAGACG

TGGTTTCGGC

ATGTTTATTA

TACTGCAGCA

TTGGAATATG

AATTGCCAGC

TGGATCAGTT

GTACTGTTGA

ATGCTGAACT

GCAGTGGATC

TTTTTGGAGA

AGAAAGGCAA

AAAGAGGAGT

ATGTATGTAG

TTTAGAAGGA

CAGACGACAG

CTCCACCTCA

AAGGCCCCAT

TAAACTTGAC

TTCCAGAGAG

CTTGTCTGTT

TTTATTAGAA

CAGTGTAGTG

CGACAACTTG

TCAAAGGATG

GATGATGCCA

TAGAATATGC

GGAGGTTCAG

GCTTTTGGAA

CTTCTACTTC

TTTATTTGAA

CGAGGCCTCA

CAAAAAAGTG

ATGTGAATAT

TTTTAAAAAC

TCCTGAGCGG

GGCTTCCTTG

CTGTTGTATG

ACT GAAAAAT

AGGAGGAGCA

CTCTGACCAA

CTCCTCAAGT

TGAAGATTTG

AGACAGATTA

TATAGTTGTT

TGAA.TTAGCA

CCATCATAAC

AGTGCCCTGT

CGTAAAATTA

GATTCAGAAG

CT GCT GCAT C

CGCTTAGCAA

ATTATTCCAA

ATCAGTAACA

AAAGTTTGTA

CCCACACTAA

GAGAGTCGAG

CTCTTTCGAA

GAAAATCTTA

CTAGAAAAGC

GAAGATTTTG

GGAGTTCTTC

AGTCAAGCCA

ACAGCAGTTC

GAGCATTTGA

AAATCAGAGC

TCTCATTTTA

CAGGTTTCCA

TGCTGTTGCA

AGCAGCACTT

GATCCCAAAT

TTTCAGCAGC

GAGATATCAC

CTAGCCCAAT

TTATCCAGTC

TTGTGGAAAT

CATAGTATAC

CAGTGGAAAT

CAT CAT TCT C

CAGTGCTTGC

CTAGTCAGGT

CCCATCGCTA

CCAATTCAGA

ATTTTTTAAC

CCAAAGAACA

GACAGGGGTC

TGAACAATT C

AGTTTGACGT

CT GAATT CCT

TAGTTTCTGC

CAGTGTTGCA

GGAAAAATTT

AGCCT GACCC T GTTTTCAAA AGATTCT CAT

TGCATTTGGT

TTCAAATGGA

AACCCCTAAT

AACTTCATCA

TGCAT CAT CA

AAAACCCATT

TTGCAGTCTG

GTGTCCACAT

CT GTAAT CAT

ATATATTGAA

CAAAAATTAA

TAGAAGAGAC

CTTCTTACAG

GGGATGCTTT

AGATTGCAAA

TATATAATTA

AACACCTAAG

CT CAGGAC CT

TTCTGCAAAA

TTCTGTAAGA

TGAAAAAGGC

ATCGTTTCCT

GACTCCTCCA

AAACACTGAC

TCCATTTGAC

TACCTTGAAT

AGCAGGCTTT

AGGTTGGACC

TCTTCTGTCA

AACTGAATTG

TTTTGCACCA

GTATCGTCAT

CTGTTGTCTT

TCAAGAATTC

ATGGCTGGTT

AAATAGT GTG TTC CATCT CT

CCGAGATCTC

TGAAGAGACT

TGCC CAT CAG

CCTATCGGGT

TCCTTTGCTC

TATCCTTAAC

AAAAGCAGCT

ACTGCAGGGA

ATTTCAAGGG

AAAGGCTTAT

TCACATTTGC

CATATTTAAT

CGTTACTTCA

GCTTCAGATT

960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 WO 97/28262 TAT GTAAAAA T GTAATGGAC

CTAAAAGCAT

CCAGATATT G

TCTTTTCATC

GGCCT CAAAG

ACAATAAACC

AGGGAGTCGG

TTAAGTCATA

ATGCACCAAG

TTCCAGAAAC

ATAATACAGA

AGTGTAAATG

GATTTATTAT

AAGAGTGCTG

GAAGTTTCAG

GAGACCTCAC

GCCAGAACTA

AGTATATTAT

GCAAAGCCTT

GAACATAATG

CCTGGTGATT

GAAGAAGAAG

GATGGGGTAG

TTACTCGAAA

AATTTGCTTT

TTGAA.AATTA

AAAAATCTTT

T GCCAGAGAG

GAGCTAACCC

CTGGGTATTC

TTCCCTTTAC

PCTIUS97/01748 CT CTGCCTAT TAAGT CAAAT

TTGAAACTCT

ATGGGATAGA

ATCAGCAAGC

AAGCTTATCC

TATTCCTCTG

CCAATGACTC

TGCTGCCATG

CAGCAGACAT

AGTTTTATAG

AACTGTTCAG

AAAACCAGGA

CTTTGGCTAT

ACAGTTTAGG

CTACTGAAGC

TTCAAAGTAT

GTCAACAGAA

TTGAAATGAG

TATTTGATGC

ATGCTATGAC

TTTCAGAAGA

GTTACGAAGC

ACT TAAAGT C

ACCTCACTCA

CTGCTAGTAA

TTAGGCAGAA

TAGGAGGGTT

TATTGGTGGA

TTCTTTTGAG

TGAAAATTAT

TGCACGCTcC

CCTGCTTAAA.

