AU723686B2 - Acetyl-CoA carboxylase compositions and methods of use - Google Patents

Description

DESCRIPTION

ACETYL-CoA CARBOXYLASE COMPOSITIONS AND METHODS OF USE 1. BACKGROUND OF THE INVENTION 1.1 Field of the Invention The present invention relates to the field of molecular biology. More specifically, it concerns nucleic acid compositions comprising cyanobacterial and plant acetyl-CoA carboxylases (ACC), methods for making and using native and recombinant ACC polypeptides, and methods for making and using polynucleotides encoding ACC polypeptides.

1.2 Description of the Related Art 1.2.1 Acetyl-CoA Carboxylase Acetyl-CoA carboxylase [ACCase; acetyl-CoA:carbon dioxide ligase (ADPforming), EC 6.4.1.2] catalyzes the first committed step in de novo fatty acid 2. biosynthesis, the addition of CO 2 to acetyl-CoA to yield malonyl-CoA. It belongs to a group of carboxylases that use biotin as cofactor and bicarbonate as a sourc, of the carboxyl group. ACC catalyzes the addition of CO 2 to acetyl-CoA to yield malonyl- CoA in two steps as shown below.

BCCP ATP HCO 3 BCCP-C02 ADP Pi (1) BCCP-CO, Acetyl-CoA BCCP malonyl-CoA (2) ^*5 WO 96/32484 PCT/US96/05095 -2- First, biotin becomes carboxylated at the expense of ATP. The carboxyl group is then transferred to Ac-CoA (Knowles, 1989). This irreversible reaction is the committed step in fatty acid synthesis and is a target for multiple regulatory mechanisms. Reaction is catalyzed by biotin carboxylase reaction by transcarboxylase BCCP biotin carboxyl carrier protein.

There are two types of ACC: prokaryotic ACC in which the three functional domains: biotin carboxylase biotin carboxyl carrier protein (BCCP) and carboxyltransferase (CT) are located on separable subunits E. coli, P.

aeruginosa, Anabaena, Synechococcus and probably pea chloroplast) and eukarvotic ACC in which all the domains are located on one large polypeptide rat, chicken, yeast, diatom and wheat).

E. coli ACC consists of a dimer of 49-kDa BC monomers, a dimer of 17-kDa BCCP monomers and a CT tetramer containing two each of 33-kDa and subunits. The primary structures of all of the E. coli ACC subunits (Alix, 1989; Muramatsu and Mizuno, 1989; Kondo etal., 1991; Li and Cronan, 1992; Li and Cronan, 1992) as well as the structure of the BC and BCCP of Anabaena 7120 (Goricki et al., 1993), and P. aeruginosa (Best and Knauf, 1993) are known, based on the gene sequences. The genes encoding the subunits of E. coli ACC are called: accA (CT a subunit), accB (BCCP), accC (BC) and accD (CT P subunit). accC and accB form one operon, while accA and accD are not linked to each other or to accCB (Li and Cronan, 1992). In cyanobacteria, accC and accB are unlinked as well (Gornicki et al., 1993).

Yeast, rat, chicken and human ACCs are cytoplasmic enzymes consisting of 250- to 280-kDa subunits while diatom ACC is most likely a chloroplast enzyme consisting of 230-kDa subunits. Their primary structure has been deduced from cDNA sequences (Al-feel et al., 1992; Lopez-Casillas et al., 1988; Takai et al., 1988; Roessler and Ohlrogge, 1993; Ha et al., 1994). In eukaryotes, homologs of the four bacterial genes are fused in the following order: accC, accB, accD and accA. Animal ACC activity varies with the rate of fatty acid synthesis or energy requirements in different nutritional, hormonal and developmental states. In the rat, ACC mRNA is WO 96/32484 PCTIUS96/05095 -3transcribed using different promoters in different tissues and can be regulated by alternative splicing. The rat enzyme activity is also allosterically regulated by a number of metabolites and by reversible phosphorylation (Ha etal., 1994 and references therein). The expression of the yeast gene was shown to be coordinated with phospholipid metabolism (Chirala, 1992; Haslacher et al., 1993).

Much less is known relating to plant ACC. Early attempts at characterization of plant ACC led to the suggestion that it consisted of low molecular weight subunits similar to those of bacteria (Harwood, 1988). More recent efforts indicate that at least one plant isozyme is composed of >200-kDa subunits, similar to the enzyme from other eukaryotes (Egin-Buhler and Ebel, 1983; Slabas and Hellyer, 1985; Gornicki and Haselkor, 1993; Egli et al., 1993; Betty et al., 1992).

While strong evolutionary conservation exists among biotin carboxylases and biotin carboxylase domains of all biotin-dependent carboxylases, BCCP domains show very little conservation outside the conserved sequence E(A/V)MKM (lysine residue is biotinylated) (Knowles, 1989; Samols et al., 1988). Although the three functional domains of the E. coli ACC are located on separate polypeptides, plant ACC is quite different, having all 3 domains on a single polypeptide.

At least one form of plant ACC is located in plastids, the primary site of fatty acid synthesis. The gene encoding it, however, must be nuclear because no corresponding sequence has been seen in the complete chloroplast DNA sequences of tobacco, liverwort or rice. The idea that in some plants plastid ACC consisted of several smaller subunits was revived by the discovery of an accD homolog in some chloroplast genomes (Li and Cronan, 1992). Indeed, it has been shown that the product of this gene in pea binds two other peptides, one of which is biotinylated. The complex may be a chloroplast isoform of ACC in pea and some other plants (Sasaki et al., 1993).

It has been shown recently that plants have indeed more than one form of ACCase (reviewed in Sasaki et al., 1995). The one located in plastids, the primary site of plant fatty acid synthesis, can be either a eukaryotic-type high molecular weight multi-functional enzyme in wheat and maize) or a prokaryotic-type WO 96/32484 PCT/US96/05095 -4multi-subunit enzyme in pea, soybean, tobacco and Arabidopsis). The other plant ACCase, located in the cytoplasm, is of the eukaryotic type.

In Graminae, genes for both cytosolic and plastid eukaryotic-type ACCase are nuclear. No ACCase coding sequence can be found in the complete sequence of rice chloroplast DNA.

In other plants, subunits of ACCase other than the carboxyltransferase subunit encoded by a homolog of the E. coli accD gene, present in the chloroplast genome (Sasaki et al., 1995; Li and Cronan, 1992), must be also encoded in the nuclear DNA.

Like the vast majority of plastid proteins, plastid ACCases are synthesized in the cytoplasm and then transported into the plastid. The amino acid sequence of the cytosolic and some subunits of the plastid ACCases from several plants have been deduced from genomic or cDNA sequences (Egli et al., 1995; Li and Cronan, 1992; Gornicki etal., 1994; Schulte etal., 1994; Shorrosh etal., 1994; Shorrosh etal., 1995; Roesler et al., 1994; Anderson et al., 1995).

There is experimental evidence suggesting that, in plants, ACCase activity controls carbon flow through the fatty acid pathway and therefore may serve as an important regulation point of plant metabolism (Page et al., 1994; Post-Beitenmiller et al., 1992; Shintani and Ohlrogge, 1995).

The possibility of different ACC isoforms, one present in plastids and another in the cytoplasm, is now accepted. The rationale behind the search for a cytoplasmic ACC isoform is the requirement for malonyl-CoA in this cellular compartment, where it is used in fatty acid elongation and synthesis of secondary metabolites. Indeed, two isoforms were found in maize, both consisting of >200-kDa subunits but differing in size, herbicide sensitivity and immunological properties. The major form was found to be located in mesophyll chloroplasts. It is also the major ACC in the endosperm and in embryos (Egli et al., 1993).

1.2.2 Cyanobacteria Unlike monocot plants, members of the cyanobacteria are resistant to these herbicide families. Cyanobacteria are prokaryotes that carry out green plant WO 96/32484 PCTIUS96/05095 photosynthesis, evolving 02 in the light. They are believed to be the evolutionary ancestors of chloroplasts. Virtually nothing is known about fatty acid biosynthesis in cyanobacteria.

Synechococcus is a unicellular obligate phototroph with an efficient DNA transformation system. Replicating vectors based on endogenous plasmids are available, and selectable markers include resistance to kanamycin, chloramphenicol, streptomycin and the PSII inhibitors diuron and atrazine. Inactivation and/or deletion of Synechococcus genes by transformation with suitable cloned material interrupted by resistance cassettes is well known in the art. Genes may also be replaced by specifically mutated versions using selection for closely linked resistance cassettes.

Anabaena differentiates specialized cells for nitrogen fixation when the culture is deprived of a source of combined nitrogen. The differentiated cells have a unique glycolipid envelope containing C26 and C28 fatty acids (Murata and Nishida, 1987), whose synthesis must start with the reaction catalyzed by ACC. Therefore ACC must be developmentally regulated in Anabaena. Powerful systems of genetic analysis exist for Anabaena as well (Golden et al., 1987).

That cyanobacteria and plants are evolutionarily-related make the former useful sources of cloned genes for the isolation of plant cDNAs. This method is well known to those of skill in the art. For example, the cloned gene for the enzyme phytoene desaturase, which functions in the synthesis of carotenoids, isolated from cyanobacteria was used as a probe to isolate the cDNA for that gene from tomato (Pecker et al., 1992).

1.2.3 Herbicide Resistance Although the mechanisms of inhibition and resistance are unknown (Lichtenthaler, 1990), it has been shown that aryloxyphenoxypropionates and cyclohexane-l,3-dione derivatives, powerful herbicides effective against monocot weeds, inhibit fatty acid biosynthesis in sensitive plants.

The aryloxyphenoxypropionate class comprises derivatives of aryloxyphenoxy-propionic acid such as diclofop, fenoxaprop, fluazifop, haloxyfop, WO 96/32484 PCTIUS96/05095 -6propaquizafop and quizalofop. Several derivatives of cyclohexane-l,3-dione are also important post-emergence herbicides which also selectively inhibit monocot plants.

This group comprises such compounds as oxydim, cycloxydim, clethodim, sethoxydim, and tralkoxydim.

Recently it has been determined that ACC is the target enzyme for both of these classes of herbicide at least in monocots. Dicotyledonous plants, on the other hand, such as soybean rape, sunflower, tobacco, canola, bean, tomato, potato, lettuce, spinach, carrot, alfalfa and cotton are resistant to these compounds, as are other eukaryotes and prokaryotes.

Important grain crops, such as wheat, rice, maize, barley, rye, and oats, however, are monocotyledonous plants, and are therefore sensitive to these herbicides.

Thus herbicides of the aryloxyphenoxypropionate and cyclohexane-1,3-dione groups are not useful in the agriculture of these important grain crops owing to the inactivation of monocot ACC by such chemicals.

1.2.4 Deficiencies in the Prior Art The genetic transformation of important commercial monocotyledonous agriculture crops with DNA segments encoding herbicide-resistant ACC enzymes would be a revolution in the farming of such grains as wheat, rice, maize, barley, rye, and oats. Moreover the availability for modulating the herbicide resistance of plants through the alteration of ACC-encoding DNA segments and the polypeptides themselves would be highly desirable. Methods of identifying and assaying the levels of ACC activity in these plants would also be important in genetically engineering grain crops and the like with desirable herbicide-resistant qualities. Likewise the availability of DNA segments encoding dicotyledonous ACC and nucleic acid segments derived therefrom would provide a much-needed means of genetically altering the activity of ACC in vivo and in vitro.

What is lacking in the prior art, therefore, is the identification of DNA segments encoding plant and cyanobacterial ACC enzymes, and the development of methods and processes for their use in creation of modified, transgenic plants which WO 96/32484 PCTIUS96/05095 -7have altered herbicide resistance. Moreover, novel methods providing transgenic plants using DNA segments encoding ACC polypeptides to modulate ACC activity, fatty acid biosynthesis in general, and oil content of plant cells in specific, are greatly needed to provide transformed plants altered in such activity. Methods for determining ACC activity in vivo and quantitating herbicide resistance in plants would also represent major improvements over the current state of the art.

2. SUMMARY OF THE INVENTION The present invention seeks to overcome these and other inherent deficiencies in the prior art by providing compositions comprising novel ACC polypeptides from plant and cyanobacterial species. The invention also provides novel DNA segments encoding eukaryotic and prokaryotic ACCs, and methods and processes for their use in regulating the oil content of plant tissues, for conferring and modulating resistance to particular herbicides in a variety of plant species, and for altering the activity of ACC in plant cells in vivo. Also disclosed are methods for determining herbicide resistance and kits for identifying the presence of plant ACC polypeptides and DNA segments.

2.1 ACC Genes and Polynucleotides The present invention provides polynucleotides and polypeptides relating to a whole or a portion of acetyl-CoA carboxylase (ACC) of cyanobacteria and plants as well as processes using those polynucleotides and polypeptides.

As used herein the term "polynucleotide" means a sequence of nucleotides connected by phosphodiester linkages. A polynucleotide of the present invention can comprise from about 2 to about several hundred thousand base pairs. Preferably, a polynucleotide comprises from about 5 to about 150,000 base pairs. Preferred lengths of particular polynucleotides are set forth hereinafter.

A polynucleotide of the present invention can be a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule. Where a polynucleotide is a DNA molecule, that molecule can be a gene or a cDNA molecule. Nucleotide bases WO 96/32484 PCTIS9605095 -8are indicated herein by a single letter code: adenine guanine thymine cytosine and uracil In one embodiment, the present invention contemplates isolated and purified polynucleotides comprising DNA segments encoding polypeptides which have the ability to catalyze the carboxylation of a biotin carboxyl carrier protein of a cyanobacterium. Preferably, the cyanobacterium is Anabaena or Synechococcus. A preferred Anabaena is Anabaena 7120. A preferred Synechococcus is Anacystis nidulans R2 (Synechococcus sp. strain PCC 7942).

Preferably, a polypeptide is a biotin carboxylase enzyme of a cyanobacterium.

This enzyme is a subunit of cyanobacterial acetyl-CoA carboxylase and participates in the carboxylation of acetyl-CoA. In a preferred embodiment, a BC polypeptide is encoded by a polynucleotide comprising an accC gene which has the nucleic acid sequence of SEQ ID NO:5 (Anabaena accC) or SEQ ID NO:7 (Synechococcus accC), or functional equivalents thereof. The BC polypeptide preferably comprises the amino acid sequence of SEQ ID NO:6 (Anabaena BC) or SEQ ID NO:8 (Synechococcus BC), or functional equivalents thereof.

In a second embodiment, the present invention contemplates isolated and purified polynucleotides comprising DNA segments encoding a biotin carboxyl carrier protein of a cyanobacterium. Preferably, the cyanobacterium is Anabaena or Synechococcus. A preferred Anabaena is Anabaena 7120. A preferred Synechococcus is Anacystis nidulans R2 (Synechococcus sp. strain PCC 7942).

Preferably, a polypeptide is a biotin carboxyl carrier protein of a cyanobacterium. This polypeptide is a subunit of cyanobacterial acetyl-CoA carboxylase and participates in the carboxylation of acetyl-CoA. In a preferred embodiment, a BCCP polypeptide is encoded by a polynucleotide comprising an accB gene which has the nucleic acid sequence of SEQ ID NO:1 (Anabaena accB) or SEQ ID NO:3 (Synechococcus accB), or functional equivalents thereof. The BCCP polypeptide preferably comprises the amino acid sequence of SEQ ID NO:2 (Anabaena BCCP) or SEQ ID NO:4 (Synechococcus BCCP), or functional equivalents thereof.

WO 96/32484 PCT/US96/05095 -9- In a third embodiment, the present invention contemplates isolated and purified polynucleotides comprising DNA segments encoding a carboxyltransferase protein of a cyanobacterium. Preferably, the cyanobacterium is Anabaena or Synechococcus. A preferred Anabaena is Anabaena 7120. A preferred Synechococcus is Anacystis nidulans R2 (Synechococcus sp. strain PCC 7942).

Preferably, a polypeptide is a carboxyltransferase c or P subunit protein of a cyanobacterium. These polypeptides are subunits of cyanobacterial acetyl-CoA carboxylase and participate in the carboxylation of acetyl-CoA. In a preferred embodiment, a CTa polypeptide is encoded by a polynucleotide comprising an accA gene which has the nucleic acid sequence of SEQ ID NO: 11 (Synechococcus accA), or a functional equivalent thereof. The CTca polypeptide preferably comprises the amino acid sequence of SEQ ID NO:12 (Synechococcus CTa), or a functional equivalent thereof.

In a fourth embodiment, the present invention contemplates isolated and purified polynucleotides comprising DNA segments encoding an acetyl-CoA carboxylase protein of a plant. Preferably, the plant is a monocotyledonous or a dicotyledonous plant. An exemplary and preferred monocotyledonous plant is wheat, rice, maize, barley, rye, oats or timothy grass. An exemplary and preferred dicotyledonous plant is soybean, rape, sunflower, tobacco, Arabidopsis, petunia, pea, canola, bean, tomato, potato, lettuce, spinach, alfalfa, cotton or carrot. A preferred monocotyledonous plant is wheat, and a preferred dicotyledonous plant is canola.

Preferably, a polypeptide is an acetyl-CoA carboxylase (ACC) protein of a plant. This polypeptide participates in the carboxylation of acetyl-CoA. In a preferred embodiment, an ACC polypeptide is encoded by a polynucleotide comprising an ACC cDNA which has the nucleic acid sequence of SEQ ID NO:9 (wheat ACC) or SEQ ID NO:19 (canola ACC), or functional equivalents thereof. The ACC polypeptide preferably comprises the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:31 (wheat ACC) or SEQ ID NO:20 (canola ACC), or functional equivalents thereof.

In yet another aspect, the present invention provides an isolated and purified DNA molecule comprising a promoter operatively linked to a coding region that WO 96/32484 PCT/US96/05095 encodes a polypeptide having the ability to catalyze the carboxylation of a biotin carboxyl carrier protein of a cyanobacterium, a biotin carboxyl carrier protein of a cyanobacterium or a plant polypeptide having the ability to catalyze the carboxylation of acetyl-CoA, which coding region is operatively linked to a transcription-terminating region, whereby said promoter drives the transcription of said coding region.

In another aspect, the present invention provides an isolated polypeptide having the ability to catalyze the carboxylation of a biotin carboxyl carrier protein of a cyanobacterium such as Synechococcus. Preferably a biotin carboxyl carrier protein gene includes the nucleic acid sequence of SEQ ID NO:2 and the polypeptide has the amino acid residue sequence of SEQ ID NO:6.

2.2 ACC Polypeptides and Anti-ACC Antibodies The present invention also provides an isolated and purified biotin carboxyl carrier protein of a cyanobacterium such as Anabaena or Synechococcus, which protein includes the amino acid residue sequence of SEQ ID NO:2 or SEQ ID NO:4, respectively; an isolated and purified biotin carboxylase of a cyanobacterium such as Anabaena or Synechococcus, which protein includes the amino acid residue sequence of SEQ ID NO:6 or SEQ ID NO:8, respectively; an isolated and purified carboxyltransferase a subunit protein of a cyanobacterium such as Synechococcus, which protein includes the amino acid residue sequence of SEQ ID NO:12; an isolated and purified monocotyledonous plant polypeptide from wheat having a molecular weight of about 220 kDa, dimers of which have the ability to catalyze the carboxylation of acetyl-CoA, which protein includes the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:31; and an isolated and purified dicotyledonous plant polypeptide from canola having the ability to catalyze the carboxylation of acetyl-CoA, which protein includes the amino acid sequence of SEQ ID Another aspect of the invention concerns methods and compositions for the use of the novel peptides of the invention in the production of anti-ACC antibodies.

WO 96/32484 PCT/US96/05095 -11- The present invention also provides methods for identifying ACC and ACC-related polypeptides, which methods comprise contacting a sample suspected of containing such polypeptides with an immunologically effective amount of a composition comprising one or more specific anti-ACC antibodies disclosed herein. Peptides that include the amino acid sequence of any of SEQ ID NO:4 through SEQ ID NO:8 and their derivatives will be preferred for use in generating such anti-ACC antibodies.

Samples which may be tested or assayed for the presence of such ACC and ACCrelated polypeptides include whole cells, cell extracts, cell homogenates, cell-free supematants, and the like. Such cells may be either eukaryotic (such as plant cells) or prokaryotic (such as cyanobacterial and bacterial cells).

In certain aspects, diagnostic reagents comprising the novel peptides of the present invention and/or DNA segments which encode them have proven useful as test reagents for the detection of ACC and ACC-related polypeptides.

2.3 ACC Transformation and Identification of Herbicide-Resistant Variants In yet another aspect, the present invention provides a process of modulating the herbicide resistance of a plant cell by a process of transforming the plant cell with a DNA molecule comprising a promoter operatively linked to a coding region that encodes a herbicide resistant polypeptide having the ability to catalyze the carboxylation of acetyl-CoA, which coding region is operatively linked to a transcription-terminating region, whereby the promoter is capable of driving the transcription of the coding region in a monocotyledonous plant.

Preferably, a polypeptide is an acetyl-CoA carboxylase enzyme and, more preferably, a plant acetyl-CoA carboxylase. In a preferred embodiment, a coding region includes the DNA sequence of SEQ ID NO:9 or SEQ ID NO:19 and a promoter is In a preferred embodiment, a cell is a cyanobacterium or a plant cell and a plant polypeptide is a monocotyledonous plant acetyl-CoA carboxylase enzyme such as wheat acetyl-CoA carboxylase enzyme. The present invention also provides a transformed cyanobacterium produced in accordance with such a process.

WO 96/32484 PCT/US96/05095 -12- The present invention still further provides a process for determining the inheritance of plant resistance to herbicides of the aryloxyphenoxypropionate or cyclohexane-1,3-dione classes, which generally involves measuring resistance to these herbicides in a parental plant line and in the progeny of the parental plant line, detecting the presence of complexes between DNA restriction fragments and the ACC gene, and then correlating the herbicide resistance of the parental and progeny plants with the presence of particular sizes of ACC gene-containing DNA fragments as an indication of the inheritance of resistance to herbicides of these classes.

Preferably, the acetyl-CoA carboxylase is a dicotyledonous plant acetyl-CoA carboxylase enzyme or a mutated monocotyledonous plant acetyl-CoA carboxylase that confers herbicide resistance or a hybrid acetyl-CoA carboxylase comprising a portion of a dicotyledonous plant acetyl-CoA carboxylase, a portion of a monocotyledonous plant acetyl-CoA carboxylase or one or more domains of a cyanobacterial acetyl-CoA carboxylase.

Where a cyanobacterium is transformed with a plant ACC DNA molecule, that cyanobacterium can be used to identify herbicide resistant mutations in the gene encoding ACC. In accordance with such a use, the present invention provides a process for identifying herbicide resistant variants of a plant acetyl-CoA carboxylase comprising the steps of: transforming cyanobacteria with a DNA molecule that encodes a monocotyledonous plant acetyl-CoA carboxylase enzyme to form transformed or transfected cyanobacteria; inactivating cyanobacterial acetyl-CoA carboxylase; exposing the transformed cyanobacteria to an effective herbicidal amount of a herbicide that inhibits acetyl-CoA carboxylase activity; identifying transformed cyanobacteria that are resistant to the herbicide; and characterizing DNA that encodes acetyl-CoA carboxylase from the cyanobacteria of step WO 96/32484 PCT/US96/05095 -13- Means for transforming cyanobacteria as well as expression vectors used for such transformation are preferably the same as set forth above. In a preferred embodiment, cyanobacteria are transformed or transfected with an expression vector comprising a coding region that encodes wheat ACC. Cyanobacteria resistant to the herbicide are identified. Identifying comprises growing or culturing transformed cells in the presence of the herbicide and recovering those cells that survive herbicide exposure. Transformed, herbicide-resistant cells are then grown in culture, collected and total DNA extracted using standard techniques. ACC DNA is isolated, amplified if needed and then characterized by comparing that DNA with DNA from ACC known to be inhibited by that herbicide.

In still yet another aspect, the present invention provides a process for identifying herbicide resistant variants of a plant acetyl-CoA carboxylase. Such methods generally involve transforming a cyanobacterium or a bacterium or a yeast cell with a DNA molecule that encodes a plant acetyl-CoA carboxylase enzyme, inactivating the host-cell acetyl-CoA carboxylase, and exposing the cells to a herbicide that inhibits monocotyledonous plant acetyl-CoA carboxylase activity.

Transformed cells may be identified which are resistant to the herbicide; and the DNA that encodes resistant acetyl-CoA carboxylase in these transformed cells may be examined and characterized.

2.4 ACC Transgenes and Transgenic Plants In yet another aspect, the present invention provides a process of altering the carboxylation of acetyl-CoA in a cell comprising transforming the cell with a DNA molecule comprising a promoter operatively linked to a coding region that encodes a plant polypeptide having the ability to catalyze the carboxylation of acetyl-CoA, which coding region is operatively linked to a transcription-terminating region, whereby the promoter is capable of driving the transcription of the coding region in the cell. The invention also provides a means of reducing the amount of ACC in plants by expression of ACC antisense mRNA.

WO 96/32484 PCTIUS96/05095 -14- Another aspect of the invention relates generally to transgenic plants which express genes or gene segments encoding the novel polypeptide compositions disclosed herein. As used herein, the term "transgenic plants" is intended to refer to plants that have incorporated DNA sequences, including but not limited to genes which are perhaps not normally present, DNA sequences not normally transcribed into RNA or translated into a protein ("expressed"), or any other genes or DNA sequences which one desires to introduce into the non-transformed plant, such as genes which may normally be present in the non-transformed plant but which one desires to either genetically engineer or to have altered expression. It is contemplated that in some instances the genome of transgenic plants of the present invention will have been augmented through the stable introduction of the transgene. However, in other instances, the introduced gene will replace an endogenous sequence.

A preferred gene which may be introduced includes, for example, the ACC DNA sequences from cyanobacterial or plant origin, particularly those described herein which are obtained from the cyanobacterial species Synechococcus or Anabaena, or from plant species such as wheat or canola, of any of those sequences which have been genetically engineered to decrease or increase the activity of the ACC in such transgenic species.

Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) and DNA segments for use in transforming such cells will, of course, generally comprise either the cDNA, gene or gene sequences of the present invention, and particularly those encoding ACC. These DNA constructs can further include structures such as promoters, enhancers, polylinkers, or even regulatory genes as desired. The DNA segment or gene may encode either a native or modified ACC, which will be expressed in the resultant recombinant cells, and/or which will impart an improved phenotype to the regenerated plant.

Such transgenic plants may be desirable for increasing the herbicide resistance of a monocotyledonous plant, by incorporating into such a plant, a transgenic DNA segment encoding a plant acetyl-CoA carboxylase enzyme which is resistant to herbicide inactivation, a dicotyledonous ACC gene. Alternatively a WO 96/32484 PCT/US96/05095 cyanobacterial ACC polypeptide-encoding DNA segment could also be used to prepare a transgenic plant with increased resistance to herbicide inactivation.

Alternatively transgenic plants may be desirable having an decreased herbicide resistance. This would be particularly desirable in creating transgenic plants which are more sensitive to such herbicides. Such a herbicide-sensitive plant could be prepared by incorporating into such a plant, a transgenic DNA segment encoding a plant acetyl-CoA carboxylase enzyme which is sensitive to herbicide inactivation, a monocotyledonous ACC gene, or a mutated dicotyledonous or cyanobacterial ACC-encoding gene.

In other aspects of the present invention, the invention concerns processes of modifying the oil content of a plant cell. Such modifications generally involve expressing in such plant cells transgenic DNA segments encoding a plant or cyanobacterial acetyl-CoA carboxylase composition of the present invention. Such processes would generally result in increased expression of ACC and hence, increased oil production in such cells. Alternatively, when it is desirable to decrease the oil production of such cells, ACC-encoding transgenic DNA segments or antisense (complementary) DNA segments to genomic ACC-encoding DNA sequences may be used to transform cells.

Either process may be facilitated by introducing into such cells DNA segments encoding a plant or cyanobacterial acetyl-CoA carboxylase polypeptide, as long as the resulting transgenic plant expresses the acetyl-CoA carboxylase-encoding transgene.

The present invention also provides a transformed plant produced in accordance with the above process as well as a transgenic plant and a transgenic plant seed having incorporated into its genome a transgene that encodes a herbicide resistant polypeptide having the ability to catalyze the carboxylation of acetyl-CoA. All such transgenic plants having incorporated into their genome transgenic DNA segments encoding plant or cyanobacterial acetyl-CoA carboxylase polypeptides are aspects of this invention.

2.5 ACC Screening and Immunodetection Kits WO 96/32484 PCT/US96/05095 -16- The present invention contemplates methods and kits for screening samples suspected of containing ACC polypeptides or ACC-related polypeptides, or cells producing such polypeptides. Said kit can contain a nucleic acid segment or an antibody of the present invention. The kit can contain reagents for detecting an interaction between a sample and a nucleic acid or antibody of the present invention.

The provided reagent can be radio-, fluorescently- or enzymatically-labeled. The kit can contain a known radiolabeled agent capable of binding or interacting with a nucleic acid or antibody of the present invention.

The reagent of the kit can be provided as a liquid solution, attached to a solid support or as a dried powder. Preferably, when the reagent is provided in a liquid solution, the liquid solution is an aqueous solution. Preferably, when the reagent provided is attached to a solid support, the solid support can be chromatograph media, a test plate having a plurality of wells, or a microscope slide. When the reagent provided is a dry powder, the powder can be reconstituted by the addition of a suitable solvent, that may be provided.

In still further embodiments, the present invention concerns immunodetection methods and associated kits. It is proposed that the ACC peptides of the present 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 ACC or ACC-related epitope-containing peptides. In general, these methods will include first obtaining a sample suspected of containing such a protein, peptide or antibody, contacting the sample with an antibody or peptide in accordance with the present invention, as the case may be, under conditions effective to allow the formation of an immunocomplex, and then detecting the presence of the immunocomplex.

In general, the detection of immunocomplex formation is quite well known in the art and may be achieved through the application of numerous approaches. For example, the present invention contemplates the application of ELISA, RIA, immunoblot dot blot), indirect immunofluorescence techniques and the like.

Generally, immunocomplex formation will be detected through the use of a label, WO 96/32484 PCT/US96/05095 -17such as a radiolabel or an enzyme tag (such as alkaline phosphatase, horseradish peroxidase, or the like). Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art.

For assaying purposes, it is proposed that virtually any sample suspected of comprising either an ACC peptide or an ACC-related peptide or antibody sought to be detected, as the case may be, may be employed. It is contemplated that such embodiments may have application in the titering of antigen or antibody samples, in the selection of hybridomas, and the like. In related embodiments, the present invention contemplates the preparation of kits that may be employed to detect the presence of ACC or ACC-related proteins or peptides and/or antibodies in a sample.

Samples may include cells, cell supematants, cell suspensions, cell extracts, enzyme fractions, protein extracts, or other cell-free compositions suspected of containing ACC peptides. Generally speaking, kits in accordance with the present invention will include a suitable ACC peptide or an antibody directed against such a protein or peptide, together with an immunodetection reagent and a means for containing the antibody or antigen and reagent. The immunodetection reagent will typically comprise a label associated with the antibody or antigen, or associated with a secondary binding ligand. Exemplary ligands might include a secondary antibody directed against the first antibody or antigen or a biotin or avidin (or streptavidin) ligand having an associated label. 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.

The container will generally include a vial into which the antibody, antigen or detection reagent may be placed, and preferably suitably aliquotted. The kits of the present invention will also typically include a means for containing the antibody, antigen, and reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

WO 96/32484 PCT/US96105095 -18- 2.6 ELISAs and Immunoprecipitation ELISAs may be used in conjunction with the invention. In an ELISA assay, proteins or peptides incorporating ACC 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, 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 milk powder. 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 about 2 to about 4 hours, at temperatures preferably on the order of about 25° to about 27 0 C. Following incubation, the antisera-contacted surface is washed so as to remove nonimmunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween®,.or borate buffer.

Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first. To provide a detecting means, the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the antisera-bound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under WO 96/32484 PCTfS96/05095 -19conditions which favor the development of immunocomplex formation incubation for 2 hours at room temperature in a PBS-containing solution such as PBS Tween®).

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-(3ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H 2 0 2 in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, using a visible spectra spectrophotometer.

The antibodies of the present invention are particularly useful for the isolation of antigens by immunoprecipitation. Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein. For the isolation of membrane proteins cells must be solubilized into detergent micelles. Nonionic salts are preferred, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations.

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, e.g. enzyme-substrate pairs.

2.7 Western Blots The compositions of the present invention will find great use in immunoblot or western blot analysis. The anti-peptide 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 WO 96/32484 PCT/US96/05095 detecting agent, or they migrate at the same relative molecular weight as a crossreacting signal.

Immunologically-based detection methods for use in conjunction with Western blotting include enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety are considered to be of particular use in this regard.

2.8 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 anti- ACC antibodies.

As used herein, the term "incorporating an epitope(s) that is immunologically cross-reactive with one or more anti-ACC 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 an ACC polypeptide. The level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against the ACC 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 ACC immunodominant epitopes, 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 WO 96/32484 PCT/US96/05095 -21- "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 8 to 20 amino acids in length, and more preferably about 8 to about 15 amino acids in length. It is proposed that shorter antigenic ACC-derived peptides 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 ACC and ACC-related sequences. These epitopic core sequences are identified herein in particular aspects as hydrophilic regions of the ACC polypeptide antigen. It is proposed that these regions represent those which are most likely to promote T-cell or B-cell stimulation, and, hence, elicit specific antibody production.

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 transferrin-binding protein 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 anticipated by the present disclosure WO 96/32484 PCT/US96/05095 -22would generally be on the order of about 8 amino acids in length, with sequences on the order of 10 to 20 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 e.g., 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 peptides in accordance with the present disclosure.

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 commercially available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptide antigens synthesized in this manner may then be aliquotted 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°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 WO 96/32484 P-T/US96/05095 -23metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled) or buffer prior to use.

2.9 DNA Segments The present invention also concerns DNA segments, that can be isolated from virtually any source, that are free from total genomic DNA and that encode the novel peptides disclosed herein. DNA segments encoding these peptide species may prove to encode proteins, polypeptides, subunits, functional domains, and the like of ACCrelated or other non-related gene products. In addition these DNA segments may be synthesized entirely in vitro using methods that are well-known to those of skill in the art.

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 an ACC peptide refers to a DNA segment that contains ACC 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.

Similarly, a DNA segment comprising an isolated or purified ACC gene refers to a DNA segment which may include in addition to peptide encoding sequences, certain other elements such as, 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, cDNA sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides.

"Isolated substantially away from other coding sequences" means that the gene of interest, in this case, a gene encoding ACC, forms the significant part of the coding WO 96/32484 PCT/US96/05095 -24region 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 cDNA 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 an ACC peptide species that includes within its amino acid sequence an amino acid sequence essentially as set forth in any 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, SEQ ID NO:20, and SEQ ID NO:31.

The term "a sequence essentially as set forth in any 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, SEQ ID and SEQ ID NO:31" means that the sequence substantially corresponds to a portion of the sequence of either SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:20 or SEQ ID NO:31, and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of any of these sequences. The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein (for example, see Preferred 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% amino acid sequence identity or functional equivalence to the amino acids of any 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, SEQ ID NO:20, and SEQ ID NO:31 will be sequences that are "essentially as set forth in any 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, SEQ ID NO:20, and SEQ ID NO:31." 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 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 WO 96/32484 PCT/US96/05095 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 internal sequences, introns, which are known to occur within genes.

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 encoding either of the peptide sequences disclosed in any 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, SEQ ID and SEQ ID NO:31, or that are identical to or complementary to DNA sequences which encode any of the peptides 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, SEQ ID NO:20, and SEQ ID NO:31, and particularly those DNA segments 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: 1l, SEQ ID NO:19, or SEQ ID NO:30. For example, DNA sequences such as about 14 nucleotides, and that are up to about 13,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, about 100, about 50, and about 14 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, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through the 200-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; 5,000-10,000, 10,000-12,000, 12,000-13,000 and up to and WO 96/32484 PCTUS96/05095 -26including sequences of about 13,000, 13,001, 13,002, or 13,003 nucleotides etc. and the like.

It will also be understood that this invention is not limited to the particular nucleic acid sequences which encode peptides of the present invention, or which encode the amino acid sequences 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, SEQ ID NO:20, and SEQ ID NO:31, including those DNA sequences which are particularly 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, SEQ ID NO:19, and SEQ ID NO:30. Recombinant vectors and isolated DNA segments may therefore variously include the peptide-coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include these peptide-coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.

The DNA segments of the present invention encompass biologicallyfunctional equivalent peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionallyequivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, to introduce improvements to the antigenicity of the protein or to test mutants in order to examine activity at the molecular level.

If desired, one may also prepare fusion proteins and peptides, where the peptide-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).

WO 96/32484 PCT1US96/05095 -27- 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 gene encoding peptides of the present invention, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCRTM 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 DNA segment encoding an ACC peptide in its natural environment. Such promoters may include promoters normally associated with other genes, and/or promoters isolated from any bacterial, viral, eukaryotic, or plant cell. Naturally, it will be 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. Appropriate promoter systems contemplated for use in high-level expression include, but are not limited to, the Pichia expression vector system (Pharmacia LKB Biotechnology).

In connection with expression embodiments to prepare recombinant proteins and peptides, it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire peptide sequence being most preferred.

However, it will be appreciated that the use of shorter DNA segments to direct the expression of ACC peptides or epitopic core regions, such as may be used to generate WO 96/32484 PCT/US96/05095 -28anti-ACC antibodies, also falls within the scope of the invention. DNA segments that encode peptide antigens from about 8 to about 50 amino acids in length, or more preferably, from about 8 to about 30 amino acids in length, or even more preferably, from about 8 to about 20 amino acids in length are contemplated to be particularly useful. Such peptide epitopes may be amino acid sequences which comprise contiguous amino acid sequences from any 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, SEQ ID NO:20, or SEQ ID NO:31.

In addition to their use in directing the expression of ACC peptides of the present invention, the nucleic acid sequences contemplated 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 DNA segment any 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, SEQ ID NO:19, and SEQ ID will find particular utility. Longer contiguous identical or complementary sequences, those of about 20, 30, 40, 50, 100, 200, 500, 1,000, 2,000, 5,000, 8,000, 10,000, 12,000, 13,000 etc. (including all intermediate lengths and up to and including full-length sequences will also be of use in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize to ACCencoding 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 DNA sequences of any 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, SEQ ID NO:19, and SEQ ID NO:30 are particularly contemplated as hybridization probes for use in, WO 96/32484 PCT/US96/05095 -29- Southern and Northern blotting. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 10-14 and about 100 or 200 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 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 and degree of specific hybrid molec;ules obtained. One will generally prefer to design nucleic acid molecules having genecomplementary stretches of 15 to 20 contiguous nucleotides, or even longer where desired.

Of course, fragments may also be obtained by other techniques such as, e.g., 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 PCRTM technology of U.S. Patents 4,683,195 and 4,683,202 (each 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 DNA 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 WO 96/32484 PCTIUS96/05095 about 0.02 M to about 0.15 M NaC1 at temperatures of about 50'C to about 70 0

C.

Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating ACCencoding DNA segments. Detection of DNA segments via hybridization is wellknown to those of skill in the art, and the teachings of U.S. Patents 4,965,188 and 5,176,995 (each incorporated herein by reference) are exemplary of the methods of hybridization analyses. Teachings such as those found in the texts of Maloy et al., 1993; Segal 1976; Proskop, 1991; and Kuby, 1991, are particularly relevant.

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 ACC-encoding 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 about 20"C to about 55°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 WO 96/32484 PCT/US96/05095 -31eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing 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 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.

2.10 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, second-generation molecule. The amino acid changes may be achieved by changing the codons of the DNA sequence, according to the codons listed in Table 1.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU PCT/US96/05095 WO 96/32484 Amino Acids Glycine Histidine Isoleucine Lysine Leucine Methionine Asparagine Proline Glutamine Arginine Serine Threonine Valine Tryptophan Tyrosine Gly His Ile Lys Leu Met Asn Pro Gin Arg Ser Thr Val Trp Tyr Codons

GGA

CAC

AUA

AAA

UUA

AUG

AAC

CCA

CAA

AGA

AGC

ACA

GUA

UGG

UAC

GGC

CAU

AUC

AAG

UUG

AAU

CCC

CAG

AGG

AGU

ACC

GUC

UAU

GGG GGU

AUU

CUA CUC CUG CUU CCG CCU

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.

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 WO 96/32484 PCT/US96/05095 -33- 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 arginne 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 WO 96/32484 PCT/US96/05095 -34- 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.

As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain 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.

2.11 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 underlying DNA. The technique 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 17 to 25 nucleotides in length is preferred, with about 5 to 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 WO 96/32484 PCT/US96/05095 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 singlestranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutationbearing 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 sequence variants.2.12 Monoclonal Antibody Generation Means for preparing and characterizing antibodies are well known in the art (See, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference). The methods for generating monoclonal antibodies (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 WO 96/32484 PCT/US96/05095 -36the 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-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.

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 may also 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.

WO 96/32484 PCTIUS96/05095 -37mAbs 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 ACC 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 frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), 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 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 5 x 10 7 to 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 then 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, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, WO 96/32484 PCT/US96/05095 -38- NS1/1.Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 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% (v/v) PEG, (Gefter et al., 1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986, pp. 71-74).

Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10 6 to 1 x 10- 8 However, this does not pose a problem, as the viable, fused hybrids are 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.

WO 96/32484 PCTIUS96/05095 -39- 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 monoclonal antibody 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 filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

WO 96/32484 PCTIUS96/05095 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. 1. Structure of the cytosolic ACCase gene from wheat. Arrows indicate fragments of the genomic clones analyzed in more detail. Sequenced fragments are marked in black. The localization of the ACCase functional domains was established by amino acid sequence comparison with other biotin-dependent carboxylases (Gomicki et al., 1994). BC, biotin carboxylase; BCC, biotin carboxyl carrier; CT, carboxyltransferase.

FIG. 2. Alignment of cDNA sequences corresponding to the 3'-end of the mRNA encoding wheat cytosolic ACCase. Only the sequence of the 3'-end of the RACE clones is shown. The putative polyadenylation signals are underlined.

Asterisks indicate identical nucleotides. Sixteen additional 3'-RACE clones were sequenced, these matched one or another of the four sequences shown.

FIG. 3. DNA sequence of the wheat genomic ACC clone. The entire sequence is given in SEQ ID FIG. 4. Deduced amino acid sequence of the wheat genomic ACC clone shown in FIG. 3. The sequence is presented in SEQ ID NO:31.

FIG. 5. Shown is the 5' flanking sequence of the ACCase 1 gene (about 3 kb upstream of the translation initiation codon, of clone 71L. The sequence is shown in SEQ ID NO:32.

FIG. 6. Shown is the 5' flanking sequence of the ACCase 2 gene designated 153. The sequence is shown in SEQ ID NO:33.

4. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS 4.1 Definitions The following words and phrases have the meanings set forth below: WO 96/32484 PCTIUS96/05095 -41- Expression: The combination of intracellular processes, including transcription and translation undergone by a coding DNA molecule such as a structural gene to produce a polypeptide.

Promoter: A recognition site on a DNA sequence or group of DNA sequences that provide an expression control element for a structural gene and to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene.

Regeneration: The process of growing a plant from a plant cell plant protoplast or explant).

Structural gene: A gene that is expressed to produce a polypeptide.

Transformation: A process of introducing an exogenous DNA sequence a vector, a recombinant DNA molecule) into a cell or protoplast in which that exogenous DNA is incorporated into a chromosome or is capable of autonomous replication.

Transformed cell: A cell whose DNA has been altered by the introduction of an exogenous DNA molecule into that cell.

Transgenic cell: Any cell derived or regenerated from a transformed cell or derived from a transgenic cell. Exemplary transgenic cells include plant calli derived from a transformed plant cell and particular cells such as leaf, root, stem, somatic cells, or reproductive (germ) cells obtained from a transgenic plant.

Transgenic plant: A plant or progeny thereof derived from a transformed plant cell or protoplast, wherein the plant DNA contains an introduced exogenous DNA molecule not originally present in a native, non-transgenic plant of the same strain.

The terms "transgenic plant" and "transformed plant" have sometimes been used in the art as synonymous terms to define a plant whose DNA contains an exogenous DNA molecule. However, it is thought more scientifically correct to refer to a regenerated plant or callus obtained from a transformed plant cell or protoplast as being a transgenic plant, and that usage will be followed herein.

Vector: A DNA molecule capable of replication in a host cell and/or to which another DNA segment can be operatively linked so as to bring about replication of the attached segment. A plasmid is an exemplary vector.

WO 96/32484 PCT/US96/05095 -42- 4.2 Polynucleotides Amino acid sequences of biotin carboxylase (BC) from Anabaena and Synechococcus show great similarity with amino acid residue sequences from other ACC enzymes as well as with the amino acid residue sequences of other biotincontaining enzymes. Based on that homology, specific nucleotide sequences were chosen for the construction of primers for polymerase chain reaction amplification of a corresponding region of the gene for ACC from wheat. Those primers have the nucleotide sequences shown below: Primer 1 5'-TCGAATTCGTNATNATHAARGC-3' (SEQ ID NO: 13); Primer 2 5'-GCTCTAGAGKRTGYTCNACYTG-3' (SEQ ID NO: 14); where N is A, C, G or T; H is A, C or T; R is A or G; Y is T or C and K is G or T. Primers 1 and 2 comprise a 14-nucleotide specific sequence based on a conserved amino acid sequence and an 8-nucleotide extension at the 5'-end of the primer to provide anchors for rounds of amplification after the first round and to provide convenient restriction sites for analysis and cloning.

In eukaryotic ACCs, a BCCP domain is located about 300 amino acids away from the end of the BC domain, on the C-terminal side. Therefore, it is possible to amplify the cDNA covering the interval between the BC and BCCP domains using primers from the C-terminal end of the BC domain and the conserved MKM region of the BCCP. The BC primer was based on the wheat cDNA sequence obtained as described above. Those primers, each with 6- or 8-base 5'-extensions, are shown below: Primer 3 5'-GCTCTAGAATACTATTTCCTG-3' (SEQ ID Primer 4 5'-TCGAATTCWNCATYTTCATNRC-3' (SEQ ID NO:16) where N, R and Y are as defined above. W is A or T. The BC primer (primer 3) was based on the wheat cDNA sequence obtained as described above. The MKM primer (primer 4) was first checked by determining whether it would amplify thefabE gene coding BCCP from Anabaena DNA. This PCR T M was primed at the other end by using a primer based on the N-terminal amino acid residue sequence as determined on WO 96/32484 PCT/US96/05095 -43protein purified from Anabaena extracts by affinity chromatography. Those primers are shown below: Primer 5 5'-GCTCTAGAYTTYAAYGARATHMG-3' (SEQ ID NO:17) Primer 4 5'-TCGAATTCWNCATYTTCATNRC-3' (SEQ ID NO:18) where H, N, R, T, Y and W are as defined above. M is A or C. This amplification (using the conditions described above) yielded the correct fragment of the Anabaena fabE gene, which was used to identify cosmids that contained the entire fabE gene and flanking DNA. An about 4-kb XbaI fragment containing the gene was cloned into the vector pBluescriptKS® for sequencing. Primers 3 and 4 were then used to amplify the intervening sequence in wheat cDNA. Again, the product of the first PCRTM was eluted and reamplified by another round of PCRTM, then cloned into the Invitrogen vector pCRII®.

The amino acid sequence of the polypeptide predicted from the cDNA sequence for this entire fragment of wheat cDNA (1473 nucleotides) was compared with the amino acid sequences of other ACC enzymes and related enzymes from various sources. Rat, chicken and yeast are more closely related to each other than to the BC subunits of bacteria, and the BC domains of other enzymes such as pyruvate carboxylase of yeast and propionyl CoA carboxylase of rat. The amino acid identities between wheat ACC and other biotin-dependent enzymes, within the BC domain are no higher than 60%, and shown below in Table 2.

WO 96/32484 PCT/US96/05095 -44- TABLE 2 identity identity with wheat ACC with rat ACC rat ACC 58 (100) chicken ACC 57 yeast ACC 56 Synechococcus ACC 32 Anabaena ACC E. coli ACC 33 rat propionyl CoA carboxylase 32 31 yeast pyruvate carboxylase 31 4.3 Probes and Primers In another aspect, DNA sequence information provided by the invention allows for the preparation of relatively short DNA (or RNA) sequences having the ability to specifically hybridize to gene sequences of the selected polynucleotides disclosed herein. In these aspects, nucleic acid probes of an appropriate length are prepared based on a consideration of a selected ACC gene sequence, a sequence such as that shown in SEQ ID NO:9 or SEQ ID NO:19, or a selected gene sequence encoding a subunit of a cyanobacterial ACC, a sequence as that shown in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO: 11. The ability of such nucleic acid probes to specifically hybridize to an ACC gene sequence lend them particular utility in a variety of embodiments. Most importantly, the probes can be used in a variety of assays for detecting the presence of complementary sequences in a given sample.

In certain embodiments, it is advantageous to use oligonucleotide primers.

The sequence of such primers is designed using a polynucleotide of the present invention for use in detecting, amplifying or mutating a defined segment of an ACC gene from a cyanobacterium or a plant using PCRTM technology. Segments of ACC genes from other organisms may also be amplified by PCRTM using such primers.

WO 96/32484 PCT/US96/05095 To provide certain of the advantages in accordance with the present invention, a preferred nucleic acid sequence employed for hybridization studies or assays includes sequences that are complementary to at least a 14 to 30 or so long nucleotide stretch of an ACC-encoding or ACC subunit-encoding sequence, such as that shown 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: 19. A size of at least 14 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective. Molecules having 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 and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 14 to 20 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCRTM technology of U.S. Patents 4, 683,195, and 4,683,202, herein incorporated by reference, or by excising selected DNA fragments from recombinant plasmids containing appropriate inserts and suitable restriction sites.

Accordingly, a nucleotide sequence of the invention can be used for its ability to selectively form duplex molecules with complementary stretches of the gene.

Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degree of selectivity of the probe toward the target sequence. For applications requiring a high degree of selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, for example, one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaC1 at temperatures of about to about 70'C. These conditions are particularly selective, and tolerate little, if any, mismatch between the probe and the template or target strand.

Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or WO 96/32484 PCT/US96/05095 -46where one seeks to isolate an ACC coding sequences for 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 about 20'C to about 55°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 is advantageous to employ a polynucleotide of the present invention in combination with an appropriate label for detecting hybrid formation. A wide variety of appropriate labels are known in the art, including radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.

In general, it is envisioned that a hybridization probe described herein is useful both as a reagent 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 nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions depend as is well known in the art on the particular circumstances and criteria required on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe). Following washing of the matrix to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantitated, by means of the label.

4.4 Expression Vectors The present invention contemplates an expression vector comprising a polynucleotide of the present invention. Thus, in one embodiment an expression WO 96/32484 PCT/US96/05095 -47vector is an isolated and purified DNA molecule comprising a promoter operatively linked to an coding region that encodes a polypeptide having the ability to catalyze the carboxylation of a biotin carboxyl carrier protein of a cyanobacterium, which coding region is operatively linked to a transcription-terminating region, whereby the promoter drives the transcription of the coding region.

As used herein, the term "operatively linked" means that a promoter is connected to an coding region in such a way that the transcription of that coding region is controlled and regulated by that promoter. Means for operatively linking a promoter to a coding region are well known in the art.

Where an expression vector of the present invention is to be used to transform a cyanobacterium, a promoter is selected that has the ability to drive and regulate expression in cyanobacteria. Promoters that function in bacteria are well known in the art. An exemplary and preferred promoter for the cyanobacterium Anabaena is the glnA gene promoter. An exemplary and preferred promoter for the cyanobacterium Synechococcus is the psbAI gene promoter. Alternatively, the cyanobacterial acc gene promoters themselves can be used.

Where an expression vector of the present invention is to be used to transform a plant, a promoter is selected that has the ability to drive expression in plants.

Promoters that function in plants are also well known in the art. Useful in expressing the polypeptide in plants are promoters that are inducible, viral, synthetic, constitutive as described (Poszkowski et al., 1989; Odell et al., 1985), and temporally regulated, spatially regulated, and spatio-temporally regulated (Chau et al., 1989).

A promoter is also selected for its ability to direct the transformed plant cell's or transgenic plant's transcriptional activity to the coding region. Structural genes can be driven by a variety of promoters in plant tissues. Promoters can be nearconstitutive, such as the CaMV 35S promoter, or tissue-specific or developmentally specific promoters affecting dicots or monocots.

Where the promoter is a near-constitutive promoter such as CaMV increases in polypeptide expression are found in a variety of transformed plant tissues callus, leaf, seed and root). Alternatively, the effects of transformation can be WO 96/32484 PCT/US96/05095 -48directed to specific plant tissues by using plant integrating vectors containing a tissuespecific promoter.

An exemplary tissue-specific promoter is the lectin promoter, which is specific for seed tissue. The Lectin protein in soybean seeds is encoded by a single gene (Lel) that is only expressed during seed maturation and accounts for about 2 to about 5% of total seed mRNA. The lectin gene and seed-specific promoter have been fully characterized and used to direct seed specific expression in transgenic tobacco plants (Vodkin et al., 1983; Lindstrom et al., 1990.) An expression vector containing a coding region that encodes a polypeptide of interest is engineered to be under control of the lectin promoter and that vector is introduced into plants using, for example, a protoplast transformation method (Dhir et al., 1991). The expression of the polypeptide is directed specifically to the seeds of the transgenic plant.

A transgenic plant of the present invention produced from a plant cell transformed with a tissue specific promoter can be crossed with a second transgenic plant developed from a plant cell transformed with a different tissue specific promoter to produce a hybrid transgenic plant that shows the effects of transformation in more than one specific tissue.

Exemplary tissue-specific promoters are corn sucrose synthetase 1 (Yang et al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989), corn light harvesting complex (Simpson, 1986), corn heat shock protein (Odell et al., 1985), pea small subunit RuBP Carboxylase (Poulsen et al., 1986; Cashmore et al., 1983), Ti plasmid mannopine synthase (Langridge et al., 1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petunia chalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein 1 (Keller et al., 1989), CaMV 35s transcript (Odell et al., 1985) and Potato patatin (Wenzler et al., 1989). Preferred promoters are the cauliflower mosaic virus (CaMV 35S) promoter and the S-E9 small subunit RuBP carboxylase promoter.

The choice of which expression vector and ultimately to which promoter a polypeptide coding region is operatively linked depends directly on the functional properties desired, the location and timing of protein expression, and the host cell WO 96/32484 PCT/US96/05095 -49to be transformed. These are well known limitations inherent in the art of constructing recombinant DNA molecules. However, a vector useful in practicing the present invention is capable of directing the expression of the polypeptide coding region to which it is operatively linked.

Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described (Rogers et al., 1987). However, several other plant integrating vector systems are known to function in plants including pCaMVCN transfer control vector described (Fromm et al., 1985). Plasmid pCaMVCN (available from Pharmacia, Piscataway, NJ) includes the cauliflower mosaic virus CaMV promoter.

In preferred embodiments, the vector used to express the polypeptide includes a selection marker that is effective in a plant cell, preferably a drug resistance selection marker. One preferred drug resistance marker is the gene whose expression results in kanamycin resistance; the chimeric gene containing the nopaline synthase promoter, Tn5 neomycin phosphotransferase II and nopaline synthase 3' nontranslated region described (Rogers et al., 1988).

RNA polymerase transcribes a coding DNA sequence through a site where polyadenylation occurs. Typically, DNA sequences located a few hundred base pairs downstream of the polyadenylation site serve to terminate transcription. Those DNA sequences are referred to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA).

Means for preparing expression vectors are well known in the art. Expression (transformation vectors) used to transform plants and methods of making those vectors are described in United States Patent Nos. 4,971,908, 4,940,835, 4,769,061 and 4,757,011, the disclosures of which are incorporated herein by reference. Those vectors can be modified to include a coding sequence in accordance with the present invention.

A variety of methods has been developed to operatively link DNA to vectors via complementary cohesive termini or blunt ends. For instance, complementary WO 96/32484 PCT/US96/05095 homopolymer tracts can be added to the DNA segment to be inserted and to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

A coding region that encodes a polypeptide having the ability to catalyze the carboxylation of a biotin carboxyl carrier protein of a cyanobacterium is preferably a biotin carboxylase enzyme of a cyanobacterium, which enzyme is a subunit of acetyl- CoA carboxylase and participates in the carboxylation of acetyl-CoA. In a preferred embodiment, such a polypeptide has the amino acid residue sequence of SEQ ID NO:6 or SEQ ID NO:8, or a functional equivalent of those sequences. In accordance with such an embodiment, a coding region comprises the entire DNA sequence of SEQ ID NO:5 or the DNA sequence of SEQ ID NO:5 comprising the Anabaena accC gene. Alternatively, a coding region comprises the entire DNA sequence of SEQ ID NO:7 or the DNA sequence of SEQ ID NO:7 comprising the Synechococcus accC gene.

In another embodiment, an expression vector comprises a DNA segment that encodes a biotin carboxyl carrier protein of a cyanobacterium. That biotin carboxyl carrier protein preferably includes the amino acid residue sequence of SEQ ID NO:2 or SEQ ID NO:4, or functional equivalents thereof. In accordance with such an embodiment, a coding region comprises the entire DNA sequence of SEQ ID NO: 1 or the DNA sequence of SEQ ID NO:1 comprising the Anabaena accB gene.

Alternatively, a coding region comprises the entire DNA sequence of SEQ ID NO:3 or the DNA sequence of SEQ ID NO:3 comprising the Synechococcus accB gene.

In another embodiment, an expression vector comprises a DNA segment that encodes a carboxyltransferase protein of a cyanobacterium. That carboxyltransferase protein preferably includes a CTa or CTP subunit, and preferably includes the amino acid residue sequence of SEQ ID NO:12, or a functional equivalent thereof. In accordance with such an embodiment, a coding region comprises the entire DNA sequence of SEQ ID NO:11 or the DNA sequence of SEQ ID NO:11 comprising the Synechococcus accA gene.

WO 96/32484 PCTIUS96/05095 -51- In still yet another embodiment, an expression vector comprises a coding region that encodes a plant polypeptide having the ability to catalyze the carboxylation of acetyl-CoA. Such a plant polypeptide is preferably a monocotyledonous or a dicotyledonous plant acetyl-CoA carboxylase enzyme. A preferred monocotyledonous plant polypeptide encoded by such a coding region is preferably wheat ACC, which ACC includes the amino acid residue sequence of SEQ ID NO: or SEQ ID NO:31 or functional equivalents thereof. A preferred coding region includes the DNA sequence of SEQ ID NO:9 or SEQ ID NO:30. Alternatively, a preferred dicotyledonous plant ACC, such as canola ACC, is also preferred. Such an ACC enzyme is encoded by the DNA segment of SEQ ID NO:19 and has the amino acid sequence of SEQ ID Polypeptides The present invention provides novel polypeptides that define a whole or a portion of an ACC of a cyanobacterium or a plant. In one embodiment, thus, the present invention provides an isolated polypeptide having the ability to catalyze the carboxylation of a biotin carboxyl carrier protein of a cyanobacterium such as Anabaena or Synechococcus. Preferably, a biotin carboxyl carrier protein from Anabaena includes the amino acid sequence of SEQ ID NO:2, with such amino acid sequence listing encoded by the DNA segment of SEQ ID NO: 1. Preferably, a biotin carboxyl carrier protein from Synechococcus includes the amino acid sequence of SEQ ID NO:4, with such amino acid sequence listing encoded by the DNA segment of SEQ ID NO:2.

In another embodiment, the present invention provides an isolated polypeptide comprising a biotin carboxylase protein of a cyanobacterium such as Anabaena or Synechococcus. Preferably, a biotin carboxylase protein from Anabaena includes the amino acid sequence of SEQ ID NO:6, with such amino acid sequence listing encoded by the DNA segment of SEQ ID NO:5. Preferably, a biotin carboxylase protein from Synechococcus includes the amino acid sequence of SEQ ID NO:8, with such amino acid sequence listing encoded by the DNA segment of SEQ ID NO:7.

WO 96/32484 PCT/US96/05095 -52- In another embodiment, the present invention provides an isolated polypeptide comprising a carboxyltransferase protein of a cyanobacterium such as Synechococcus.

Preferably, a carboxyltransferase protein comprises a CTa or CTP subunit and includes the amino acid sequence of SEQ ID NO: 12, with such amino acid sequence listing encoded by the DNA segment of SEQ ID NO: 11.

In another embodiment, the present invention contemplates an isolated and purified plant polypeptide having a molecular weight of about 220 kDa, dimers of which have the ability to catalyze the carboxylation of acetyl-CoA. Such a polypeptide preferably includes the amino acid residue sequence of SEQ ID NO: 10 or SEQ ID NO:31, with such amino acid sequence listing encoded by the DNA segment of SEQ ID NO:9 or SEQ ID NO:30. Alternatively the present invention provides an isolated and purified plant polypeptide from canola which has the ability to catalyze the carboxylation of acetyl-CoA. Such a polypeptide preferably includes the amino acid residue sequence of SEQ ID NO:20, with such amino acid sequence listing encoded by the DNA segment of SEQ ID NO: 19.

4.6 Transformed or Transgenic Cells or Plants A cyanobacterium, a yeast cell, or a plant cell or a plant transformed with an expression vector of the present invention is also contemplated. A transgenic cyanobacterium, yeast cell, plant cell or plant derived from such a transformed or transgenic cell is also contemplated. Means for transforming cyanobacteria and yeast cells are well known in the art. Typically, means of transformation are similar to those well known means used to transform other bacteria or yeast such as E. coli or Saccharomyces cerevisiae. Synechococcus can be transformed simply by incubation of log-phase cells with DNA. (Golden et al., 1987) Methods for DNA transformation of plant cells include Agrobacteriummediated plant transformation, protoplast transformation, gene transfer into pollen, injection into reproductive organs, injection into immature embryos and particle bombardment. Each of these methods has distinct advantages and disadvantages.

Thus, one particular method of introducing genes into a particular plant strain may not WO 96/32484 PCT/US96/05095 -53necessarily be the most effective for another plant strain, but it is well known which methods are useful for a particular plant strain.

There are many methods for introducing transforming DNA segments into cells, but not all are suitable for delivering DNA to plant cells. Suitable methods are believed to include virtually any method by which DNA can be introduced into a cell, such as by Agrobacterium infection, direct delivery of DNA such as, for example, by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993), by desiccation/inhibition-mediated DNA uptake, by electroporation, by agitation with silicon carbide fibers, by acceleration of DNA coated particles, etc. In certain embodiments, acceleration methods are preferred and include, for example, microprojectile bombardment and the like.

Technology for introduction of DNA into cells is well-known to those of skill in the art. Four general methods for delivering a gene into cells have been described: chemical methods (Graham and van der Eb, 1973; Zatloukal etal., 1992); (2) physical methods such as microinjection (Capecchi, 1980), electroporation (Wong and Neumann, 1982; Fromm et al., 1985) and the gene gun (Johnston and Tang, 1994; Fynan etal., 1993); viral vectors (Clapp, 1993; Lu etal., 1993; Eglitis and Anderson, 1988a; 1988b); and receptor-mediated mechanisms (Curiel et al., 1991; 1992; Wagner et al., 1992).

4.6.1 Electroporation The application of brief, high-voltage electric pulses to a variety of animal and plant cells leads to the formation of nanometer-sized pores in the plasma membrane.

DNA is taken directly into the cell cytoplasm either through these pores or as a consequence of the redistribution of membrane components that accompanies closure of the pores. Electroporation can be extremely efficient and can be used both for transient expression of clones genes and for establishment of cell lines that carry integrated copies of the gene of interest. Electroporation, in contrast to calcium phosphate-mediated transfection and protoplast fusion, frequently gives rise to cell lines that carry one, or at most a few, integrated copies of the foreign DNA.

WO 96/32484 PCT/US96/05095 -54- The introduction of DNA by means of electroporation, is well-known to those of skill in the art. In this method, certain cell wall-degrading enzymes, such as pectindegrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells. Alternatively, recipient cells are made more susceptible to transformation, by mechanical wounding. To effect transformation by electroporation one may employ either friable tissues such as a suspension culture of cells, or embryogenic callus, or alternatively, one may transform immature embryos or other organized tissues directly. One would partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wounding in a controlled manner. Such cells would then be recipient to DNA transfer by electroporation, which may be carried out at this stage, and transformed cells then identified by a suitable selection or screening protocol dependent on the nature of the newly incorporated DNA.

4.6.2 Microprojectile Bombardment A further advantageous method for delivering transforming DNA segments to plant cells is microprojectile bombardment. In this method, particles may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like.

An advantage of microprojectile bombardment, in addition to it being an effective means of reproducibly stably transforming monocots, is that neither the isolation of protoplasts (Cristou et al., 1988) nor the susceptibility to Agrobacterium infection is required. An illustrative embodiment of a method for delivering DNA into maize cells by acceleration is a Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with corn cells cultured in suspension. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectiles WO 96/32484 PCT/US96/05095 aggregate and may contribute to a higher frequency of transformation by reducing damage inflicted on the recipient cells by projectiles that are too large.

For the bombardment, cells in suspension are preferably concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. If desired, one or more screens are also positioned between the acceleration device and the cells to be bombarded. Through the use of techniques set forth herein one may obtain up to 1000 or more foci of cells transiently expressing a marker gene. The number of cells in a focus which express the exogenous gene product 48 hours post-bombardment often range from 1 to 10 and average 1 to 3.

In bombardment transformation, one may optimize the prebombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment are important in this technology. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the flight and velocity of either the macro- or microprojectiles. Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmids. It is believed that pre-bombardment manipulations are especially important for successful transformation of immature embryos.

Accordingly, it is contemplated that one may wish to adjust various of the bombardment parameters in small scale studies to fully optimize the conditions. One may particularly wish to adjust physical parameters such as gap distance, flight distance, tissue distance, and helium pressure. One may also minimize the trauma reduction factors (TRFs) by modifying conditions which influence the physiological state of the recipient cells and which may therefore influence transformation and integration efficiencies. For example, the osmotic state, tissue hydration and the subculture stage or cell cycle of the recipient cells may be adjusted for optimum WO 96/32484 PCT/US96/05095 -56transformation. The execution of other routine adjustments will be known to those of skill in the art in light of the present disclosure.

Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art. See, for example, the methods described (Fraley et al., 1985; Rogers et al., 1987). Further, the integration of the Ti-DNA is a relatively precise process resulting in few rearrangements. The region of DNA to be transferred is defined by the border sequences, and intervening DNA is usually inserted into the plant genome as described (Spielmann et al., 1986; Jorgensen et al., 1987).

Modem Agrobacterium transformation vectors are capable of replication in E.

coli as well as Agrobacterium, allowing for convenient manipulations as described (Klee et al., 1985). Moreover, recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate construction of vectors capable of expressing various polypeptide coding genes. The vectors described (Rogers et al., 1987), have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes. In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant strains where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.

Agrobacterium-mediated transformation of leaf disks and other tissues such as cotyledons and hypocotyls appears to be limited to plants that Agrobacterium naturally infects. Agrobacterium-mediated transformation is most efficient in dicotyledonous plants. Few monocots appear to be natural hosts for Agrobacterium, although transgenic plants have been produced in asparagus using Agrobacterium vectors as described (Bytebier et al., 1987). Therefore, commercially important cereal grains such as rice, corn, and wheat must usually be transformed using alternative WO 96/32484 PCTfUS96/05095 -57methods. However, as mentioned above, the transformation of asparagus using Agrobacterium can also be achieved (see, for example, Bytebier et al, 1987).

A transgenic plant formed using Agrobacterium transformation methods typically contains a single gene on one chromosome. Such transgenic plants can be referred to as being heterozygous for the added gene. However, inasmuch as use of the word "heterozygous" usually implies the presence of a complementary gene at the same locus of the second chromosome of a pair of chromosomes, and there is no such gene in a plant containing one added gene as here, it is believed that a more accurate name for such a plant is an independent segregant, because the added, exogenous gene segregates independently during mitosis and meiosis.

More preferred is a transgenic plant that is homozygous for the added structural gene; a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains a single added gene, germinating some of the seed produced and analyzing the resulting plants produced for enhanced carboxylase activity relative to a control (native, non-transgenic) or an independent segregant transgenic plant.

It is to be understood that two different transgenic plants can also be mated to produce offspring that contain two independently segregating added, exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both added, exogenous genes that encode a polypeptide of interest. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated.

Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, for example, Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1986; Uchimiya et al., 1986; Callis et al., 1987; Marcotte et al., 1988).

Application of these systems to different plant strains depends upon the ability to regenerate that particular plant strain from protoplasts. Illustrative methods for the WO 96/32484 PCT/US96/05095 -58regeneration of cereals from protoplasts are described (Fujimura et al., 1985; Toriyama et al., 1986; Yamada et al., 1986; Abdullah et al., 1986).