AAT CGAATTA

GATAATCAGC

CATTGAACAG

TTATTCAGAT

AAAGAGACGG

TGTGGCTTTT

AGAAGATACT

TATATCTCTC

TTGGTCTATG

GCTTGGTGGT

AAGTCACAAA

TTTAAACAGA

AAAAAkGTGAC

TAAATTAGAG

CGCTCCCGAG

ACGACTTTTG

GATGGAATTG

GGACCATCTT

CCTGCTTCGA

TGAGAAGAGT

AGCTGAGGAT

AGATAGTGAA

TGAAACAGAA

AGGGGAAATA

AGCCAAACTT

AGAAAAGAAT

CTT GAGTATT

TCTTTTGGTA

AATATTTCTG

TGAAAGTGAT

AAATTTAAGC

TCCAGGGTAA

AATT GCTTAA

CTAGGGGAGC

AAGGAGTTGT

TCTCCTCAGA

AAGACTGTTA

TTATGCGTAA

TCTGGCTATG

GAGAGCCTTG

TGTCGTTGGA

TTCCGAGTAT

GAGGAGCAAG

ATTTCTCAAC

CCCATACCAT

TTACAGCATA

GAAGCAAAGC

GAAGCCCTTC

GAGTTACCTA

T CCCAGTCAA

GTTGCCCTCG

CAT CAATCTG

TCTCAGTGTT

AGCAATCCTG

GGTTTCAGTG

ATTTATCCT C

GATGTGCTTG

GTTTTTCTGC

TTAACACAGG

TCTTTGATGA

GAGAAATCTC

ACTACTAT GA

AACGGTGTTT

TAAGAGATTT

ATGGTATTCG

AACAGAAAGA

CCTCTGTACA

GTCTCAGCAA

ACCAAGATGT

GTAAAGAAGC

ACAGCACAGC

TCTTGCCTTC

TCTACATGTT

GCCATAAGTT

GAAAAAAGGA

CTAAGAGAAC

CAGAACTAGG

TTTCTTCCAT

TATTTACAAG

TGGCCATTTG

AT CAGAACTT

AGGTGATTGA

GGAATTATTC

CAGAAGAATT

GTAGTTTTAA

JAAGATGGCGA

CAT CAAGCAG

AGATTTGTAT

CC CAT GTATT TCAT GCAACA AT GATT CTGA

GTTCAAGAAC

CTTGTACAAA

GCCCTTCACA

CAT CACAAAA

GTTTTTGAGT

AAGTCATTCT

TGCCTCAGTT

TGTGGGTACT

ATTTTATGCT

TCATATCAAC

AGAGTCTGAC

CAGCGAGCCT

TCCTGAACAT

GAGTTCAGTG

AATATTTATG

GGGAGATACA

TAT GAAGGAA

TAGTCTAAAA

AAATGTGGAA

TCAAGAAAGT

TCTTCATGGT

GTCTGTGGAA

AACACAACTA

AGCAGATTTT

GTCATCCCAG

ACTTTTAGTT

AACCCAGGAT

TCCAAATGAC

GCTGGAATTA

TGAGAGTTTT

GGGAACTGTG

TTTTCAAGCA

ATGTTCAGAA

AATTCTTCTT

GTATCTAACC

GTATCCTGGG

2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 WO 97/28262 PCTIUS97/01748 ATTTTAAACA GTAAGGCCAT GGGTTTATTG

GAGGCTGATA

CACCTGCCGT

CTGTGGTTTA

ATAAAGAAAA

GACAGACCAG

ATTCATATAA

AATGCCACTC

CTAGCACAGG

CTCACCTACT

GTCTCTGGAC

TTGTCATCTG

GAGTTTTTGC

AAGGTTGGTT

ATGCCATGTA

TTGCGATGTG

TTAATTGGAG

GAACCAGTGA

TCAAAAGAAG

GAATATAAAA

CTCTTTGCCA

GTTATACTCT

GGACTTTCTA

TTGAAGACCC

GAGTTTAAGT

CTATTGCTTG

GCAGCTCTAG

TTATTGAAAG

AAAGAGGGGC

GGTCAATCAT

TTATGGTAAT

ATATTATTCC

CTACTTCTTC

GAGAGAGTTT

TGCTGGGGCA

ATGTGGAGTG

GAAACAAATC

AAGGT GCAGA

TTTCACTGGG

TTATCTTTCG

TTGAATCACA

TACAGCAGCC

AGAGGAAGCC

ATAGCAATAA

AGTTGGCTGG

CACAAGAGGC

AGTATGGCAA

AACAAAT GAG

TCTTTCAGTT

TTAGACTTAA

TTCAGAGCAT

CTATTCAAGG

GGGTTGTTGA

CAT TAAT TAA

TGATTCATCA

TT CTTGAAGG

TGGATGTAGA

ACT GGAAGAT

AAGTCCTCAT

CTCAAGTGGT

AACTCACACC

GTACCTGAGG

AAGCCCATCT

ACAACAGATG

ACTACTAACG

TCCCCATCGC

AAACTGCTGCG

TAT CCAT GAA

ATTAATTTTA

GTACATAAAT

ATCCAAAGCG

TGTGTGCATG

GGAGAATATT

CCAAGGGAAA

TGATGTTACT

AACATTTTGC

AAAATGGGAC

CTTTTATCTG

GCCAGTCAAT

AGAATTTTTT

GTTTATACAA

AGGTCAAATG

CTTATTAGAA

CATTCTGCAC

ACTCAGTAGC

ATACAACCAA

GGTGTTGATc

ATGCTGTGGT

CTCTAATGCT

ATGGAGTAAA

CAGAGCAGAT

TCATCACTTT

CAT GCC CCGA

CATTCCAGCA

GGTTTTACTG

GCCGCCCATA

CAAkTCACAAA )w,

AGAAGAGCAC

CTGCTTTCAT

CCACACCTAT

GCTGAGAGTA

CCAGATAGCA

CCTGGTGAAA

TT GATGATCC

GATTCAAATG

TT CCT CCCAA

AGGAGGATTC

TT GGATTTTA

ATGATTGGCC

CTGGGAAATT

TATGCTTGTG

GACTACTCCA

ATGACCAAGA

CTTACTGTCC

AAAACCCAAC

CCTCATCATC

GAAATTGGTG

TGTGAAGAAA

CAAAGAGTAC

AAACAAAAAT

GAAGATATTA

ATAATCCAAG

GCAGAGCAAG

CACCACCAGC

CTACTGACTT

GAGATGGCAA

GTGGAGGAAG

CTTCACCATA

TGCTGCGTTC

AACTGACTGG

GAGTTTCACG

CTTGGCACAT

CAGAAGGTTT

CTACAGAAAA

GTTTTGATGG

GACTCATAGA

AAGTGTGGGC

ATGACATGAA

GCAAATGGCA

ATGGGAAAAT

TGCTTCCAAG

ATTGTTTATC

TGCTTCTCTT

GACCCAACCA

AATATATTAA

AAGATGTGGA

TGCTCCAGTA

TCTCTCAAAG

TAAAGAATCT

GAACTGGCAT

CTCAAGCATT

ATGAATTAGA

GCATTGTTGG

TTTATATGAA

AT GTTAAG CT

GTGTTTGGGA

AGATGTTTAA

GTCAGGTTTT

GATCTTTCAG

GTCCCTTATG

TGAAGGAGAG

TAGAAGCCTA

AAGTTTGGGT

GAGCAAGAAA

AGCCCCAGTC

CAGTGTTTCC

AGGAAAGAAG

TACAGAGAGC

AGAAGGATGT

TGATCCCCAC

AGCTGTTTTA

ACATTTAGTA

CTCCATATGG

AAAAACAAGT

AT C CCAAGAA

CAACGGAGCT

TACATCTGTA

TAAAGAAATT

TATTGGTCTC

TACCATCTAT

ACCCTTCAGC

CCAACCTACT

ATTTGTTTTT

AGCACTGCGA

AAATTGTAAT

GTTTTACATT

TGAGAATGGA

GTTAGAGGAA

AACTTTGCTA

TATTAAGCAG

GCAGGAATAC

GAGAAAGTGC

AGCCCTGGAT

AATTCCTCTA

C GAG CATTC C

TGTAGTATCG

4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 WO 97/28262 PCTfUS97/01748 I OL ACAGGTTACA AAATATTGCA GATACTTATG.TTGCCACCCA ATCAAAGAAA CAAAATTCTT 6840 TGGGGAGTTC CGACACACTG AAAAAAGGCA AAGAGGACGC ATTCATCAGT AGCTGTGAGT 6900 CTGCAAAAAC TGTTTGTGAA ATGGAAGCTG TCCTCTCAGC CCAGGTCTCT GTCAGTGATG 6960 TCCCAAAGGG AGTGCTGGGA TTTCCAGTGG TCAAAGCAGA TCATAAACAG TTGGGAGCAG 7020 AACCCAGGTC AGAAGATGAC AGTCCTGGGG ATGAGTCCTG CCCACGCCGA GCCCTATGCA 7080 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 2001 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Met Ser Thr Asp Ser Asn Ser Leu Ala Arg Glu Phe Leu Thr Asp Val 1 5 10 Asn Arg Leu Cys Asn Ala Val Val Gin Arg Val Glu Ala Arg Glu Glu 25 Glu Glu Glu Glu Thr His Met Ala Thr Leu Gly Gin Tyr Leu Val His 40 Gly Arg Gly Phe Leu Leu Leu Thr Lys Leu Asn Ser Ile Ile Asp Gin 55 Ala Leu Thr Cys Arg Glu Glu Leu Leu Thr Leu Leu Leu Ser Leu Leu 70 75 Pro Leu Val Trp Lys Ile Pro Val Gin Glu Glu Lys Ala Thr Asp Phe 90 Asn Leu Pro Leu Ser Ala Asp Ile Ile Leu Thr Lys Glu Lys Asn Ser 100 105 110 Ser Ser Gin Arg Ser Thr Gin Glu Lys Leu His Leu Glu Gly Ser Ala 115 120 125 Leu Ser Ser Gin Val Ser Ala Lys Val Asn Val Phe Arg Lys Ser Arg 130 135 140 Arg Gin Arg Lys Ile Thr His Arg Tyr Ser Val Arg Asp Ala Arg Lys 145 150 155 160 Thr Gin Leu Ser Thr Ser Asp Ser Glu Ala Asn Ser Asp Glu Lys Gly 165 170 175 Ile Ala Met Asn Lys His Arg Arg Pro His Leu Leu His His Phe Leu 180 185 190 Thr Ser Phe Pro Lys Gin Asp His Pro Lys Ala Lys Leu Asp Arg Leu 195 200 205 Ala Thr Lys Glu Gin Thr Pro Pro Asp Ala Met Ala Leu Glu Asn Ser 210 215 220 Arg Glu Ile Ile Pro Arg Gn Gly Se Asn Thr Asp Ile Leu Se Glu Arg Glu Ile Ile Pro Arg Gin Gly Ser Asn Thr Asp Ile Leu Ser Glu WO 97/28262 225 PCT/US97/01748 Pro Ala Ala Leu Ser Val Ile Ser Leu Cys Val Thr Leu Thr 290 Asn Leu 305 Ala Leu Val Asp Leu Thr Lys Gin 370 Val Gin 385 Leu Pro Gin Ser Vai Gin Val Leu 450 Aia Ser 465 Ser Thr Thr Arg His Arg Ser Lys 530 Giu Arg 545 Leu Gin His Leu 275 Glu Giu Ile Vai Glu 355 Pro Giu Gi u Ala Glu 435 Gin Giu Val1 Lys Asp 515 As n Cys Gin Vai 260 As n Phe Ser Gin Her 340 Leu Asp Asp Leu Al a 420 Phe Met His Lys Arg 500 Leu Pro Cys Aila 245 Leu Leu His Asn Leu Ala Arg Vai 310 Arg Met 325 Thr Ala Leu Arg Pro Phe Phe Val 390 Leu Glu 405 Ser Asn Ile Gin Glu Trp Leu Lys 470 Lys Vai 485 His Arg Ser Gly Phe Glu Cys Ile 550 Ser Leu 565 Ser Ser Gi y 295 Val Leu GI u Al a Al a 375 Phe Gi y Pro His Leu 455 Al a Lys Arg Leu Giu 535 Al a Ser Leu Pro 280 Phe Ser Phe Met Al a 360 Pro Ser Val Phe His 440 Val Leu Her Cys Leu 520 Th r Val Ser Asn Leu 265 Leu Gly Ala Arg Met 345 Leu Arg Lys Leu Tyr 425 Gi y Leu Ile Giu Giu 505 Val Al a Cys Thr Met 250 Giu Ala Asp Gi y Thr 330 Pro Lys Gin Tyr Gin 410 Phe Phe Arg As n Gin 490 Tyr Ser Asp Al a Cys 570 Asn Lys Al a Cys T rp 315 Val1 Gi u Ile Lys Arg 395 Ile Ser As n Asp Ser 475 Leu Her Al a Gi y His 555 Val1 As n Val1 Ser Cys 300 Thr Leu As n Arg Lys 380 His Leu Gin Leu Gi y 460 Val His His Phe Asp 540 Gin Gin Ser Cys Val 285 S er Giu His Leu Ile 365 Thr Arg Ile Al a Phe 445 Val1 Met His Phe Lys 525 Val Cys Ile Pro Lys 270 Val1 Leu Glu Leu Arg 350 Cys Leu Al a Cys Met 430 Giu Pro Lys Ser Met 510 Asn Tyr Leu Leu Phe 255 Phe Pro Ser Pro Leu 335 Lys Leu Gin Leu Cys 415 Asp Thr Pro Ile Met 495 His Gin Tyr Arg Ser 575 Asp Asp Thr Asp Val 320 Ser As n Gi u Glu Leu 400 Leu Leu Al a Glu Met 480 Cys His Val Pro Leu 560 Gi y WO 97/28262 M4 PCT/US97/01748 Val His Asn Ile Gly Ile Cys Cys Cys Met Asp Pro Lys Ser Val Ile 580 Ile Gin Gly 625 Thr Asn Arg Lys Arg 705 Lys Pro Ser Leu Leu 785 Ser Leu Asp Leu Ser 865 Ala Thr Pro His 610 Ala Val Leu Phe Trp 690 Leu Gly Val Val Pro 770 Lys Gin Lys Ala Ser 850 Asp Tyr Ile Leu 595 Ile Glu Asp Cys Gin 675 Asp His Asn Leu Thr 755 Gin Ser Ile Ala Ser 835 Ser Ser Pro Asn Leu Leu Ile Ser Asp 660 Gly Ala Ser Ile Gin 740 Ser Asp Arg Ile Phe 820 Val Val Pro Lys Leu 900 His Asn Ser Asp 645 Ala Ile Leu Ile Val 725 Arg Ala Val Val Glu 805 Glu Pro His Gin Arg 885 Phe Ala Ile Pro 630 Gin Glu Leu Lys Gin 710 Val Gly Gin Leu Ile 790 Leu Thr Asp Val Ser 870 Arg Leu Phe Leu 615 Lys Leu Leu Pro Ala 695 Ile Gin Val Ser Gin 775 Arg Asn Leu Ile Gly 855 Leu Lys Cys Lys 600 Asn Ile Ala Ser Ser 680 Tyr Ala Trp Glu His 760 Ile Asp Cys Ile Asp 840 Thr Ser Thr Val 585 Leu Pro Ala Lys Leu Ile Lys Lys Ala 635 Gin Leu Glu 650 Ser Ser Leu 665 Ser Gly Ser Gin Asn Phe Asn His Ile 715 Lys Leu Tyr 730 Leu Ala His 745 Val Cys Ser Tyr Val Lys Leu Phe Leu 795 Leu Asn Gly 810 Ile Ser Leu 825 Gly Ile Asp Ser Phe His Lys Phe Tyr 875 Val Asn Gin 890 Ala Phe Leu 905 Leu Leu 620 Ala Glu Ser Glu Val 700 Cys Asn His His Thr 780 Ser Ile Gly Ile His 860 Ala Asp Cys Lys 605 Asp Cys Thr Ser Asp 685 Phe Asn Tyr Cys His 765 Leu Cys Arg Glu Glu 845 Gin Gly Val Val 590 Asn Gin Asn Leu Pro 670 Leu Gly Leu Ile Gin 750 Asn Pro Asn Ser Gin 830 Gin Gin Leu His Ser 910 Phe Leu Ile Gin 655 Ser Leu Glu Ile Phe 735 His Gin Ile Gly His 815 Gin Lys Ala Lys Ile 895 Lys Gin Gly Cys 640 Gly Tyr Trp Asp Gin 720 Asn Leu Cys Leu Val 800 Ser Lys Glu Tyr Glu 880 Asn Glu Ala Glu Ser Asp Arg Glu Ser Ala Asn Asp Ser Glu Asp Thr Ser Gly 915 920 925 WO 97/28262 PCT/US97/01748 Tyr Asp Ser Thr Ala Ser Glu.Pro Leu Ser His Met Leu Pro Cys Ile 930 935 940 Ser Leu Glu Ser Leu Val Leu Pro Ser Pro Glu His Met His Gin Ala 945 950 955 960 Ala Asp Ile Trp Ser Met Cys Arg Trp Ile Tyr Met Leu Ser Ser Val 965 970 975 Phe Gin Lys Gin Phe Tyr Arg Leu Gly Gly Phe Arg Val Cys His Lys 980 985 990 Leu Ile Phe Met Ile Ile Gin Lys Leu Phe Arg Ser His Lys Glu Glu 995 1000 1005 Gin Gly Lys Lys Glu Gly Asp Thr Ser Val Asn Glu Asn Gin Asp Leu 1010 1015 1020 Asn Arg Ile Ser Gin Pro Lys Arg Thr Met Lys Glu Asp Leu Leu Ser 1025 1030 1035 1040 Leu Ala Ile Lys Ser Asp Pro Ile Pro Ser Glu Leu Gly Ser Leu Lys 1045 1050 1055 Lys Ser Ala Asp Ser Leu Gly Lys Leu Glu Leu Gin His Ile Ser Ser 1060 1065 1070 Ile Asn Val Glu Glu Val Ser Ala Thr Glu Ala Ala Pro Glu Glu Ala 1075 1080 1085 Lys Leu Phe Thr Ser Gin Glu Ser Glu Thr Ser Leu Gin Ser Ile Arg 1090 1095 1100 Leu Leu Glu Ala Leu Leu Ala Ile Cys Leu His Gly Ala Arg Thr Ser 1105 1110 1115 1120 Gin Gin Lys Met Glu Leu Glu Leu Pro Asn Gin Asn Leu Ser Val Glu 1125 1130 1135 Ser Ile Leu Phe Glu Met Arg Asp His Leu Ser Gin Ser Lys Val Ile 1140 1145 1150 Glu Thr Gin Leu Ala Lys Pro Leu Phe Asp Ala Leu Leu Arg Val Ala 1155 1160 1165 Leu Gly Asn Tyr Ser Ala Asp Phe Glu His Asn Asp Ala Met Thr Glu 1170 1175 1180 Lys Ser His Gin Ser Ala Glu Glu Leu Ser Ser Gin Pro Gly Asp Phe 1185 1190 1195 1200 Ser Glu Glu Ala Glu Asp Ser Gin Cys Cys Ser Phe Lys Leu Leu Val 1205 1210 1215 Glu Glu Glu Gly Tyr Glu Ala Asp Ser Glu Ser Asn Pro Glu Asp Gly 1220 1225 1230 Glu Thr Gin Asp Asp Gly Val Asp Leu Lys Ser Glu Thr Glu Gly Phe 1235 1240 1245 Ser Ala Ser Ser Ser Pro Asn Asp Leu Leu Glu Asn Leu Thr Gin Gly 1250 1255 1260 Glu Ile Ile Tyr Pro Glu Ile Cys Met Leu Glu Leu Asn Leu Leu Ser WO 97/28262 PCT/US97/01748 1265 1270 1275 1280 Ala Ser Lys Ala Lys Leu Asp Val Leu Ala His Val Phe Glu Ser Phe 1285 1290 1295 Leu Lys Ile Ile Arg Gin Lys Glu Lys Asn Val Phe Leu Leu Met Gin 1300 1305 1310 Gin Gly Thr Val Lys Asn Leu Leu Gly Gly Phe Leu Ser Ile Leu Thr 1315 1320 1325 Gin Asp Asp Ser Asp Phe Gin Ala Cys Gin Arg Val Leu Val Asp Leu 1330 1335 1340 Leu Val Ser Leu Met Ser Ser Arg Thr Cys Ser Glu Glu Leu Thr Leu 1345 1350 1355 1360 Leu Leu Arg Ile Phe Leu Glu Lys Ser Pro Cys Thr Lys Ile Leu Leu 1365 1370 1375 Leu Gly Ile Leu Lys Ile Ile Glu Ser Asp Thr Thr Met Ser Pro Ser 1380 1385 1390 Gin Tyr Leu Thr Phe Pro Leu Leu His Ala Pro Asn Leu Ser Asn Gly 1395 1400 1405 Val Ser Ser Gin Lys Tyr Pro Gly Ile Leu Asn Ser Lys Ala Met Gly 1410 1415 1420 Leu Leu Arg Arg Ala Arg Val Ser Arg Ser Lys Lys Glu Ala Asp Arg 1425 1430 1435 1440 Glu Ser Phe Pro His Arg Leu Leu Ser Ser Trp His Ile Ala Pro Val 1445 1450 1455 His Leu Pro Leu Leu Gly Gin Asn Cys Trp Pro His Leu Ser Glu Gly 1460 1465 1470 Phe Ser Val Ser Leu Trp Phe Asn Val Glu Cys Ile His Glu Ala Glu 1475 1480 1485 Ser Thr Thr Glu Lys Gly Lys Lys Ile Lys Lys Arg Asn Lys Ser Leu 1490 1495 1500 Ile Leu Pro Asp Ser Ser Phe Asp Gly Thr Glu Ser Asp Arg Pro Glu 1505 1510 1515 1520 Gly Ala Glu Tyr Ile Asn Pro Gly Glu Arg Leu Ile Glu Glu Gly Cys 1525 1530 1535 Ile His Ile Ile Ser Leu Gly Ser Lys Ala Leu Met Ile Gin Val Trp 1540 1545 1550 Ala Asp Pro His Asn Ala Thr Leu Ile Phe Arg Val Cys Met Asp Ser 1555 1560 1565 Asn Asp Asp Met Lys Ala Val Leu Leu Ala Gin Val Glu Ser Gin Glu 1570 1575 1580 Asn Ile Phe Leu Pro Ser Lys Trp Gin His Leu Val Leu Thr Tyr Leu 1585 1590 1595 1600 Gin Gin Pro Gin Gly Lys Arg Arg Ile His Gly Lys Ile Ser Ile Trp 1605 1i r i c IV V 15L WO 97/28262 PCT/US97/01748 1167 Val Ser Gly Gin Arg Lys Pro Asp Val Thr Leu Asp Phe Met Leu Pro 1620 1625 1630 Arg Lys Thr Ser Leu Ser Ser Asp Ser Asn Lys Thr Phe Cys Met Ile 1635 1640 1645 Gly His Cys Leu Ser Ser Gin Glu Glu Phe Leu Gin Leu Ala Gly Lys 1650 1655 1660 Trp Asp Leu Gly Asn Leu Leu Leu Phe Asn Gly Ala Lys Val Gly Ser 1665 1670 1675 1680 Gin Glu Ala Phe Tyr Leu Tyr Ala Cys Gly Pro Asn His Thr Ser Val 1685 1690 1695 Met Pro Cys Lys Tyr Gly Lys Pro Val Asn Asp Tyr Ser Lys Tyr Ile 1700 1705 1710 Asn Lys Glu Ile Leu Arg Cys Glu Gin Ile Arg Glu Phe Phe Met Thr 1715 1720 1725 Lys Lys Asp Val Asp Ile Gly Leu Leu Ile Gly Val Phe Gin Leu Phe 1730 1735 1740 Ile Gln Leu Thr Val Leu Leu Gin Tyr Thr Ile Tyr Glu Pro Val Ile 1745 1750 1755 1760 Arg Leu Lys Gly Gin Met Lys Thr Gin Leu Ser Gin Arg Pro Phe Ser 1765 1770 1775 Ser Lys Glu Val Gin Ser Ile Leu Leu Glu Pro His His Leu Lys Asn 1780 1785 1790 Leu Gin Pro Thr Glu Tyr Lys Thr Ile Gin Gly Ile Leu His Glu Ile 1795 1800 1805 Gly Gly Thr Gly Ile Phe Val Phe Leu Phe Ala Arg Val Val Glu Leu 1810 1815 1820 Ser Ser Cys Glu Glu Thr Gin Ala Leu Ala Leu Arg Val Ile Leu Ser 1825 1830 1835 1840 Leu Ile Lys Tyr Asn Gin Gin Arg Val His Glu Leu Glu Asn Cys Asn 1845 1850 1855 Gly Leu Ser Met Ile His Gin Val Leu Ile Lys Gin Lys Cys Ile Val 1860 1865 1870 Gly Phe Tyr Ile Leu Lys Thr Leu Leu Glu Gly Cys Cys Gly Glu Asp 1875 1880 1885 Ile Ile Tyr Met Asn Glu Asn Gly Glu Phe Lys Leu Asp Val Asp Ser 1890 1895 1900 *Asn Ala Ile Ile Gin Asp Val Lys Leu Leu Glu Glu Leu Leu Leu Asp 1905 1910 1915 1920 Trp Lys Ile Trp Ser Lys Ala Glu Gin Gly Val Trp Glu Thr Leu Leu 1925 1930 1935 Ala Ala Leu Glu Val Leu Ile Arg Ala Asp His His Gin Gin Met Phe 1940 1945 1950 Asn Ile Lys Gin Leu Leu Lys Ala Gin Val Val His His Phe Leu Leu 1955 1960 1965 WO 97/28262 PCT[US97/01748 Thr Cys Gin Val Leu Gin Glu .Tyr Lys Glu Gly Gin Leu Thr Pro Met 1970 1975 1980 Pro Arg Glu Met Ala Arg Ser Phe Arg Arg Lys Cys Gly Gin Ser Cys 1985 1990 1995 2000 Thr INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 5221 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CGCAAGGGCT TCTAAGAAGC CATCCCAATG ACCTTTTGGC TTTGAGAAGA GCAGTCCTCA