To transform plant strains that cannot be successfully regenerated from protoplasts, other ways to introduce DNA into intact cells or tissues can be utilized.

For example, regeneration of cereals from immature embryos or explants can be effected as described (Vasil, 1988). In addition, "particle gun" or high-velocity microprojectile technology can be utilized. (Vasil, 1992) Using that latter technology, DNA is carried through the cell wall and into the cytoplasm on the surface of small metal particles as described (Klein et al., 1987; Klein et al., 1988; McCabe et al., 1988). The metal particles penetrate through several layers of cells and thus allow the transformation of cells within tissue explants.

Thus, the amount of a gene coding for a polypeptide of interest a polypeptide having carboxylation activity) can be increased in monocotyledonous plants such as corn by transforming those plants using particle bombardment methods (Maddock et al., 1991). By way of example, an expression vector containing an coding region for a dicotyledonous ACC and an appropriate selectable marker is transformed into a suspension of embryonic maize (corn) cells using a particle gun to deliver the DNA coated on microprojectiles. Transgenic plants are regenerated from transformed embryonic calli that express ACC. Particle bombardment has been used to successfully transform wheat (Vasil et al., 1992).

DNA can also be introduced into plants by direct DNA transfer into pollen as described (Zhou et al., 1983; Hess, 1987; Luo et al., 1988). Expression of polypeptide coding genes can be obtained by injection of the DNA into reproductive organs of a plant as described (Pena et al., 1987). DNA can also be injected directly into the cells of immature embryos and the rehydration of desiccated embryos as described (Neuhaus et al., 1987; Benbrook et al., 1986).

The development or regeneration of plants from either single plant protoplasts or various explants is well known in the art (Weissbach and Weissbach, 1988). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of WO 96/32484 PCT/US96/05095 -59embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign, exogenous gene that encodes a polypeptide of interest introduced by Agrobacterium from leaf explants can be achieved by methods well known in the art such as described (Horsch et al., 1985). In this procedure, transformants are cultured in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant strain being transformed as described (Fraley et al., 1983).

This procedure typically produces shoots within two to four months and those shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Shoots that rooted in the presence of the selective agent to form plantlets are then transplanted to soil or other media to allow the production of roots. These procedures vary depending upon the particular plant strain employed, such variations being well known in the art.

Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants, as discussed before. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important, preferably inbred lines. Conversely, pollen from plants of those important lines is used to pollinate regenerated plants.

A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art. Any of the transgenic plants of the present invention can be cultivated to isolate the desired ACC or fatty acids which are the products of the series of reactions of which that catalyzed by ACC is the first.

A transgenic plant of this invention thus has an increased amount of an coding region gene) that encodes a polypeptide of interest. A preferred transgenic plant is an independent segregant and can transmit that gene and its activity to its progeny.

A more preferred transgenic plant is homozygous for that gene, and transmits that gene to all of its offspring on sexual mating.

WO 96/32484 PCT/US96/05095 Seed from a transgenic plant is grown in the field or greenhouse, and resulting sexually mature transgenic plants are self-pollinated to generate true breeding plants.

The progeny from these plants become true breeding lines that are evaluated for, by way of example, herbicide resistance, preferably in the field, under a range of environmental conditions.

The commercial value of a transgenic plant with increased herbicide resistance or with altered fatty acid production is enhanced if many different hybrid combinations are available for sale. The user typically grows more than one kind of hybrid based on such differences as time to maturity, standability or other agronomic traits. Additionally, hybrids adapted to one part of a country are not necessarily adapted to another part because of differences in such traits as maturity, disease and herbicide resistance. Because of this, herbicide resistance is preferably bred into a large number of parental lines so that many hybrid combinations can be produced.

4.7 Process of Increasing Herbicide Resistance Herbicides such as aryloxyphenoxypropionates and cyclohexane-l ,3-dione derivatives inhibit the growth of monocotyledonous weeds by interfering with fatty acid biosynthesis of herbicide sensitive plants. ACC is the target enzyme for those herbicides. Dicotyledonous plants, other eukaryotic organisms and prokaryotic organisms are resistant to those compounds.

Thus, the resistance of sensitive monocotyledonous plants to herbicides can be increased by providing those plants with ACC that is not sensitive to herbicide inhibition. The present invention therefore provides a process of increasing the herbicide resistance of a monocotyledonous plant comprising transforming the plant with a DNA molecule comprising a promoter operatively linked to a coding region that encodes a herbicide resistant polypeptide having the ability to catalyze the carboxylation of acetyl-CoA, which coding region is operatively linked to a transcription-terminating region, whereby the promoter is capable of driving the transcription of the coding region in a monocotyledonous plant.

WO 96/32484 PCT/US96/05095 -61- Preferably, a herbicide resistant polypeptide, a dicotyledonous plant polypeptide such as an acetyl-CoA carboxylase enzyme from soybean, rape, sunflower, tobacco, Arabidopsis, petunia, canola, pea, bean, tomato, potato, lettuce, spinach, alfalfa, cotton or carrot, or functional equivalent thereof. A promoter and a transcription-terminating region are preferably the same as set forth above.

Transformed monocotyledonous plants can be identified using herbicide resistance. A process for identifying a transformed monocotyledonous plant cell involves transforming the monocotyledonous plant cell with a DNA molecule that encodes a dicotyledonous acetyl-CoA carboxylase enzyme, and determining the resistance of the plant cell to a herbicide and thereby the identification of the transformed monocotyledonous plant cell. Means for transformirg a monocotyledonous plant cell are the same as set forth above.

The resistance of a transformed plant cell to a herbicide is preferably determined by exposing such a cell to an effective herbicidal dose of a preselected herbicide and maintaining that cell for a period of time and under culture conditions sufficient for the herbicide to inhibit ACC, alter fatty acid biosynthesis or retard growth. The effects of the herbicide can be studied by measuring plant cell ACC activity, fatty acid synthesis or growth.

An effective herbicidal dose of a given herbicide is that amount of the herbicide that retards growth or kills plant cells not containing herbicide-resistant ACC or that amount of a herbicide known to inhibit plant growth. Means for determining an effective herbicidal dose of a given herbicide are well known in the art. Preferably, a herbicide used in such a process is an aryloxyphenoxypropionate or cyclohexanedione herbicide.

4.8 Process of Altering ACC Activity ACC catalyzes the carboxylation of acetyl-CoA. Thus, the carboxylation of acetyl-CoA in a cyanobacterium or a plant can be altered by, for example, increasing an ACC gene copy number or changing the composition nucleotide sequence) of an ACC gene. Changes in ACC gene composition may alter gene expression at either WO 96/32484 PCT/US96/05095 -62the transcriptional or translational level. Alternatively, changes in gene composition can alter ACC function activity, binding) by changing primary, secondary or tertiary structure of the enzyme. By way of example, certain changes in ACC structure are associated with changes in the resistance of that altered ACC to herbicides. The copy number of such a gene can be increased by transforming a cyanobacterium or a plant cell with an appropriate expression vector comprising a DNA molecule that encodes ACC.

In one embodiment, therefore, the present invention contemplates a process of altering the carboxylation of acetyl-CoA in a cell comprising transforming the cell with a DNA molecule comprising a promoter operatively linked to a coding region that encodes a polypeptide having the ability to catalyze the carboxylation of acetyl- CoA, which coding region is operatively linked to a transcription-terminating region, whereby the promoter is capable of driving the transcription of the coding region in the cyanobacterium.

In a preferred embodiment, a cell is a cyanobacterium or a plant cell, a polypeptide is a cyanobacterial ACC or a plant ACC. Exemplary and preferred expression vectors for use in such a process are the same as set forth above.

4.9 Determining Herbicide Resistance Inheritability In yet another aspect, the present invention provides a process for determining the inheritance of plant resistance to herbicides of the aryloxyphenoxypropionate or cyclohexanedione class. That process involves measuring resistance to herbicides of the aryloxyphenocypropionate or cyclohexanedione class in a parental plant line and in progeny of the parental plant line and detecting the presence of a DNA segment encoding ACC in such plants.

The inheritability of phenotypic traits such as herbicide resistance can be determined using RFLP analysis. Restriction fragment length polymorphisms (RFLPs) are due to sequence differences detectable by lengths of DNA fragments generated by digestion with restriction enzymes and typically revealed by agarose gel electrophoresis. There are large numbers of restriction endonucleases available, WO 96/32484 PCT/US96/05095 -63characterized by their recognition sequences and source. From these studies, it is possible to correlate herbicide resistance with a particular DNA fragment and analyze the inheritance of such resistance in progeny plants.

In a preferred embodiment, the herbicide resistant variant of acetyl-CoA carboxylase is a dicotyledonous plant acetyl-CoA carboxylase enzyme or a portion thereof. In another preferred embodiment, the herbicide resistant variant of acetyl- CoA carboxylase is a mutated monocotyledonous plant acetyl-CoA carboxylase that confers herbicide resistance or a hybrid acetyl-CoA carboxylase comprising a portion of a dicotyledonous plant acetyl-CoA carboxylase, a portion of a monocotyledonous plant acetyl-CoA carboxylase or one or more domains of a cyanobacterial acetyl-CoA carboxylase.

Restriction fragment length polymorphism analyses are conducted, for example, by Native Plants Incorporated (NPI). This service is available to the public on a contractual basis. For this analysis, the genetic marker profile of the parental inbred lines is determined. If parental lines are essentially homozygous at all relevant loci they should have only one allele at each locus), the diploid genetic marker profile of the hybrid offspring of the inbred parents should be the sum of those parents, if one parent had the allele A at a particular locus, and the other parent had B, the hybrid AB is by inference.

Probes capable of hybridizing to specific DNA segments under appropriate conditions are prepared using standard techniques well known to those skilled in the art. The probes are labelled with radioactive isotopes or fluorescent dyes for ease of detection. After restriction fragments are separated by size, they are identified by hybridization to the probe. Hybridization with a unique cloned sequence permits the identification of a specific chromosomal region (locus). Because all alleles at a locus are detectable, RFLP's are co-dominant alleles. They differ from some other types of markers, from isozymes, in that they reflect the primary DNA sequence, they are not products of transcription or translation.

WO 96/32484 PCT/US96/05095 -64- 4.10 Oil Content of Seeds Manipulation of the oil content and quality of seeds may benefit from knowledge of this gene's structure and regulation. Understanding the basis of resistance to herbicides, on the other hand, will be useful for future attempts to construct transgenic grasses and to provide crop plants such as wheat with selective resistance.

Genes of the present invention may be introduced into plants, particularly monocotyledonous plants, particularly commercially important grains. A wide range of novel transgenic plants produced in this manner may be envisioned depending on the particular constructs introduced into the transgenic plants. The largest use of grain is for feed or food. Introduction of genes that alter the composition of the grain may greatly enhance the feed or food value.

The introduction of genes encoding ACC may alter the oil content of the grain, and thus may be of significant value. Increases in oil content may result in increases in metabolizable-energy-content and -density of the seeds for uses in feed and food.

The introduction of genes such as ACC which encode rate-limiting enzymes in fatty acid biosynthesis, or replacement of these genes through gene disruption or deletion mutagenesis could have significant impact on the quality and quantity of oil in such transgenic plants.

Likewise, the introduction of the ACC genes of the present invention may also alter the balance of fatty acids present in the oil providing a more healthful or nutritive feedstuff. Alternatively, oil properties may also be altered to improve its performance in the production and use of cooking oil, shortenings, lubricants or other oil-derived products or improvement of its health attributes when used in the food-related applications. Such changes in oil properties may be achieved by altering the type, level, or lipid arrangement of the fatty acids present in the oil. This in turn may be accomplished by the addition of genes that encode enzymes that catalyze the synthesis of novel fatty acids and the lipids possessing them or by increasing levels of native fatty acids while possibly reducing levels of precursors.

WO 96/32484 PCT/US96/05095 Alternatively, introduction of DNA segments which are complementary to the DNA segments disclosed herein into plant cells may bring about a decrease in ACC activity in vivo and lower the level of fatty acid biosynthesis in such transformed cells.

Therefore, transgenic plants containing such novel constructs may be important due to their decreased oil content in such cells. Introduction of specific mutations in either the DNA segments disclosed, or in their complements, may result in transformed plants having intermediate ACC activity.

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 inventor 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.

EXAMPLES

5.1 EXAMPLE 1 Cloning and Sequencing of the Anabaena acc Genes 5.1.1 Biotin Carboxylase (accC) The gene for the BC subunit was cloned with a fragment of the E. colifabG gene as a heterologous hybridization probe. Southern analysis of Anabaena sp. strain PCC 7120 DNA digested with various restriction enzymes, carried out at low stringency (57°C, 1 M NaC1, GeneScreen Plus® membrane [DuPont]) in accordance with the manufacturer's protocol, with an SstlI-PstI fragment consisting of -90% of the coding region of thefabG gene from E. coli as a probe revealed, in each case, only one strongly hybridizing restriction fragment. The 3.1-kb HindI fragment identified by this probe in the Anabaena sp. strain PCC 7120 DNA digest was purified by gel electrophoresis and then was digested with NheI, yielding a 1.6-kb NheI-HindI fragment that hybridized with the samefabG probe. The 1.6-kb fragment was purified WO 96/32484 PCT/US96/05095 -66by gel electrophoresis and cloned into XbaI-HindII-digested pUC 18. The ends of the insert were sequenced.

A fragment of an open reading frame coding for a polypeptide with very high similarity to an internal sequence of E. coli BC was found at the NheI end of the insert. This result indicated that the 3.1-kb HindR fragment contained the entire Anabaena sp. strain PCC 7120 BC gene. The 1.6-kb Anabaena sp strain PCC 7120 DNA fragment was then used as a probe to screen, at high stringency (65'C, 1 M NaC1), a cosmid library of Anabaena sp. strain PCC 7120 DNA in the cosmid vector pWB79 (Charng et al., 1992), constructed by W.J. Buikema (University of Chicago) with a sized partial HindlII digest of chromosomal DNA. Five cosmids containing overlapping fragments of Anabaena sp. strain PCC 7120 DNA were found in the 1,920-member bank, all of which contained the same size HindIl and NheI fragments as those identified by the E. coli probe previously. From one of the cosmids, the 3.1-kb HindIII fragment was subcloned into pUC 18 and sequenced.

Nucleotide sequences of both strands were determined on double-stranded templates by the dideoxy chain termination method with Sequenase (United States Biochemicals). Sets of nested deletions generated with an Erase-a-Base kit (Promega) as well as specific primers were used for sequencing. The 3065-nucleotide DNA segment comprising the Anabaena accC gene is given in SEQ ID NO:5. The 477amino acid translation of the accC gene encoding the Anabaena BC protein is given in SEQ ID NO:6.

5.1.2 Biotin Carboxyl Carrier Protein (accB) A different approach had to be used to clone the Anabaena sp. strain PCC 7120 BCCP gene. An earlier attempt to clone the gene with a fragment of E. coli DNA containing the fabE gene as a heterologous hybridization probe failed.

Furthermore, analysis of the sequence located upstream of the Anabaena sp.

strain PCC 7120 BC gene revealed no open reading frame corresponding to BCCP, in contrast to the E. coli gene organization in which the BCCP gene is located WO 96/32484 PCT/US96/05095 -67immediately upstream of the BC gene. The BCCP gene was cloned by PCR T M amplification.

The N-terminal amino acid sequence of BCCP was used to design an upstream PCRTM primer. The downstream primer was targeted to the conserved sequence encoding the biotinylation site. The primers had the following structure: Amino acid sequence: LDFNEIR (SEQ ID NO:22) Primer I 5'-GCTCTAGAYTTYAAYGARATHMG-3' (SEQ ID NO:23) Amino acid sequence: NMKMX (SEQ ID NO:24) V or A) Primer II 3'-CRNTACTTYTACNWCTTAAGCT-5' (SEQ ID where Y= T or C; R= A or G; M= C or A; H= A, C, or T; W= A or T; N= T, C, A, or G.

PCRTM was carried out as described in the GeneAmp® kit manual (Perkin-Elmer Cetus). All components of the PCRTM except the Taq DNA polymerase were incubated for 3 to 5 min at 95 0 C. The PCR T M was then initiated by the addition of polymerase. Amplification was for 45 cycles, each 1 min at 95 0 C, 1 min at 42 to and 2 min at 72 0 C, with 0.5 to 1.0 tg of template DNA per ml and 50 gLg of each primer per ml. The PCR T M amplification yielded a product -450 bp in size the correct size for the anticipated fragment of the Anabaena sp. strain PCC 7120 BCCP gene deduced from the E. coli sequence and allowing for a 60- to addition due to the polypeptide length difference). The PCR T M product was cloned into the Invitrogen vector pCR1000 with the A/T tail method and was sequenced to confirm its identity.

The fragment of the Anabaena sp. strain PCC 7120 BCCP gene was then used as a probe to identify cosmids that contain the entire gene and flanking DNA. Three such cosmids were detected in a 1,920-member library (same as described above). A 4.2-kb XbaI fragment containing the BCCP gene was subcloned into pBluescriptII®, and its HindHl-NheI fragment was sequenced with specific primers as described above. The 1458-nucleotide DNA segment comprising the Anabaena accB gene is WO 96/32484 PCT/US96/05095 -68given in SEQ ID NO:1. The 182-amino acid translation of the accB gene encoding the Anabaena BCCP is given in SEQ ID NO:2.

The amino acid sequence deduced from the DNA sequence of the BCCP gene exactly matches the N-terminal sequence obtained for purified protein. Likely translation initiation codons were identified by comparison with E. coli. For the BC gene, the AUG start codon is not preceded by an obvious ribosome-binding site.

There is a stop codon in the same open reading frame one codon upstream from the AUG codon, excluding the possibility of additional amino acids at the N terminus.

The GUG start codon for BCCP immediately precedes codons for the amino acids identified by protein sequencing of the N terminus of purified BCCP. A putative ribosome-binding site, GAGGU, is located 11 nucleotides upstream of the GUG codon. The open reading frame extends further upstream of the GUG codon (for about 60 codons), but there are no AUG or GUG codons that could serve as start sties from translation. This excludes the possibility that the purified BCCP polypeptide lacks more than one amino acid (Met) because of rapid proteolytic degradation.

Structural similarities deduced from the available amino acid sequences suggest strong evolutionary conservation among BCs (Al-Feel et al., 1992; Knowles, 1989; Lopez-Casillas etal., 1988; Samols etal., 1988; Takai etal., 1988).

Comparison of the amino acid sequence of the BC domain defined as the part of the sequence between amino acids Lys-5 and Phe-432 of Anabaena sp. strain PCC 7120 BC, the two outermost amino acids present in all or all but one of the compared sequences, revealed that all highly conserved amino acid residues identified before are present in Anabaena sp. strain PCC 7120 BC, including the ATP binding site motif and the conserved sequence including Cys-230 as a part of the bicarbonate binding site. The identity between the amino acid sequence of the Anabaena sp. strain PCC 7120 BC domain (based on the best multiple alignment) and that of rat (Lopez-Casillas et al., 1988), chicken (Takai et al., 1988), yeast (Al-Feel et al., 1992), and wheat ACCs was no more than 32 to 37%. Mitochondrial enzymes, rat propionyl-CoA carboxylase (Browner et al., 1989) and yeast pyruvate carboxylase WO 96/32484 PCT/US96/05095 -69- (Lim et al., 1988), are only 45 to 47% identical. Similarities with carbamoyl-phosphate synthetases observed for other BCs (Knowles, 1989; Li and Cronan, 1992; Lopez-Casillas et al., 1988; Samols et al., 1988; Takai et al., 1988) are also evident for Anabaena sp. strain PCC 7120 BC.

Anabaena sp. strain PCC 7120 BCCP is unique with its biotinylation site, the result of a single A-to-C base change resulting in a Met-to-Leu substitution. This base change explains the highly variable yield of the PCR T M amplification with primer II.

The structure of this part of the BCCP gene was confirmed by sequencing the corresponding PCRTM-cloned fragment of Anabaena sp. strain PCC 7120 DNA. The result is not entirely surprising, because in vitro analysis of mutants of the 1.3S subunit of transcarboxylase from Propionibacterium shermanii, in which the same Met-to-Leu change was introduced, showed that this methionine residue is not essential for efficient biotinylation of the apoprotein (Shenoy et al., 1992). Urea carboxylase contains Ala at this position. The conserved motif may be required for some other functions. Furthermore, it was suggested that the distance between the biotinylated lysine residue and the C terminus and the structure of the last two amino acids (hydrophobic one followed by acidic one) are important determinants for the modification of at least some BCCP apoproteins (Shenoy et al., 1992). Two amino acids with the same properties are also found at an analogous position (with respect to the distance from the biotinylation site) of large eukaryotic biotin-dependent carboxylases. Anabaena sp. strain PCC 7120 BCCP also contains those amino acids, but they are separated from the biotinylation site by two additional amino acids.

Anabaena sp. strain PCC 7120 BCCP is about 30 amino acids longer than the E. coli protein, including a 21-amino-acid insertion near the N terminus. The moderate conservation of the amino acid sequence is reflected by rather low conservation at the nucleotide level (Table which explains why the E. coli BCCP specific probe failed to identify the Anabaena sp. strain PCC 7120 gene.

Comparison of the amino acid sequence encoded by the additional short open reading frame located upstream of the BCCP gene and transcribed in the same WO 96/32484 PCT/US96/05095 direction and sequences deposited in GenBank (release 75) revealed no similar proteins.

5.1.3 Northern analysis of the BCCP message The size of Anabaena sp. strain PCC 7120 BCCP mRNA was established by Northern (RNA) analysis with the PCR

TM

-amplified fragment of the gene as a probe.

The major hybridizing mRNA is 1.45-kb in size. The two minor species are 1.85 and 2.05-kb in size. All of these are long enough to include the BCCP coding region. The amount of all three mRNAs seems to be higher (about twofold) in cells grown in the absence of combined nitrogen. The 24-h induction time correlates with the onset of nitrogen fixation in heterocysts, differentiated cells that fix nitrogen and have a unique glycolipid envelope containing C 26 and C 28 fatty acids (Murata and Nishida, 1987). If the increase of the level of the BCCP mRNA is heterocyst specific, it must be significant because heterocysts in Anabaena sp. strain PCC 7120 filaments are formed only at -10-cell intervals. This result suggests that ACC may be developmentally regulated in Anabaena sp. strain PCC 7120. Results of some recent experiments indicate that, in bacteria, modulation of ACC activity may indeed play an important role in the overall regulation of the biosynthesis of the cell lipids. It has been demonstrated that the level of transcription of the ACC genes is correlated in E. coli with the rate of cellular growth and nutritional upshifts and downshifts (Li and Cronan, 1993). Mutations in the E. colifabGE operon which decrease the rate of phospholipid biosynthesis suppress a null mutation in the htrB gene by restoring the balance between phospholipid biosynthesis and cell growth (Karow et al., 1992).

Northern analysis with the 1.6-kb NheI-Hindml fragment as a BC-specific probe repeatedly gave a smeared band pattern which could not be interpreted.

Unlike the BCCP and BC genes of E. coli where they are cotranscribed, the BCCP and BC genes of the present invention are separated by at least several kilobases (no overlapping cosmids were seen when the cosmid library was screened with probes specific for BCCP and BC).

WO 96/32484 PCT/US96/05095 -71- 5.2 EXAMPLE 2 Purification and Characterization ofAnabaena BCCP Western immunoblot analysis of Anabaena sp. strain PCC 7120 proteins with 35 S-streptavidin revealed one biotinylated polypeptide -25 kDa in size. Although the presence of other, much less abundant biotinylated proteins cannot be strictly ruled out, this result strongly suggests that ACC is the only biotin-dependent enzyme in Anabaena sp. strain PCC 7120, with the BCCP subunit of 19 kDa, the calculated size; kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE).

The polypeptide shows a slightly lower mobility than E. coli BCCP (-22.5 kDa), suggesting that Anabaena sp. strain PCC 7120 BCCP is longer by 20 to amino acids. However, the unusual electrophoretic properties of the E. coli protein (Li and Cronan, 1992) make an accurate prediction of the polypeptide length difficult.

Separation of Anabaena sp. strain PCC 7120 proteins for Western analysis or sequencing) was by SDS-PAGE with 12.5% separating gels (Sambrook et al., 1989) followed by transfer onto polyvinylidene difluoride membrane (Immobilon-P®; Millipore) in 10 mM sodium 3-(cyclohexylamino)-l-propane-sulfonate buffer (pH 11)-10% methanol. Western blots were blocked with 3% bovine serum albumin solution in 10 mM Tris-HCl (pH 7.5) and 0.9% NaCI and then were incubated for 3 to 16 h with 35 S-streptavidin (Amersham). The blots were washed at room temperature with 0.5% Nonidet P-40TM in 10 mM Tris-HCl (pH 7.5) and 0.9% NaC1.

WO 96/32484 PCTIUS96/05095 -72- TABLE 3 COMPARISON OF BC AND BCCP SUBUNITS FROM Anabaena AND E. coli ACC subunita No. of amino acids (mol wt) Identity (similarity) Anabaena sp. strain E. colic PCC 7120

BC

Protein 447 (49,076) 449 57 (74) DNAd 58

BCCP

Protein 182 (19,126) 156 39(65) DNAd 41 a The genes for the two subunits of ACC are unlinked in Anabaena sp. strain PCC 7120; in E. coli they are in one operon.

b Molecular weight was calculated from amino acid composition.

c From Li and Cronan, 1992.

d On the basis of amino acid alignment.

BCCP from Anabaena sp. strain PCC 7120 was purified starting with cells from a 3-liter culture grown on BGl1 medium (Rippka et al., 1979). Cells were broken by sonication at 0°C in 30 ml of 0.5 m NaCl-0.1 M Tris-HC1 (pH 7.5)-14 mM P-mercaptoethanol-0.2 mM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 31,000 x g for 30 min, and the soluble protein fraction containing BCCP was precipitated by adding solid ammonium sulfate saturation). The pellet was resuspended in 15 ml of 0.2 M NaCl-50 mM Tris-HCl (pH 7.5)-10% glycerol-0.5% SDS and then mixed at room temperature for about 18 h with ml of streptavidin-agarose suspension (GIBCO BRL). The mixture was loaded onto a column, was washed with about 30 ml of 0.25 M NaCl-50 mM Tris-HCl (pH 7.5)-0.5 mM EDTA-0.2% SDS, and then was washed with 5 ml of water.

Biotinylated peptides were eluted with 3 ml of 70% formic acid, dried under vacuum, WO 96/32484 PCT/US96/05095 -73and separated by SDS-PAGE. The N-terminal sequence of the biotin-containing polypeptide was determined by Edman degradation after transfer to Immobilon-P® as described above. The sequence was PLDFNEIRQL (SEQ ID NO:21).

5.3 EXAMPLE 3 Characterization of the Synechococcus ace Genes and Purification of the Synechococcus BCCP 5.3.1 Biotin Carboxylase (accC) All carboxylases have a conserved amino acid motif that constitutes the ATP-binding site. A 1.2-kb Sstll-PstI fragment (containing the ATP-binding motif) within the E. coli accC gene was used as a probe to examine the Synechococcus PCC 7942 genomic DNA by Southern hybridization at 58 0 C. A strongly hybridizing 0.8-kb BamHI-PstI fragment was detected and subsequently cloned by a two-stage size fractionation method.

Synechococcus PCC 7942 genomic DNA was first digested with BamHI and electrophoresed on an agarose gel. The gel region containing DNA of sizes between 1.6-kb and 3-kb was cut out and purified (using Geneclean II Kit from BiolOl). The purified DNA was then digested with PstI and electrophoresed on an agarose gel. The gel region containing DNA of sizes between 0.5-kb and 2-kb was cut out and purified.

DNA samples (from each step of purification) were electrophoresed, transferred onto a Genescreen Plus membrane, hybridized with the E. coli accC probe to confirm that the homologous DNA fragment was not lost during each purification step. A library of fragments between 0.5-kb and 2-kb was created by cloning the purified fraction of Synechococcus PCC 7942 DNA into vector pBluescript® KS. Ampicillin-resistant and white with insert) colonies were selected by plating on LB plates containing ampicillin, X-Gal and IPTG.

A total of 287 ampicillin-resistant, white clones were screened; the plasmid DNA mixture (from pools of 5 white clones per pool) were prepared, doubly-digested with PstI and BamHI, electrophoresed, transferred onto a Genescreen Plus membrane, then hybridized with the E. coli accC probe at 58 0 C. Positive signals appeared on 8 WO 96/32484 PCT/US96/05095 -74pools. Twelve positive individual clones were identified at the second round of screening. Two (of the 12) positive clones, each with a single fragment inserted, had the inserts sequenced. Both clones had identical inserts. Sequence comparison indicated only about 60% identity at the nucleotide level between the E. coli accC gene and the cloned Synechococcus PstI-BamHI fragment. This cloned fragment was then used as a probe to screen a Synechococcus cosmid library. Hybridization of the cosmid library was performed at 65 0 C. One hybridizing clone was identified and a 2.4-kb BamHI-NheI fragment from this cosmid clone was isolated and sequenced.

The 1362-nucleotide DNA segment comprising the Synechococcus accC gene is given in SEQ ID NO:7. Only one significant open reading frame (ORF) was found.

This ORF potentially encodes a protein of 453 amino acids. The complete translated amino acid sequence of the Synechococcus accC gene encoding BC is given in SEQ ID NO:8.

5.3.2 Biotin Carboxyl Carrier Protein (accB) In Synechococcus PCC 7942, the accB gene is not immediately upstream of accC, as it is in E. coli. Gene-specific DNA probes from both E. coli and Anabaena PCC7120 accB failed to hybridize with the Synechococcus genomic DNA by Southern analysis. A different approach was necessary.

Since biotin carboxyl carrier protein is biotinylated and streptavidin has a strong specific affinity for biotin, streptavidin was used to identify the number of biotin-containing proteins in Synechococcus PCC 7942. The proteins (from a crude whole protein extract) of Synechococcus PCC 7942 were first separated by standard SDS-PAGE method, then transferred onto an Immobilon-P® transfer membrane, which was subsequently incubated with 35 S-streptavidin. Only one radioactive band (corresponding to a protein of about 25 kDa) appeared on the autoradiogram. This result suggests that there is only one biotin-containing protein in Synechococcus and its mass is similar to the reported mass of E. coli biotin carboxyl carrier protein, 22,500 Da.

WO 96/32484 PCT/US96/05095 This biotin-containing protein was purified Synechococcus cells were first broken by sonication in a buffer containing NaC1, Tris, glycerol and SDS. The supernatant was separated from cell debris by centrifugation, then followed by a

(NH

4 2 S04 precipitation. The precipitate was dissolved in the same buffer, and was allowed to bind to streptavidin agarose beads. The bound agarose beads were washed and the bound proteins were eluded with 70% formic acid. The formic acid-eluted portion was dried and washed with water before loading onto an acrylamide gel. After electrophoresis, the proteins were transferred from the gel to an Immobilon-P® transfer membrane. The membrane was stained briefly with Coomassie Brilliant blue dye, destained in a mixture of methanol and acetic acid, and soaked in water for na hour or so before air drying. The band corresponding to the streptavidin-bound protein was cut out and its N-terminal amino acid sequence was determined.

Based on the amino acid sequence from the N-terminus of the Synechococcus biotin-containing protein and the amino acid sequence around the biotinylation site in all other known BCCPs, degenerate oligonucleotide primers were designed for PCRTM amplification studies with Synechococcus genomic DNA. The pair of primers were: primer LE8 5'-GCTCTAGACNCARYTNAAYTT-3' (SEQ ID NO:26) primer LE7 3'-CRNTACTTYGACNWCTTAAGCT-5' (SEQ ID NO:27) PCRTM was performed for 40 cycles (each with 1 minute at 95 0 C, 1 minute at 48 0 C, 2 minutes at 72 0 with Cetus Taq polymerase, 0.5 mg/ml of template DNA, mg/ml of primer LE8, 40 mg/ml of primer LE7 and with 1 mM Mg2+ final concentration. Under these conditions, a specific PCRTM produce was identified.

Sequence analysis of this cloned PCRTM product indicated that it encoded a region of conserved amino acids within accB of Synechococcus PCC 7942 (compared to the amino acid sequences of the biotin carboxyl carrier protein from Anabaena PCC 7120 and E. coli). Using this PCRTM fragment as a probe in Southern hybridization, a positive clone was identified from the Synechococcus cosmid library. A 1.6-kb PstI fragment from this positive cosmid clone was isolated and sequenced.

A 477-nucleotide DNA segment comprising the Synechococcus accB gene is given in SEQ ID NO:3. Only one significant ORF was found. The deduced amino WO 96/32484 PCT/US96/05095 -76acid sequence at the N-terminus of this ORF matches the earlier determined N-terminal amino acid sequence of the purified Synechococcus biotin-containing protein. The 158-amino acid sequence of the Synechococcus BCCP is given in SEQ ID NO:4. Sequence alignment indicated that the translational product of accB from Synechococcus PCC 7942 is closer to that from Anabaena PCC 7120 than that from E. coli (53% versus 31% amino acid identity).

5.3.3 Carboxyltransferase a Subunit (CTa, accA) A 0.9-kb ClaI-MluI fragment of the E. coli accA gene was used as a probe to examine the Synechococcus PCC 7942 genomic DNA by Southern hybridization at 0 C. A strongly hybridizing 1.6-kb PstI fragment was detected and subsequently cloned.

Synechococcus PCC 7942 genomic DNA was digested with PstI and electrophoresed on an agarose gel. The gel region containing DNA of sizes between 1.6 and 2.5-kb was cut out and purified. A size library between 1.6-kb and 2.5-kb was created by cloning the purified fraction of Synechococcus PCC 7942 DNA into vector pBR322. Tetracycline-resistant, but ampicillin-sensitive, colonies with insert) were selected by first plating on LB plates containing tetracycline, then scored on plates containing ampicillin.

A total of 800 tetracycline-resistant, but ampicillin-sensitive, clones were screened: the plasmid DNA was prepared, digested (in pools of 5 clones per pool) with PstI, electrophoresed, transferred onto a Genescreen Plus membrane, then hybridized with the E. coli accA probe at 60 0 C. Positive signals appeared on 3 pools.

One positive individual clone, with 2 fragments inserted, was identified at the second round of screening. The positive fragment was isolated and re-cloned. This cloned 1.6-kb PstI fragment was then used as a probe to screen the Synechococcus cosmid library where 9 positive clones were identified. A 5-kb BamHI fragment from one of these 9 clones was isolated and sequenced. DNA sequence analysis of the region indicated a cluster of three ORFs in the same orientation.