TACCAGAGTG

TGTATTCCGA

TCCTCGGGTG

GTTTGACTCA

GAACAGAAGC

GAACCAGTTT

TCCTTGCTCA

GTTTCATCTA

GACTGCTCGG

AACCGGCTTT

ACGCACATGG

AAGCTAAATT

CTGTCTCTCC

AACCTACCGC

TCCACTCAGG

GTAAATGTTT

GATGCAAGAA

ATAGCAATGA

AAACAAGACC

GATCCTATGG

ATTTTAAGTG

TTTGGGGTTT

GGTTTCTGTG

ACATCAGAGT

ACCGTGTGTG

CAGGATCTAA

CCTTGCAGCT

TCTAAAAGAG

GTTTATGAGT

TCATGAGCAC

GCAATGCAGT

CAACCCTTGG

CTATAATT GA

TTCCACTGGT

TCTCAGCAGA

AAAAATTACA

TTCGAAAAAG

AGACACAGCT

ATAAGCATAG

AC CCCAAAGC

CTTTGGAAAA

AGCCAGCTGC

TGGCCTCTTT

GTGCAT GAAG

ACAGCAGTAT

TTCCTCAATG

CTGCAAACAA

ACCTGGGAAG

GCAATCACCG

CCAAAT GATA

CGACAGTAAC

GGTCCAGAGG

ACAGTACCTT

TCAGGCATTG

ATGGAAGATA

TATAAT CCTG

TTTAGAAGGA

CAGACGACAG

CTCCACCTCA

AAGGCCCCAT

TAAACTTGAC

TTCCAGAGAG

CTTGTCTGTT

CAGTGTTTAT

CTTTTGCCTT

TTTCCCTTGC

CCAGGGGAAT

GAGACCCAGC

ACGGTTGCTA

TTCAGGTGAA

TAGACTGTAA

TCACTGGCAC

GTGGAGGCCA

GTCCATGGTC

ACATGTAGAG

CCTGTCCAAG

ACCAAAGAAA

AGTGCCCTGT

CGTAAAATTA

GATTCAGAAG

CTGCTGCATC

CGCTTAGCAA

ATTATTCCAA

ATCAGTAACA

TCATT CTTAC CACCAT CTGT CAT CTAAT GG PAT CCTACCC

TTGCTTAACA

ATTAGCCTGC

GCTTTGTTCT

ATGTCACAGC

GTGAATTTCT

GGGAGGAAGA

GAGGATTTCT

AAGAACTCCT

AAGAAAAGGC

AGAACTCAAG

CTAGTCAGGT

CCCATCGCTA

CCAATT CAGA

ATTTTTTAAC

CCAAAGAACA

GACAGGGGTC

TGAACAATTC

GTGGGAAAGT

TCCCGTGTCT

GGTTTGGGCT

TAAGTCAGCT

GCATGGAAGA

AACAAAGAGT

AAGAATATTT

AGTGGTGAAA

GACCGATGTC

AGAGGAGGAG

ATTACTTACC

GACTCTTCTT

AACAGATTTT

TTCACAAAGA

TTCTGCAAAA

TTCTGTAAGA

TGAAAAAGGC

ATCGTTTCCT

GACTCCTCCA

AAACACTGAC

TCCATTTGAC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 WO 97/28262 PCTIUS97/01748