WO 96/32484 PCT/US96/05095 -77- The 984-nucleotide DNA segment comprising the Synechococcus accA gene is given in SEQ ID NO:11. The first open reading frame encodes the a subunit of the carboxyltransferase. The 327-amino acid sequence of the Synechococcus ORF is 54% identical to that of the E. coli accA gene. The amino acid sequence of the Synechococcus accA gene encoding CTa is given in SEQ ID NO: 12.

5.3.4 Carboxyltransferase 3 Subunit (CTP, accD) Oligonucleotide primers, for polymerase chain reaction (PCRTM) amplification experiments with Synechococcus genomic DNA, were based on the sequence of ORF326 (which is a homolog of the E. coli accD) from a different cyanobacterium, Synechocystis PCC 6803. he pair of primers were: LE39 5'-GAAGATCTTTATGGGCGGTAGTATG-3' (SEQ ID NO:28) 3'-GGTCGAAACGGTACAACCTAGGC-5' (SEQ ID NO:29) PCRTMwas run for 40 cycles (each with 1 minute at 95 0 C, 1 minute at 50 0 C, 2 minutes at 72 0 with Boehringer-Mannheim Taq polymerase, 0.5 mg/ml of template DNA, 5 mg/ml of each primer and with 1 mM Mg2+ final concentration. Under these conditions, a specific PCRTM product of 256 bp was identified. Sequence analysis of this cloned PCRTM fragment showed a significant similarity between the Synechococcus and Synechocystis genomic DNAs in the region between the primers.

Using this cloned PCRTM product as a probe, 5 positive cosmid clones were identified from the Synechococcus cosmid library by Southern hybridization.

5.4 EXAMPLE 4 Isolation and Characterization of the Wheat ACC Enzyme Biotin-containing (streptavidin-binding) proteins in extracts prepared from leaves of two-week old seedlings of wheat and pea, both total protein and protein from intact chloroplasts (prepared by centrifugation on Percoll gradients as described previously in Fernandez and Lamppa, 1991), and from wheat germ (Sephadex G-100 fraction prepared as described below) were analyzed by western blotting with WO 96/32484 PCT/US96/05095 -78- 35 S-Streptavidin. Proteins were separated by SDS-PAGE using a 7.5% separating gel (Maniatis etal., 1982), and then were transferred onto a PVDF membrane (Immobilon-P®, Millipore) in 10 mM 3-(cyclohexylamino)-l-propanesulfonic acid buffer (pH 11), 10% methanol, at 4°C, 40 V, overnight. The blots were blocked with 3% BSA solution in 10 mM Tris-HCl pH 7.5 and 0.9% NaCI and then incubated for 3-16 h with 35 S-Streptavidin (Amersham). The blots were washed at room temperature with 0.5% Nonidet-P40TM in 10 mM Tris-HCl pH 7.5 and 0.9% NaC1.

In wheat, the 220-kDa protein was present in both total and chloroplast protein. It was the major biotinylated polypeptide in the chloroplast protein (traces of smaller biotinylated polypeptides, most likely degradation products of the large one, could also be detected). ACC consisting of 220-kDa subunits is the most abundant biotin-dependent carboxylase present in wheat chloroplasts. In pea chloroplasts the biotinylated peptides are much smaller, probably due to greater degradation of the 220-kDa peptide, which could be detected only in trace amounts in some chloroplast preparations. The amount of all biotinylated peptides, estimated from band intensities on western blots (amount of protein loaded was normalized for chlorophyll content).

is much higher in pea than in wheat chloroplasts.

Purification of wheat germ ACC was carried out at 4 0 C or on ice. 200 g of wheat germ (Sigma) were homogenized (10 pulses, 10 s each) in a Waring blender with 300 ml of 100 mM Tris-HCl pH 7.5, 7 mM 2-mercaptoethanol. Two 0.3 ml aliquots of fresh 0.2 M solution of phenylmethyl-sulfonyl fluoride (Sigma) in 100% ethanol were added immediately before and after homogenization. Soluble protein was recovered by centrifugation for 30 min at 12000 rpm. 1/33 volume of poly(ethyleneimine) solution (pH 7.5) was added slowly and the mixture was stirred for 30 min (Egin-Buhler et al., 1980), followed by centrifugation for 30 min at 12000 RPM to remove the precipitate. ACC in the superatant was precipitated by adding solid ammonium sulfate to 50% saturation.

The precipitate was collected by centrifugation for 30 min at 12000 rpm, dissolved in 200 ml of 100 mM KC1, 20 mM Tris-HC1 pH 7.5, 20% glycerol, 7 mM 2-mercaptoethanol, mixed with 0.2 ml of phenylmethylsulfonyl fluoride solution (as WO 96/32484 PCT/US96/05095 -79above) and loaded on a 5 cm x 50 cm Sephadex G-100 column equilibrated and eluted with the same buffer. Fractions containing ACC activity (assayed as described below using up to 20 gtl aliquots of column fractions) were pooled and loaded immediately on a 2.5 cm x 40 cm DEAE-cellulose column also equilibrated with the same buffer.

The column was washed with 500, 250 and 250 ml of the same buffer containing 150, 200 and 250 mM KC1, respectively. Most of the ACC activity was eluted in the last wash. Protein present in this fraction was precipitated with ammonium sulfate saturation), dissolved in a small volume of 100 mM KC1, 20 mM Tris-HCl pH glycerol, 7 mM 2-mercaptoethanol, and separated in several portions on two Superose columns connected in-line (Superose 6 and 12, Pharmacia). 1 ml fractions were collected at 0.4 ml/min flow rate. Molecular mass standards were thyroglobulin, 669-kDa; ferritin, 440-kDa; aldolase, 158-kDa; albumin, 67-kDa (Pharmacia).

ACC-containing fractions were concentrated using Centricon-100 concentrators (Amicon) and the proteins were separated by SDS-PAGE as described above.

By gel filtration, active ACC had an apparent molecular mass of 500-kDa and the individual polypeptides have a molecular mass of 220-kDa. The 220-kDa polypeptide was the major component of this preparation as revealed by Coomassie staining of proteins separated by SDS-PAGE. This preparation also contained several smaller biotin-containing peptides as revealed by western blotting with 35 S-Streptavidin, most likely degradation products of the ca. 220-kDa peptide, which retained their ability to form the -500-kDa complex and therefore co-purified with intact ACC. The ACC preparations were active only when they contained intact 220kDa biotinylated polypeptide. It is not possible to estimate the recovery of the active ACC, due to continuous degradation of the 220-kDa peptide during purification and to increased recovery of ACC activity in more purified preparations, probably due to separation of the enzyme from inhibitors in the cruder extracts.

The 220-kDa wheat peptide isolated as a dimer according to the above protocol was finally purified by SDS-PAGE and transferred to Immobilon-P® for sequencing. The N-terminus of the peptide appeared to be blocked. A mixture of amino acids was detected only after the protein was cleaved chemically with CNBr.

WO 96/32484 PCT/US96/05095 The 220-kDa protein was therefore purified on an SDS gel, cleaved with CNBr, and the resulting peptides were fractionated by gel electrophoresis basically as described (Jahnen-Dechent and Simpson, 1990), with the following modifications. A slice of gel containing about 20 gg of the 220-kDa polypeptide was dried under vacuum to about half of its original volume and then incubated overnight in 0.5 ml of formic acid containing 25 mg of CNBr. The gel slice was dried again under vacuum to about half of its original volume and was equilibrated in 1 ml of 1 M Tris-HCl (pH The CNBr peptides were separated by inserting the gel piece directly into a well of a tricine gel (as described in Jahnen-Dechent and Simpson, 1990; but without a spacer gel). Gels used to separate peptides for sequencing were pre-run for 30 min with 0.1 mM thioglycolic acid in the cathode buffer. Peptides were transferred to Immobilon-P for sequencing by the Edman degradation method as described above.

Several bands of peptides, ranging in size from 4 to 16-kDa, with a well-resolved single band at about 14-kDa, were obtained. Attempts to sequence the smaller peptides failed, but the 14-kDa peptide yielded a clean results for residues 3-13.

EXAMPLE 5 Effects of the Herbicide Haloxyfop on Wheat ACC The effect of haloxyfop, one of the aryloxyphenoxypropionate herbicides has been tested, on the activity of ACC from wheat germ and from wheat seedling leaves.

For the in vitro assay of ACC activity, 1-8 gl aliquots of ACC preparations were incubated for 45 min at 37°C with 20 gl of 100-200 mM KC1, 200 mM Tris-HCl pH 10 mM MgCl 2 2 mM ATP, 2 mM DTT, 2 mM 4 C-NaHCO 3 and where indicated 1 mM Ac-CoA, in a final volume of 40 pl. The reaction was stopped by adding 4 gl of concentrated HC1 30-40 gl aliquots of the reaction mixture were spotted on filter paper and dried, and acid-stable radioactivity was measured using scintillation cocktail. Haloxyfop was added as the Tris salt of the acid, generously supplied by J. Secor of Dow-Elanco.

For the in vivo assay of ACC activity, 2-week old seedlings of wheat (Triticum aestivum cv. Era) were cut about 1 cm below the first leaf and transferred to a 1.5 ml WO 96/32484 PCTIUS96/05095 -81micro tube containing 14C-sodium acetate and haloxyfop (Tris salt) for 4-6 h. The leaves were then cut into small pieces and treated with 0.5 ml of 40% KOH for 1 h at 0 C, and then with 0.3 ml of H 2

SO

4 and 20 pl of 30% TCA on ice. Fatty acids were extracted with three 0.5 ml aliquots of petroleum ether. The organic phase was washed with 1 ml of water. Incorporation of 1 4 C-acetate into fatty acids is expressed as the percentage of the total radioactivity taken up by the seedlings, present in the organic phase.

As expected, the enzyme from wheat germ or from wheat chloroplasts was sensitive to the herbicide at very low levels. 50% inhibition occurs at about 5 and 2 gpM haloxyfop, respectively. For comparison, the enzyme from pea chloroplasts is relatively resistant (50% inhibition occurs at >50 :M haloxyfop). Finally, the in vivo incorporation of 1 4 C-acetate into fatty acids in freshly cut wheat seedling leaves is even more sensitive to the herbicide (50% inhibition occurs at <1 :M haloxyfop), which provides a convenient assay for both ACC and haloxyfop.

5.6 EXAMPLE 6 Cloning and Sequencing of Triticum aestivum ACC cDNA 5.6.1 Materials and Methods 5.6.1.1 PCR T M Amplification Degenerate PCRTM primers were based on the alignment of amino acid sequences of the following proteins (accession numbers in brackets): rat (J03808) and chicken (J03541) ACCs; E. coli (M80458, M79446, X14825, M32214), Anabaena 7120 (L14862, L14863) and Synechococcus 7942 BCs and BCCPs; rat (M22631) and human (X14608) propionyl-coenzyme A carboxylase subunit); yeast (J03889) pyruvate carboxylase; Propionibacterium shermanii (Ml1738) transcarboxylase (1.3S subunit) and Klebsiella pneumonia (J03885) oxaloacetate decarboxylase (a subunit). Each primer consisted of a 14-nucleotide specific sequence based on the amino acid sequence and a 6- or 8-nucleotide extension at the Poly(A)' RNA from 8-day old plants (Triticum aestivum var. Era) was used for the synthesis of the first strand of cDNA with random hexamers as primers for AMV reverse transcriptase (Haymerle etal., 1986). Reverse transcriptase was WO 96/32484 PCT/US96/05095 -82inactivated by incubation at 90 0 C and low molecular weight material was removed by filtration. All components of the PCRTM (Cetus/Perkin-Elmer), except the Taq DNA polymerase, were incubated for 3-5 min at 95 0 C. The PCRTM was initiated by the addition of polymerase. Conditions were optimized by amplification of the BC gene from Anabaena 7120. Amplification was for 45 cycles, each 1 min at 95 0 C, 1 min at 42-46 0 C and 2 min at 72 0 C. MgCl 2 concentration was 1.5 mM. Both the reactions using Anabaena DNA and the single-stranded wheat cDNA as template yielded the expected 440-bp products. The wheat product was separated by electrophoresis on LMP-agarose and reamplified using the same primers and a piece of the LMP-agarose slice as a source of the template. That product, also 440-bp, was cloned into the Invitrogen vector pCR1000 using their A/T tail method, and sequenced.

In eukaryotic ACCs, the BCCP domain is located about 300 amino acids downstream from the end of the BC domain. Therefore, it was possible to amplify the cDNA encoding that interval between the two domains using primers, one from the Cterminal end of the BC domain and the other from the conserved biotinylation site.

The expected 1.1-kb product of the first low yield PCR T M with primers II and IV was separated by electrophoresis on LMP-agarose and reamplified by another round of PCRTM, then cloned into the Invitrogen vector pCRII® and sequenced. The PCRTM conditions were the same as those described above.

5.6.1.2 Isolation and Analysis of ACC cDNA A wheat cDNA library (Triticum aestivum, var. Tam 107, Hard Red Winter, 13-day light grown seedlings) was purchased from Clontech. This 8gtl1 library was prepared using both oligo(dT) and random primers. Colony ScreenPlus® (DuPont) membrane was used according to the manufacturers' protocol (hybridization at in 1 M NaCI and 10% dextran sulfate). The library was first screened with the 1.1-kb

PCR

T

-amplified fragment of ACC-specific cDNA. Fragments of clones 39-1, 45-1 and 24-3 were used in subsequent rounds of screening. In each case, -2.5 x 106 plaques were tested. More than fifty clones containing ACC-specific cDNA WO 96/32484 PCT/US96/05095 -83fragments were purified, and EcoRI fragments of the longest cDNA inserts were subcloned into pBluescriptSK® for further analysis and sequencing. A subset of the clones was sequenced on both strands by the dideoxy chain termination method with Sequenase® (United States Biochemicals) or using the Perkin Elmer/Applied Biosystems Taq DyeDeoxy Terminator cycle sequencing kit and an Applied Biosystems 373A DNA Sequencer.

5.6.1.3 RNA and DNA Total RNA from 10-day old wheat plants was prepared as described in (Haymerle et al., 1986). RNA was separated on a glyoxal denaturing gel (Sambrook et al., 1989). GeneScreen Plus® (DuPont) blots were hybridized in 1M NaCl and dextran sulfate at 65 0 C (wheat RNA and DNA) or 58-60 0 C (soybean and canola DNA). All cloning, DNA manipulation and gel electrophoresis were as described (Sambrook et al., 1989).

5.6.2 Results 5.6.2.1 PCR T M Cloning of the Wheat (Triticum aestivum) ACC cDNA A 440-bp cDNA fragment encoding a part of the biotin carboxylase domain of wheat ACC and a 1.1-kb cDNA fragment encoding the interval between the biotin carboxylase domain and the conserved biotinylation site were amplified. These fragments were cloned and sequenced. In fact, three different 1.1-kb products, corresponding to closely related sequences that differ from each other by were identified. The three products most likely represent transcription products of three different genes, the minimum number expected for hexaploid wheat. These two overlapping DNA fragments (total length of 1473 nucleotides) were used to screen a wheat cDNA library.

5.6.2.2 Isolation and Sequence Analysis of Wheat ACC cDNAs A set of overlapping cDNA clones covering the entire ACC coding sequence was isolated and a subset of these clones has been sequenced. The nucleotide WO 96/32484 PCT/US96/05095 -84sequence within overlapped regions of clones 39-1, 20-1 and 45-1 differ at 1.1% of the nucleotides within the total of 2.3 kb of the overlaps. The sequence within the overlap of clones 45-1 and 24-3 is identical. The sequence contains a 2257-amino acid reading frame encoding a protein with a calculated molecular mass of 251 kDa.

In wheat germ the active ACC has an apparent molecular mass of -500 kDa and the individual polypeptides have an apparent molecular mass (measured by SDS-PAGE) of about 220 kDa (Gornicki and Haselkor, 1993). The 220-kDa protein was also present in both total leaf protein and protein from intact chloroplasts. In fact, it was the major biotinylated polypeptide in the chloroplast protein. The cDNAs (total length 7.4 kb) include 158 bp of the 5'-untranslated and 427 bp of the 3'-untranslated sequence.

The 7360-nucleotide DNA segment comprising the wheat ACC cDNA is given in SEQ ID NO:9. The 2257-amino acid translated wheat ACC sequence is given in SEQ ID NO: 5.6.2.3 Northern Analysis of ACC mRNA Northern blots with total RNA from 10-14 day old wheat leaves were probed using different cDNA fragments (the 1.1-kb PCR

TM

-amplified fragment and parts of clones 20-1, 24-3 and 01-4). In each case the only hybridizing mRNA species was 7.9 kb in size. This result shows clearly that all the cDNA clones correspond to mRNA of large, eukaryotic ACC and that there are no other closely related biotin-dependent carboxylases, consisting of small subunits that are encoded by smaller mRNAs, in wheat.

Northern analysis of total RNA prepared from different sectors of 10-day old wheat seedlings indicates very high steady-state levels of ACC-specific mRNA in cells of leaf sectors I and II near the basal meristem. The ACC mRNA level is significantly higher in sectors I and II than in sectors II-VI. This cannot be explained by dilution of specific mRNA by increased levels of total RNA in older cells. Based on published results (Dean and Leech, 1982), the increase in total RNA between sectors I and VI is expected to be only about two-fold.

WO 96/32484 PCT!US96/05095 All cell division occurs in the basal meristem and cells in other sectors are in different stages of development. Differences between these young cells and the mature cells at the tip of the leaf include cell size, number of chloroplasts and amount of total RNA and protein per cell (Dean and Leech, 1982). Expression of some genes is correlated with the cell age Lampa et al., 1985). It is not surprising that the level of ACC-specific mRNA is highest in dividing cells and in cells with increasing number of chloroplasts. The burst of ACC mRNA synthesis is necessary to supply enough ACC to meet the demand for malonyl-coenzyme A. The levels of ACC mRNA decrease significantly in older cells where the demand is much lower. The same differences in the level of ACC specific mRNA between cells in different sectors were found in plants grown in the dark and in plants illuminated for one day at the end of the dark period.

5.6.2.4 Southern Analysis of Plant DNA Hybridization, under stringent conditions, of wheat total DNA digests with wheat ACC cDNA probes revealed multiple bands. This was expected due to the hexaploid nature of wheat (Triticum aestivum). Some of the wheat cDNA probes also hybridize with ACC-specific DNA from other plants. The specificity of this hybridization was demonstrated by sequencing several fragments of canola genomic DNA isolated from a library using wheat cDNA probe 20-1 and by Northern blot of total canola RNA using one of the canola genomic clones as a probe. The Northern analysis revealed a large ACC-specific message in canola RNA similar in size to that found in wheat.

5.6.2.5 ACC mRNA The putative translation start codon was assigned to the first methionine of the open reading frame. An in-frame stop codon is present 21 nucleotides up-stream from this AUG. The nucleotide sequence around this AUG fits quite well with the consensus for a monocot translation initiation site derived from the sequence of 93 genes, except for U at position +4 of the consensus which was found in only 3 of the WO 96/32484 PCT/US96/05095 -86- 93 sequences. The ACC mRNA stop codon UGA is also the most frequently used stop codon found in monocot genes, and the surrounding sequence fits the consensus well.

5.6.2.6 Homologies with Other Carboxylases A comparison of the wheat ACC amino acid sequence with other ACCs shows sequence conservation among these carboxylases. The sequence of the polypeptide predicted from the cDNA described above was compared with the amino acid sequences of other ACCs, and about 40% identity are with the ACC of rat, diatom and yeast (about Less extensive similarities are evident with subunits of bacterial ACCs. The amino acid sequence of the most highly conserved domain, corresponding to the biotin carboxylases of prokaryotes, is about 50% identical to the ACC of yeast, chicken, rat and diatom, but only about 27% identical to the biotin carboxylases of E. coli and Anabaena 7120. The biotin attachment site has the typical sequence of eukaryotic ACCs. Several conserved amino acids found in the carboxyltransferase domains previously identified (Li and Cronan, 1992) are also present in the wheat sequence. Surprisingly, none of the four conserved motifs containing serine residues, which correspond to phosphorylation sites in rat, chicken and human ACCs (Ha et al., 1994), is present at a similar position in the wheat polypeptide.

5.6.2.7 Lack of Targeting Sequence in Wheat ACC cDNA The wheat cDNA does not encode an obvious chloroplast targeting sequence unless this is an extremely short peptide. There are only 12 amino acids preceding the first conserved amino acid found in all eukaryotic ACCs (a serine residue). The conserved core of the BC domain begins about 20 amino acids further down-stream.

The apparent lack of a transit peptide poses the question of whether and how the ACC described in this paper is transported into chloroplasts. It was shown recently that the large ACC polypeptide purifies with chloroplasts of wheat and maize (Goricki and Haselkor, 1993; Egli et al., 1993). No obvious chloroplast transit peptide between WO 96/32484 PCTIUS96/05095 -87the ER signal peptide and the mature protein was found in diatom ACC either (Roessler and Ohlrogge, 1993).

The number of ACC genes in wheat have been assessed by Southern analysis and by sequence analysis of the and 3'-untranslated portions of ACC cDNA representing transcripts of different genes. These cDNA fragments may be obtained by PCR T M amplification using the and 3'-RACE methodology. The genome structure of wheat (Triticum aestivum) suggests the presence of at least three copies of the ACC gene, i.e. one in each ancestral genome. Sequence analysis of the untranscribed parts of the gene may determine whether any familiar promoter and regulatory elements are present. The structure of introns within the control region and in the 5'-fragment of the coding sequence is also of interest.

The plant ACC genes are full of introns and their transcripts undergo alternative splicing. In some plant genes, introns have been found both within the sequence encoding the transit peptide, and at the junction between the transit peptide and the mature protein.

In plants, variant cytoplasmic and plastid isoenzymes could arise, for example, by alternative splicing or by transcription of two independent genes. This problem is especially intriguing as it was not possible to identify a transit peptide in the sequences of wheat ACC obtained so far. The two possibilities can be distinguished by sequence analysis of the appropriate fragment of the ACC genes (clones from genomic library) and mRNAs (as cDNA). The sequence of these and 3'-untranscribed and untranslated fragments of the gene are usually significantly different for different alleles so they may also be used as specific probes to follow expression of individual genes.

5.7 EXAMPLE 7 DNA Compositions Comprising a Wheat Cytosolic ACC This example describes the cloning and DNA sequence of the entire gene encoding wheat (var. Hard Red Winter Tam 107) acetyl-CoA carboxylase (ACCase).

Comparison of the 12-kb genomic sequence (SEQ ID NO:30) with the 7.4-kb cDNA sequence reported in Example 6 revealed 29 introns. Within the coding region (SEQ WO 96/32484 PCT/US96/05095 -88- ID NO:31), the exon sequence is 98% identical to the wheat cDNA sequence (SEQ ID NO:XX). A second ACCase gene was identified by sequencing fragments of genomic clones that include the first two exons and the first intron. Additional transcripts were detected by and 3'-RACE analysis. One set of transcripts had 5'-end sequence identical to the cDNA found previously and another set was identical to the gene reported here. The 3'-RACE clones fall into four distinguishable sequence sets, bringing the number of ACCase sequences to six. None of these cDNA or genomic clones encode a chloroplast targeting signal. Identification of six different sequences suggests that either the cytosolic ACCase genes are duplicated in the three chromosome sets in hexaploid wheat or that each of the six alleles of the cytosolic ACCase gene has a readily distinguishable DNA sequence.

5.7.1 Materials and Methods 5.7.1.1 Isolation and Analysis of ACCase Genomic Clones A wheat genomic library aestivum, var. Hard Red Winter Tam 107, 13-day light grown seedlings) was purchased from Clontech. This 8 EMBO3 library was prepared from genomic DNA partially digested with Sau3A. Colony ScreenPlus (DuPont) membrane was used according to the manufacturers' protocol (hybridization at 65°C in 1M NaCI and 10% dextran sulfate). The library was screened with a 440-bp PCR

TM

-amplified fragment of ACCase-specific cDNA and with cDNA clone 24-3 (Gomicki et al., 1994). In each case, -1.2 x 106 plaques were tested. 24 clones containing ACCase-specific DNA fragments were purified and mapped. Selected restriction fragments of these genomic clones were subcloned into pBluescriptSK® for further analysis and sequencing. The 3'-terminal fragment of the gene (clone 145) was amplified by PCRTM using wheat genomic DNA as a template. Primers were based on the sequence of genomic clone 233, CGCTATAGGGAAACGTTAGAAGGATGGG-3' (SEQ ID NO:34) and 3'-RACE clone 4, 5'-ATCGATCGGCCTCGGCTCCAATTTCATT-3' (SEQ ID All PCRTM components except Taq polymerase were incubated for 5 min. at 95 0 C. The reactions were initiated by the addition of the polymerase followed by WO 96/32484 PCT/US96/05095 -89cycles of incubation at 94C for Imin, 55°C for 2 min and 72 0 C for 2 min. A 1.8-kb PCRTM product was gel-purified, reamplified using the same primers, cloned into the Invitrogen vector pCRITM and sequenced.

5.7.1.2 Analysis of mRNA by rapid amplification of cDNA ends (RACE) Two sets of 15 and 20 cDNA fragments corresponding to mRNA and 3'-ends, respectively, were prepared by T/A cloning of RACE products into the vector pCRII. Total RNA from 15-day old wheat (Triticum aestivum var. Tam 107, Hard Red Winter) plants was prepared as described in Chirgwin etal. (1979). A Gibco BRL 5'-RACE kit was used according to the manufacturers' protocol. For the amplification, the first strand of cDNA was prepared using a gene-specific primer: 5'-GTTCCCAAAGGTCTCCAAGG-3' (SEQ ID NO:36); followed by the addition of a homopolymeric dA-tail.

dT-Anchor primer: 5'-GCGGACTCGAGTCGACAAGC11"1TT ITI"TITITI T -3' (SEQ ID NO:37); and a gene-specific primer, GGTGCGGATGCTGCGCATG-3' (SEQ ID NO:38) were used in the first round of

PCR

T M Universal primer, 5'-GCGGACTCGAGTCGACAAGC-3' (SEQ ID NO:39) and another gene-specific primer, TTGTTGGCAACC-3' (SEQ ID NO:40) were used for reamplification. The gene-specific primers were targeted to a stretch of 5'-end coding sequence identical in clones 39 and 71 that were available.

Clone 71 was isolated from a 8gt 1 cDNA library as described before using a fragment of cDNA 39 as probe (Example The same dT-anchor primer and universal primer together with a gene specific primer 5'-GACTCATTGAGATCAAGTTC-3' (SEQ ID NO:41) were used for the first strand cDNA synthesis and 3'-end amplification. The latter primer was targeted to the 3'-end of the ACCase open reading frame.

All cloning, DNA manipulations and gel electrophoresis were as described (Sambrook et al., 1989). DNA was sequenced on both strands by the dideoxy chain WO 96/32484 PCT/US96/05095 termination method using 35 S-[dATP] with Sequenase (United States Biochemicals) or using the Perkin Elmer/Applied Biosystems Taq DyeDeoxy Terminator cycle sequencing kit and an Applied Biosystems 373A DNA Sequencer.

5.7.2 Results 5.7.2.1 Analysis of wheat cytosolic ACCase genes Two cDNA fragments, one encoding a part of the biotin carboxylase domain of wheat ACCase and the other a part of the carboxyltransferase, were used to isolate a set of overlapping DNA fragments covering the entire ACCase gene. Some of these genomic fragments were sequenced as indicated in FIG. 1. Where they overlap, the nucleotide sequences of clones 31, 191 and 233 are identical. These obviously derive from the same gene. cDNA clone 71 (see below) represents the transcription product of this gene (430-nucleotide identical sequence). The sequence of clone 145 obtained by PCRTM to cover the remaining 3'-end part of the gene differs from clone 233 by of 400 nucleotides of the overlap located within the long exon 28 (FIG. It must therefore derive from a different copy of the ACCase gene. 3'-RACE clone 4 see below) differs at 6 of 490 nucleotides in the overlap.

The sequence was deposited in GenBank (as accession number U39321), and is a composite of these three very closely related sequences. Its 5'-end corresponds to the 5'-end of clone 71 and the 3'-end corresponds to the poly(A) attachment site of the 3'-RACE clone 4. It was assumed that no additional introns are present at the very end of the gene.

Comparison of the genomic sequence with the cDNA sequence in Example 4 revealed 29 introns. Intron location is conserved among all three known plant ACCase genes except for two introns not present in wheat but found in rape (Schulte et al., 1994), A. thaliana (Roesler et al., 1994) and soybean (Anderson et al., 1995) (FIG. The nucleotide sequence at splice sites fits well with the consensus for monocot plants. The A+T content of the gene exons and introns is 52% and 63%, respectively, compared to 42% and 61% found for other monocot plant genes (White etal., 1992). The exon coding sequence is 98% identical to that of the cDNA WO 96/32484 PCT/US96/05095 -91sequence reported earlier. This is the same degree of identity as found previously for different transcripts of the cytosolic ACCase genes in hexaploid wheat (Example 4).

The 11-amino acid sequence obtained previously for a CNBr-generated internal fragment of purified 220-kDa wheat germ ACCase (Goricki and Haselkorn, 1993) differs from the sequence encoded by these cDNA and genomic clones at one position, but it is identical with the corresponding cDNA sequence of the plastid ACCase from maize (Egli et al., 1995), excluding one amino acid which could not be assigned unambiguously in the sequence.

Two additional genomic clones, 153 and 231, were also partially sequenced (FIG. The sequenced fragments include parts of the first two exons and the first intron. Although cDNA corresponding exactly to genomic clone 153 is not available, the boundaries of the first intron could easily be identified by sequence comparison with cDNA clone 71 (corresponding to genomic clone 31). Clone 153 encodes a polypeptide that differs by only one out of the first 110 amino acids of the ACCase open reading frame. The sequence of the 5'-leader was also well conserved but the of the first intron of clone 153 is significantly different from that of genomic clone 31.

On the other hand, only the 3'-splice site of an intron could be identified by sequence comparison in this part of clone 231. The sequence immediately upstream of the 3'-splice site and that of the following exon is identical to that of clone 31. No sequence related to that found upstream of the first intron of clone 191 could be identified in clone 231 by hybridization (including a -6 kb fragment upstream of the ACCase open reading frame) or by sequencing 2 kb of the upstream fragment). It is possible that the first intron in this gene is much larger (additional upstream introns can not be excluded) or that the upstream exon(s) and untranscribed part of the gene has a completely different sequence. A cloning artifact can not be ruled out. Indeed clone 31 contained such an unrelated sequence at its 5'-end (probably a ligation artifact).

Identification of three additional genomic clones with sequence closely related to the other ACCase genes but containing no introns at several tested locations WO 96/32484 PCT/US96/05095 -92suggests the existence of a pseudogene in wheat. A fragment of clone 232 that was sequenced is represented in the diagram shown in FIG. 1. It is 93% and 96% identical with clone 233 at the nucleotide and amino acid level, respectively.

Shown in FIG. 5 is the 5' flanking sequence of the ACCase 1 gene (about 3 kb upstream of the translation initiation codon, of clone 71L (SEQ ID NO:32). The flanking sequence of the ACCase 2 gene designated 153 (SEQ ID NO:33) is shown in FIG. 6.

5.7.2.2 Analysis of mRNA ends In the original library screen (Gornicki etal., 1994) it was not possible to isolate any cDNA clones corresponding to the very ends of the ACCase mRNA. With the new sequence available it became possible to generate the missing pieces by RACE. Two sets of 5'-end RACE clones, 71L and 39L, were identified. Their sequence is identical to the sequence of cDNA clones 71 (this work) and 39 (Gornicki et al., 1994), respectively. The two sequences extend 239 and 312 nucleotides upstream of the ACCase initiation codon and define an approximate position of the transcription start site. None of the genomic clones corresponds to 39L. The presence of the first intron in the corresponding gene could not therefore be confirmed. All three coding sequences are very similar (they differ by only one three-amino acid deletion or one E to D substitution found within the first 110 amino acids) and none of them encodes additional amino acids at the N-terminus, none of them encodes a potential chloroplast transit peptide.

The sequences of the 5'-leaders differ significantly although they share some distinctive structural features. They are relatively long (at least 239-312 nucleotides as indicated by the lengths of 39L and 71L, respectively), G+C rich and contain upstream AUG codons. The open reading frames found in the leaders are 70-90 amino acids long and they end within a few nucleotides of the ACCase initiation codon. A similar arrangement was found in the sequence of genomic clone 153. The three upstream AUG codons are conserved and the presence of deletions, most of which are a multiple of three nucleotides, suggests at least some conservation of the WO 96/32484 PCT/US96/05095 -93open reading frames at the amino acid level. This arrangement, found in the cytosolic ACCase genes, contrasts with the majority of 5'-untranslated leaders found in plants.

Although much longer leader sequences containing upstream AUG codons have been reported in plants Shorrosh et al., 1995), they are rare. In most cases, the first AUG codon is the site of initiation of translation of the major gene product. The upstream AUGs are believed to affect the efficiency of mRNA translation and as such may be important in the regulation of expression of some genes (Roesler et al., 1994; Anderson et al., 1995). They are often found in mRNAs encoding transcription factors, growth factors and receptors, all important regulatory proteins (Kozak, 1991).

They are also found in some plant mRNAs encoding heat shock proteins (Joshi and Nguyen, 1995). The -800 nucleotide long leader intron found in both genes (clones 153 and 191) may also be important for the level and pattern of gene expression Fu et al., 1995).

Four different sequences and two different polyadenylation sites -300 and -500 nucleotides downstream of the translation stop codon, respectively, were detected among the 3'-end RACE clones (FIG. The sequence of the cDNA reported previously (Gomicki et al., 1994) and the sequence of genomic clone 145 are also different in this region, bringing the total number of different sequences to six.

3-14 nucleotide differences were found in pairwise comparisons among these six sequences within two stretches that include 282 nucleotides at the 5'-end of the 3'-RACE clones and 204 nucleotides at the 3'-end (FIG. 2).

5.7.2.3 Cytosolic ACC A gene encoding eukaryotic-type cytosolic ACCase from wheat, very similar in sequence to the cDNA in Example 4, was cloned and sequenced. Nucleotide identity between the cDNA and the gene within the coding sequence is 98%. The putative translation start codon was assigned in the original cDNA sequence to the first methionine of the open reading frame. An in-frame stop codon is present 21 nucleotides upstream from this AUG and the conserved core of the biotin carboxylase domain begins about 20 amino acids further down-stream. The gene, shown in FIG. 3 WO 96/32484 PCT/US96/05095 -94- (SEQ ID NO:30), encodes a 2260-amino acid protein with a calculated molecular mass of 252 kDa (FIG. 4 and SEQ ID NO:31). The wheat cDNA did not encode an obvious chloroplast targeting sequence. The same is true for all the cDNA and genomic sequences described in this paper. The cDNA for maize plastid ACCase, reported recently (Egli et al., 1995), does encode a chloroplast transit peptide.