TTATGTCATG-

CATAATT CT C

GGGGACTGCT

GAAGAACCGG

GTAGATGTTA

CTTAGAGCAG

AGACAAAAGA

AGAGCCCTTC

CAGAGTGCAG

ATTCAGCATC

TTAAGAGATG

ATGAAAATAA

ACAAGAAAAA

TCAGGTCTTC

GCAGATGGAG

TGCTTGCGCT

GTTCATAACA

CATGCTTTTA

AAACTTATTT

TGTAATATTT

AACTTATGTG

AT CCTGCC CA

CAGAACTTTG

AATTTAATCC

CCTGTGCTCC

GCTCAAAGTC

TATGTAAAAA

TGTAATGGAG

CTAAAAGCAT

CCAGATATTG

TCTTTTCATC

GGCCTCAAAG

ACAATAAACC

TTTTGTTATC

CTTTAGCAGC

GCAGTCTGAG

TGGCTTTGAT

*GTACTGCAGA

CTTTAAAAAT

AAACACTGCA

TTTTACCTGA

CTTCAAATCC

ATGGATTTAA

GAGTTCCTCC

TGAGCACTGT

GGCACAGACG

TGGTTTCGGC

ATGTTTATTA

TACT GCAGCA

TTGGAATATG

AATTGCCAGC

TGGATCAGTT

GTACTGTTGA

ATGCTGAACT

GCAGTGGATC

TTTTTGGAGA

AGAAAGGCAA

AAAGAGGAGT

ATGTATGTAG

CTCTGCCTAT

TAAGTCAAAT

TTGAAACTCT

ATGGGATAGA

ATCAGCAAGC

AAGCTTAT CC TATTCCT CTG

TTTATTAGAA

CAGTGTAGTG

CGACAACTTG

TCAAAGGATG

GATGATGCCA

TAGAATATGC

GGAGGTTCAG

GCTTTTGGAA

CTTCTACTTC

TTTATTTGAA

C GAGGC CTCA

CAAAAAAGTG

ATGTGAATAT

TTTTAAAAAC

TCCTGAGCGG

GGCTTCCTTG

CTGTTCTATG

ACT GAAAAAT

AGGAGGAGCA

CTCTGACCAA

CTCCTCAAGT

TGAAGATTTG

AGACAGATTA

TATAGTTGTT

TGAATTAGCA

CCATCATAAC

CCTGCTTAAA

AATCGAATTA

GATAATCAGC

CATTGAACAG

TTATTCAGAT

AAAGAGACGG

TGTGGCTTTT

AAAGTTTGTA

CCCACACTAA

GAGAGTCGAG

CTCTTT CGAA GAAAAT CTTA

CTAGAAAAGC

GAAGATTTTG

GGAGTTCTTC

ACTCAAGCCA

ACAGCAGTTC

GAGCATTTGA

AAATCAGAGC

TCTCATTTTA

CAGGTTT CCA

TGCTGTTGCA

AGCAGCACTT

GATCCCAAAT

TTTCAGCAGC

GAGATATCAC

CTAGCCCAAT

TTATCCAGTC

TTGTGGAAAT

CATAGTATAC

CAGTGGAAAT

CATCATTGTC

CAGTGCTTGC

TCCAGGGTAA

AATTGCTTAA

CTAGGGGAGC

AAGGAGTTGT

TCTCCTCAGA

AAGACTGTTA

TTATGCGTAL

AGTTTGACGT

CTGAATTCCT

TAGTTTCTGC

CAGTGTTGCA

GGAAAAATTT

AG CCT GAGCC

TGTTTTCAAA

AGATTCTGAT

TGGATTTGGT

TTCAAATGGA

AAGCCCTAAT

AACTTCATCA

TGCATCATCA

AAAACCCATT

TTGCAGTGTG

GTGTCCAGAT

CTGTAATCAT

ATATATTGAA

CAAAAATTAA

TAGAAGAGAC

CTT CTTACAG

GGGATGCTTT

AGATTGCAAA

TATATAATTA

AACACCTAAG

CTCAGGACGT

TAAGAGATTT

ATGGTATTCG

AACAGAAAGA

CCTCTGTACA

GTCTCAGCAA

ACCAAGATGT

GTAAAGAAGC

TACCTTGAAT

AGCAGGCTTT

AGGTTGGACC

TCTTCTGTCA

AACTGAATT G

TTTTGCACCA

GTATCGTCAT

CTGTTGTCTT

TCAAGAATT C ATGC T GGTT

AAATAGTGTG

TTCGATGTGT

CCGAGATCTC

TGAAGAGACT

TGCCCAT GAG

CCTATCGGGT

TCCTTTGCTC

TATCCTTAAC

AAAAGCAGCT

ACTGCAGGGA

ATTT CAAGGG

AAAGGCTTAT

TCACATTTGC

CATATTTAAT

CGTTACTTCA

GCTTCAGATT

GTTTTTGAGT

AAGTCATTCT

TGCCTCAGTT

TGTGGGTACT

ATTTTATGCT

TCATATCAAC

AGAGTCTGAC

1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 WO 97/28262 PTU9/14 PCTIUS97/01748 AGAAGATACT -TCTGGCTATG AGGGAGTCGG CCAATGACTC ACAGCACAGC CAGCGAGCCT

TTAAGTCATA

ATGCACCAAG

TTCCAGAAAC

ATAATACAGA

AGTGTAAATG

GATTTATTAT

AAGAGTGCTG

GAAGTTTCAG

GAGACCTCAC

GCCAGAACTA

AGTATATTAT

GCAAAGCCTT

GAACATAAT C

CCTGGTGATT

GAAGAAGAAG

GATGGGGTAG

TTACTCGAAA

AATTT GCTTT

TTGAAAATTA

AAAAATCTTT

TGCCAGAGAG

GAGCTAACCC

CTGGGTATTC

TTCCCTTTAC

ATTTTAA.ACA

GAGGCTGATA

CACCTGCCGT

CTGTGGTTTA

ATAAAGAAAA

ATGACAGGAT

CGTAAAAAT G

TGCTGCCATG

CAGCAGACAT

AGTTTTATAG

AACTGTTCAG

AAAACCAGGA

CTTTGGCTAT

ACAGTTTAGG

CTACTGAAGC'