Comparison of the ACCase sequence encoded by the gene reported in this paper with the sequence of the wheat ACCase of Example 4 and with other representative biotin-dependent carboxylases is shown in Table 4. Wheat ACCase is most similar to other eukaryotic-type plant ACCases. Identity with other eukaryotic carboxylases is also significant. The core sequence of the most conserved ACCase domain, biotin 0 TABLE 4 Amino Acid Identities Between T. aestivumn Cytosolic ACCase and Some Other Representative Biotin-Dependent Carboxylases Specimen Location Full Biotin References Length Carboxylase Domain Eukaryotic type carboxylases T. aestivum I cytosolic A. thaliana cytosolic M. sativa cytosolic B. napus2 Z. mays plastid R. ratus cytosolic C cryptica 2 S. cerevisiae cytosolic Prokaryotic type carboxylases E. coli 3 bacterial Anabaena 71 203 bacterial Gornicki et at., 1994 Roesler et at., 1994 Shorrosh et at., 1994 Schulte et at., 1994 Egli et al, 1995 Lopez-Casillas et at., 1988 Roessler and Ohlrogge, 1993 Al-Feel etal, 1992 Li and Cronan, 1992 Gornicki et at., 1993 Specimen Location Full Biotin References Length Carboxylase Domain M. Ieprae 4 bacterial 32 Norman et al., 1994 N. tabacum 3 plastid 32 Shorrosh et al., 1995 R. ratus pCC 5 mitochondrial 34 Browner et al., 1989 S. cerevisiae pC 6 mitochondrial 32 Lim et al., 1988 A. thaliana mitochondrial 34 Weaver et al., 1995 MCCase 7 'Sequence deduced from cDNA sequence reported previously (product of a different allele or gene).

2 Cellular localization uncertain.

3 Biotin carboxylase subunit of ACCase.

4 Biotin carboxylase-biotin carboxyl carrier subunit of ACCase.

Biotin carboxylase-biotin carboxyl carrier subunit of propionyl-CoA carboxylase 6 Pyruvate carboxylase.

7 Biotin carboxylase-biotin carboxyl carrier subunit of methylcrotonyl-CoA carboxylase.

WO 96/32484 PCTIUS96/05095 -97carboxylase, is well conserved in both eukaryotic and prokaryotic biotin-dependent carboxylases. The other functional domains are less conserved (Example Among plant eukaryotic-type ACCases, the wheat cytosolic ACCase is no more similar to the maize plastid ACCase (both monocots) than it is to cytosolic ACCases from dicot plants. Clearly, cytosolic and plastid eukaryotic-type ACCases are quite distinct proteins. Another wheat ACCase for which partial sequence is available (Elborough et al., 1994) is most likely a plastid isozyme. It is more similar to the maize plastid ACCase than to the wheat cytosolic enzyme. The plant prokaryotic-type plastid enzyme is more similar to bacterial, most notably cyanobacterial ACCases and to biotin-dependent carboxylases found in mitochondria, than to any of the plant cytosolic ACCases.

Sequence comparison of fragments of cDNA and genomic clones from the 3Nend of the gene brings the total number of different genes encoding cytosolic ACCase in wheat to six, indicating that in hexaploid wheat there are at least two distinguishable coding sequences for the cytosolic ACCase in each of the three ancestral chromosome sets. Those two sequences might correspond to the alleles of the ACCase gene present in each ancestral chromosome set. On the other hand, it is possible that each pair of alleles has identical sequences, since the bread wheat studied is extensively inbred. If that is the case, then one or more ancestral genes has been duplicated.

5.8 EXAMPLE 8 Developmental Analysis of ACC Genes Methods have been developed for analyzing the regulation of ACC gene expression on several levels. With the cDNA clones in hand, the first may be obtained by preparing total RNA from various tissues at different developmental stages from different segments of young wheat plants, then probing Northern blots to determine the steady-state level of ACC mRNA in each case. cDNA probes encoding conserved fragments of ACC may be used to measure total ACC mRNA level and gene specific probes to determine which gene is functioning in which tissue.

WO 96/32484 PCTIUS96/05095 -98- In parallel, the steady-state level of ACC protein (by western analysis using ACC-specific antibodies and/or using labeled streptavidin to detect biotinylated peptides) and its enzymatic activity may be measured to identify the most important stages of synthesis and reveal mechanisms involved in its regulation. One such study evaluates ACC expression in fast growing leaves (from seedlings at different age to mature plants), in the presence and in the absence of light.

5.9 EXAMPLE 9 Isolation of Herbicide-Resistant Mutants Development of herbicide-resistant plants is an important aspect of the present invention. The availability of the wheat cDNA sequence facilitates such a process.

By insertion of the complete ACC cDNA sequence into a suitable yeast vector in place of the yeast ACC coding region, it is possible to complement a FAS3 mutation in yeast using procedures well-known to those of skill in the art (see Haslacher et al., 1993). Analysis of the function of the wheat gene in yeast depends first on tetrad analysis, since the FAS3 mutation is lethal in homozygotes.

Observation of four viable spores from FAS3 tetrads containing the wheat ACC gene may confirm that the wheat gene functions in yeast, and extracts of the complemented FAS3 mutant may be prepared and assayed for ACC activity. These assays may indicate the range of herbicide sensitivity, and in these studies, haloxyfop acid and clethodim may be used as well as other related herbicide compounds.

Given that the enzyme expressed in yeast is herbicide-sensitive, the present invention may be used in the isolation of herbicide-resistant mutants. If spontaneous mutation to resistance is too infrequent, chemical mnutagenesis with DES or EMS may be used to increase such frequency. Protocols involving chemical mutagenesis are well-known to those of skill in the art. Resistant mutants, strains capable of growth in the presence of herbicide, may be assayed for enzyme activity in vitro to verify that the mutation to resistance is within the ACC coding region.

Starting with one or more such verified mutants, several routes may lead to the identification of the mutated site that confers resistance. Using the available restriction map for the wild-type cDNA, chimeric molecules may be constructed WO 96/32484 PCT/US96/05095 -99containing half, quarter and eighth fragments, etc. from each mutant, then checked by transformation and tetrad analysis whether a particular chimera confers resistance or not.

Alternatively a series of fragments of the mutant DNA may be prepared, endlabeled, and annealed with the corresponding wild-type fragments in excess, so that all mutant fragments are in heterozygous molecules. Brief S1 or mung bean nuclease digestion cuts the heterozygous molecules at the position of the mismatched base pair.

Electrophoresis and autoradiography is used to locate the position of the mismatch within a few tens of base pairs. Then oligo-primed sequencing of the mutant DNA is used to identify the mutation. Finally, the mutation may be inserted into the wild-type sequence by oligo-directed mutagenesis to confirm that it is sufficient to confer the resistant phenotype.

Having identified one or more mutations in this manner, the corresponding parts of several dicot ACC genes may be sequenced (using the physical maps and partial sequences as guides) to determine their structures in the corresponding region, in the expectation that they are now herbicide resistant.

5.10 EXAMPLE 10 Isolation and Sequence Analysis of Canola ACC cDNA Wheat ACC cDNA probes were used to detect DNA encoding canola ACC.

Southern analysis indicated that a wheat probe hybridizes quite strongly and cleanly with only a few restriction fragments that were later used to screen canola cDNA and genomic libraries (both libraries provided by Pioneer HiBred Co [Johnson City, IA]).

About a dozen positive clones were isolated from each library.

Sequence analysis was performed for several of these genomic clones.

Fragments containing both introns and exons were identified. One exon sequence encodes a polypeptide which is 75% identical to a fragment of wheat ACC. This is very high conservation especially for this fragment of the ACC sequence which is not very conserved in other eukaryotes. The 398-nucleotide DNA segment comprising a portion of the canola ACC gene is given in SEQ ID NO:19. The 132-amino acid WO 96/32484 PCTIS96/05095 -100translated sequence comprising a portion of the canola ACC polypeptide is given in SEQ ID One of the other genomic clones (6.5 kb in size) contains the 5' half of the canola gene, and additional screening of the genomic library may produce other clones which contain the promoter and other potential regulatory elements.

5.11 EXAMPLE 11 Methods for Obtaining ACC Mutants In E. coli, only conditional mutations can be isolated in the acc genes. The reason is that although the bacteria can replace the fatty acids in triglycerides with exogenously provided ones, they also have an essential wall component called lipid A, whose $-hydroxy myristic acid can not be supplied externally.

One aspect of the present invention is the isolation of Anacystis mutants in which the BC gene is interrupted by an antibiotic resistance cassette. Such techniques are well-known to those of skill in the art (Golden et al., 1987). Briefly, the method involves replacing the cyanobacterial ACC with wheat ACC, so it is not absolutely necessary to be able to maintain the mutants without ACC. The wheat ACC clone may be introduced first and then the endogenous gene can be inactivated without loss of viability.

By replacing the endogenous herbicide resistant ACC in cyanobacteria with the wheat cDNA, resulting cells are sensitive to the herbicides haloxyfop and clethodim, whose target is known to be ACC. Subsequently, one may isolate mutants resistant to those herbicides. These methods are known to those of skill in the art (Golden et al., 1987).

The transformation system in Anacystis makes it possible to pinpoint a very small DNA fragment that is capable of conferring herbicide resistance. DNA sequencing of wild type and resistant mutants then reveals the basis of resistance.

Alternatively, gene replacement may be used to study wheat ACC activity and herbicide inhibition in yeast. Mutants may be selected which overcome the normal sensitivity to herbicides such as haloxyfop. This will yield a variant(s) of wheat ACC that are tolerant/resistant to the herbicides. The mutated gene (cDNA) present on the WO 96/32484 PCT/US96/05095 -101plasmid can be recovered and analyzed further to define the sites that confer herbicide resistance. As for the herbicide selection, there is a possibility that the herbicide may be inactivated before it can inhibit ACCase activity or that it may not be transported into yeast. There are general schemes for treatment of yeast with permeabilizing antibiotics at sublethal concentrations, which are known to those of skill in the art.

Such treatments allow otherwise impermeable drugs to be used effectively. For these studies haloxyfop acid and clethodim may be used.

Characterization of the site(s) conferring herbicide resistance generally involves assaying extracts of the complemented ACC1 mutant for ACCase activity.

Both spontaneous mutation and chemical mutagenesis with DES or EMS, may be used to obtain resistant mutants, strains capable of growth in the presence of herbicide. These may be assayed for enzyme activity in vitro to verify that the mutation to resistance is within the ACCase coding region. Starting with one or more such verified mutants, the mutated site that confers resistance may be analyzed. Using the available restriction map for the wild-type cDNA, chimeric molecules may be constructed which containing half, quarter and eighth fragments, etc., from each mutant, and then checked by transformation and tetrad analysis to determine whether a particular chimera confers resistance or not.

An alternative method involves preparing a series of fragments of the mutant DNA, end-labeling, and annealing with the corresponding wild-type fragments in excess, so that all mutant fragments are in heterozygous molecules. Brief S 1 or mung bean nuclease digestion cuts the heterozygous molecules at the position of the mismatch within a few tens of base pairs. Then oligo-primed sequencing of the mutant DNA is used to identify the mutation. Finally, the mutation can be inserted into the wild-type sequence by oligo-directed mutagenesis to confirm that it is sufficient to confer the resistant phenotype. Having identified one or more mutations in this manner, the corresponding parts of several dicot ACCase genes to determine their structures in the corresponding region, in the expectation that they would be "resistant".

WO 96/32484 PCT/US96/05095 -102- Another method for the selection of wheat ACCase mutants tolerant or resistant to different herbicides involves the phage display technique. Briefly, in the phage display technique, foreign peptides can be expressed as fusions to a capsid protein of filamentous phage. Generally short (6 to 18 amino acids), variable amino acid sequences are displayed on the surface of a bacteriophage virion (a population of phage clones makes an epitope library). However, filamentous bacteriophages have also been used to construct libraries of larger proteins such as the human growth hormone, alkaline phosphatase (Scott, 1992) or a 50-kDa antibody Fab domain (Kang et al., 1991). In those cases, the foreign inserts were spliced into the major coat protein pVllI of the M13 phagemid. A complementary helper phage supplying wild-type pVUl has to be cotransferred together with the phagemid. Such "fusion phages" retained full infectivity and the fused proteins were recognized by monoclonal antibodies. These results demonstrate that foreign domains displayed by phage can retain at least partial native folding and activity.

Phage libraries displaying wild-type fragments of the wheat ACCase of 250 to 300 amino acids in size may be constructed without "panning" for phage purification.

The mechanism of purifying phages by panning involves reaction with biotinylated monoclonal antibodies, then the complexes are diluted, immobilized on streptavidin-coated plates, washed extensively and eluted. Generally, a few rounds of panning are recommended.

Instead, fragments bearing the ATP-binding site may be obtained by using Blue Sepharose CL-6B affinity chromatography, which was shown to bind plant ACCs (Betty et al., 1992; Egin-Buhler et al., 1980). Herbicides bound to Sepharose serve for capturing those phages which display amino acid fragments involved in herbicide binding. Such herbicide affinity resins may also be employed. After identifying peptide fragments that bind herbicides, ATP or acetyl-CoA the phages bearing those peptides may be subjected to random mutagenesis, again using phage display and binding to the appropriate support to select the interesting variants.

Sequence analysis then is used to identify the critical residues of the protein required for binding.

WO 96/32484 PCT/US96/05095 -103- 5.12 EXAMPLE 12 Preparation of ACC-specific antibodies Another aspect of the present invention is the preparation of antibodies reactive against plant ACC for use in immunoprecipitation, affinity chromatography, and immunoelectron microscopy. The antisera may be prepared in rabbits, using methods that are well-known to those of skill in the art (see Schneider and Haselkorn, 1988).

Briefly, the procedure encompasses the following aspects. Gel-purified protein is electroeluted, dialyzed, mixed with complete Freund's adjuvant and injected in the footpad at several locations. Subsequent boosters are given with incomplete adjuvant and finally with protein alone. Antibodies are partially purifiecL by precipitating lipoproteins from the serum with 0.25% sodium dextran sulfate and mM CaCl 2 Immunoglobulins are precipitated with 50% saturating ammonium sulfate, suspended in phosphate-buffered saline at 50 mg/ml and stored frozen. The antisera prepared as described may be used in Western blots of protein extracts from wheat, pea, soybean, canola and sunflower chloroplasts as well as total protein.

5.13 EXAMPLE 13 Protein Fusions, Transgenic Plants and Transport Mutants Analysis of promoter and control elements with respect to their structure as well as tissue specific expression, timing etc., is performed using promoter fusions with the GUS gene) and appropriate in situ assays. Constructs may be made which are useful in the preparation of transgenic plants.

For identifying transport of ACC, model substrates containing different length N-terminal fragments of ACC may be prepared by their expression (and labeling) in E. coli or by in vitro transcription with T7 RNA polymerase and translation (and labeling) in a reticulocyte lysate. Some of the model substrates will include the functional biotinylation site (located -800 amino acids from the N-terminus of the mature protein; the minimum biotinylation substrate will be defined in parallel) or native ACC epitope(s) for which antibodies will be generated as described above.

WO 96/32484 PC2TIUS96IO5095 -104- Adding an antibody tag at the C-terminus will also be very helpful. These substrates will be purified by affinity chromatography (with antibodies or streptavidin) and used for in vitro assays.

For modification of ACC protein transport, model substrates consisting of a transit peptide (or any other chloroplast targeting signals) to facilitate import into chloroplasts, fused to different ACC domains that are potential targets for modification, may be used. Modified polypeptides from cytoplasmic and/or chloroplast fractions will be analyzed for modification. For example, protein phosphorylation (with 32 p) can be followed by immunoprecipitation or by PAGE.

Antibodies to individual domains of ACC may then be employed. The same experimental set-up may be employed to study the possible regulation of plant ACC by phosphorylation Witters and Kemp, 1992). Biotinylation may be followed by Western analysis using 35 S-streptavidin for detection or by PAGE when radioactive biotin is used as a substrate.

5.14 EXAMPLE 14 Expression Systems for Preparation of ACC Polypeptides The entire plant ACC cDNA and its fragments, and BC, BCCP and the CT gene clones from cyanobacteria may be used to prepare large amounts of the corresponding proteins in E. coli. This is most readily accomplished using the T7 expression system. As designed by Studier, this expression system consists of an E.

coli strain carrying the gene for T7 lysozyme and for T7 RNA polymerase, the latter controlled by a lac inducible promoter. The expression vector with which this strain can be transformed contains a promoter recognized by T7 RNA polymerase, followed by a multiple cloning site into which the desired gene can be inserted (Ashton et al., 1994).

Prior to induction, the strain grows well, because the few molecules of RNA polymerase made by basal transcription from the lac promoter are complexed with T7 lysozyme. When the inducer IPTG is added, the polymerase is made in excess and the plasmid-bore gene of interest is transcribed abundantly from the late T7 promoter.

-105- This system easily makes 20% of the cell protein the product of the desired gene. A benefit of this system is that the desired protein is often sequestered in inclusion bodies that are impossible to dissolve after the cells are lysed. This is an advantage in the-present invention, because biological activity of these polypeptides is not required for purposes of raising antisera. Moreover, other expression systems are also available (Ausubel et al., 1989).

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

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WO 96/32484 PCT1US96/05095 113 7. SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: ARCH DEVELOPMENT CORPORATION STREET: 1101 East 58th Street CITY: Chicago STATE: Illinois COUNTRY: United States of America POSTAL CODE (ZIP): 60637 (ii) TITLE OF INVENTION: NUCLEIC ACID COMPOSITIONS ENCODING ACETYL-CoA CARBOXYLASE AND USES

THEREFOR

(iii) NUMBER OF SEQUENCES: (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 Unknown FILING DATE: 05-MAR-1996 (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/422,560 FILING DATE: 14-APR-1995 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 1458 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AAGCTTCATG ATTTCTAGTA ACGATTTTCG ACCTGGTGTA TCCATTGTCT TAGATGGGTC TGTATGGCGA GTGATAGATT TCCTTCACGT TAAGCCAGGT AAGGGTTCTG CCTTTGTACG 120 GACAACTCTG AAGAACGTCC AAAGCGGCAA AGTTTTAGAA AAAACCTTCC GGGCTGGGGA 180 AACTGTTCCA CAAGCTACTT TAGAAAAAAT TACAATGCAG CATACCTATA AAGAGGGCGA 240 TGAGTTCGTC TTTATGGATA TGGAAAGCTA TGAAGAAGGA CGACTCAGCG CCGCACAAAT 300 TGGCGATCGC GTCAAATACC TCAAGGAAGG TATGGAAGTG AACGTCATTC GTTGGGGTGA 360 GCAAGTGCTA GAGGTGGAAC TGGCTAATTC TGTAGTCTTG GAAGTTATAC AAACTGATCC 420 AGGTGTCAAG GGTGACACGG CTACAGGTGG CACGAAACCA GCAATTGTCG AAACTGGTGC 480 I I

AACTGTGATG

TGATAAATAC

TCCCTATTTC

AAAACTGGAT

ATGAAAAATT

ATGAAATCCG'

AAAGTGATGA

1s CGGTTGTGAC

TTGTAGCCCA

ATGCTGTCAC

AAGTGGCTTC

GTTCCTTTGT

TTAGGCAGGG

AGGGATTAAA

TCCCTTAAGG

CTTTATTTAC

TCAACTGCTG

TTTTGAACTA

AGCACCCTTG

TGCTGCCCCA

GAGTTCTGGC

CCCAATGGTG

TTATTTCTCA AGGAGAGCGA ATTAAAATTG ATACCCGTGA AATAGGTTTT ATCTCATCCG AGAACAAATC CCGATTTCAA TCCCTGCCAC ACTTAGGCCA ATTCAAAATT CAAAATTCAA TTTCTGAGTC TCAATGGTAG GGACTGGTCG AGGTAATAAA ACAACTATTG CACAAACAGA ACGGTGCGTA AAGCTGTTGG AGTGGTGTGQ TAGGTTCGGG TCTCCATCTC CAGAGCCGGG TCACAGCCAG GAGCAAAAAT GGAACATTTT ACCGCGCTCC ATGGATTTTG GAGAGTTGGT AACTGTGCCA TTGGACTTTA TATCGCGGAA GTAACGCTCA TGTGAATAAT AGTGTTGTGC ATTGCCATCG GCTATACCGA AACAAGCCGT GCTGCTGATC CATTGACCAA AAATTAGCAG TGCACCAGGT GAAGCGGTAT 0 540 600 660 720 780 840 900 I'J 960 1020 1080 1140 TTGTGGAAGT CGGCGATCGC ATCCGTCAAG GTCAAACCGT CTGCATCATC GAAGCAATGA 1200 I I w AGCTGATGAA TGAAATTGAG GCTGATGTT CTGGGCAAGT (GATCGAMAT CTCGTCCAA.A 1260 ACGGCGAACC TGTAGAATAT AATCAACCTT TGATGAGAAT TAAACCAGAT TAAGTATTAA 1320 TGTATATAGG TGAGTCATTA CTAACTCAAG TTGCTAGTTA TGTTTGGTAA TTGGTAAC!TG 1380 GTGATTGCTA ATTGGTAATT GAGAAAAATT TTACTCATTA. CCCATCACCC ATTACCAGTT 1440 CTTAAATTGA TAGCTAGC 1458 INFORMATION FOR SEQ ID NO: 2:

CF)

Wi SEQUENCE CHARACTERISTICS: LENGTH: 182 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Pro Leu Asp Phe Asn Glu Ile Arg Gin Leu Leu Thr Thr Ile Ala 1 5 10 I I s Gln Thr Asp Ile Ala Glu Val Thr Leu Lys Ser Asp Asp Phe Glu Leu Thr Val Arg Lys Ala Val Gly Val Asn Asn Ser Val Val Pro Val Val Thr Ala Pro Leu Ser Gly Val Val Gly Ser Gly Leu Pro Ser Ala Ile Pro Ile Val Ala His Ala Ala Pro Ser Pro Ser Pro Glu Pro Gly Thr Ser Arg Ala Ala Asp His Ala Val Thr Ser Ser Gly Ser Gin Pro Gly Ala Lys Ile Ile Asp Gin Lys Leu Ala Glu Val Ala Ser Pro Met Val 100 105 110 Gly Thr Phe Tyr Arg Ala Pro Ala Pro Gly Glu Ala Val Phe Val Glu 115 120 125 Val Gly Asp Arg Ile Arg Gin Gly Gin Thr Val Cys Ile Ile Glu Ala 130 135 140 0 Met Lys Leu Met Asn Glu Ile Glu Ala Asp Val Ser Gly Gin Val Ile 145 150 155 160 Glu Ile Leu Val Gin Asn Gly Glu Pro Val Glu Tyr Asn Gin Pro Leu 165 170 175 Met Arg Ile Lys Pro Asp 180 INFORMATION FOR SEQ ID NO: 3:

H

SEQUENCE CHARACTERISTICS: LENGTH: 477 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GTGCAACTGA ACTTCAGCCA ACTGCAAGAG CTGCTGACCG TGCTGAGTGA CTCAGACATC GCTGAGTTTG ACCTCAAAGG TACGGATTTT GAGTTGCACG TGAAGCGCGG CTCGACCGGC 120 GACCCGATCG TCATTGCGGC TCCCACCACG TTACCCGCTC CAACCCCTGC GGCAGCACCG CTTGAGATTA CGGCGCCGAT GGTGGGCACC CCCTTCGTCA. ATGTTGGCGA TCGCATTCAG ATGAAGCTGA TGAACGAGTT GGAGTCGGAG CAGAACGGCG AACCGGTGGA GTTTAATCAG CCCGTTGCTG TCGCTCCCGT CCTGCTGGAC CTCTGGGTGG TTCTATCGCG CTCCAGCACC GTGGGACAGA CCGTCTGCAT GTGACGGGGG AAGTCGTCGA CCCCTGTTCC GGTTGCGGCC

GCCCGCTCCC

CGAGAAGTTC

GGAAGAACCG

CCTCGAAGCG

GATTCTGGTC

TCTCTGA

0 180 240 300 360 420 477 INFORMATION FOR SEQ ID NO: 4: ()SEQUENCE CHARACTERISTICS: LENGTH: 158 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: I I Met Gin Leu Asn Phe Ser Gin Leu Gin Glu Leu Leu Thr 1 5 10 Asp Ser Asp Ile Ala Glu Phe Asp Leu Lys Gly Thr Asp 25 His Val Lys Arg Gly Ser Thr Gly Asp Pro Ile Val Ile 40 Thr Thr Pro Val Ala Val Ala Pro Val Pro Ala Pro Leu 55 Thr Pro Ala Ala Ala Pro Pro Ala Gly Pro Leu Gly Gly 70 75 Leu Glu Ile Thr Ala Pro Met Val Gly Thr Phe Tyr Arg 90 Pro Glu Glu Pro Pro Phe Val Asn Val Gly Asp Arg Ile 100 105 Gin Thr Val Cys Ile Leu Glu Ala Met Lys Leu Met Asn 115 120 125 Val Leu Ser Phe Glu Leu Ala Ala Pro Pro Ala Pro Glu Lys Phe Ala Pro Ala Gin Val Gly 110 Glu Leu Glu

I

Ser Glu Val Thr Gly Val Val Val Glu Ile Leu Val GisnAsn Gly Glu 130 135 140 Pro Val Glu Phe Asn Gin Pro Leu Phe Arg Leu Arg Pro Leu 145 150 155 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 3065 base pairs TYPE: nucleic acid STRANDEDNESS: single i TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AAGCTTTTAT ATTTTGCCAT TTCTAGAACT TAGCTGCATC GGCCCCAAGT ATTTTGTCAA ATATGGCGAA AAGACTTCAT AAATCAAGGT TAAAGGTTGA CCGTGATGCC AAAACAGGTA 120 ATGGCGACCC CAGAAAGGCC CATCCACGCC AAAACCTAAT TGCAAGGCCT CTGAATTTCC 180 GTAATAAATA CCCCGCACAT CCCGATACAA CTCCGTGCGA AGACGAGCTA GACTTGCCCA 240

AATTGGTAAT

TGCATAGGCG

CGCTGGAACA

ATCAATGACA

GCCATCACCT

TTTAATTACT

TGTTGAGTTG

TTATATCAAA

CCCCCTCCC

TATCACTTTT

GAGACTCGAA

GAACGGTTTT

AGTCGTTGGC

GATTGGATTA

TCGACTTCTG

AAGAGT.AGTA

TCTTCCCCAC

TTGCTTGGAG

GCATAAAAAA

CCTACTTCCC

GACCACTGAC

CTCGCAAGGC

GCAAATACTC

CAGAGCGTGT

AATCCGGCGC

TTGGCTCACC

GTTGCCACGC

CTTGCCAAAT

TAAAAGTGGC

TTAATTAGTT

TCCTCTGCCC

CTTTCACAAT

AAAGCCACAC

GTCTACATGG

ACGTAGCCAT

ACTATCTAAA

AGTAAGTAAT

TGGAGCCAGC

AGGAGTGAGG

AGTCAATGTT

GTCAGTTGTC

AATAATTAGA

TGACTATAGT

GCACCTCAAG

CTGGCTTCCC

ACCTGTCGCC

TCCAAACCAA

TCTAAATGCC

TGAGTGTGAG

CGATGCCATC

CTTTACAAAA

ATTGGTTATT

AAGGTCAGGA

CACTAAAAAA

CGTGCGCGTA

ACCATGAGGT

GCAGTCTTGG

TCAATGACAT

TTGTGGGTGA

GCAAACTATG

CGGCTGGCAG

GTTCACCTAT

CTTCTTTGCT

GTCAAAAACT

TGCGGATGGC

TACCAATTCC

300 360 420 480 540 600 660 720 780 840 900 960 GCCACATCCG CACGGGTTGT ACAAGAAGAT ATACTAGCAC AAAAAAATTG CATAAAACAA 0 1 GGTAA.AACTA TATTTGCCAA CGAAGAGGAT ACGAGACTAA TGGGGGTTTT CTTCCCTTAC TTGGTAAGTC CGTTTCCTGC TTTGAGATGA AGCGATGCCT GACAATTTCT ATCTGCTGGT AAATATTAAT TGCCAATCGG TGGGGATTGC GACGATCGCA TTGCTGACGA AGCGGTTTGT CCAATATTAT TGCTGCGGCT TTTTATCTGA AAATGCCAAA GCCCCACCCC AGAAGCTATC ACTTTATGGA AAATTTATCT CAGAAATGTA GTATCGCCAC ACCCTTAAAC CCTCACACCC AATTTATTTA AAGATGAGCC AAGGCGGCTA CGCTACGCGC ACTGATACTG ATATCGAAAA GGAGAAATAG CGCTGCGCAT GTTCATTCGA CTGTTGACCG ATTGGCGAAC CTGCTAGCGC TTAACGCGCA ATGCCAGTGC TTTGCGGAAA TCTGTGCTGA CGCCTCATGG GGGACAAATC TGCTAAATAT ACAAATTTCC AAGTGATATT AAAGGGGGTA CACCTCCATG AAAAATCTTG TGGGGTATCT CCTGTCATAA TAAAAGCAAC TTGGATGGGA CTAGAAAATG AAGTTTGACA TCTCCGCGCC TGTGAGGAAA GAATGCTCTT CATGTCCAAC TAAAAGTTAT TTGAATATTC TATTCATCCT GGGTATGGCT CCATCACATT GCATTCATTG CACTGCCAAG GAAACCATGC 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 I I AAAAAGCTGG TGTACCGACA GTACCGGGTA GTGAAGGTTT GGTAGAGACA GAGCAAGAAG

GATTAGAACT

GCGG CCGGGG

CCCAAGGTGA

AACGTCCGCG

TGGGTGAGAG

GCCCAGCCTT

AGTTTATCAA

TTTACTTTAT

CTGGAGTGGA

CTCAAGACCA

CAGACCACGA

GGCGAAAGAT ATTGGCTACC CAGTGATGAT TATGCGACTG GTGCGATCGC CAGATGAATT AGCTGGTGCA GCCTTTGGTA ATGCTGGCGT CCACATTGAA TTTCAAATTT TGGCTGATAA GGATTGCTCA ATTCAGCGTC GTAACCAAAA GGACTCAGAC CTAAGGGAAA AAATGGGACA TTACGCCGGG GCAGGTACTA TCGAGTTTTT GGAGATGAAC ACCCGGATTC AAGTAGAACA TTTATTGGTT GAGCAAATCA GAATTGCCCA AGTAGTTTTA CGCGGTCATG CGATCGAATG TTTCCGCCCA GCACCCGGAC GCATTAGCGG

CAAAGCCACG

TGTCAAACTG

TTATATAGAA

TTACGGCAAT

GTTACTAGAA

AGCGGCGGTG

GCTAGATAGA

TCCCGTAACT

AGGGGAAAGA

TCGCATCAAT

TTATCTTCCC

GCTGGTGGTG

TTCTTAGCCG

AAATTTATTG

GTGATTCACT

GAAGCCCCCA

AAAGCGGCTC

TCCGGTCAGT

GAGATGGTTA

CTTAGACTAA

GCCGAAGACC

CCTGGCGGCC

1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400

I

CTGGCGTGCG

CCTTAATTGG

AACGCGCCCT

GAATTATGGA

TGAATAAATA

AATTCCCTAA

TCGCAACAGG

TGGGATGATT

GGGCAAACGG

AAATGTCATC

GATTGACTCC

TAAATTGATC

CAGGGAATGC

AAATCCCCAA

GGGTAATGGG

CTCATCCGTG

CTAAAAATAC

TTTCGCAAGG

TTCAGATCGA

ACTCCCAATA

CACGTTTACA

GTTTGGGGCC

GCCATCACTG

TTTTTACAAG

TAATGGGTAA

CCAACATCGT

CAACACACAC

GAATGAGAAA

CTTGCGGATA

CAGGGCCAAG

CGGATTACCA

CTGATCGCGC

GATTACCTAC

GTAATGTGTC

TGGGTAATAG

CAGTAATCCT

AATGGGGGTG

TGGTTCAGTC

CCAGGTCAGA

AATCCAAACG

AATTCCGCCC

TACTGCTATT

AACCATTGGG

TACTAGTTTT

AGTTTCAATC

TGCTGGCCTA

ATATCAACAC

GGCCAAGCAA

ATGATACGGA

CTCAGGTTAA

TACTACGATT

AACCGCATGA

TTTCATCAAA

GTGCAGGAGA

ACCAATTACC

GAAGAACTTC

CACCTATTGG

TTAAGTTGAA

AAATAAACAG

CACCAGTCAT

2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3065

CGATCTAAGC

AGCTT

TACTATTTTG TGAATTTACA AAAAACTGCA AGCAAAAGCT GAAAATTTTA 0 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 447 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Met Lys Phe Asp Lys Ile Leu Ile Ala Asn Arg Gly Glu Ile Ala Leu 1 5 10 Arg Ile Leu Arg Ala Cys Glu Glu Met Gly Ile Ala Thr Ile Ala Val 25 His Ser Thr Val Asp Arg Asn Ala Leu His Val Gin Leu Ala Asp Glu 40 Ala Val Cys Ile Gly Glu Pro Ala Ser Ala Lys Ser Tyr Leu Asn Ile 55 60 0u' Pro Asn Ile Ile Ala Ala Ala Leu Thr Arg Asn Ala Ser Ala Ile His Pro Gly Tyr Gly Phe Leu Ser Glu Asn Ala Lys Phe Ala Glu Ile Cys Ala Asp His His Ile Ala Phe Ile Gly Pro Thr Pro Glu Ala Ile Arg 100 105 110 Leu Met Gly Asp Lys Ser Thr Ala Lys Glu Thr Met Gin Lys Ala Gly 115 120 125 Val Pro Thr Val Pro Gly Ser Glu Gly Leu Val Glu Thr Glu Gin Glu 130 135 140 Gly Leu Glu Leu Ala Lys Asp Ile Gly Tyr Pro Val Met Ile Lys Ala 145 150 155 160 Thr Ala Gly Gly Gly Gly Arg Gly Met Arg Leu Val Arg Ser Pro Asp 165 170 175 Glu Phe Val Lys Leu Phe Leu Ala Ala Gin Gly Glu Ala Gly Ala Ala 180 185 190 Phe Gly Asn Ala Gly Val Tyr Ile Glu Lys Phe Ile Glu Arg Pro Arg 195 200 205 His Ile Glu Phe Gin Ile Leu Ala Asp Asn Tyr Gly Asn Val Ile His 210 215 220 Leu Gly Glu Arg Asp Cys Ser Ile Gin Arg Arg Asn Gin Lys Leu Leu 225 230 235 240 Glu Glu Ala Pro Ser Pro Ala Leu Asp Ser Asp Leu Arg Glu Lys Met 245 250 255