TT CAAAGTAT

GTCAACAGAA

TTGAAATGAG

TATTTGATGC

ATGCTATGAC

TTTCAGAAGA

GTTACGAAGC

ACT TAAAGT C

ACCTCACTCA

CTGCTAGTAA

TTAGGCAGAA

TAGGAGGGTT

TATTGGTGGA

TTCTTTTGAG

TGAAAATTAT

TGCACGCT CC

GTAAGGCCAT

GAGAGAGTTT

TGCTGGGGCA

ATGTGGAGTG

GAAACAAATC

TATCTGATTT

TGGTAGTGTT

TATATCT CTC

TTGGTCTATG

GCTTGGTGGT

AAGTCACAAA

TTTAAACAGA

AAAAAGTGAC

TAAATTAGAG

CGCTCCC GAG

ACGACTTTTG

GATGGAATTG

GGACCATCTT

C CT GCTT CGA

TGAGAAGAGT

AGCTGAGGAT

AGATAGTGAA

TGAAACAGAA

AGGGGAAATA

AGCCAAACTT

AGAAAAGAAT

CTTGAGTATT

TCTTTTGGTA

AATATTTCTG

TGAAAGTGAT

AAATTTAAGC

GGGTTTATTG

TCCCCATCGG

AAACTGCTGG

TAT CCATGAA

ATTAATTTTA

GTACACAAAG

CTTAACACTC

GAGAGCCTTG

TGTCGTTGGA

TTCCGAGTAT

GAGGAGCAAG

ATTTCTCAAC

CCCATAC CAT

TTACAGCATA

GAAGCAAAGC

GAAGCCCTTC

GAGTTACCTA

TCCCAGTCAA

GTTGCCCTCG

CATCAATCTG

TCTCAGTGTT

AGCAATCCTG

GGTTTCAGTG

ATTTAT CCT C

GATGTGCTTG

GTTTTTCTGC

TTAACACAGG

TCTTTGATGA

GAGAAAT CT C

ACTACTATGA

AACGGTGTTT

AGAAGAGCAC

CTG CTTT CAT

CCACACCTAT

GCT GAGAGTA

CCAGATAGCA

ATTGTTTTCA

TTAACATGTT

TCTTGCCTTC

TCTACATGTT

GCCATAAGTT

GAAAAAAGGA

CTAAGAGAAC

CAGAACTAGG

TTTCTTCCAT

TATTTACAAG

TGGCCATTTG

AT CAGAACTT

AGGTGATTGA

GGAATTATTC

CAGAAGAATT

GTAGTTTTAA

AAGATGGCGA

CATCAAGCAG

AGATTTGTAT

CCCATGTATT

TCATGCAACA

ATCAT TCT GA

GTTCAAGAAC

CTTGTACAAA

GCCCTTCACA

CATCACAAAA

GAGTTTCACG

CTTGGCACAT

CAGAAGGTTT

CTACAGAAAA

GTTTT GATGG

GACTATAATT

ATTCACCTTA

TCCTGAACAT

GAGTTCAGTG

AATATTTATG

GGGAGATAGA

TAT GAAGGAA

TAGTCTAAAA

AAATGTGGAA

TCAAGAAAGT

TCTTCATGGT

GTCTGTGGAA

AACACAACTA

AGCAGATTTT

GT CAT CC CAG

ACTTTTAGTT

AACCCAGGAT

TCCAAATGAC

GCTGGAATTA

TGAGAGTTTT

GGGAACTGT G

TTTTCAAGCA

ATGTTCAGAA

AATTCTTCTT

GTATCTAACC

GTATCCTGGG

GAG CAAGAAA

AGCCCCAGTC

CAGTGTTTCC

AGGAAAGAAG

TACAGGTATG

TTCCTTGAGC

AAGATCCTAC

3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 WO 97/28262 PCTJUS97/01748 5221 11 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 1531 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Ser Thr Asp Ser Asri Ser Leu Ala Arg Glu Phe Leu Thr Asp Val Asn Giu Gi y Al a Pro As n Ser Leu Arg 145 Thr Ile Thr Al a Arg 225 Pro Leu Leu Glu Gi y Th r Val Pro Gin 115 Ser Arg Leu Met Phe 195 Lys Ile Al a His As n Thr Leu Arg Lys Ser Val Ile Thr 165 Lys Lys Gin Pro Ser 245 Leu Al a His Leu Giu 70 Ile Al a Thr Ser Thr 150 Ser His Gin Thr Arg 230 Val Leu Val Met Leu Giu Pro Asp Gin Al a 135 His Asp Arg Asp Pro 215 Gin Ile Ser Vai Al a 40 Thr Leu Val Ile Gi u 120 Lys Arg Ser Arg His 200 Pro Gi y Se r Leu Gin Thr Lys Leu Gin Ile 105 Lys Val Tyr Glu Pro 185 Pro Asp Ser As n Leu Arg Leu Leu Th r Glu Leu Leu As n Ser Al a 170 His Lys Al a As n Met 250 Glu Val1 Gly As n Leu dl u Thr His Val Val 155 As n Leu Al a Met Thr 235 As n Lys dl u Gin Ser Leu Lys Lys Leu Phe 140 Arg Ser Leu Lys Al a 220 Asp As n Val Al a Tyr Ile Leu Al a Gi u Gi u 125 Arg Asp Asp His Leu 205 Leu Ile Se r Cys Arg Leu Ile Ser Thr Lys 110 Gi y Lys Al a Gi u His 190 Asp Giu Leu Pro Lys 270 di u Val1 Asp Leu Asp Asn Ser Ser Arg Lys 175 Phe Arg As n Ser Phe 255 Phe 260 265 Val Thr Leu Asn His Asn Ser Pro Leu Ala Ala Ser Vai Val Pro Thr WO 97/28262 PCTIUS97/01748 275 280 285 Leu Thr Glu Phe Leu Ala Gly Phe Gly Asp Cys Cys Ser Leu Ser Asp 290 295 300 Asn Leu Glu Ser Arg Val Val Ser Ala Gly Trp Thr Glu Glu Pro Val 305 310 315 320 Ala Leu Ile Gin Arg Met Leu Phe Arg Thr Val Leu His Leu Leu Ser 325 330 335 Val Asp Val Ser Thr Ala Glu Met Met Pro Glu Asn Leu Arg Lys Asn 340 345 350 Leu Thr Glu Leu Leu Arg Ala Ala Leu Lys Ile Arg Ile Cys Leu Glu 355 360 365 Lys Gin Pro Asp Pro Phe Ala Pro Arg Gin Lys Lys Thr Leu Gin Glu 370 375 380 Val Gin Glu Asp Phe Val Phe Ser Lys Tyr Arg His Arg Ala Leu Leu 385 390 395 400 Leu Pro Glu Leu Leu Glu Gly Val Leu Gin Ile Leu Ile Cys Cys Leu 405 410 415 Gin Ser Ala Ala Ser Asn Pro Phe Tyr Phe Ser Gin Ala Met Asp Leu 420 425 430 Val Gin Glu Phe Ile Gin His His Gly Phe Asn Leu Phe Glu Thr Ala 435 440 445 Val Leu Gin Met Glu Trp Leu Val Leu Arg Asp Gly Val Pro Pro Glu 450 455 460 Ala Ser Glu His Leu Lys Ala Leu Ile Asn Ser Val Met Lys Ile Met 465 470 475 480 Ser Thr Val Lys Lys Val Lys Ser Glu Gin Leu His His Ser Met Cys 485 490 495 Thr Arg Lys Arg His Arg Arg Cys Glu Tyr Ser His Phe Met His His 500 505 510 His Arg Asp Leu Ser Gly Leu Leu Val Ser Ala Phe Lys Asn Gin Val 515 520 525 Ser Lys Asn Pro Phe Glu Glu Thr Ala Asp Gly Asp Val Tyr Tyr Pro 530 535 540 Glu Arg Cys Cys Cys Ile Ala Val Cys Ala His Gin Cys Leu Arg Leu 545 550 555 560 Leu Gin Gin Ala Ser Leu Ser Ser Thr Cys Val Gin Ile Leu Ser Gly 565 570 575 Val His Asn Ile Gly Ile Cys Cys Cys Met Asp Pro Lys Ser Val Ile 580 585 590 Ile Pro Leu Leu His Ala Phe Lys Leu Pro Ala Leu Lys Asn Phe Gin 595 600 605 Gin His Ile Leu Asn Ile Leu Asn Lys Leu Ile Leu Asp Gin Leu Gly 610 615 620 WO 97/28262 PCT/US97/01748 /73 Gly Ala Glu Ile Ser Pro Lys Ile Lys Lys Ala Ala Cys Asn Ile Cys 625 630 635 640 Thr Val Asp Ser Asp Gin Leu Ala Gin Leu Glu Glu Thr Leu Gin Gly 645 650 655 Asn Leu Cys Asp Ala Glu Leu Ser Ser Ser Leu Ser Ser Pro Ser Tyr 660 665 670 Arg Phe Gin Gly Ile Leu Pro Ser Ser Gly Ser Glu Asp Leu Leu Trp 675 680 685 Lys Trp Asp Ala Leu Lys Ala Tyr Gin Asn Phe Val Phe Gly Glu Asp 690 695 700 Arg Leu His Ser Ile Gin Ile Ala Asn His Ile Cys Asn Leu Ile Gin 705 710 715 720 Lys Gly Asn Ile Val Val Gin Trp Lys Leu Tyr Asn Tyr Ile Phe Asn 725 730 735 Pro Val Leu Gin Arg Gly Val Glu Leu Ala His His Cys Gin His Leu 740 745 750 Ser Val Thr Ser Ala Gin Ser His Val Cys Ser His His Asn Gin Cys 755 760 765 Leu Pro Gin Asp Val Leu Gin Ile Tyr Val Lys Thr Leu Pro Ile Leu 770 775 780 Leu Lys Ser Arg Val Ile Arg Asp Leu Phe Leu Ser Cys Asn Gly Val 785 790 795 800 Ser Gin Ile Ile Glu Leu Asn Cys Leu Asn Gly Ile Arg Ser His Ser 805 810 815 Leu Lys Ala Phe Glu Thr Leu Ile Ile Ser Leu Gly Glu Gin Gin Lys 820 825 830 Asp Ala Ser Val Pro Asp Ile Asp Gly Ile Asp Ile Glu Gin Lys Glu 835 840 845 Leu Ser Ser Val His Val Gly Thr Ser Phe His His Gin Gin Ala Tyr 850 855 860 Ser Asp Ser Pro Gin Ser Leu Ser Lys Phe Tyr Ala Gly Leu Lys Glu 865 870 875 880 Ala Tyr Pro Lys Arg Arg Lys Thr Val Asn Gin Asp Val His Ile Asn 885 890 895 Thr Ile Asn Leu Phe Leu Cys Val Ala Phe Leu Cys Val Ser Lys Glu 900 905 910 Ala Glu Ser Asp Arg Glu Ser Ala Asn Asp Ser Glu Asp Thr Ser Gly 915 920 925 Tyr Asp Ser Thr Ala Ser Glu Pro Leu Ser His Met Leu Pro Cys Ile 930 935 940 Ser Leu Glu Ser Leu Val Leu Pro Ser Pro Glu His Met His Gin Ala 945 950 955 960 Ala Asp Ile Trp Ser Met Cys Arg Trp Ile Tyr Met Leu Ser Ser Val 965 970 975 WO 97/28262 PCT/US97/01748 Phe Gin Lys Gin Phe Tyr Arg.Leu Gly Gly Phe Arg Val Cys His Lys 980 985 990 Leu Ile Phe Met Ile Ile Gin Lys Leu Phe Arg Ser His Lys Glu Glu 995 1000 1005 Gin Gly Lys Lys Glu Gly Asp Thr Ser Val Asn Glu Asn Gin Asp Leu 1010 1015 1020 Asn Arg Ile Ser Gin Pro Lys Arg Thr Met Lys Glu Asp Leu Leu Ser 1025 1030 1035 1040 Leu Ala Ile Lys Ser Asp Pro Ile Pro Ser Glu Leu Gly Ser Leu Lys 1045 1050 1055 Lys Ser Ala Asp Ser Leu Gly Lys Leu Glu Leu Gin His Ile Ser Ser 1060 1065 1070 Ile Asn Val Glu Glu Val Ser Ala Thr Glu Ala Ala Pro Glu Glu Ala 1075 1080 1085 Lys Leu Phe Thr Ser Gin Glu Ser Glu Thr Ser Leu Gin Ser Ile Arg 1090 1095 1100 Leu Leu Glu Ala Leu Leu Ala Ile Cys Leu His Gly Ala Arg Thr Ser 1105 1110 1115 1120 Gin Gin Lys Met Glu Leu Glu Leu Pro Asn Gin Asn Leu Ser Val Glu 1125 1130 1135 Ser Ile Leu Phe Glu Met Arg Asp His Leu Ser Gin Ser Lys Val Ile 1140 1145 1150 Glu Thr Gin Leu Ala Lys Pro Leu Phe Asp Ala Leu Leu Arg Val Ala 1155 1160 1165 Leu Gly Asn Tyr Ser Ala Asp Phe Glu His Asn Asp Ala Met Thr Glu 1170 1175 1180 Lys Ser His Gin Ser Ala Glu Glu Leu Ser Ser Gin Pro Gly Asp Phe 1185 1190 1195 1200 Ser Glu Glu Ala Glu Asp Ser Gin Cys Cys Ser Phe Lys Leu Leu Val 1205 1210 1215 Glu Glu Glu Gly Tyr Glu Ala Asp Ser Glu Set Asn Pro Glu Asp Gly 1220 1225 1230 Glu Thr Gin Asp Asp Gly Val Asp Leu Lys Ser Glu Thr Glu Gly Phe 1235 1240 1245 Ser Ala Ser Ser Ser Pro Asn Asp Leu Leu Glu Asn Leu Thr Gin Gly 1250 1255 1260 Glu Ile Ile Tyr Pro Glu Ile Cys Met Leu Glu Leu Asn Leu Leu Ser 1265 1270 1275 1280 Ala Ser Lys Ala Lys Leu Asp Val Leu Ala His Val Phe Glu Ser Phe 1285 1290 1295 Leu Lys Ile Ile Arg Gin Lys Glu Lys Asn Val Phe Leu Leu Met Gin 1300 1305 1310 Gin Gly Thr Val Lys Asn Leu Leu Gly Gly Phe Leu Ser Ile Leu Thr WO 97/28262 PCTIUS97/01748 1315 1320 1325 Gin Asp Asp Ser Asp Phe Gin Ala Cys Gin Arg Val Leu Val Asp Leu 1330 1335 1340 Leu Val Ser Leu Met Ser Ser Arg Thr Cys Ser Glu Glu Leu Thr Leu 1345 1350 1355 1360 Leu Leu Arg Ile Phe Leu Glu Lys Ser Pro Cys Thr Lys Ile Leu Leu 1365 1370 1375 Leu Gly Ile Leu Lys Ile Ile Glu Ser Asp Thr Thr Met Ser Pro Ser 1380 1385 1390 Gin Tyr Leu Thr Phe Pro Leu Leu His Ala Pro Asn Leu Ser Asn Gly 1395 1400 1405 Val Ser Ser Gin Lys Tyr Pro Gly Ile Leu Asn Ser Lys Ala Met Gly 1410 1415 1420 Leu Leu Arg Arg Ala Arg Val Ser Arg Ser Lys Lys Glu Ala Asp Arg 1425 1430 1435 1440 Glu Ser Phe Pro His Arg Leu Leu Ser Ser Trp His Ile Ala Pro Val 1445 1450 1455 His Leu Pro Leu Leu Gly Gin Asn Cys Trp Pro His Leu Ser Glu Gly 1460 1465 1470 Phe Ser Val Ser Leu Trp Phe Asn Val Glu Cys Ile His Glu Ala Glu 1475 1480 1485 Ser Thr Thr Glu Lys Gly Lys Lys Ile Lys Lys Arg Asn Lys Ser Leu 1490 1495 1500 Ile Leu Pro Asp Ser Ser Phe Asp Gly Thr Gly Met Met Thr Gly Leu 1505 1510 1515 1520 Ser Asp Leu Tyr Thr Lys Ile Val Phe Arg Leu 1525 1530 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 1979 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ATACTTCTGA TGTAAAGGAA CTAATTCCAG AGTTCTACTA CCTACCAGAG ACAGTAATGG ATATAATCTT GGAGTCAGAG AAGATGAAGT AGTGGTAAAT TTCCCCCTTG GGCAAAAAAA CCTGAAGACT TTGTGCGGAT CAACAGGATG GTGAATTTGT TTCTTGCCAA CTTCATCAGT GGATCGACCT TATATTTGGC GAGGACCAGA AGCAGTTCGT GCTCTGAATG TTTTTCACTA CTTGACTTAT TGAACCTGGA TAGTATCACT GATCCTGTGC TCAGGGAGGC CATGGAGGCA ACTTTGGACA GACGCCATCT CAGTTGCTTA TTGAGCCACA TCCGCCTCGG

ATGTTTGTCA

GATGTTGATC

GCCCTAGAAA

TATAAGCAGC

GAAGGCTCTG

CAGATACAGA

AACTCTGCCA

WO 97/28262 WO 9728262PCT[US97/01748

TGCACCTGTG

TGATAATGGT

TGCCCCACTT

ATAGATGGCA

ACCATCTTCC

AGATCACAGA

CAGATAATCG

CAGAAACAGG

CCAGGTCCGA

CCCTGCTGCT

GTGACTATCC

CTGTCTGTGC

ACAC CAT CAC

GCTTGATATC

GTAATTTCAG

C CAT TCTC CT

AGGTCTGGCA

ATTAGAGCAA

AGCATTGTAG

TGAAGATAAA

AGGCAATATC

CACCCAGCAA

ACTGAACACC

GTTTTGGTAA

AGAGACTTTT

ACCTAACCTG

T TTCC TT CCA

GCTGAAGTTT

GAC CAT CC CC

CAACACAGTA

CATTGAAATG

CCTCGTTGAC

CTATATTCTT

GAAATTGACT

GTCATACATT

CTGGTACTGG

GGCACCAAGA

AGAACTTGGG

TGGAGATTTG

TGTCTCCAGC

CATTAATGGG

GAGCAGTGAC

GGCCTGTGAC

TGGACTTGTC

CTTTTAATAT

GGAACAACCA

TCTGGTGGAA

TAGCTGTACA

AGCTGCTATC

TTATTTCATA

GACAATT CTG CAT CCCATTT

CAGAGTCCGC

CCTTCAAATT

GCAGTGGTGA

GGCCTCAGAG

GATCCATTAA

CAGAGTATAC

ATCTGTGGAT

CAGATTGTAT

GGTGGGGACT

AGTGGGCGGC

GCCGTCCTCA

CTTGTTATCA

CT GAGAG CC C

GAAGGCCACT

AAACTTTTGG

GGCCAGAACC

TTCAAGCAAC

CCATGACCAG

AGATTTTAAT

AAAGCCAAGT

AAAATTCGTC

TTGTAGTCAG

AAGCAAGCTT

TATTGTTGTT

AGGAACCTTG

CCAGCCTCTT

-TCATGTTTAA

CTCCAGTAAC

CAGTGACTTG

GAGCTCCAGG

TAGCCAATAA

AAATCAATGC

TCTGGGATAA

TTGGCCATTG

GCTACATCGT

ACCATAT CAT

CAGGCCATGA

GTGGTGCTAA

TTGAAGGACC

GTATCATATA

CTCAAATGGA

TGGTCACCGG

TGTACATTTA

AGGACT CTGA

CGGTGGCATT

TAAAGCTGAG

TACATCGACC

CAACCATTTT

ATATCATGTA

TATTGAGAAA

TGTCCAGTTG

TT CAAGCTGA AGAT CAGATG C CATCT GG CA

CAGCCGACTC

ATACTCCTTG

TTCAGGTGTA

ACATTGTTTT

GAGCTT CAGA

GGATGTGGTC

GTCCGGATCT

AGGAGACAAC

CCATGAAGTT

AGAGGGCCCT

AGAAAACTGC

CTATGAACGA

GATCAATGAT

AGGGGACAAT

ACCCTGGATG

TCACTGGCAT

ATGAGCATCA

GGCACAAGTG

TCCGTTTGTA

ACTTTGTGTG

AATTATATGA

AGGTTGTAGG

TTACAAAGTT

CAACAGGATG

GCCAACACTC

TTTGCAGTGA

GATCAAGCCC

AACAAACGGC

GTGGTAACAG

GTTTATACTA

ACTTGCTTGG

CGAGATGCCA

CCTAACAGCA

GTCTGTGTTT

TGCCTTGTCC

TTATTCCCAC

GGGCGATTCA

TCAACACGGG

GGGGTAGTAG

TGATGCTGGC

GGCTTCTGGT

GAACAGATAC

CTGCATGGAA

CATTC CAT CA

TTTTTTCACG

ATTAGGAGAT

ATGTGTCACA

TAAGCTTTGA

480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1979 GAAAAAAAAA AAAAAAAAA INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 472 amino acids TYPE: amino acid

STP.ANDEDNESS:

TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Thr Ser Asp Val Lys Glu Leu Ile Pro Glu Phe Tyr Tyr Leu Pro Glu WO 97/28262 PCT/US97/01748 tri Met Phe Val Asn Ser Asn Gly Tyr Val Asp Cys Gly Glu Ala Leu Leu 145 Ile Ala Cys Arg Glu 225 Ile Val Lys Val Tyr 305 Leu Val Phe Gin Pro Gly Met Ile 130 Pro Met Asn Ser Gly 210 Met Thr Val Ser Phe 290 Ile Leu SVal Val Leu Glu Ser Glu 115 Glu Gin Val Thr Arg 195 Ala Asp Asp Thr Phe 275 Gly Gly Leu Asn Arg His Ala Val 100 Ala Pro Ser Leu Leu 180 Leu Pro Pro Leu Ala 260 Arg His Gly Trp Asn 25 Leu Gly Val Arg Glu Asp Glu Asp Ile Gin Val Asn Gin His Pro Lys 165 Pro Phe Gly Leu Val 245 Asp Val Trp Asp Tyr 325 Val Asn Trp 70 Arg Leu Ile Pro Leu 150 Phe His Ala Tyr Ile 230 Asp Asn Tyr Asp Cys 310 Trp Asp Arg 55 Ile Ala Asp Gin Pro 135 Met Pro Leu Val Ser 215 Ala Gin Arg Thr Val 295 Tyr Ser Leu 40 Met Asp Leu Ser Asn 120 Arg Phe Ser Thr Asn 200 Leu Asn Ser Tyr Thr 280 Val Ile Gly Pro Pro Ala Leu Leu Ile Asn Val 90 Ile Thr 105 Phe Gly Asn Ser Lys Asp Asn Ser 170 Ile Pro 185 Arg Trp Asp Gin Asn Ser Ile Gin 250 Ile Leu 265 Glu Thr Thr Cys Val Ser Arg His I 330 Trp Glu Phe 75 Phe Asp Gin Ala Gin 155 Pro Ala His Ala Gly 235 Ile Ile Gly Leu Gly 315 His Ala Ser SGly His Pro Thr Met 140 Met Val Val Asn His 220 Val Asn Cys Lys Ala 300 Ser Ile Lys Glu Tyr Tyr Val Pro 125 His Gin Thr Val Thr 205 His Asn Ala Gly Leu 285 Arg Arg Ile Lys Phe Lys Leu Leu 110 Ser Leu Gin His Thr 190 Val Leu Lys His Phe 270 Thr Ser Asp Gly Pro Glu Val Ser Gin Arg Thr Tyr Arg Glu Gin Leu Cys Phe Asp Val 160 Val Ala 175 Val Thr Gly Leu Pro Ile Arg Gin 240 Cys Phe 255 Trp Asp Gin Ile Glu Ser Ala Thr 320 Asp Asn 335 Pro Asn Ser Ser Asp Tyr Pro Ala Pro Arg Ala Val Leu 340 345 Thr Gly His 350 WO 97/28262 PCT/US97/01748 Asp His Glu Val 355 Ile Ser Gly Ala 370 Asp Leu Leu Arri Val Cys Val Ser .360 Lys Glu Gly Pro 375 Ala Leu Glu Gly Val Cys Ala Giu Leu Gly Leu Val 365 Thr Ile Thr Gly Cys Leu Val His 380 Cys 385 Leu Pro Glu Ile Ser Val 390 Ser Giu Phe Ser Gly His Cys 410 Ile Asn Gly As n 395 Ile Leu Phe Pro Ile Tyr Tyr Giu Arg 415 Gly Arg Phe Ser 420 Giu Ile Asn Asp 435 Asn Leu Val Thr Lys Leu Leu 425 Ile Ser Thr Arg A 440 Leu Leu Ser Ala Gin Met 430 Asp Gly Gin Trp Gin Ala 450 Cys Asp 465 Gly Gly Asp Asn 455 Gin Leu Tyr Ile 470 Gly Val Val Gi u 460 Phe Lys INFORMATION FOR SEQ ID NO: 13: SEQUENCE

CHARACTERISTICS:

LENGTH: 2543 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GCAGCAGGGC GAACCGGACC TCTGTGATGT TTAATTTTCC

TGACCAAGCA

AAGTTGTCTA

GGAGGATATC

GGCAAAGAAG

GGACGTATAA

CAGAGGAGTT

CTTTGAACCC

AAAGCCCACC

TTGTTCGGAT

ATCCAGAC CG

CCGATGTCAA

ATGGGTACCA

C CT GGG CCAA

TTGTTTCTTG

CAGCTTGCCT

ACTGGCCACT

GGAAAT CT CC

TGATCTGAAC

GGACCTGACT

GAAGAGAGCA

CTTCCACTAC

TGAGCCATTC

AACCTTCTCA

GGAACTAATT

TCTTGGAGTG

GAAGCCAGAA

CCAACTCCAT

CGGGTTGGAG

CCTCGACAGC

AACTTTGAGT

CAGTATCCTG

CTCCCAGGAA

GTGTTTTACG

AACACACATT

ACAACCTTCT

TCCATTGCA

CCAGAGTTCT

AGGGAGGACG

GACTTTGTGC

CAATGGATTG

T GGGGACCAG

TGTATAAGTC

ATTTGATGTT

TGTTTCCATG

ACTTCAGGCA

CAGAGCGCTA

ACTCAACGGC

TCCTCAATGC

GGTCATGGAG

ATTACGTACC

AAGTGGTGGT

GGATCAACAG

ACCTTATATT

CTATGGTTTG

TTCCAATATG

TCTCAACACG

GGTGTTAACA

TCTGTCAAAG

TGAGACATGG

GACTTCCCCC

AAATGATGGG

AAC CAGT GAG

AGAGATGTTT

TAATGATGTG

GATGG C CCT G

TGGCTACAAA

ACAGTTAAAA

CCACAAGCCA

ACTCAGCGCT

ATAGCAGGTC

AAC TAT GAAT

CCAAAAGGTG

GAGGAGGATC

CTTTCATGGC

AAATTTGACC

AGAGATACAT

GTCAACAGCA

GACCTGCCCC

GAAAGTGAAT

CAGCGAGGGC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 CAGAGGCAGT CCGTGCTCTC AATGTTTTCC ACTACTTGAC CTACGAAGGC TCTGTAAACC WO 97/28262 PTU9/14 PCT[US97/01748

TGGACAGCAT

CC CAGAC GC C

TGTGTTCCCT

TGGTGCTGAA

ACOT GAC CAT

GGCACAACAC

TTCCCATTGA

CAGACCTTGT

ATCGCTACAT

CAGGGAAACT

CGGAGTCCTA

TTCTCTGGTA

AT CCT GC GC C

GTGCAGAACT

TCACTGGAAA

TTTCGGTATC

TCAGCATCAA

TCCTGAGCAG

GGCAGGCCTG

CGATGGATTT

TACTTTTAAT

GCAGCAGAAG.