H

Gly Gin Ala Ala Val Lys Ala Ala Gin Phe Ile Asn Tyr Thr Gly Ala 260 265 270 Gly Thr Ile Glu Phe Leu Leu Asp Arg Ser Gly Gin Phe Tyr Phe Met 275 280 285 Glu Met Asn Thr Arg Ile Gin Val Glu His Pro Val Thr Glu Met Val 290 295 300 Thr Gly Val Asp Leu Leu Val Glu Gin Ile Arg Ile Ala Gin Gly Glu 305 310 315 320 Arg Leu Arg Leu Thr Gin Asp Gin Val Val Leu Arg Gly His Ala Ile 325 330 335 Glu Cys Arg Ile Asn Ala Glu Asp Pro Asp His Asp Phe Arg Pro Ala 340 345 350 Pro Gly Arg Ile Ser Gly Tyr Leu Pro Pro Gly Gly Pro Gly Val Arg 355 360 365 Ile Asp Ser His Val Tyr Thr Asp Tyr Gin Ile Pro Pro Tyr Tyr Asp 370 375 380 Ser Leu Ile Gly Lys Leu Ile Val Trp Gly Pro Asp Arg Ala Thr Ala 385 390 395 400 Ile Asn Arg Met Lys Arg Ala Leu Arg Glu Cys Ala Ile Thr Gly Leu 405 410 415 Pro Thr Thr Ile Gly Phe His Gin Arg Ile Met Glu Asn Pro Gin Phe 420 425 430 Leu Gin Gly Asn Val Ser Thr Ser Phe Val Gin Glu Met Asn Lys 435 440 445 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 1362 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: ATGCGTTTCA ACAAGATCCT GATCGCCAAT CGCGGCGAAA TCGCCCTGCG CATTCTCCGC ACTTGTGAAG AACTCGGGAT CGGCACGATC GCCGTTCACT CCACTGTGGA TCGCAACGCG 120 o CTCCATGTGC AGTTAGCGGA CGAAGCGGTC TGTATTGGCG AAGCGGCCAG CAGCAAAAGC 180 TATCTCAATA TCCCCAACAT CATTGCGGCG GCCCTGACCC GTAATGCCAG CGCCATTCAC 240 CCCGGCTATG GCTTCTTGGC GGAGAATGCC CGCTTTGCAG AAATCTGCGC CGATCACCAT 300 CTCACCTTTA TTGGCCCCAG CCCCGATTCG ATTCGAGCCA TGGGCGATAA ATCCACCGCT 360 AAGGAAACAA TGCAGCGGGT CGGCGTTCCG ACGATTCCGG GCAGTGACGG TCTGCTGACG 420 GATGTTGATT CGGCTGCCAA AGTTGCTGCC ACGGCGGGGG GCGGTGGTCG CGGTATGCGG CTGTTCCTTG CTGCCCAAGG AGAAGCCGAG GAAAAATTTA TCGATCGCCC ACGCCACGTT AATGTAGTGC ATCTAGGCGA GCGCGATTGC GAAGAAGCCC CCAGTCCGGC GCTATCGGCA GTCAAAGTCG CTCAAGCGAT CGGCTACATC GCGACCGGCA ACTTCTACTT CATGGAGATG ACAGAAATGA TTACGGGACT GGACTTGATT GCGCTGCGCT TCCGGCAAGC CGATATTCAA AATGCGGAAG ATCCGGAATA CAATTTCCGG CCGCCCGGCG GCCCCGGCGT TCGTGTCGAT

GAGATCGGCT

CTGGTGCGTG

GCAGCTTTTG

GAATTTCAGA

TCCATTCAAC

GACCTGCGGC

GGTGCCGGCA

AATACCCGCA

GCGGAGCAGA

CTGCGCGGCC

ATCCCGTCAT GATCAAAGCG AGCCTGCAGA TCTGGAAAAA GGAATCCAGG ACTGTATCTC TCTTGGCCGA TGCCTACGGC GTCGTCACCA AAAGCTGCTC AGAAAATGGG CGATGCCGCC CCGTGGAGTT TCTGGTCGAT TCCAAGTCGA GCATCCAGTC TTCGGATTGC CCAAGGCGAA ATGCGATCGA ATGCCGTATC 480 540 600 660 720 780 840 900 960 1020 1080 1140 CCGAATCCTG GCCGCATTAC AGGCTATTTA TCCCATGTTT ATACCGACTA CGAAATTCCG CCCTATTACG ATTCGCTGAT TGGCAAATTG ATTGTCTGGG GTGCAACACG GGAAGAGGCG 1200 ATCGCGCGGA TGCAGCGTGC TCTGCGGGAA TGCGCCATCA CCGGCTTGCC GACGACCCTT 1260 AGTTTCCATC AGCTGATGTT GCAGATGCCT GAGTTCCTGC GCGGGGAACT CTATACCAAC 1320 TTTGTTGAGC AGGTGATGCT ACCTCGGATC CTCAAGTCCT AG 1362 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS:H LENGTH: 453 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Met Arg Phe Asn Lys Ile Leu Ile Ala Asn Arg Gly Giu Ile Ala Leu 1 5 10 Arg Ile Leu Arg Thr Cys Glu Glu Leu Gly Ile Gly Thr Ile Ala Val His Ser Thr Val Asp Arg Asn Ala Leu His Val Gin Leu Ala Asp Glu Ala Val Cys Ile Gly Glu Ala Ala Ser Ser Lys Ser Tyr Leu Asn Ile Pro Asn Ile Ile Ala Ala Ala Leu Thr Arg Asn Ala Ser Ala Ile His Pro Gly Tyr Gly Phe Leu Ala Glu Asn Ala Arg Phe Ala Glu Ile Cys Ala Asp His His Leu Thr Phe Ile Gly Pro Ser Pro Asp Ser Ile Arg 100 105 110 Ala Met Gly Asp Lys Ser Thr Ala Lys Glu Thr Met Gin Arg Val Gly 115 120 125 Val Pro Thr Ile Pro Gly Ser Asp Gly Leu Leu Thr Asp Val Asp Ser 130 135 140 Ala Ala Lys Val Ala Ala Glu Ile Gly Tyr Pro Val Met Ile Lys Ala 145 150 155 160 Thr Ala Gly Gly Gly Gly Arg Gly Met Arg Leu Val Arg Glu Pro Ala 165 170 175 Asp Leu Glu Lys Leu Phe Leu Ala Ala Gln Gly Glu Ala Glu Ala Ala 180 185 190 Phe Gly Asn Pro Gly Leu Tyr Leu Glu Lys Phe Ile Asp Arg Pro Arg 195 200 205 His Val Glu Phe Gin Ile Leu Ala Asp Ala Tyr Gly Asn Val Val His 210 215 220 Leu Gly Glu Arg Asp Cys Ser Ile Gin Arg Arg His Gin Lys Leu Leu 225 230 235 240 Glu Glu Ala Pro Ser Pro Ala Leu Ser Ala Asp Leu Arg Gin Lys Met 245 250 255 Gly Asp Ala Ala Val Lys Val Ala Gin Ala Ile Gly Tyr Ile Gly Ala 260 265 270 1. i Gly Thr Val 275 Glu Met Asn 290 Glu Phe Leu Val Asp 280 Thr Arg Ile Gin Val 295 Thr 305 Gly Leu Asp Leu Ile Ala Glu 310 Ala Leu Arg Glu Cys Arg Pro Gly Arg 355 Phe Arg Gin Ala Asp 325 Ile Asn Ala Glu Asp 340 Ile Thr Gly Tyr Leu 360 His Val Tyr Thr Asp 375 Ala Thr Gly Asn Phe Tyr Phe Met 285 Glu His Pro Val Thr Glu Met Ile 300 Gin Ile Arg Ile Ala Gin Gly Glu 315 320 Ile Gin Leu Arg Gly His Ala Ile 330 335 Pro Glu Tyr Asn Phe Arg Pro Asn 345 350 Pro Pro Gly Gly Pro Gly Val Arg 365 Tyr Glu Ile Pro Pro Tyr Tyr Asp 380 Val Asp Ser 370 Ser Leu Ile Gly Lys Leu Ile Val Trp Gly Ala Thr Arg Glu Glu Ala 385 390 395 400 Ile Ala Arg Met Gin Arg Ala Leu Arg Glu 405 410 Pro Thr Thr Leu Ser Phe His Gin Leu Met 420 425 Leu Arg Gly Glu Leu Tyr Thr Asn Phe Val 435 440 Cys Ala Ile Thr Gly Leu 415 Leu Gin Met Pro Glu Phe 430 Glu Gin Val Met Leu Pro 445 Arg Ile Leu Lys Ser 450 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 7360 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

ATCTCTTTCA

TGGATCTCCA

CGGGCGGACT

ACGGGAGGAT

ACAGCGTGCT

CCTGGGCCTT

CAGAGGACCT

CTGGTGGGAC

GAACTCGGGT

CAGACGCGCT

CACTAGGCGA

ACTTGGATAC

TCTCTCCTTC

CACGTGCTGA

GTCCTCGGTC

GGTTGCCAAC

GGAGACCTTT

CAGGATTAAT

GAATAACAAC

TTCTGCAGTT

CATGGAAAAG

TAAGATTGGT

CAGGCCGTTG

GCGGCGCGGC

AGGTTGGAGG

GACGAGTTCT

AATGGGATGG

GGGAA.CGAGA

GCGGAGCACA

AACTATGCAA

TGGCCTGGCT

GGAATCATTT

TCTTCTCTTA

CCTCCGCCGC

ATTCCGTCGA

GGGCAATAAT

GTAAAGCGCT

CTGCGGTCAA

AGGCCATTCT

TAAGAATCGC

ATGTACAGCT

GGGGTCATGC

TTCTTGGGCC

TTGCACAAGC

CGCCGCCTGC

ACGCCTCCGC

GGTGGAATCT

CGGGGGCGAC

GTTCATGCGC

CTTGGTGGCT

CGACCAGTTC

CATAGTGGAG

TTCTGAGAAC

ACCATCAGCC

AGCAGGAGTT

CTGCCTCTCC

GGCGCGCCTC

GACCAGATAA

TCGCCGATAC

AG CAT CCG CA

ATGGCAACTC

TTAGAAGTTC

ATAGCAGAGA

CCAGAACTTC

GCGATGGGGG

CCAACTCTTC

120 180 240 300 360 420 480 600 660 720 CATGGAGCGG GTCACATGTG AAAGTTCCGC AAGAAACCTG CCACTCAATA CCTGAGGAGA

TCTATAAGAA

GGTATCCTGC

ACAATGATGA

CTATATTTAT

ACAAGCATGG

AAAAGAT CAT

AGCAGGCGGC

ATCTGTACAG

TAGAACACCC

TAGGAATGGG

ATGGAGGTGG

ACAAAGCACA

CGCTTGTGTT

AATGATCAAG

TGAGGTCAGA

TATGAAGGTG

CAACGTGGCA

TGAGGAGGGA

AAGGCGACTA

CATGGAAACA

TGTGACCGAA

CATACCACTC

CTATCACGCT

GTCTGTAAAG

TCAACTACAG

GCATCATGGG

GCATTGTTTA

GCATCTCAGA

GCACTGCACA

CCAATTACAG

GCTAAATGTG

GGCGAATACT

TGGATTGCTG

TACAATATTC

TGGAAGGAAA

CCAAAGGGTC

ACGAAGCAGT

GTGGGGGTGG

AGCAAGTGCA

GCCGACATCT

GTCGAGACTG

TTGCTCCTCC

TGCAATATCA

ATTTCCTGGA

AAATTAACTT

CAGAGATCAG

TATCAGCTGT

ATTGTGTAGC

CGCTAGTTGT

TAAAGGAATA

AGGAGAGGTC

AGAGGTTCAG

TAGTGTTCAA

AGAAACAATT

GGGTGCTGCT

GCTTAATCCA

ACCTGCATCT

ACGCTTTTAT

AGCAACTAAA

AGTTAGAGTT

CAGGTGGTGG

AGGAAGGTAC

CCCGGATCGC

TTGCTCTGTG

AGAAGGCACC

AAAGAGCTTG

ACAGTGGAAT

AGGTTGCAGG

CAAGTTGTAG

GGAATAGAAC

TTTGATTTGG

ACTAGCGAGG

780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 ATCCAGATGA TGGGTTTAAG AACCCAATGT TTGGGCCTAC ATTCCCAGTT TGGTCATGTT TGGTACTTGG GTTAAAAGAG CTGTGGATCT CTTGAATGCT ACAGCAGAAT AGCTATGCGT GTGGAGCTCT ATATGAAGCA ATCTCAGTIAA AGGTCAAATA TGAATATAGA TGGGGGCAAA AATTAAGAAT TAATGAATCA TCTTAATGCA GTTGGATGGA CGCGCCTTCT AATCAATGGG CCTACCAGTG GAAGAGTGGA TTCTCCGTTA AGTCCGGAGG

TTTGCTTTTG

ATCCAAATTC

GCAGAGTACC

GTTAGAGCAG

TCAAGCAGGA

CCACCAAAGC

TATACGATTG

GAGGTTGAAG

AACAGTCATG

AGAACATGCT

GGGAATCTAG

GTGGAGAGAT

GAGAAAATAA

AGAGG CC CC C

GCTCGAGCGT

ACATCTCTCT

AGACAGTACG

CAGAGATACA

TAATTTACGC

TATTACAGAA

AGAGCTGAAC TTTAAAAGCA TGCAATTCAT GAGTTCTCTG GTCATTGGCA ATAGCCAATA ACGCACTAAT GTTGACTACA GATTCACACT GGTTGGCTAG ATGGTACCTT TCAGTTGTTG TGTAACCGAT TATGTTGGTT TGTCAATTTG ACTGTGACAC AGGTGGACCC CGTAGCTACA TTCTCTGCGA GATGGCGGAC CGAGACAGAA GCTGCTGGCA AGAGCATGAT CCTTCCAGGT 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 TGTTGGCTGA TACACCGTGC AAACTTCTTC TTGCTGATAC GCCATATGCT GAGGTGGAGG CGGCCTCTGG TGTCATTCAC TTTGTCATGC TGATAGCAAG GTTGGATCTT GATGACCCAT GCACCTTTCC AAAACTTGGA CCTCCTACTG CTGCAAGTGT GAATTCTGCC CACATGATCC TTGTACAAGA TTTGCTAAAC TGCCTAGACA AACTCATGTC CGTTTTGGCA ACCCGACTCC AGTACAAGGA GTATGAGTTG AATQCTGACT TGCTAAGGGG AGTCATTGAG GCTAATCTTG GTGAGAGGCT TGTAGAGCCA CTTATGAGCC GCCATGCTCG TGCGGTTGTC AAGTCTCTGT

GGTTTTTGGT

TTATGAAAAT

CTGAGGGTCA

CTTCTGTGAG

CTATTTCTGG

TTGCAGGATA

GCCCTGAGCT

CGAAAGATCT

TCCGGAAGAG

CATACTGTTC

TGGTCAAGTC

TTGAGGAGTA

CGCGGATGGT TCTCATGTGG GTGCATGCCA CTGTTACTAC GGCCATGCAG GCAAGTGATC AAGAGCTGAA CCATTTCATG CAAAGTTCAC CAAAAGTTTG TGAACATAAC ATCAATCATG CCCTTTCCTA CAGTGGCAAG TAGGAATGAG TTGGATGCTA CAAGGATTTC CCTGCCAAGT CGAGAAGGAT AGGGTTACTA ATATGAGGGT GGAAGAGAAA TTTATCTGTT GAAGAACTCT 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880

TCAGCGATGA

TTGAGAAGGT

TACTTCGGCT

TTCGCTTTTC

TTCTTGAGCA

TGGAGATGTT

ATGAAAGGAT

TGTTTGATCA

TGTATCAGCA

TTGCTTTATG

TT CTAAAGCC

AGTCTCTTCA

CATTCAGTCT

CGTATATATT

TATGGAAGCA

TGCCCTTAAC

CACTAAATTG

TACTGAGGAA

GGAAGATTTA

CAGTGATCCT

TTATCTTGTA

GGAATTCTCT

ACAAGTAGAG

GCTTCTGTCA

GATGTGATAG

GTGTTCTCCC

TTGGTCTATC

CATACAGCAT

AGTGAACTCC

GGAGAGCGGA

GTATGTGCCC

ACTCTTCAGC

AGGGGCAGTG

GAGGAACATA

GATCCAATTG

ACTGCAATTG

AACGTCTACG

ACCAGGGCGT

CAAATCCATC

ACTCTGGGCT

GCACAAGCAT

TTTCAACACC

CGGTTGCAGT

GGAGAGTTGT

TCCGGATGCA

TTGAACAAAG

GCAGGCGATG

AAGCTGCATT

ACTTCAACAT

GAAAAGTAAA

TGCGTACAGG

GGCGCTTAAA

AGCAAGAAGC

TAGGAGGAAG

TGAAGACGCC

TGAGACATAC

ATGGCACAGG

AAATGGGCAA

GGGTGTAATG

AAAGGAGACT

GCAAAAGACC

AATAAATTAA

GACCAGTTGA

GCAAGCCAAC

CTTTCAGAGC

ATGGCTATCA

CTTGTGGCTT

ATACGCAGAT

TCTGGTCTAA

TCTGCGTCAC

GTTGTAATCA

TCACATTACG

2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 GAGCAGGTGT TGGAGGTGTC TCAAATGGTA TGCATATTGC TTTGGTTGGT ATCAACAATC AGGATCAAGC GCAAGAAAGG ATCAACAAAC CATCACATCT CAATGGTGCT GGTGTTAGGG GGCGTTCACC AATGCGCCAC TCCTTCAAAT ACCCGATGCT CCGCCATGTG GAACCTCCTT ATTTAGAAGG TTACAATGAC GCGAAATACA ACACACTAGT AAAGAACAAG AAAGATCCGA

ATCCTATAAA

AGATGAGCAC

TCTCCAAGAT

TTGTCAGCTG

GGTCATCTGA

TGTCCACCTT

CCCCATCACG

GATCAAATGA

TTCTAACAGT AGCAATATGC TCTTCAAGAC AGTGGTGATG TTTGAAGGAT AACACTATAA CATTATCCAA AGAGATGAAG CAAGTTATAT TATGAGGAGG CCTTGAATTG GACAAAGTGA TGATCGCCAG TGGCACATGT CCAAAGGATG TTTCTTCGTA TCGAAGTATT GATAATGAAG ATCATTGATG GCAGCGCTAG AGGCCACTCC CACATGTATC ATCTTCAAGG ATGACGAATG 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 CCATAGTCAG ACAGCCAAGT GTGACCAATG GGTTTTTGTT TTCAAGCCTC ATCATCATTC ACATCTAACA GCATACTCAG AAGAAATAGA GTTGCGCGCT CACAGTGAGA CTGGGATGTC TGTGCATAAT GAGAGAACAG CGGTTGTTTG ATCTAATTCC AAGTTGGTCA AGATGAGAAG ACAGCATGCA CATTATTGAA TGAGCATGTG GTGTCAGGAT GCATCGCTTT CTGTGTGCCA TGGATTGTGA TGGGCAGGCT AATGGTGCTT GGAGAGTTGT ATACCTGCAC TGTTGATATT TACCGAGAAG TGGAGGACCC ACCGCTCTGC CACACCCACA GCTGGTCCTT TGCATGGCAT AACCTTTGGA TGCTATTGAC CTGAAACGTG CCGCTGCTAG GCTATGATTT CCCATTGGCA TTTGAAACAG CATTGAAGAA CACATGTTGC AGAATCTAAC GAGCATAACC AGCGGTATGC TTGCTCATTC AACTGGATCA TGCGGTACTC CTTTGGTTCC GCAACAATTT TGGTGTTGTT GCTTGGAACA TGAAGCTCTC GCCGGGAGAT TATAGTTGTT GCAAATGATG TGACATTTAA GAGAAGATGC ATTCTTTGAT GCTGTCACCA ATCTTGCTTG GCATATGGGT ATGATATATA GTGGGAAGTG AAGCTATGGT TGTTACCAGT GTAACTGGGC CAATACACAT CAGCTTTTCT TGCATTGCAT GAGCCATACA GAAAAATGAA ACCACATACT GTCATGGGAA TCTGGTATTT TGAAGTGACA GAGCTTATAT AGTTGAGCGT CCTCCAGGTA CACACCAGAA TTTCCAGGCG AGCTGGGTCT TTTGGTCCTA TGAGAGGAAA ATTCCTCTAA 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 TTTACTTGTC AGCAACTGCT TCCATGTTGG ATGGTCTGAT CTGAACAAGA TTATTCACGT AAAGCGGAGA AACCAGATGG GTGAGAATCT ACATGGAAGT CCTTTACTCT GACATTTGTG TAGGAATGCG GTGTATACAA TGAACAAGCT CCTGGGGCGC TCATGGCTAC AAATGGAGTT CTATCTTGAA ATGGCTCAGC AATCTCTTGA TCCACCAGAG GTGCCGCCAT CTGTGGCATC GGTGCTAGGC TCGGTGTAGC GACCAGAGCC CTGAACGTGG CTAAGCTCTT CAGTTATAGC GTTGTTGATA CCATTGTTGG GGTGCCATTG CCAGTGCCTA ACTGGGCGAG CTATTGGAAT CGTCTTGATC AACCAATTAT GAGGTGTATA GCTCTCAGAT GTCCATCTCA CTGTGTCAGA TATGTTCCTC CCTATGTTGG AGAGCTGTAA CATATTTCCC CAGGACACTC AAGGAGGCAA AGAGGAAATA AAGGCGTGCT TTTTCACTAC ATTTACCTCA CCATGAGCTA AAAGTACCGG GAAAGAGGAC GGACTTGGTT CTCTAAGGCA TACAGAGAGA TGGGGCTTAT CTTGCTCGGT TTTGACTGGG TATTCTGCAC GCAACTGGGT GGCCCCAAAA TGATCTTGAA GGTGTTTCTG CGGTCCTCTT CCTATTGTGA AGAGAATTCA TGTGATGCCC GTGGTTGGAT GGTATGTTTG 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 ACAGAGAAAG CTTTGTGGAA ACATTAGAAG GATGGGCCAA AACTGTTATT ACTGGAAGGG CAAAGCTAGG TGGGATTCCA TAATCCCTGC TGACCCTGGT AGGTGTGGTT CCCAGATTCG AAGAGCTCCC ATTGTTCATA TGTTTGAAGG AATCCTTCAG AGCCTGCTTT TGTGTACATA TGGACAGCAA GATCAATCCT ATGTCCTTGA GGCACCAGGA GTATGCTAAG GCTTGACCCT GTGCTAGCCC TAGTCCTTGG CTGCTCGGAG GAAGCAGCTG

GTTGGTATCA

CAGCTTGATT

GCCGCAAAAA

CTTGCTAACT

GCTGGCTCTA

CCAAAGGCTG

GAGCACATTG

CTCATTGAGA

GAGTTGATCA

GAAACGGCGG

ATGCCCATAT

TAGCTGTGGA

CTGCCGAGCG

CGGGCCAGGC

GGAGAGGCTT

TGATTGTTGA

GAGAGCTGCG

AGATGTATGC

TCAAGTTCAA

GCCTCAATGC

CGGCGGCGGA

ATACTCAGGT

AACCGAGACA GTGATGCAAG TGTAGTCCCT CAAGCTGGAC ACTGCTGGAT TTCAACCGTG TTCTGGTGGG CAAAGGGATC GAATCTGAGG ACGTATAAGC TGGAGGTGCA TCGGTTGTGG CGAGAGGACT GCGAGAGGGA GCCAAATGAA CTGGAAGAGA CAAACTCCTC AAAGAAACTA GACCATCAGG AGGAGCATGG TGCCACCCGG TTTGCTGAGT 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480

TGCACGACAC

AGGAGTCCCG

CCAAACAAGT

AATGCATCAA

ATGAAGCCTT

TGAAAGCCGA

CCTTGCCCAA

TTATGGATGG

CATCCATTCA

GTGGACTGGG

CTACAGGAGG

TCATCTCAGG

CTCTGCGAGA

AGCCTTCTTC

CAGAGAAGC C

GAAATGGTAC

CTTCGCCTGG

GAGAGCGTCT

CGGTCTATCG

CCTCAGGCAG

GCCAGCATAA

TACCAACGGA

GGGAGGTTTT

ACAATTTGTA

ATGGCTGCCA

TACAGGAGAC

GCCGGCGAGC

CTGGCCTCTC

AAAGATGATC

ACACTGCTGT

CTC CT CCT CA

CTTCTTGGTT

ACCGGCCTTG

AGCGCAGACG

TCAACTGAAA

TGTTTATTGT

AAGGCGTGAT

TGCGAAGGAG

AGCAGATGCC

AGGGAGGAGA

CTGACAAGTA

CGCATCTCGC

GCAAAATGGA

GATGACTGGC

CTTGTTGCCA

ACGACAAGCA

CACATTGTTT

TATTACAGAT

CAGTAAGGTG

GCTTGCCGAG

CACTCACAGA

CGGCGAGAAG

TGGCAAGTAT

TGAAACCTCT

TCCTGCAAAG

CCACCCTTTG

CCAAGCAAGT

AATTTTACTT

GCACATAGGT

AGGTACACAC

GTGGACTGGG

GACTCGCTCG

TCGGCCTTGG

TGGGGAGACG

CTTGAGGAGC

GATGCCAAGG

AGGGAGCAGG

ATAACGGGAG

CCTGTCTATG

GCGTGGCGAG

AGGAGGCATC

AAAGCATATG

6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 0 TATGCTGGAT AGATATTCGG TGTGAGTTGT TGCAATGCAA GATTCATCAT CTTAATTTAC 7260 GAGATACGTG TGATGGTCGA TGTGATAGTC CTAGTTTCCT CGGTGGCGAG GAACGCTGAG 7320 TTTCCTTTTG CTGCAGTTAT GTGATGTATA CCCTGAGAAC 7360 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 2257 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Val Glu Ser Asp Gin Ile Asn Gly Arg Met Ser Ser Val Asp Glu 1 5 10 Phe Cys Lys Ala Leu Gly Gly Asp Ser Pro Ile His Ser Val Leu Val 25 us Ala Asn Asn Gly Met Ala Ala Val Lys Phe Met Arg Ser Ile Arg Thr Trp Ala Leu Glu Thr Phe Gly Asn Glu Lys Ala Ile Leu Leu Val Ala Met Ala Thr Pro Glu Asp Leu Arg Ile Asn Ala Glu His Ile Arg Ile Ala Asp Gin Phe Leu Glu Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gin Leu Ile Val Glu Ile Ala Glu Arg Thr Arg Val Ser 100 105 110 Ala Val Trp Pro Gly Trp Gly His Ala Ser Glu Asn Pro Glu Leu Pro 115 120 125 Asp Ala Leu Met Glu Lys Gly Ile Ile Phe Leu Gly Pro Pro Ser Ala 130 135 140 Ala Met Gly Ala Leu Gly Asp Lys Ile Gly Ser Ser Leu Ile Ala Gin 145 150 155 160 Ala Ala Gly Val Pro Thr Leu Pro 165 Cys His Ser Ile Pro Gin Glu Thr 180 Cys Val Ser 195 Thr Thr Asp Glu Ala 200 Tyr Pro 210 Arg Lys 225 Ala Met Ile Lys Ala Ser 215 Val His Asn Asp Asp Glu 230 Trp Ser Gly Ser His Val Lys Val 170 175 Pro Glu Glu Ile Tyr Lys Asn Ala 185 190 Val Ala Ser Cys Gin Val Val Gly 205 Trp Gly Gly Gly Gly Lys Gly Ile 220 Val Arg Ala Leu Phe Lys Gin Val 235 240 Ile Phe Ile Met Lys Val Ala Ser 250 255 Leu Leu Cys Asp Lys His Gly Asn 265 270 Cys Ser Val Gin Arg Arg His Gin 285 Gin Gly Glu Gin Ser Arg Val Ala Ala 275 Val Pro Gly Ser Pro 245 His Leu Glu Val Gin 260 Leu His Ser Arg Asp 280 Lys Ilie Ilie Giu Glu Gly Pro Ile Thr Val Ala Pro Pro Giu Thr Ile 290 295 300 Lys Giu Leu Glu Gin Ala Ala Arg Arg Leu Ala Lys Cys Val Gin Tyr 305 310 315 320 Gin Gly Ala Ala Thr Val Giu Tyr Leu Tyr Ser Met Glu Thr Gly Giu 325 330 335 Tyr Tyr Phe Leu Glu Leu Asn Pro Arg Leu Gin Val Glu His Pro Val 340 345 350 Thr Giu Trp Ile Ala Glu Ile Asn Leu Pro Ala Ser Gin Val Val Val 355 360 365 Gly Met Gly Ile Pro Leu Tyr Asn Ile Pro Glu Ile Arg Arg Phe Tyr 370 375 380 Gly Ile Giu His Gly Gly Gly Tyr His Ala Trp Lys Giu Ile Ser Ala 385 390 395 400 Vai Ala Thr Lys Phe Asp Leu Asp Lys Ala Gin Ser Vai Lys Pro Lys 405 45410 415 Gly His Phe Lys Cys Val Ala Val Arg Val Thr Ser Glu Asp Pro Asp Asp Gly 420 425 430 Pro Thr Ser Gly Arg Val Glu Glu Leu Asn Phe Lys Ser Lys 435 440 445 Val Trp Ala Tyr Phe Ser Val Lys Ser Gly Gly Ala Ile His 455 460 Pro Asn 450 Glu 465 Phe Ser Asp Ser Gin Phe Gly His Val Phe Ala Phe Gly Glu 470 475 Ser 480 Gin Arg Ser Leu Ala Ile Ala Asn Met Val Leu Gly Leu Lys Glu Ile 485 490 495 Ile Arg Asn Ala Gly Glu Ile Arg Thr Asn Val Asp Tyr Thr Val Asp Leu Leu 500 505 510 Ala Glu Tyr Arg Glu Asn Lys Ile His Thr Gly Trp Leu Asp 515 520 525 Ile Ala Met Arg Val Arg Ala Glu Arg Pro Pro Trp Tyr Leu 535 540 Ser Arg 530 Ser Val Val 545 Val Val Thr Gly Gly Asp Tyr 565 Ser Leu 580 Ala Leu Tyr Giu Ala Ser 550 555 Val Gly Tyr Leu Ser Lys 570 Val Asn Leu Thr Val Thr 585 Lys His Ile Gly Lys Tyr 595 Leu Arg Ile 610 Thr Ile Giu Thr Val Arg Gly Gly 600 Asn Glu Ser Giu Val Glu Ala Glu 615 Ser Arg Ser Ser Ser 560 Gly Gin Ile Pro Pro 575 Leu Asn Ile Asp Gly 590 Pro Arg Ser Tyr Lys 605 Ile His Ser Leu Arg 620 Ser His Val Ile Tyr 640 Ile Asn Giy Arg Thr 655 Leu Leu Ala Asp Thr 670 Asp Giy Giy Leu Leu 625 Ala Giu Thr Giu Ala 645 Gin Lys 660 Met Gin Leu Asp Giy Asn 630 635 Ala Gly Thr Arg Leu Leu 650 Giu His Asp Pro Ser Arg 665 Cys Leu Leu Pro Cys Lys Leu Leu Arg Phe Leu 675 680 Ala Asp Thr Pro Tyr Ala Glu Val 690 695 Leu 705 Leu Leu Pro Ala Ser Gly Val 710 Gin Ala Met Gin Ala Ser Asp Leu 725 Pro Ser Ser Val Arg Arg Ala Glu 740 Leu Gly Pro Pro Thr Ala Ile Ser 755 760 Ala Ser Val Asn Ser Ala His Met 770 775 Val Ala Asp Gly Ser His Val Val 685 Glu Val Met Lys Met Cys Met Pro 700 Ile His Phe Val Met Pro Glu Gly 715 720 Ile Ala Arg Leu Asp Leu Asp Asp 730 735 Pro Phe His Gly Thr Phe Pro Lys 745 750 Gly Lys Val His Gin Lys Phe Ala 765 Ile Leu Ala Gly Tyr Glu His Asn 780 Leu Asn Cys Leu Asp Ser Pro Glu Ile 785 Asn His Val Val Gin Asp Leu 790 795 800 Leu Pro Phe Leu Gin Trp Gin Glu Leu Met Ser Val 805 810 Leu Pro Lys Asp Leu Arg Asn Glu Leu Asp Ala Lys 820 825 Glu Leu Asn Ala Asp Phe Arg Lys Ser Lys Asp Phe 835 840 Leu Arg Gly Val Ile Glu Ala Asn Leu Ala Tyr Cys 850 855 860 Arg Val Thr Ser Glu Arg Leu Val Glu Pro Leu Met 865 870 875 Ser Tyr Glu Gly Gly Arg Glu Ser His Ala Arg Ala 885 890 Leu Phe Glu Glu Tyr Leu Ser Val Glu Glu Leu Phe 900 905 Gin Ser Asp Val Ile Glu Arg Leu Arg Leu Gin His 915 920 Leu Ala Thr Arg 815 Tyr Lys Glu Tyr 830 Pro Ala Lys Leu 845 Ser Glu Lys Asp Ser Leu Val Lys 880 Val Val Lys Ser 895 Ser Asp Asp Ile 910 Ala Lys Asp Leu 925 :0 Giu Lys Val Val Tyr Ile Val Phe Ser His Gin Gly Val Lys Ser Lys 930 935 940 Asn Lys Leu Ile Leu Arg Leu Met Glu Ala Leu Val Tyr Pro Asn Pro 945 950 955 960 Ser Ala Tyr Arg Asp Gin Leu Ile Arg Phe Ser Ala Leu Asn His Thr 965 970 975 Ala Tyr Ser Gly Leu Aia Leu Lys Ala Ser Gin Leu Leu Glu His Thr 980 985 990 Hn Lys Leu Ser Glu Leu Arg Thr Ser Ile Ala Arg Ser Leu Ser Giu Leu LI!U 995 1000 1005 Glu Met Phe Thr Giu Giu Giy Giu Arg Ile Ser Thr Pro Arg Arg Lys 1010 1015 1020 Met Ala Ilie Asn Giu Arg Met Giu Asp Leu Vai Cys Aia Pro Vai Aia 1025 1030 1035 1040 Vai Giu Asp Ala Leu Vai Ala Leu Phe Asp His Ser Asp Pro Thr Leu 0 1045 1050 1055