GTGGGACGCT

ACATCATAGT

AGCAAGCTTA

ATCGCCGTTT

GGAACCTTGT

CAGCCTCGGG

CACAGACCCT

AT CTCAGTTG

TCCACAGAGC

GTTTCCATCC

CCCTGCAGTG

AGTCGGCCTC

GATGGACCCA

AGACCAGAGC

C CT CAT CT GT

GACACAGATT

CATTGGTGGA

CTGGAGTGGG

CAGAGCTGTC

CGGACTCGTT

T CTGCTGAAG

CAGTGAAGGC

TGGGAAACTT

CGATGGACAG

T GACTTTAAG

ATCCCATGAC

ATAGATTTTA

CCACATTCAA

GGTCCACATC

CGGGAGCCAT

TAT CAT GTAA

ATTGAGAAAA

GCCCAGTTGT

CTTCACTCGT

GTGCTCCGGG

CTCATTGAGC

CCACTCATGT

AATTCTCCTG

GTGACAGTGA

AGAGGAGCCC

TTAATCGCAA

AT CCAGAT CA

GGGTTTTGGG

GTATTTGGCC

GACTGCTACA

CGTCACCACA

CT CACAGGC C

ATCAGTGGTG

GCCCTGGAAG

CACTGCATCA

TTGGCTCAAA

AACCTGGTGA

CACCTGTACA

CAAAGGACTC

ATCGGTGGC.A

GTGAGAGCAC

GGCCTCTGCT

TTCACCCTGT

ATTATCTGAA

GGTTGTAGGA

TACAAAGTTT

GCC

AGGCCATGGT

CGCAT CCGCC

TCAAAGATCA

TGACTCATGT

CCTGCAGCCG

CCGGATACTC

ATAACTCTGG

ATGCCCACTG

ATAAAAkGTTT

ACTGGGATGT

TAGTGTCTGG

TCATCGGAGA

ATGACCATGA

CTAAAGAGGG

GACCAGAAAA

TATAT TAT GA

TGGAGATCAA

CTGGAGGGGA

TTTACCCAGG

T GATCACTGG TTAT GAG CAT

AAGTGCTTCT

T GTACATCCA

TTTTCCAGGA

TTAGGAGCCG

AGCCTCACAA

AAGCTTTGAA

TGCACAGATA CAGAACTTTG

TAGGACTTCA

GATGCAGCAG

GGCTGCCAAC

ACTGTTTGCA

CTTGGATCAA

TGTGAACAAG

CTT CGT GGTC

CAGAGTTTAC

TGTCACATGC

ATCTCGGGAC

CAACCCCAAT

AGTTGTCTGT

CCCTTGCCTC

CT GCTTATTT

GCGAGGACGG

TGATTCCACT

CAATGGTGTG

ATGTGATGCT

CAT GG CTTC C

CAGAACAGTA

GTGGAAAGGC

TCCCACCCAG

CTGAACACCA

TTTTGGTAAT

GAGACTTTTG

CCTAACTTGC

GCCATGCATC

GATGTGATCA

AC CCT GCC CC

GTGAACAGAT

GCACACCATC

CGGCAGATCA

ACAGCTGATA

TCGACAGAAA

CTGGCCAGGT

GCCACCTTGC

AGCAGTGACT

GTCTCCGTCT

GTT CATACCA

CCACGCCTAA

TTTAGCAACT

AGGGCTATTC

GTGGAGGTCT

GGCATTAGAG

GGCAGCATTG

CTGAAGAGAA

AGTATCTCTG

CAGTCGCCGA

GCTGCTGTCA

TATTTCATAT

ACAATT CTGA AT C CCAT TTC 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2543 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 703 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear WO 97/28262 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: PCTIUS97/01748 Ser Arg Ala Asn Arg Thr Ser Vai Met Phe Asn Phe Pro Asp Gin Ala Th r Ser Gin Ile Thr As n His Tyr His 145 Val Lys Arg Phe Gi y 225 T rp Glu Phe Phe Asp 305 Gin Val Tyr Leu Ser Tyr Tyr Leu Al a 130 Tyr Arg Phe Thr Tyr 210 Val Ala Ser Gly His 290 Pro Thr Lys Gi y Tyr As n Asn Glu Ser 115 Glu Asn Ile Asp Ser 195 Tyr Arg Lys Giu Tyr 275 Tyr Val Pro Lys Leu Lys Ph e Asp Ser 100 Lys Ar g Th r Gi u His 180 Gin Val Giu Lys Phe 260 Lys Leu Leu Ser Val Pro Ser Giu Leu Gi u Pro Tyr His Pro 165 Pro Arg Pro Asp Pro 245 Val Gin Thr Arg Gin 325 Val1 Gin Ser Tyr 70 As n Giu Lys Glu Tyr 150 Phe Asp Asp Glu Gi u 230 Giu Ser Arg Tyr Glu 310 Leu Tyr Al a As n 55 Leu Gin Leu Gi y Thr 135 Ser Thr Arg Thr Met 215 Val Asp Cys Gi y Glu 295 Ala Leu Ser Arg 40 Met Met Tyr Asp Al a 120 Trp Thr Thr Tb r Ser 200 Phe Val Phe Gin Pro 280 Gi y Met Ile Leu 25 Arg Thr Phe Pro Leu 105 Leu Giu Ala Phe Phe 185 Asp Val Val Val Leu 265 Giu Ser Val1 Glu Pro Ile Gin Leu Val 90 Thr Asn Giu Thr Phe 170 Ser Val As n Asn Arg 250 His Al a Val Ala Pro 330 Arq Val Ser Leu Arg Trp Asn Thr 75 Phe Pro Leu Pro Pro Lys Asp Gin 140 Ser Pro 155 Leu Asn Ser Ile Lys Glu Ser Asn 220 Asp Val 235 Ile Asn Gin Trp Val Arq Asn Leu 300 Gin Ile 315 His Pro Gly Al a Gin Ile Trp Giy Arg 125 Ser Leu Al a Al a Leu 205 Gi y Asp Arg Ile Al a 285 Asp Gin Pro Vai Thr Arg Al a Val As n 110 Aila Pro Ser As n Arg 190 Ile Tyr Leu Met Asp 270 Leu Ser As n Arg Gi y Pro Arg Gly Leu Phe Val Pro T rp Asp 175 Ser Pro His Pro Al a 255 Leu Asn Ile Phe Thr 335 Thr Arg Glu Arg Thr Arg Phe Phe Leu 160 Gi y T rp Gi u Leu Pro 240 Leu Ile Val1 Thr Al a 320 Ser WO 97/28262 Ala Met His Leu Cys Ser Leu Pro Gin Se'r Pro Leu Met Phe 'CTJIS97/01748 340 Gin Pro Ala 385 His Ala Gly Ile Ile 465 Gly Leu Gly His Ala 545 Ala Val Asn Ile Lys 625 Leu Val Met Val 370 Val Asn His Val Asn 450 Cys Lys Ala Ser Ile 530 Val Glu His Cys Ile 610 Leu Ser Glu Gin 355 Thr Val Thr His Asn 435 Ala Gly Leu Arg Arg 515 Ile Leu Leu Thr Leu 595 Tyr Leu Ser Val Gin His Thr Val Leu 420 Lys His Phe Thr Ser 500 Asp Gly Thr Gly Ile 580 Phe Tyr Ala Asp Trp 660 Asp Val Val Gly 405 Pro Arg Cys Trp Gin 485 Glu Ala Asp Gly Leu 565 Thr Pro Glu Gin Gly 645 Gin Val Ala Thr 390 Leu Ile Gin Phe Asp 470 Ile Ser Thr Asn His 550 Val Gly Arg Arg Met 630 Gin Ala Ile Ala 375 Cys Arg Glu Ile Val 455 Lys Val Tyr Leu Pro 535 Asp Ile Asn Leu Gly 615 Glu Asn Cys Met 360 Asn Ser Gly Met Thr 440 Val Ser Phe Ile Leu 520 Asn His Ser Leu Ile 600 Arg Ile Leu Asp 345 Val Thr Arg Ala Asp 425 Asp Thr Phe Gly Gly 505 Leu Ser Glu Gly Leu 585 Ser Phe Asn Val Phe 665 Leu Leu Leu Pro 410 Pro Leu Ala Arg His 490 Gly Trp Ser Val Ala 570 Lys Val Ser Asp Thr 650 Lys Lys Pro Phe 395 Gly Leu Val Asp Val 475 Trp Asp Tyr Asp Val 555 Lys Ala Ser Asn Ser 635 Gly Gin Phe His 380 Ala Tyr Ile Asp Asn 460 Tyr Asp Cys Trp Tyr 540 Cys Glu Leu Ser Phe 620 Thr Gly Leu Pro 365 Leu Val Ser Ala Gin 445 Arg Ser Val Tyr Ser 525 Pro Val Gly Glu Glu 605 Ser Arg Asp Tyr 350 Ser Thr Asn Leu Asn 430 Ser Tyr Thr Val Ile 510 Gly Ala Ser Pro Gly 590 Gly Ile Ala Asn Ile 670 Lys Asn Ile Arg Asp 415 Asn Ile Ile Glu Thr 495 Val Arg Pro Val Cys 575 Pro His Asn Ile Gly 655 Tyr Asp Ser Pro Trp 400 Gin Ser Gin Leu Thr 480 Cys Ser His Arg Cys 560 Leu Glu Cys Gly Leu 640 Val Pro Gly Cys Asp 675 Ala Gly Ile Arg Ala Met Asp Leu Ser His 685 Asp Gin Arg WO 97/28262 PCT/US97/01748 Thr Leu Ile Thr Gly Met Ala Ser Gly Ser Ile Val Leu Leu Ile 690 695 700 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CATTCTTTAT TGACAGTGTT INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GTGAACCCTA CCATATCT 18 INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: ATGCCATTCT TTATTGACAG INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: CATTGGCACA GGAAACAAC 19 INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/28262 PCTIUS97/01748 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: AAACCCTGTC TCGAAACAA 19 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GAATTCCCAA GGACAGGT 18 INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: TAGGAGGTGT GGCCTTG 17 INFORMATION FOR SEQ ID NO: 22: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: AGAGACGGCG GACACTTA 18 INFORMATION FOR SEQ ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: TAAATATGAG GCGGGCAG 18 INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/28262 PCTUS97/01748 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: ATACTCTAAG TAAGATACAC INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TGTTTTAACT GTTTGCTAA 19 INFORMATION FOR SEQ ID NO: 26: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: ACGCAGTGGG CATGCTG 17 INFORMATION FOR SEQ ID NO: 27: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: CACAGGCTGT GACTGGAA 18 INFORMATION FOR SEQ ID NO: 28: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: AGTAGCCACA GGCCCTA 17 INFORMATION FOR SEQ ID NO: 29: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/28262 PCTIS97/01748 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: GAGATTACCC CAATAGTA 18 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AGTGGAAGGA GGCTGTC 17 INFORMATION FOR SEQ ID NO: 31: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: CCATGGCGAT GAAGCGG 17 INFORMATION FOR SEQ ID NO: 32: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: ATGATGCAAA GAACCCAG 18 INFORMATION FOR SEQ ID NO: 33: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: TTCAACTACA TAGTGAATT 19 INFORMATION FOR SEQ ID NO: 34: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/28262 PCT/US97/01748 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: TCCCTAACAC ATCCCTAA 18 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CCTCATGTTA GGGTAGAG 18 INFORMATION FOR SEQ ID NO: 36: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: CCGTTAGTGT GTAGTCTC 18 INFORMATION FOR SEQ ID NO: 37: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: CACATTGGCA CAGGAAAC 18 INFORMATION FOR SEQ ID NO: 38: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: TTACGAATGT GCCTGGTG 18 INFORMATION FOR SEQ ID NO: 39: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/28262 PCTfUS97/01748 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: TCCAAACACA CTAAACCTG 19 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CATGCCATTC TTTATTGACA INFORMATION FOR SEQ ID NO: 41: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: CTCAGTAGAC TATACGAG 18 INFORMATION FOR SEQ ID NO: 42: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: GAGTTCAAGG TCATCCTC 18 INFORMATION FOR SEQ ID NO: 43: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: GCCTAAGCCC ATTATCG 17 INFORMATION FOR SEQ ID NO: 44: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/28262 PC'TITfT7/1i 748 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: TAAATGCTGC CATAAACTCC INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AGAAGTGACT TCAGGTAATA INFORMATION FOR SEQ ID NO: 46: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: AGTCTCTCAC ACTTACAC 18 INFORMATION FOR SEQ ID NO: 47: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: GACCCAAGTC AGCTTTC 17 INFORMATION FOR SEQ ID NO: 48: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48: CCAGTGTGTC ACTTAAGC 18 INFORMATION FOR SEQ ID NO: 49: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/28262 PCT/US97/n1 748 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: AGGGAGATGT ATCATCTGC 19 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AGGGATCACC ATGCTTTG 18 INFORMATION FOR SEQ ID NO: 51: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: ACTTGGTCTT GGGGTCC 17 INFORMATION FOR SEQ ID NO: 52: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52: GAAAGCAGTG TAATGAGG 18 INFORMATION FOR SEQ ID NO: 53: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: TGCCTCTACA TGGGAGC 17 INFORMATION FOR SEQ ID NO: 54: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/28262 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: GCAAGCATTT AGTTAAACG INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CTTGTTCTTG TATATCTG INFORMATION FOR SEQ ID NO: 56: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56: ACAATGAAAT CCTCCACC INFORMATION FOR SEQ ID NO: 57: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57: GTGACTTGAT CCAGACTG INFORMATION FOR SEQ ID NO: 58: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: CTTGCTCTCA CTGTTCTC INFORMATION FOR SEQ ID NO: 59: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear PCT/US97/01748 WO 97/28262 PCT/US97/01748 I1q1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: CAGGTGGAGA TGCTGTTC 18 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GAGATGCCTT CAGGCAGT 18 INFORMATION FOR SEQ ID NO: 61: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: CCGTTAGTGT GTAGTCTC 18 INFORMATION FOR SEQ ID NO: 62: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62: CTTGCTCTCA CTGTTCTC 18 INFORMATION FOR SEQ ID NO: 63: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: TGGATGGGCT GTCTGAACGC INFORMATION FOR SEQ ID NO: 64: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/28262 PCT/US97/01748 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: TGCTGGCAGA TGCTGGCATA INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CCAAGATGAA AGCAGCCGAT GGGGAAAACT INFORMATION FOR SEQ ID NO: 66: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66: TCAGCCTCTT TCTTGCTCCG TGAAACTGCT INFORMATION FOR SEQ ID NO: 67: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: AGTTTATGAG TCCAAATGAT INFORMATION FOR SEQ ID NO: 68: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68: GAATGATGAA GTTGCTCTGA INFORMATION FOR SEQ ID NO: 69: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/28262 PCT/US97/01748 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69: CAGCAGTTCT TCAGATGGA 19 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ATCTTTCTGT TGTTCCCCTA INFORMATION FOR SEQ ID NO: 71: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: TAGGGGAGCA ACAGAAAGAT INFORMATION FOR SEQ ID NO: 72: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72: GCTCATAGTA GTATCACTTT INFORMATION FOR SEQ ID NO: 73: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73: CGCACATGGC AACCCTT 17 INFORMATION FOR SEQ ID NO: 74: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/28262 PCT/US97/01748 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74: GCACATGGGC AACCCTT 17 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CCAGGACACC AGGGCTACAG AG 22 INFORMATION FOR SEQ ID NO: 76: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76: CCCGAGTGCT GGGATTAAAG INFORMATION FOR SEQ ID NO: 77: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77: GTTGTAAAAC GACGGCCAGT GGCAAGTTCA GCCTGGTTAA G 41 INFORMATION FOR SEQ ID NO: 78: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78: CACAGGAAAC AGCTATGACC AGAGTATTTC TTCCAGGGTA An WO 97/28262 PCT/US97/01748 All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved.

All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Accordingly, the exclusive rights sought to be patented are as described in the claims below.

Claims (15)

1. A protein comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14.

2. An isolated nucleic acid segment encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14.

3. A nucleic acid segment according to claim 2 that has the sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID ND:13.

4. A nucleic acid segment that is the complement of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID ND:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13. A nucleic acid segment that hybridises under stringent conditions of high selectivity to the sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13 or to the complement of said sequences.

6. A nucleic acid segment according to claim 4, further 25 defined as an RNA segment.

7. A nucleic acid segment according to claim 4, further defined as a DNA segment.

8. A DNA segment according to claim 7 that encodes a protein or peptide that includes a contiguous amino acid sequence of about to about 50 amino acids from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14. 35 9. A DNA segment according to claim 8, wherein the contiguous amino acid sequence is about 50 to about 150 amino acids. A DNA segment according to claim 8, wherein the contiguous amino acid sequence is about 1185 amino acids.

11. A DNA segment according to claim 7 that has a contiguous nucleic acid sequence of about 14 to about 10000 nucleotides from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13. H:\Emma\Keep\Spcis\18562.97.doc 160200196 197

12. contiguous

13. contiguous

14. contiguous contiguous

16. contiguous

17. contiguous

18. contiguous A DNA segment according to claim 11, wherein the nucleic acid sequence is about 20 nucleotides. A DNA segment according to claim 11, wherein the nucleic acid sequence is about 30 nucleotides. A DNA segment according to claim 11, wherein the nucleic acid sequence is about 50 nucleotides. A DNA segment according to claim 11, wherein the nucleic acid sequence is about 100 nucleotides. A DNA segment according to claim 11, wherein the nucleic acid sequence is about 200 nucleotides. A DNA segment according to claim 11, wherein the nucleic acid sequence is about 500 nucleotides. A DNA segment according to claim 11, wherein the nucleic acid sequence is about 1000 nucleotides. A DNA segment according to claim 11, wherein the nucleic acid sequence is about 5000 nucleotides. A DNA segment according to claim 11, wherein the nucleic acid sequence is about 4000 nucleotides. A DNA segment according to claim 11, wherein the 00 r* 0 *000 esf p. 9 *e 0 S

090.. 00*5 S. 0 *0 0 S 0 I 0 0040 00 S 05 0 19. contiguous 20. contiguous 21. contiguous nucleic acid sequence is about 3000 nucleotides. 22. contiguous A DNA segment according to claim 11, wherein the nucleic acid sequence is about 3514 nucleotides. 23. A DNA segment according to that is a recombinant vector. any one of claims 11 to 22 24. vector pCH. A DNA segment according to claim 23 that is recombinant A DNA segment according to wherein the DNA is operatively linked expressing the DNA segment. any one of claims 11 to 22, to a promoter, said promoter H:\Era\Keep\pecis\18562.97.d 16/02/00197 198 26. A recombinant host cell comprising the DNA segment according to any one of claims 11 to 22, wherein said host cell is a prokaryotic cell, a eukaryotic cell, a bacterial cell, a yeast cell, a rammalian cell, an animal cell or a human cell. 27. A method for detecting a nucleic acid sequence encoding a polypeptide having the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID N:14, comprising the steps of: obtaining sample nucleic acids suspected of encoding a polypeptide having the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14; contacting said sample nucleic acids with an isolated nucleic acid segment complementary to at least one of said sample nucleic acids under stringent conditions of high selectivity effective to allow hybridisation with sample nucleic acid; and detecting the hybridised nucleic acid. 25 28. A method according to claim 27, wherein the sample nucleic acids are located within a cell. 29. A method according to claim 27, wherein the sample nucleic acids are separated from a cell prior to the contacting. 30. A method according to claim 27, further comprising a detectable label attached to said isolated nucleic acid segment. 31. A nucleic acid detection kit, comprising in suitable S 35 container means the nucleic acid segment according to any one of claims 2 to 25 and a detection reagent. 32. A nucleic acid detection kit according to claim 31, further comprising a restriction enzyme. 33. A peptide composition comprising a protein that includes the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14, or includes a 15 to contiguous amino acid sequence from said sequence. H:\Emma\Keep\Specis\18562.97.doc 16/02/00198 199 34. A peptide composition according to claim 33, wherein the contiguous amino acid sequence is about 50 to about 150 amino acids. 35. A peptide composition according to claim 33, wherein the contiguous amino acid sequence is about 150 to about 300 amino acids. 36. A composition according to any one of claims 33 to wherein the protein or peptide is a recombinant protein or peptide. 37. An antibody that specifically binds with a polypeptide that comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14. 38. An antibody according to claim 37, linked to a detectable label identified as a radioactive label, a fluorogenic label, a nuclear magnetic spin resonance label, biotin, or an enzyme that generates a colored product upon contact with a chromogenic substance. 39. An antibody according to claim 37, linked to an alkaline phosphatase, hydrogen peroxidase or glucose oxidase enzyme. 40. An antibody according to claim 37 which is a monoclonal antibody. 41. Use of a nucleic acid segment according to any one of 30 claims 2 to 25, or a polypeptide having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID SEQ ID NO:12 or SEQ ID NO:14, or an antibody according to any one of claims 37 to 40 to determine the presence of Chediak-Higashi oe Syndrome in a patient, coprising identifying said polypeptide, nucleic acid segment or antibody in a clinical sample from said patient suspected of having said syndrome. 42. A method for diagnosing Chediak-Higashi Syndrome, comprising identifying a nucleic acid segment according to claim 3 within a clinical sample from a patient suspected of having such a syndrome. 43. A method for diagnosing Chediak-Higashi Syndrome, comprising identifying a protein or peptide according to claim 1 H:\Emma\Keep\Specis\18562.97.doc 16/02/00199 200 within a clinical sairple from a patient suspected of having such a syndrome. 44. A transgenic non-humn animal having incorporated into its genomae a transgene that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14. Dated this 16th day of February 2000 UNIVERSITY OF FLORIDA By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H:\Emma\KeeP\Specis\j8562.97.doc 16/02/00 200