L

0 Gin Arg Arg Vai Val Glu Thr Tyr Ile Arg Arg Leu Tyr Gin His Tyr 1060 1065 1070 Leu Val Arg Gly Ser Val Arg Met Gin Trp His Arg Ser Gly Leu Ile 1075 1080 1085 Ala Leu Trp Glu Phe Ser Giu Giu His Ile Glu Gin Arg Asn Giy Gin 1090 1095 1100 Ser Ala Ser Leu Leu Lys Pro Gin Val Glu Asp Pro Ile Gly Arg Arg 1105 1110 1115 1120 Trp Gly Val Met Val Val Ile Lys Ser Leu Gin Leu Leu Ser Thr Ala 1125 1130 1135 Ile Giu Ala Ala Leu Lys Glu Thr Ser His Tyr Gly Ala Giy Vai Gly 1140 1145 1150 Gly Val Ser Asn Gly Asri Pro Ile Asn Ser Asn Ser Ser Asn Met Leu 1155 1160 1165 His Ile Aia Leu Val Gly Ile Asn Asn Gin Met Ser Thr Leu Gin Asp 1170 1701175 1180 0 Ser Gly Asp Glu Asp Gin Ala Gin Glu Arg Ile Asn Lys Leu Ser Lys 1185 1190 1195 1200 Ile Leu Lys Asp Asn Thr Ile Thr Ser His Leu Asn Gly Ala Gly Val 1205 1210 1215 Arg Val Val Ser Cys Ile Ile Gin Arg Asp Glu Gly Arg Ser Pro Met 1220 1225 1230 Arg His Ser Phe Lys Trp Ser Ser Asp Lys Leu Tyr Tyr Glu Glu Asp 1235 1240 1245 Pro Met Leu Arg His Val Glu Pro Pro Leu Ser Thr Phe Leu Glu Leu 1250 1255 1260 Asp Lys Val Asn Leu Glu Gly Tyr Asn Asp Ala Lys Tyr Thr Pro Ser 1265 1270 1275 1280 Arg Asp Arg Gin Trp His Met Tyr Thr Leu Val Lys Asn Lys Lys Asp 1285 1290 1295 Pro Arg Ser Asn Asp Gin Arg Met Phe Leu Arg Thr Ile Val Arg Gin 1300 1305 1310 Pro Ser Val Thr Asn Gly Phe Leu Phe Giy Ser Ile Asp Asn Glu Val 1315 1320 132S Gin Ala Ser Ser Ser Phe Thr Ser Asn Ser Ile Leu Arg Ser Leu Met 1330 1335 1340 Ala Ala Leu Giu Giu Ile Glu Leu Arg Aia His Ser Giu Thr Gly Met 1345 1350 1355 1360 Ser Gly His Ser His Met Tyr Leu Cys Ile Met Arg Giu Gin Arg Leu 1365 1370 1375 Phe Asp Leu Ile Pro Ser Ser Arg Met Thr Asn Giu Val Gly Gin Asp 1380 1385 1390 Giu Lys Thr Ala Cys Thr Leu Leu Lys His Met Gly Met Ile Tyr Met 1395 1400 1405 Ser Met Trp Cys Gin Asp Ala Ser Leu Ser Val Cys Gin Trp Glu Val 1410 1415 1420 Lys Leu Trp Leu Asp Cys Asp Giy Gin Ala Asn Gly Ala Trp Arg Val 1425 1430 1435 1440 Val Val Thr Ser Val Thr Gly His Thr Cys Thr Val Asp Ile Tyr Arg 1445 1450 1455 Glu Val Glu Asp Pro Asn Thr His Gin Leu Phe Tyr Arg Ser Ala Thr 1460 1465 1470 Pro Thr Ala Gly Pro Leu His Gly Ile Ala LeuHis Giu Pro Tyr Lys 1475 1480 1485 Pro Leu Asp Ala Ile Asp Leu Lys Arg Ala Ala Ala Arg Lys Asn Glu 1490 1495 1500 Thr Thr Tyr Cys Tyr Asp Phe Pro Leu Ala Phe Glu Thr Ala Leu Lys 1505 1510 1515 1520 Lys Ser Trp Giu Ser Gly Ile Ser His Val Ala Glu Ser Asn Glu His 1525 1530 1535 Asn Gin Arg Tyr Ala Glu Val Thr Glu Leu Ile Phe Ala Asp Ser Thr 1540 1545 1550 Gly Ser Trp Gly Thr Pro Leu Val Pro Val Glu Arg Pro Pro Gly Ser 1555 1551560 1565 Asn Asn Phe Gly Vai Val Ala Trp Asn Met Lys Leu Ser Thr Pro Glu 1570 1575 1580 Phe Pro Gly Gly Arg Giu Ile Ile Val Vai Ala Asn Asp Val Thr Phe 1585 1590 1595 1600 Lys Ala Gly Ser Phe Gly Pro Arg Giu Asp Ala Phe Phe Asp Ala Vai 1605 1610 1615 Thr Asn Leu Ala Cys Glu Arg Lys Ile Pro Leu Ile Tyr Leu Ser Ala 1620 1625 1630 Thr Ala Gly Ala Arg Leu Giy Val Ala Giu Glu Ile Lys Ala Cys Phe 1635 1640 1645 His Val Gly Trp Ser Asp Asp Gln Ser Pro Glu Arg Giy Phe His Tyr 1650 16S5 1660 Ile Tyr Leu Thr Glu Gin Asp Tyr Ser Arg Leu Ser Ser Ser Val Ile 1665 1670 1675 1680 Ala His Giu Leu Lys Val Pro Glu Ser Gly Glu Thr Arg Trp Val Val 1685 1851690 1695 Asp Thr Ile Val Gly Lys Giu Asp Gly Leu Gly Cys Giu Asn Leu His 1700 1705 1710 Gly Ser Gly Ala Ile Ala Ser Ala Tyr Ser Lys Ala Tyr Arg Giu Thr 1715 1720 1725 Phe Thr Leu Thr Phe Val Thr Gly Arg Ala Ile Gly Ile Gly Ala Tyr 1730 1735 1740 Leu Ala Arg Leu Gly Met Arg Cys Ile Gin Arg Leu Asp Gin Pro Ile 1745 1750 1755 1760 Ile Leu Thr Giy Tyr Ser Ala Leu Asn Lys Leu Leu Gly Arg Giu Val 1765 1770 1775 Tyr Ser Ser Gin Met Gin Leu Gly Gly Pro Lys Ile Met Ala Thr Asn 1780 1785 1790 Gly Val Val His Leu Thr Val Ser Asp Asp Leu Giu Gly Val Ser Ala 1795 1800 1805 Ile Leu Lys Trp Leu Ser Tyr Val Pro Pro Tyr Val Gly Gly Pro Leu 1810 1101815 1820 Pro Ile Val Lys Ser Leu Asp Pro Pro Glu Arg Ala Val Thr Tyr Phe 1825 1830 1835 1840 Pro Giu Asn Ser Cys Asp Ala Arg Ala Ala Ile Cys Gly Ile Gin Asp 1845 1850 1855 Thr Gin Giy Gly Lys Trp Leu Asp Gly Met Phe Asp Arg Giu Ser Phe 1860 1865 1870 Val Giu Thr Leu Giu Giy'Trp Ala Lys Thr Vai Ile Thr Giy Arg Aia 1875 1880 1885 Lys Leu Gly Gly Ile Pro Val Gly Ile Ile Aia Vai Giu Thr Giu Thr 1890 1895 1900 Val Met Gin Vai Ile Pro Ala Asp Pro Giy Gin Leu Asp Ser Ala Giu 1905 1910 1915 1920 Arg Vai Val Pro Gin Ala Gly Gln Vai Trp, Phe Pro Asp Ser Ala Aia 1925 1930 1935 Lys Thr Gly Gin Ala Leu Leu Asp Phe Asn Arg Giu Giu Leu Pro Leu 1940 1401945 1950 Phe Ile Leu Ala Asn Trp Ar Gly PeSer Gl Gly Gin Arg Asp Leu 1955 1960 1965 Phe Glu Gly Ile Leu Gin Ala Gly Ser Met Ile Val Giu Asn Leu Arg 1970 1975 1980 Thr Tyr Lys Gin Pro Ala Phe Val Tyr Ile Pro Lys Ala Gly Giu Leu 1985 1990 1995 2000 Arg Gly Gly Ala Trp Val Val Val Asp Ser Lys Ile Asn Pro Glu His 2005 2010 2015 Ile Glu Met Tyr Ala Glu Arg Thr Ala Arg Gly Asn Val Leu Glu Ala 2020 2025 2030 Pro Gly Leu Ile Glu Ile Lys Phe Lys Pro Asn Glu Leu Glu Glu Ser 2035 2040 2045 Met Leu Arg Leu Asp Pro Glu Leu Ile Ser Leu Asn Ala Lys Leu Leu 2050 2055 2060 Lys Giu Thr Ser Ala Ser Pro Ser Pro Trp, Giu Thr Ala Ala Ala Ala 2065 2070 2075 2080 9: Glu Thr Ile Arg Arg Ser Met Ala Ala Arg Arg Lys Gin Leu Met Pro 2085 2090 2095 Ile Tyr Thr Gin Val Ala Thr Arg Phe Ala Glu Leu His Asp Thr Ser 2100 2105 2110 Ala Arg Met Ala Ala Lys Gly Val Ile Ser Lys Val Val Asp Trp Glu 2115 2120 2125 Glu Ser Arg Ala Phe Phe Tyr Arg Arg Leu Arg Arg Arg Leu Ala Glu 2130 2135 2140 Asp Ser Leu Ala Lys Gin Val Arg Glu Ala Ala Gly Glu Gin Gin Met 2145 2150 2155 2160 Pro Thr His Arg Ser Ala Leu Glu Cys Ile Lys Lys Trp Tyr Leu Ala 2165 2170 2175 Ser Gin Gly Gly Asp Gly Glu Lys Trp Gly Asp Asp Glu Ala Phe Phe 2180 2185 2190 Ala Trp Lys Asp Asp Pro Asp Lys Tyr Gly Lys Tyr Leu Glu Glu Leu 2195 2200 2205 Lys Ala Glu Arg Ala Ser Thr Leu 2210 2215 Asp Ala Lys Ala Leu Pro Asn Gly 2225 2230 Asp Pro Ala Lys Arg Glu Gln Val 2245 Leu Ser His Leu Ala 2220 Leu Ser Leu Leu Leu 2235 Met Asp Gly Leu Arg 2250 Glu Thr Ser Ser Lys Met 2240 Gln Leu Leu 2255 Gly INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 984 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ATGGCTGCAC CTGTCACGAA GAAGCCAATT CTGCTGGAGT TTGAAAAGCC CCTAGTTGAG CTGGAGGAAC GGATCACGCA AATCCGCACC CTCGCAGCGG ACAACCAGGT GGATGTGAGC GGCCAAATTC AGCAACTGGA CTCTCGCCAG CCCAGCGCAT TACATCCAAG CGATCAGCGA GACCTCGCAC TCGTCGGTGG CACCAAAAGG GGCGCGACAC GGCGGCTATC GCAAGG'CACT CTGACCTTTA TCGATACACC GGTGAGGCAA TCGCAGTCAA ACAGTGATTG GCGAAGGCGG CTGATGTTTG AGCATTCCGT TGGCGTGATG CGGGCAAGGC

AGCCCGGGCG

CCAAGTGGCG

CGAGTGGATT

TGTTGGTGCG

CAAGGACAAC

GCGTTTGATG

CGGTGCTTAC

CCTGCGCGAA

TTCGGGCGGG

CTACACTGTT

AGCCCAGGCG

ATTCAACTGC

CGTCATCCCC

GAATTACACG

CTCGACGGCC

GTGCTGCGCA

GAGCATGCCG

GCTGGGGTCA

ATGTTCCGCT

GCCTTGGGCA

GCCAGTCCCG

GCAGAAGCGC

GGCGAGAAAT

GACGTCCGAG

GCGATCGCAA

AGCCAGTCGT

ACTTCGGGAT

ATCGCTTCGG

GTGCTGAAGA

TCTCGGTGCC

TTGGCGTCGG

AAGCCTGCGC

TCAAGATTAC

TTTTAGTAAT

TACCTTGGAC

CGGTAGTGAT

TTTCTTGGGC

GGCTTCACCC

GATGCCGATT

ACTGGGTCAA

GATTCTCTGC

CGATCGCCTG

ATCAATTCTC

GGCGCGAGAC

120 180 240 300 360 420 480 540 600 660 720 780 CTCAAGCAAT TAGGCATCCT TGACGAAATC ATCACCGAAC CTTTGGGCGG TGCCCATTCT 840 GCACCGCTGG AAACGGCCCA GAGTTTGCGT CAGGTTTTGC TGCGCCATCT GAAGGATTTG 900 CAAGCCCTCA GTCCGGCTCA GTTGCGCGAG CAGCGTTATC AAAAGTTTCG CCAGCTCGGG 960 GTGTTTCTGG AAAGCAGTGA CTAA 984 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 327 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Met Ala Ala Pro Val Thr Lys Lys Pro Ile Leu Leu Glu Phe Glu Lys 1 5 10

U,

Pro Leu Val Glu Leu Glu Glu Arg Ile Thr Gin Ile Arg Thr Leu Ala Ala Asp Asn Gin Val Asp Val Ser Gly Gin Ile Gin Gin Leu Glu Ala Arg Ala Ile Gin Leu Arg Arg Glu Ile Phe Ser Asn Leu Ser Pro Ala Gin Arg Ile Gin Val Ala Arg His Pro Arg Arg Pro Ser Thr Leu Asp Tyr Ile Gin Ala Ile Ser Asp Glu Trp Ile Glu Leu His Gly Asp Arg Asn Gly Ser Asp Asp Leu Ala Leu Val Gly Gly Val Gly Ala Leu Asp 100 105 110 Gly Gin Pro Val Val Phe Leu Gly His Gin Lys Gly Arg Asp Thr Lys 115 120 125 Asp Asn Val Leu Arg Asn Phe Gly Met Ala Ser Pro Gly Gly Tyr Arg 130 135 140

I

Lys Ala Leu Arg Leu Met Glu His Ala Asp Arg Phe Gly Met Pro Ile 145 150 155 160 Leu Thr Phe Ile Asp Thr Pro Gly Ala Tyr Ala Gly Val Ser Ala Glu 165 170 175 Glu Leu Gly Gin Gly Glu Ala Ile Ala Val Asn Leu Arg Glu Met Phe 180 185 190 Arg Phe Ser Val Pro Ile Leu Cys Thr Val Ile Gly Glu Gly Gly Ser 195 200 205 Gly Gly Ala Leu Gly Ile Gly Val Gly Asp Arg Leu Leu Met Phe Glu 210 215 220 His Ser Val Tyr Thr Val Ala Ser Pro Glu Ala Cys Ala Ser Ile Leu 225 230 235 240 Trp Arg Asp Ala Gly Lys Ala Ala Gin Ala Ala Glu Ala Leu Lys Ile 245 250 255 Thr Ala Arg Asp Leu Lys Gin Leu Gly Ile Leu Asp Glu Ile Ile Thr 260 265 270 Glu Pro Leu Gly Gly Ala His Ser 275 280 Leu Arg Gin Val Leu Leu Arg His 290 295 Pro Ala Gin Leu Arg Glu Gin Arg 305 310 Val Phe Leu Glu Ser Ser Asp 325 Ala Pro Leu Glu Thr Ala Gin Ser 285 Leu Lys Asp Leu Gin Ala Leu Ser 300 Tyr Gin Lys Phe Arg Gin Leu Gly 315 320 INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: modified_base WO 96/32484 PCTUS96/05095 171 LOCATION:one-of(11, 14) OTHER INFORMATION:/modbase= OTHER /note= "N A, C, G, or T" (ix) FEATURE: NAME/KEY: modifiedbase OTHER INFORMATION:/modbase= OTHER /note= "R A or G" (ix) FEATURE: NAME/KEY: modifiedbase LOCATION:17 OTHER INFORMATION:/modbase= OTHER /note= "H A, C, or T" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TCGAATTCGT NATNATHAAR GC 22 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: modified base LOCATION:one-of(3, 9) OTHER INFORMATION:/mod_base= OTHER /note= "Y C or T" (ix) FEATURE: WO 96/32484 PCT/US96/05095 172 NAME/KEY: modifiedbase LOCATION:6 OTHER INFORMATION:/modbase= OTHER /note= "N A, C, G, or T" (ix) FEATURE: NAME/KEY: modifiedbase LOCATION:13 OTHER INFORMATION:/modbase= OTHER /note= "K G or T" (ix) FEATURE: NAME/KEY: modified base LOCATION:12 OTHER INFORMATION:/mod_base= OTHER /note= "R A or G" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GTYCANCTYG TRKGAGATCT CG 22 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GCTCTAGAAT ACTATTTCCT G 21 INFORMATION FOR SEQ ID NO: 16: WO 96/32484 PCT/US96/05095 173 SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: modified base LOCATION:one-of(3, 9) OTHER INFORMATION:/mod_base= OTHER /note= "Y C or T" (ix) FEATURE: NAME/KEY: modified base LOCATION:6 OTHER INFORMATION:/mod_base= OTHER /note= "N A, C, G, or T" (ix) FEATURE: NAME/KEY: modified base LOCATION:12 OTHER INFORMATION:/mod_base= OTHER /note= "R A or G" (ix) FEATURE: NAME/KEY: modified base LOCATION:13 OTHER INFORMATION:/mod_base= OTHER /note= "K G or T" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GTYCANCTYG TRKGAGATCT CG 22 INFORMATION FOR SEQ ID NO: 17: WO 96/32484 PCT/US96/05095 174 SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: modifiedbase LOCATION:one-of(9, 11, 14) OTHER INFORMATION:/mod_base= OTHER /note= "Y C or T" (ix) FEATURE: NAME/KEY: modifiedbase LOCATION:18 OTHER INFORMATION:/mod_base= OTHER /note= "R A or G" (ix) FEATURE: NAME/KEY: modifiedbase LOCATION:21 OTHER INFORMATION:/modbase= OTHER /note= "H A, C, or T" (ix) FEATURE: NAME/KEY: modified base LOCATION:22 OTHER INFORMATION:/mod_base= OTHER /note= "M A or C" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: GCTCTAGAYT TYAAYGARAT HMG 23 INFORMATION .FOR SEQ ID NO: 18: WO 96/32484 PCT/US96/05095 175 SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: modified base LOCATION:2 OTHER INFORMATION:/mod_base= OTHER /note= "R A or G" (ix) FEATURE: NAME/KEY: modifiedbase LOCATION:one-of(3, 13) OTHER INFORMATION:/mod_base= OTHER /note= "N A, C, G, or T" (ix) FEATURE: NAME/KEY: modifiedbase LOCATION:9 OTHER INFORMATION:/mod_base= OTHER /note= "Y C or T" (ix) FEATURE: NAME/KEY: modifiedbase LOCATION:14 OTHER INFORMATION:/mod_base= OTHER /note= "W A or T" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: CRNTACTTYT ACNWCTTAAG CT 22 INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 398 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: AAAATTATCA TAGTCGCCAA TGACGTTACC TTCAAAGCTG GGTCTTTTGG TCCTAGAGAG GACGCGTTTT TCCTCGCTGT GACTGAACCC TTGTGCGCGG AGAAGCTTCC CTTGATTTAC 120H TTAGCAGCAA ACTCTGGCGC CCGGCTAGGG GTGGCTGAAG AAGTCAAAGC CTGCTTTAAA 180 GTTGGATGGT CGGATGAAGT TTCCCCGGAG AATGGTTTTC AGTATATATA CCTAAGCCCT 240 GAGGATCACG AAAGGATTGG ATCATCTGTC ATTGCGCACG AAATAAAGCT GCCCAGCGGG 300 GAAACGAGGT GGGTCATTGA TACAATCGTT GGTAAAGAAG ATGGTATTGG CGTAGAGAAT 360 CTAACGGGAA GCGGGGCAAT AGCGGGTGCT TACTCGAG 398 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 132 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Lys Ile Ile Ile Val Ala Asn Asp Val Thr Phe Lys Ala Gly Ser Phe 1 5 10 Gly Pro Arg Glu Asp Ala Phe Phe Leu Ala Val Thr Glu Pro Leu Cys 20 25 Ala Glu Lys Leu Pro Leu Ile Tyr Leu Ala Ala Asn Ser Gly Ala Arg 40 Leu Gly Val Ala Glu Glu Val Lys Ala Cys Phe Lys Val Gly Trp Ser 55 Asp Glu Val Ser Pro Glu Asn Gly Phe Gin Tyr Ile Tyr Leu Ser Pro 70 75 Glu Asp His Glu Arg Ile Gly Ser Ser Val Ile Ala His Glu Ile Lys 85 90 Leu Pro Ser Gly Glu Thr Arg Trp Val Ile Asp Thr Ile Val Gly Lys 100 105 110 Glu Asp Gly Ile Gly Val Glu Asn Leu Thr Gly Ser Gly Ala Ile Ala 115 120 125 Gly Ala Tyr Ser 130 INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear WO 96/32484 PCT/US96/05095 179 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: Pro Leu Asp Phe Asn Glu Ile Arg Gin Leu 1 5 INFORMATION FOR SEQ ID NO: 22: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: Leu Asp Phe Asn Glu Ile Arg 1 INFORMATION FOR SEQ ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: modified_base LOCATION:one-of(9, 11, 14) OTHER INFORMATION:/mod_base=

OTHER

/note= "Y C or T" (ix) FEATURE: NAME/KEY: modified_base WO 96/32484 PCT/US96/05095 180 LOCATION:18 OTHER INFORMATION:/modbase= OTHER /note= "R A or G" (ix) FEATURE: NAME/KEY: modifiedbase LOCATION:21 OTHER INFORMATION:/mod_base= OTHER /note= "H A, C, or T" (ix) FEATURE: NAME/KEY: modified base LOCATION:22 OTHER INFORMATION:/mod_base= OTHER /note= "M A or C" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: GCTCTAGAYT TYAAYGARAT HMG 23 INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: Asn Met Lys Met Xaa 1 INFORMATION FOR SEQ ID NO: WO 96/32484 PCT/US96/05095 181 SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: modifiedbase LOCATION:2 OTHER INFORMATION:/mod_base=

OTHER

/note= "R A or G" (ix) FEATURE: NAME/KEY: modified_base LOCATION:one-of(3, 13) OTHER INFORMATION:/mod_base=

OTHER

/note= "N A, C, G, or T" (ix) FEATURE: NAME/KEY: modifiedbase LOCATION:9 OTHER INFORMATION:/mod_base=

OTHER

/note= "Y C or T" (ix) FEATURE: NAME/KEY: modified_base LOCATION:14 OTHER INFORMATION:/mod_base=

OTHER

/note= "W A or T" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CRNTACTTYT ACNWCTTAAG CT 22 INFORMATION FOR SEQ ID NO: 26: WO 96/32484 PCTIUS96/05095 182 SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: modifiedbase LOCATION:one-of(10, 16) OTHER INFORMATION:/mod_base= OTHER /note= "N A, C, G, or T" (ix) FEATURE: NAME/KEY: modifiedbase LOCATION:13 OTHER INFORMATION:/mod_base= OTHER /note= "R A or G" (ix) FEATURE: NAME/KEY: modifiedbase LOCATION:one-of(14, 19) OTHER INFORMATION:/mod_base= OTHER /note= "Y C or T" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: GCTCTAGACN CARYTNAAYT T 21 INFORMATION FOR SEQ ID NO: 27: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 96/32484 PCT/US96/05095 183 (ix) FEATURE: NAME/KEY: modifiedbase LOCATION:2 OTHER INFORMATION:/mod_base=

OTHER

/note= "R A or G" (ix) FEATURE: NAME/KEY: modifiedbase LOCATION:one-of(3, 13) OTHER INFORMATION:/mod_base=

OTHER

/note= "N A, C, G, or T" (ix) FEATURE: NAME/KEY: modifiedbase LOCATION:9 OTHER INFORMATION:/mod_base=

OTHER

/note= "Y C or T" (ix) FEATURE: NAME/KEY: modifiedbase LOCATION:14 OTHER INFORMATION:/mod_base=

OTHER

/note= "W A or T" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: CRNTACTTYG ACNWCTTAAG CT 22 INFORMATION FOR SEQ ID NO: 28: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 96/32484 PCT/US96/05095 184 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: GAAGATCTTT ATGGGCGGTA GTATG INFORMATION FOR SEQ ID NO: 29: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: GGTCGAAACG GTACAACCTA GGC 23 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 11994 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: modified_base LOCATION:10357 OTHER INFORMATION:/modbase=

OTHER

/note= "R A or G" (ix) FEATURE: NAME/KEY: modified_base LOCATION:one-of(10198, 10472, 10501, 11698) OTHER INFORMATION:/mod_base=

OTHER

/note= 11Y C or T" (ix) FEATURE: NAME/KEY: modified-base LOCATION:10321 OTHER INFORMATION:/mod base= OTHER /nlote= "K G or T" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GCCCGCCCAA CCAGGGCCAT GCGGCCCAAC TACCCGTCGT CCCCGTCTAG ACCACGCCCC CCACCTGCCC CGCCCCACCC CACCCCCAAC TCCTCCATGA ATGCACGCAT TTCATCGCTC CAACCACAAC GCAGCAGCCC CAGCACCAGC GGCCTCGGCG ACGCGGCGCG CATTTATACC ACGCAATTCC ATCTGGATCT- CGGCATTCCG TCGAACGGCT TGCCCTTGCT ATGCCCCTGC TCCGCCTATG CGTGCGGGCG CCACCTGGCC GCAGCACGGG TTTCCTCCTC CCTCCCCGCG TGGCGGCGCG CCTCCGGACG GACCCACGGT AAGCTCCCCC TTCTGCACGC ATCTTCCGAT TTTCGCTGGA GCGCTCCGCC ATTGACTGGG CCGGACTTGC CATGGACTCG TACTGACCAG 120 180 240 300 360 420 TGATGTACTC GCTCGCTAGC CTCTCCGCCC ACGCCGGCCT CAAATCGAGC GCGCGTAGGC

TGCCTCCAGG

CGCCAGGAGA

TGCCGGGTTC

GGTTTTGGGC

CCCTCTTTGC

CGTATTTTTA

TCTGCACTCT

AGTGCCACCA

ACACACATGC

AAATGATTGA

GAGGGGGCAA

CCC CAAT CCA

TCACGGGACC

GCCCCGTCTG

ACACTTGTTG

TCCTGCTTGG

AT GTGAT CT C

CCCATGGCAC

TGTTGTTGTT

CAAACCGAAA

TGCGCTAACG

TAATGGTGGA

AGCAGCGCAG

AGATACCACT

GCATTACGTC

CTTACTTCCT

ACTAACCAGT

TGGTCTTGCT

TCTTGCCGGA

TTCTGTAGTA

AGAAATATCC

GACGGACTTG

ATCTGACCAA

CGCAGGGCCT

GCTAGCAGTC

GAGCGGGTGG

TCTGCTGAAT

CCC CTAGTGT

CTTCTGGTTC

GGAATTTCCC

CCATTTTAGC

CAGTGCTGCA

TTCTTTTGCT

ATAAACGGGA

TCCTGCTGAT

GACCCGTGCC

TGGGCGCGCG

GCCGGAATTC

GGACTACAGC

TGCTGGTTGT

GATTTAGCTA

ATCTGGTACA

ATTCTACGCT

TTTCCCAGCG

CGCCCAACAG

TCTCTCTCAG

GTCGCCGGAT

CGACTGGCCG

AAGTCCATTT

ATTTTTTTCG

TGACTAGAAT

GCCGTTAXTT

GAAAAAGGGC

AATCGGACAT

CTGAAGGTTG

GATGTCCTCG

480 540 600 660 720 780 840 900 960 1020 1080 1140 GTCGATGAAT TCTGTAAAGC GCTCGGGGGT GACTCGCCGA TACACAGCGT GCTGGTTGCC AACAATGGGA TGGCTGCGGT CAAATTCATG TTTGGGAACG AGAAGGCCAT TCTCTTGGTG AATGCGGAGC ACATAAGAAT CGCCGACCAG AACAACTATG CAAATGTACA GCTCATAGTG CCTACTACTT ATGGATTACC ATGTTCATTA CTGATTCACC TGTCCTGTCA CAGATAGCAG GCTGGGGTCA TGCTTCTGAG AACCCAGAAC TTTTTCTTGG GCCACCATCA GCCGCGATGG TTATTGCACA AGCAGCAGGA GTTCCAACTC CTTGTCCTAT TTCTTTATGG TTTTGCTCTT

CGCAGCATCC

GCTATGGCAA

TTCTTAGAAG

GAGGTTAGTG

TGCTGGATAC

AGAGAACTCG

TTCCAGACGC

GGGCACTAGG

TTCCATGGAG

CTGTTTTTCT

GCACCTGGGC

CTCCAGAGGA

TTCCTGGTGG

CAGTTGATCA

TTGACTAGTT

GGTTTCTGCA

GCTCATGGAA

CGATAAGATT

CGGGTCACAT

CTCCACCACT

CTTGGAGACC

CCTCAGGATA

AACGAACAAT

TCCTTTTTCA

ATTAATCTTT

GTTTGGCCTG

AAGGGAATCA

GGTTCTTCTC

GTATGTATAC

GTGTATTTCT

1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 CAAAACTAAA TCAATACACG CTGTAGGTGA AAGTTCCGCA AGAAACCTGC CACTCAATAC

CTGAGGAGAT

AGGTGGTGGG

GGAAGGTTGG

CTACTAGAAA

GCAATTATTG

TAATGATGAT

TATATTTATT

TTTCTGTTGT

ATATAGTTCA

GAGGTTCAGT

AGTGTTCAAA

CTATAAGAAC

GTATCCTGCA

TATTCTTTTC

CAATTACATG

TATATTAACC

GAGGTCAGAG

ATGAAGGTGG

ATCTTTGTGT

TTTAGCTAAA

TGCTCTGTGA

GAAGGCACCA

GCTTGTGTTT

ATGATCAAGG

ATCTTTTCAA

TAATTTCCAC

AAAGATGTTT

CATTGTTTAA

CATCTCAGGT

TACTCATGTT

ATCTTGCCTT

CAAGCATGGC

AAAGGTTAGT

CAACTACAGA

CATCATGGGG

TTCATCTCTA

TGTTCATTTG

TTTATGTCAT

GCAAGTGCAA

GATACGTGAT

CAGTATTCAG

TCTGTACTTT

AACGTGGCAG

TATTCTCCTG

CGAAGCAGTT

CGGGGGTGGT

CCTTAGTTAT

AAATGAAGTC

CAAATGGTTT

GGAGAAGTCC

AAGCTGATAA

CGAGTGCTTC

CTTTGTAGAG

CACTGCACAG

AAGCATTGGG

GCTAGTTGTC

AAAGGAATAA

ATGGAATGCT

CAAGTTTTCT

.TATAGGTACA

CCGGATCGCC

CAGCCATTAT

TTCTGTACTG

CCGACATCTA

TCGAGACTGT

TTGTTCAATA

1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 TCAGTTTTGT TGGAATTAGT CTTAGCCAAA CATTTGTGTA GTGAGTACTG GTAGAAGTTC TACAGCTTCA GGGGAATAAA AACTTCATTG GACAATGTAG CAATCATATA GTACTGTTTA GCAAAGTGCA AAATGTTGCA GAATCATTTA TTACTGTTAG GAATTCAAGA ACAAACTTTT AACTCAGAAT ATTGAAAAGT TTAACTAGTG CAAGACAGAT TCTCACGATA TATATACTTG TCCAGAAACA ATTAAAGAGC TCAGGGTGCT GCTACAGTAG GGAGCTTAAT CCAAGGTTGC CTTACCTGCA TCTCAAGTTG GCCAGTTGTC CAACTTGATG

GGAGCTATAC

TTATACTTAT

TCTTCCTATA

CTAGTGACTG

AATAGCAGAG

TGAAACAGAT

TTGAGCAGGC

AATATCTGTA

AGGTAGAACA

TAGTAGGAAT

GTTGATGATA

CAAATTTATG TCGTGGCATT TTCTTAAATG ACTGTACTAA ATAGTTGAAT GTTGCATTTT GTGATATATG TGTTGTACTT GAAGTTTTTG TATTACAGAT TATTTTGTAA CCAAAAAAAT AAGTCTTAGC AAAATTATAT TTATTTTACT CATTGAGGAG GGACCAATTA CAGTTGCTCC AGCAAGGCGG CTTGCTAAAT GTGTGCAATA CAGCATGGAA ACAGGCGAAT ACTATTTCCT CCCTGTGACC GAATGGATTG CTGAAATAAA GGGCATACCA CTCTACAACA TTCCAGGTAG TTATCTCTTT CCCCCCACAC TAATCAATAT 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 AAGGATAACT GCAGAGATCA GACGCTTTTA TGGAATAGAA CATGGAGGTG TTGGAAGGAA ATATCAGCTG TTGCAACTAA ATTTGATCTG GACAAAGCAC GCCAAAGGGT CATTGTGTAG CAGTTAGAGT TACTAGCGAG GATCCAGATG GCCTACAAGT GGAAGAGTGG AAGAGCTGAA CTTTAAAAGT AAACCCAATG TTTCTCTGTT AAGGCAAGTT TGCATCCATG CAGAATGATC TTTGATACCA CACAACAGCT GCAGCTTATC ATTACCCTTG AGTTTTCCTG TTTCTTATGT CCTGGTTAAA AACTGTATCT TGTGTGGCAA ACCTAACCTG AATCATCGTT GTCCGGAGGT GCAATTCATG AGTTCTCTGA TTCCCAGTTT GGTAAGTGAT TTCTGTTTCC TCATATATCT CATGATGATG CTTCTCTTAA ACAGCATGCC GGTCATGTTT TTGCTTTTGG GGAATCTAGG TCATTGGCAA TAGCCAATAT TTAAAAGAGA TCCAAATTCG TGGAGAGATA CGCACTAATG TTGACTACAC TTGAATGTAA GATAACCCCA CAGTAAACAT GTTCTCTGAT TACATGGTAC

GCTATCACGC

AGTCTGTAAA

ATGGGTTTAA

TTTGGGCCTA

CATGACATGT

CGATAAATTT

TTTTGTTTCA

GTGCGTAAAT

TTTTTTCGCA

GGTACTTGGG

TGTGGATCTC

ATTTATTAAG

3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020

AAAAACATGG

TGCAGAGTAC

TACAATTTTG TGTGTGTAAT CGAGAAAATA AGATTCACAC TTATGTTCAA AATTTTTCAT TGGTTGGCTA GACAGCAGAA

ATCTCCAGGC

TAGCAATGCG

TGTTAGAGCA GAGAGGCCCC ATTTCTTTTT CTGGGGAACT TTTTCCATCC TCAAATACTG TGTTGTAACC GATTATGTTG TATATGATGA ATGTTCTTAC TCTTTCCGTT CACAGCACAT AGCAAATATA CGGTAATTAT TAATCCAGTT GGTATCCTTG AGTACGAGGT GGACCCCGTA GATACATTCG CTGCGAGATG

CATGGTACCT

ATGATTTATT

TGTTTCTTAT

GTTATCTCAG

TGTTTATATT

CTCTCTTGTC

TTCAGTTGTT GGTGGAGCTC TATATGTATG AGGTGGTTAT GAGCTTTCAT ACAAGATCCA ATTTCAGGAA GCATCAAGCA GGAGCTCGAG TAAAGGTCAA ATACCACCAA AGGTACATAC CCAATTTCTA TATGAATAAA ACTGTCTAAC AATTTGACTG TAACACTGAA TATAGATGGG 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 CTATAATTTT CTCTTTAATC TTATCCATGC CATACCCATC TCACATCTGC TAATTATTAT TTTCTTCTGC AGATTGAGAC GCTACAAATT AAGAATTAAT GAATCAGAGG TTGAAGCAGA GCGGACTCTT AATGCAGGTA GATATATCTA CCAAGTTTTT ATACAAG'CGC AATCTATCTA ATTTTCTTTT TATTTGGAAA TGGTCTGACC AATTTTCAAT

TGTGAATTTT

GCACGCGTCT

TACTCTCCTC

ATTGTATGAA

GATCCTTCCA

GGTTCTCATG

CCACTGTTAC

CAGGTTCCTC

TGGTTCATGC

GCAGGCGAGT

CTAGTTGGAT

TCTAATCAAT

TTGGTTCCTA

AATACATAAA

GGTTGTTGGC

TGGTTGCTGA

TACCGGCCTC

CCCCTCCTCT

TCGTAATATA

GATCTGATAG

GGAAACAGTC

GGGAGAACAT

TGTAATATAG

TTAATTATGT

TGATACACCA

TACGCCATAT

TGGTGTCATT

GTTTGCAGCA

CGTGCATCTT

CAAGGTTGGA

ATGTAATTTA CGCCGAGACA GCTTATTACA GGTGAAGATA GGGTTGTTTC AGTTGTAACT CCTCTGAATG ATATATTACA TGCAAGCTTC TTCGGTTTTT GCTGAGGTGG AGGTGATGAA CACTTTGTCA TGCCTGAGGG

GAAGCTGCTG

GCTAGATCTG

CTAGCTGCAA

GAAAGAG CAT

GGTCGCGGAT

AATGTGCATG

TCAGGCCATG

4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460

CTAGATGTAC

TTAAATAGTA

TCTTGATGAC

ATTCTGACAA AAGTACTATA GCTGAAATGG CTGTCTTTGT CCATCTTCTG TGAGAAGGGC TGAACCATTT CATGGCACCT TTCCAAAACT TGGACCTCCT ACTGCTATTT CTGGCAAAGT TCACCAAAAG TTTGCTGCAA GTGTGAATTC TGCCCACATG ATCCTTGCAG GATATGAACA

TAACATCAAT

TTTCCCTTGT

ACTAACCTCT

TACCTTTTCT

TTCCTGCAGT

AATGAGGTGA

CATTTCGGCT

CAAGGAGTAT

AAGGGGAGTC

TCATGTTGTT

GATAGGGTCA

CATGTAAGGC

CTATCACATT

TAATTGTGAC

TTCAGGTTGT

GGCAAGAACT

ATAAGTATTC

GCATATCAAA

GAGTTGAATG

ATTGAGGTCA

TCCTTACAAA

CTAGTGAGAG

ACATCAAACT

GCCATGGGAA

AAACTATACC

ACAAGATTTG

CATGTCCGTT

AAGTTATATT

TGGATAACTG

CTGACTTCCG

GTTTGAGACT

GTCATCATTG

GCTTGTAGAG

GTCAGTGTAT

AACAGAG CAT

ATCTTTCTTC

CTGAACTGCC

TTGGCAACCC

TTTTTATCTT

ATTTACCTGT

GAAGAGCAAG

GTTACTTGGC

CAGGCTAATC

CCACTTATGA

ACTTGTTCTT

GAGTTCTTCT

AATCAATAAG

TAGACAGCCC

GACTCCCGAA

AGAGTTATTA

TCTCAGTTGG

GATTTCCCTG

AT C CCTTC CT

TTGCATACTG

GTCTGGTCAA

CCACTTTTCT

ACAGAGAGAA

TTCCTGACTG

TGAGCTCCCT

AGATCTTAGG

TTCCATTTTT

ATGCTAAGTA

CCAAGTTGCT

TTTTTATGTG

TTCCGAGAAA

GTCATATGAG

5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 GGTGGAAGAG AAAGCCATGC TCGTGCGGTT GTCAAGTCTC TGTTTGAGGA GTATTTATCT

GTTGAAGAAC

GATCGATGCT

TCTCAGTCTG

GTATATATTG

TGCAAAATTT

TTAATACTAC

TTGATTCGCT

GATCTGCATC

TTGTAATTGA

GAACTCCGCA

TCTTCAGCGA

CACTTTCTGA

ATGTGATAGA

TGTTCTCCCA

TCGTGCTGAC

GGCTTATGGA

TCTCTGCC CT

TATTTATTTT

TGCAGCTGGC

CAAGCATAGC

TGACATTCAG

CCAAAACGTG

ACGTCTACGA

CCAGGTAATG

AATTTGTGTT

AGCATTGGTC

GAACCATACA

GCACATTGAT

GCTTAAAGCA

AAGAAGCCTT

GTAACTATTT

CTAAACCGTT

CTTCAACATG

TCTTCTATTG

CTTTTGAAGG

TATCCAAATC

GCATACTCGG

ATGATAGTCT

AGCCAACTTC

TCAGAGCTGG

ATAATTGCTT

GTGCTTTTTT

CAAAAGACCT

TGCAATCTGT

GTGTGAAAAG

CATCTGCATA

GGGTAAAATT

AGAAAAATAA

TTGAGCACAC

AGATGTTTAC

GGAATGGTTT

GTTTTTATAT

TGAGAAGGTC

TGACTTGATA

TAAAAATAAA

CAGGGACCAG

GAGTTTGGAT

AATAAATCTA

CAAATTGAGT

TGAGGAAGGA

6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 GAGCGGATTT CAACACCTAG GAGGAAGATG GCTATCAATG AAAGGATGGA AGATTTAGTA

TGTGCACCGG

CTTCAGCGGA

TTTTTTACTA

CACAATCACC

TGGTCTAATT

TGCGTCACTT

TGTAATCAAG

ACACTACGGA

CAATATGCTG

GTTTGTTTAC

CTGTGAACTG

TTGCAGTTGA

GAGTAGTCGA

CACTCTTTCT

TTCCAGCATT

GCTTTATGGG

CTAAAGCCAC

TCTCTTCAGC

GCAGGTGTTG

CACATTGCTC

ACTCTATTCT

GTCGTTAATT

AGACGCCCTT

GACATACATA

TGAGACAACT

ATCTTGCAAG

AATTCTCTGA

AAGTAGAGGA

TTCTGTCAAC

GAAGTGTCTC

TGGTTGGTAT

TATGTGGTTT

TCATGATTTT

GTGGCTTTGT

CGCAGATTGT

AGAACATTAA

GGGCAGCGTC

AGAGCATATT

TCCAATTGGC

TGCAATTGAA

AAATGGTAAT

CAACAATCAG

GTTGTTATTG

TTAGTTACCT

TTGATCACAG

ATCAGGTATC

CAAATTTATG

CGGATGCAAT

GAACAAAGAA

AGGCGATGGG

GCTGCATTAA

CCTATAAATT

ATGAGCACTC

CACAGGAGAC

CTTCCACTCT

TGATCCTACT

ACTGATTTTT

CCGGCTAACT

GGCATAGGTC

ATGGGCAATC

GTGTAATGGT

AGGAGACTTC

TGAACGGCAG

TTCAAGACAG

GAGTGTGATT

GTTTTCTCTT

6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 TATAGTGGTG ATGAGGATCA AGCGCAAGAA AGGATCAACA AACTCTCCAA GATTTTGAAG GATAACACTA TAACATCACA CAAAGAGATG AAGGGCGTTC TATTATGAGG AGGACCCGAT TTGGTATTCA GCTTTTGTTT GTTTATGTGT AAATACAGGA CCATCACGTG ATCGCCAGTG TCAAATGACC AAAGGATGTT TTTTTGTTTG GAAGTATTGA ATACTCAGAT CATTGATGGC GGGATGTCAG GCCACTCCCA CTAATTCCAT CTTCAAGGTC TAAATATGTT CATAGATGTT TCTCAATGGT GCTGGTGTTA GGGTTGTCAG CTGCATTATC ACCAATGCGC CACTCCTTCA AATGGTCATC TGACAAGTTA GCTCCGCCAT GTGGAATCTC CTTTGTCCAC CTTCCTTGAA TGGCTTATGT TCCCTTCAAT AATACCAGTA CCTCTTAACA CAAAGTGAAT TTAGAAGGTT ACAATGACGC GAAATACACC GCACATGTAC ACACTAGTAA AGAACAAGAA AGATCCGAGA TCTTCGTACC ATAGTCAGAC AGCCAAGTGT GACCAATGGG TAATGAAGTT CAAGCCTCGT CATCATTCAC ATCTAACAGC AGCTCTAGAA.GAAATAGAGT TGCGTGCTCA CAGTGAGACT CATGTATCTG TGCATAATGA GAGAACAACG GTTGTTTGAT AGTCAAAATT TATTTATGTT CTCAACAGAT TATATTGCAT CACTTGGTTT TTGCTTCTCA TTATGTTAGG ATGACGAATG 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 AAGTTGGTCA AGATGAGAAG ACAGCATGCA CACTATTGAA GCATATGGTT ATGAATATAT

ATGAGCATGT

GGTTGGATTG

GCAATACCTG

CATCTACAGT

ATTTTGTTAG

ATGGGTAATC

TAAGTACCAG

ATTTACCGAG

ACAGCTGGTC

GACCTGAAAC

GTGCGTTAGC

TGGTGTCAGG

TGATGGGCAG

CACTGTTGAT

TTTGCAAATA

CTTATCTAGT

TCTCAATTTT

GTACCATTTT

AAGTGGAGGA

CTTTGCATGG

GTGCCGCTGC

TACATCTCTT

ATGCATCGCC

GCTAATGGTG

GTAAGTTACC

CTACCTCTGA

TAGTGAATAG

TGCCTTTAAA

CTCTTTATTG

CCC CAATACA

CATTGCATTG

TAGGAAAAAT

TTCTTTTTTT

TTTCCGTGTG

CTTGGAGAGT

TTAGCTATTG

TGGATAAAGC

AAAATGTTCA

AGTTCTATTA

CTCTTATGCT

CATAAGCTTT

CATGAGCCAT

GAAACCACAT

CTCTACAATT

CCAGTGGGAA

TGTTGTTACC

CACTGCTACG

CCCACAGATC

TCACCCCCAT

AACACTACTT

TGAATTATTT

TCTATCGCTC

ACAAACCTTT

ACTGCTATGA

GGTTAACATG

GTGAAGCTAT

AGTGTAACTG

CGAGCATTAT

ATCAAATATG

TATGAGTGTA

AAAAGACTTG

TGACTTTCAG

TGCCACACCC

GGATGCTATT

TTTCCCATTG

ATTAACTAAG

8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 9000 9060 ATTGG3TAATA

TGGTATTTCA

GCTTATATTT

TCCAGGTAGC

TCCAGGTGGC

TGGTCCTAGA

TCCTCTAATC

GGCATGCTTC

TTACCTCACT

AGTACCAGAA

ACTTGGTTGT

TAGAGAGACA

ATACTCTGTC

CATGTTGCAG

G:CTGATTCAA

AACAATTTTG

CGGGAGATTA

GAAGATGCAT

TACTTGTCAG

CATGTTGGAT

GAACAAGATT

AGCGGAGAAA

GAGAATCTAC

TTTACTCTGA

CGCAGGCATT

AATCTAATGA

CTGGATCATG

GTGTTGTTGC

TAGTTGTTGC

TCTTTGATGC

CAACTGCTGG

GGTCTGATGA

ATTCACGTCT

CCAGATGGGT

ATGGAAGTGG

CATTTGTGAC

TGAAACAGCA

GCATAACCAG

GGGTACTCCT

TTGGAACATG

AA.ATGATGTG

TGTCACAAAT

TGCAAGGCTC

CCAGAGCCCT

AAGCTCTTCA

TGTTGATACC

TGCCATTGCC

TGGCCGAGCT

TTGAAGAAGT

CGGTATGCTG

TTGGTTCCAG

AAGCTCTCCA

ACATTTAAAG

CTTGCTTGTG

GGTGTAGCAG

GAACGTGGTT

GTTATAGCCC

ATTGTTGGGA

AGTGCCTACT

ATTGGAATTG

CATGGGAATC

AAGTGACAGA

TTGAGCGTCC

CACCAGAATT

CTGGGTCTTT

AGAGGAAAAT

AGGAAATAAA

TTCACTACAT

ATGAGCTAAA

AAGAGGACGG

CTAAGGCATA

GGGCCTATCT

9120 9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 TGCTCGGTTA GGAATGCGGT TTCTGCACTG AACAAGCTCC CCCCAAAATC ATGGCTACAA TGTTTCTGCT ATCTTGAAAT TATTGTAAAA TCTCTTGATC TGATGCCCGT GCTGCCATCT GTTTGACAGA GAAAGCTTTG AAGGGCAAAG CTGGGTGGGA GCAAGTAATC CCTGCTGACC KGGACAGGTG TGGTTCCCAG CCGTGAAGAG CTCCCGTTGT GGATCTGTTT GAAGGAATCC

GTATACAACG

TGGGGCGCGA

ATGGAGTTGT

GGCTCAGCTA

CACCAGAGAG

GTGGCATTCA

TGGAAACGTT

TTCCAGTTGG

CTGGTCAGCT

ATTCGGCCGC

TCATACTTGC

TTCAGGCTGG

TCTTGATCAA

GGTTTATAGC

TCATCTCACT

TGTTCCTCCC

AGCTGTAACA

GGACACTCAA

AGAAGGATGG

TATCATAGCT

TGATTCTGCC

AAAAACRGCC

TAACTGGAGA

TYCTATGATT

CCAATTATTT

TCTCAGATGC

GTGTCAGATG

TATGTTGGTG

TACTTTCCAG

GGCAAGTGGT

GCCAAA.ACTG

GTGGAAACCG

GAGCGTGTAG

CAGGCACTGC

GGCTTTTCTG

GTTGAGAATC

TGACTGGGTA

AACTGGGTGG

ATCTTGAAGG

GTCCTCTTCC

AGAATTCATG

TGAGTGGTAT

TTATTACYGG

AGACAGTGAT

TCCCTCAAGC

TGGATTTCAA

GTGGGCAAAG

TGAGGACGTA

9840 9900 9960 10020 10080 10140 10200 10260 10320 10380 10440 10500

YAAGCAGCCT

TGTGGTGGAC

AGGGAATGTC

AGAGAGTATG

AACTAGTGCT

CATGGCTGCT

TGAGTTGCAC

CTGGGAGGAG

GCTCGCCAAA

CTTGGAGTGC

GCTTTTGTGT

AGCAAGATCA

CTTGAGGCAC

CTAGGGCTGG

AGCCCTAGCC

CGGAGGAAGC

GACACCTCCG

TCCCGGGCCT

CAAGTCAGAG

ATCAGGAAAT

ACATACCAAA

ATCCGGAGCA

CGGGACTCAT

ACCCTGAGTT

CTTGGGAAAC

AGCTGATGCC

CAAGAATGGC

TCTTCTACAG

AAGCCGCCGG

GGTACCTGGC

GGCTGGAGAG CTGCGTGGAG GTGCATGGGT CATTGAGATG TATGCCGAGA GGACTGCGAG TGAGATCAAA TTCAAGCCAA ATGAATTGGA GATCAGCCTC AATGCTAAAC TCCTCAAAGA GGCGGCGGCG GCAGAGACCA TCAGGAGGAG CATATATACT CAGGTTGCCA CCCGGTTTGC TGCCAAAGGC GTGATCAGTA AGGTGGTGGA GAGACTGCGA AGGAGGCTTG CCGAGGACTC CGAGCAGCAG ATGCCCACTC ACAGATCAGC CTCTCAAGGA GGAGACGGCG AGAAGTGGGG 10560 10620 10680 10740 10800 10860 10920 10980 11040 11100 11160 11220 CGATGATGAA GCCTTCTTCA GGAGCTGAAA GCCGAGAGAG CCTGGAAAGA TGATCCTGAC AAGTATGGCA AGTATCTTGA CGTCTACACT GCTGTCGCAT CTCGCTGAAA CCTCGGACGC CAAGGCCTTG CCCAACGGTC TCTCGCTCCT CCTCAGCAAA GTAAGTTTCT AGTATTTGTT TGTTCTTGTA TACATTTCCT AATAAGTTTC TTTTGCTTCT GTTCTTGTAT AGTTTTCCTA ATTAAATTCT TTCTGTCCCT AAGTTCATCT TACATTTGAT TGATTGTACA GATGGATCCT GCAAAGAGGG AGCAGGTTAT AGGCAGCTTC TTGGTTGATT ACTGGCCCGC GCCCTTTGAT AACGCATCCA ATAAATCGGC CTTGCTTGTT GCCACCAAGC AAGTCCTGTC TATGGTGGGC TGGAACAAGC AAATTTTACT TGCGTGGCGA GCTACAGGAG GGGGAGGATT GAAAACTGAA ACACATTGTT TGCACATAGG TAGGAGGCAT CTCATCTCAG ATGTTTATTG TCATTACAGA TAGGTACACA CAAAGCATAT GTATGCTGGA GTGTGAGTTG TTGCAATGCA AGATTCATCA TCTTAATTTA CGAGATACGA GQTCGATGTG GTAGTTGTAG TTTCCTCAGT GGCAGGGAAT GCCGAGTTTC AGTTATGTGA TATGTAAACC CTGAGAACTT TGGGGTGATA TGATGGACGT

TTTGCTTATT

TCTTTTCTTT

CCCTGATACA

GGATGGCCTC

TTCAGCCAGC

TGGGTACCAG

TTCAGCGGAA

GACAATCYGT

TAGATATTCG

TGTGATGATC

CTTACGCTGC

TTTATCAGTT

11280 11340 11400 11460 11520 11580 11640 11700 11760 11820 11880 11940 0 TCATGAGAAA TGAAATTGGA GCCGAGGCCC CTTACATCAG TTTTTTTTCT TCTA 11994 INFORMATION FOR SEQ ID NO: 31: SEQUENCE CHARACTERISTICS: LENGTH: 2260 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear 0 o (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: Met Val Glu Ser Asp Gin Ile Asn Gly Thr Pro Asn Arg Met Ser Ser 1 5 10 Val Asp Glu Phe Cys Lys Ala Leu Gly Gly Asp Ser Pro Ile His Ser 25 Val Leu Val Ala Asn Asn Gly Met Ala Ala Val Lys Phe Met Arg Ser 40 §0 ui Ile Arg Leu Val Thr Trp Ala Leu Glu Thr Phe Gly Asn Glu Lys Ala Ile Leu 55 Ala Met Ala Thr Pro Glu Asp Leu Arg Ile Asn Ala Glu His 70 75 Ile Arg Ile Ala Asp Gin Phe Leu Glu Val Pro Gly Gly Thr Asn Asn 90 Asn Asn Tyr Ala Asn Val Gin Leu Ile Val Glu Ile Ala Glu Arg Thr 100 105 110 Arg Val Ser Ala Val Trp Pro Gly Trp Gly His Ala Ser Glu Asn Pro 115 120 125 Glu Leu 130 Pro Ser 145 Pro Asp Ala Leu Met Glu Lys Gly Ile Ile Phe Leu Gly Pro 135 140 Ala Ala Met Gly Ala Leu Gly Asp Lys Ile Gly Ser Ser Leu 150 155 160 Ile Ala Gin Ala Ala Gly Val Pro Thr Leu Pro Trp Ser Gly Ser His 165 170 175 Val Lys Val Pro Gln Glu Thr Cys His Ser Ile 180 185 Lys Asn Ala Cys Val Ser Thr Thr Asp Glu Ala 195 200 Val Val Gly Tyr Pro Ala Met Ile Lys Ala Ser 210 215 Lys Gly Ile Arg Lys Val His Asn Asp Asp Glu 225 230 235 Lys Gin Val Gln Gly Glu Val Pro Gly Ser Pro 245 250 Val Ala Ser Gin Ser Arg His Leu Glu Val Gin 260 265 His Gly Asn Val Ala Ala Leu His Ser Arg Asp 275 280 Arg His Gin Lys Ile Ile Glu Glu Gly Pro Ile 290 295 Pro Glu Val Ala 205 Trp Gly 220 Glu Ile Tyr 190 Ser Cys Gin Gly Gly Gly Val Arg Ala Leu Phe 240 Ile Phe Ile Met Lys 255 Leu Leu Cys Asp Lys 270 Cys Ser 285 Val Gin Arg Thr Val Ala Pro Pro 300 Glu Thr Ile Lys Giu Leu Giu Gin Ala Ala Arg Arg Leu Aia* 305 310 315 Val Gin Tyr Gin Gly Ala Ala Thr Val Glu Tyr Leu Tyr Ser 325 330 Thr Gly Giu Tyr Tyr Phe Leu Giu Leu Asn Pro Arg Leu Gin 340 345 350 His Pro Val Thr Glu Trp Ile Ala Giu Ile Asn Leu Pro Ala 355 360 365 Val Val Val Gly Met Gly Ile Pro Leu Tyr Asn Ile Pro Glu 370 375 380 Arg Phe Tyr Gly Ile Giu His Gly Gly Gly Tyr His Ala Trp 385 390 395 Ile Ser Ala Val Ala Thr Lys Phe Asp Leu Asp Lys Ala Gin 405 410 Lys Pro Lys Gly His Cys Val Ala Val Arg Val Thr Ser Giu 420 425 430 Lys Cys 320 Met Glu 335 Val Giu Ser Gin Ile Arg Lys Glu 400 Ser Val 415 Asp Pro Asp Asp Gly Phe Lys Pro Thr Ser 435 440 Lys Ser Lys Pro Asn Val Trp Ala 450 455 Ala Ile His Glu Phe Ser Asp Ser 465 470 Gly Glu Ser Arg Ser Leu Ala Ile 485 Glu Ile Gin Ile Arg Gly Glu Ile 500 Asp Leu Leu Asn Ala Ala Glu Tyr 515 520 Trp Leu Asp Ser Arg Ile Ala Met 530 535 Trp Tyr Leu Ser Val Val Gly Gly 545 550 Gly Arg Val Glu Glu Leu 445 Tyr Phe Ser Val Lys Ser 460 Gin Phe Gly His Val Phe 475 Ala Asn Met Val Leu Gly 490 Arg Thr Asn Val Asp Tyr 505 510 Arg Glu Asn Lys Ile His 525 Arg Val Arg Ala Glu Arg 540 Ala Leu Tyr Glu Ala Ser 555 Asn Phe Gly Gly Ala Phe 480 Leu Lys 495 Thr Val Thr Gly Pro Pro Ser Arg 560 Ser Ser Ser Val Val Thr Asp 565 Tyr Val Gly Tyr Leu Ser Lys 570 Leu Val Asn Leu Thr Val Thr 585 590 Gly Gin 575 Leu Asn Ilie Pro Pro Lys His Ilie Ser 580 Ile Asp Gly Ser Lys Tyr Thr 595 Ser Tyr Lys Leu Arg Ile Asn 610 615 Ser Leu Arg Asp Gly Gly Leu 625 630 Val Ile Tyr Ala Glu Thr Giu 645 Giy Arg Thr Cys Leu Leu Gin 660 Ala Asp Thr Pro Cys Lys Leu 675 Ile Giu Thr Val Arg Gly Gly Pro Arg 600 605 Giu Ser Giu Val Giu Ala Giu ,620 Leu Met Gin Leu Asp Gly Asn 635 Ala Ala Gly Thr Arg Leu Leu 650 Lys Glu His Asp Pro Ser Arg 665 670 Ile His Ser His 640 Ile Asn 655 Leu Leu Leu Arg Phe Leu Val Ala Asp Gly Ser 680 685 His Val Val Ala Asp Thr Pro Tyr Ala Glu Val Glu Val Met Lys Met 690 695 700 Cys Met Pro Leu Leu Leu Pro Ala Ser Gly Val Ile His Phe Val Met 705 710 715 720 Pro Glu Gly Gin Ala Met Gin Ala Ser Asp Leu Ile Ala Arg Leu Asp 725 730 735 Leu Asp Asp Pro Ser Ser Val Arg Arg Ala Glu Pro Phe His Gly Thr 740 745 750 o Phe Pro Lys Leu Gly Pro Pro Thr Ala Ile Ser Gly Lys Val His Gin 755 760 765 Lys Phe Ala Ala Ser Val Asn Ser Ala His Met Ile Leu Ala Gly Tyr 770 775 780 Glu His Asn Ile Asn His Val Val Gin Asp Leu Leu Asn Cys Leu Asp 785 790 795 800 Ser Pro Glu Leu Pro Phe Leu Gin Trp Gin Glu Leu Met Ser Val Leu 805 810 815 Ala Thr Arg Leu Pro Lys Asp Leu Arg Asn Glu Leu Asp Ala Lys Tyr 820 825 830 Lys Glu Tyr Glu Leu Asn Ala Asp Phe Arg Lys Ser Lys Asp Phe Pro 835 840 845 Ala Lys Leu Leu Arg Gly Val Ile Glu Ala Asn Leu Ala Tyr Cys Ser 850 855 860 Glu Lys Asp Arg Val Thr Ser Glu Arg Leu Val Glu Pro Leu Met Ser 865 870 875 880 Leu Val Lys Ser Tyr Glu Gly Gly Arg Glu Ser His Ala Arg Ala Val 885 890 895 Val Lys Ser Leu Phe Glu Glu Tyr Leu Ser Val Glu Glu Leu Phe Ser 900 905 910 Asp Asp Ile Gin Ser Asp Val 915 Ile Glu Arg Leu Arg Leu Gin His Ala 920 925 Tyr Ile Val Phe Ser His Gin Gly Val 940 Lys Asp Leu Glu Lys Val 930 Val 935 Lys Ser Lys Asn Lys Leu Ile Leu Arg Leu Met Glu Ala Leu Val Tyr 0* 945 950 955 960 i; Pro Asri Pro Ser Ala Tyr Arg Asp Gin Leu Ile Arg Phe Ser Ala Leu 965 970 975 Asn His Thr Ala Tyr Ser Giy Leu Ala Leu Lys Ala Ser Gin Leu Leu 980 985 990 Giu His Thr Lys Leu Ser Giu Leu Arg Thr Ser Ile Ala Arg Ser Leu 995 1000 1005 Ser Giu Leu Giu Met Phe Thr Giu Giu Gly Giu Arg Ile Ser Thr Pro 1010 1015 1020 Arg Arg Lys Met Ala Ile Asn Giu Arg Met Giu Asp Leu Vai Cys Ala 1025 1030 1035 1040 Pro Val Ala Val Giu Asp Ala Leu Val Ala Leu Phe Asp His Ser Asp 1045 1050 1055 Pro Thr Leu Gin Arg Arg Val Val Giu Thr Tyr Ile Arg Arg Leu Tyr 1060 1065 1070 0 Gin His Tyr Leu Ala Arg Gly Ser Val Arg Met Gin Trp His Arg Ser 1075 1080 1085 Gly Leu Ile Ala Leu Trp Glu Phe Ser Glu Glu His Ile Giu Gin Arg 1090 1095 1100 Asn Gly Gin Ser Ala Ser Leu Leu Lys Pro Gin Val Giu Asp Pro Ile 1105 1110 ill5 1120 Gly Arg Arg Trp Gly Val Met Val Val Ile Lys Ser Leu Gin Leu Leu 1125 1130 1135 Ser Thr Ala Ile Glu Ala Aia Leu Lys Giu Thr Ser His Tyr Giy Ala 1140 1145 1150 Gly Val Gly Ser Val Ser Asn Giy Asn Pro Ile Asn Leu Asn Giy Ser 1155 1160 1165 Asn Met Leu His Ilie Ala Leu Val Gly Ile Asn Asn Gin Met Ser Thr 1170 1175 1180 Leu Gin Asp Ser Gly Asp Glu Asp Gin Ala Gin Giu Arg Ile Asn Lys 1185 1190 1195 1200 Leu Ser Lys Ile Leu Lys Asp Asn Thr Ile Thr Ser His Leu Asn Gly 1205 1210 1215 Ala Gly Val Arg Val Val Ser Cys Ile Ile Gin Arg Asp Giu Gly Arg 1220 1225 1230 Ser Pro Met Arg His Ser Phe Lys Trp, Ser Ser Asp Lys Leu Tyr Tyr 1235 1240 1245 Glu Glu Asp Pro Met Leu Arg His Val,,Glu Ser Pro Leu Ser Thr Phe 1250 1255 1260 Leu Giu Leu Asp Lys Vai Asn Leu Giu Gly Tyr Asn Asp Ala Lys Tyr 1265 1270 1275 1280 Thr Pro Ser Arg Asp Arg Gin Trp His Met Tyr Thr Leu Val Lys Asn 1285 1290 129S Lys Lys Asp Pro Arg Ser Asn Asp Gin Arg Met Phe Leu Arg Thr Ile 1300 1305 1310 Val Arg Gin Pro Ser Vai Thr Asn Gly Phe Leu Phe Gly Ser Ile Asp 131S 1151320 1325 Asn Glu Val Gin Ala Ser Ser Ser Phe Thr Ser Asn Ser Ile Leu Arg 1330 1335 1340 Ser Leu Met Ala Ala Leu Giu Glu Ile Glu Leu Arg Ala His Ser Glu 1345 1350 1355 1360 Thr Giy Met Ser Gly His Ser His Met Tyr Leu Cys Ile Met Arg Giu 1365 1370 1375 Gin Arg Leu Phe Asp Leu Ile Pro Ser Ser Arg Met Thr Asn Giu Val 1380 1385 1390 Gly Gin Asp Giu Lys Thr Ala Cys Thr Leu Leu Lys His Met Vai Met 1395 1400 1405 Asn Ile Tyr Glu His Val Gly Val Arg Met His Arg Leu Ser Val Cys 1410 1415 1420 Gin Trp Glu Val Lys Leu Trp Leu Asp Cys Asp Gly Gin Ala Asn Gly 1425 1430 1435 1440 Ala Trp Arg Val Val Val Thr Ser Val Thr Gly Asn Thr Cys Thr Val 1445 1451450 1455 Asp Ile Tyr Arg Glu Val Glu Asp Pro Asn Thr His Lys Leu Phe Tyr 1460 1465 1470 Arg Ser Ala Thr Pro Thr Ala Gly Pro Leu His Gly Ile Ala Leu His 1475 1480 1485 Giu Pro Tyr Lys Pro Leu Asp Ala Ile Asp Leu Lys Arg Ala Ala Ala 1490 1495 1500 Arg Lys Asn Glu Thr Thr Tyr Cys Tyr Asp Phe Pro Leu Ala Phe Giu 1505 1510 1515 1520 Thr Ala Leu Lys Lys Ser Trp Giu Ser Gly Ile Ser His Val Ala Glu 1525 1530 1535 Ser Asn Glu His Asn Gin Arg Tyr Ala Glu Val Thr Glu Leu Ile Phe 1540 1545 1550 Ala Asp Ser Thr Gly Ser Trp Gly Thr Pro Leu Val Pro Val Glu Arg 1555 1560 1565 Pro Pro Gly Ser Asn Asn Phe Gly Val Val Ala Trp Asn Met Lys Leu 1570 1701575 1580 Ser Thr Pro Glu Phe Pro Giy Gly Arg Giu Ile Ile Val Val Ala Asn 1585 1590 1595 1600 Asp Val Thr Phe Lys Ala Gly Ser Phe Gly Pro Arg Glu Asp Ala Phe 1605 1610 1615 Phe Asp Ala Val Thr Asn Leu Ala Cys Glu Arg Lys Ile Pro Leu Ile 1620 1625 1630 Tyr Leu Ser Ala Thr Ala Gly Ala Arg Leu Gly Val Ala Glu Giu Ile 1635 1640 1645 Lys Ala Cys Phe His Val Gly Trp Ser Asp Asp Gin Ser Pro Glu Arg 1650 1655 1660 Gly Phe His Tyr Ile Tyr Leu Thr Glu Gin Asp Tyr Ser Arg Leu Ser 1665 1670 1675 1680 Ser Ser Val Ile Ala His Giu Leu Lys Val Pro Glu Ser GlyGiu Thr 1685 1690 1695 Arg Trp Val Val Asp Thr Ile Val Gly Lys Giu Asp Giy Leu Gly Cys 1700 1001705 1710 Glu Asn Leu His Gly Ser Gly Ala Ile Ala Ser Ala Tyr Ser Lys Ala 1715 1720 1725 Tyr Arg Giu Thr Phe Thr Leu Thr Phe Val Thr Gly Arg Ala Ile Giy 1730 1735 1740 Ile Gly Ala Tyr Leu Ala Arg Leu Gly Met Arg Cys Ile Gin Arg Leu 1745 1750 1755 1760 Asp Gin Pro Ile Ile Leu Thr Gly Tyr Ser Ala Leu Asn Lys Leu Leu 1765 1770 1775 Gly Arg Giu Vai Tyr Ser Ser Gin Met Gin Leu Gly Gly Pro Lys Ile 1780 1785 1790 Met Ala Thr Asn Gly Va, Val His Leu Thr Val Ser Asp Asp Leu Giu 1795 1800 1805 Gly Val Ser Ala Ile Leu Lys Trp Leu Ser Tyr Val Pro Pro Tyr Val 1810 1815 1820 Gly Gly Pro Leu Pro Ile Val Lys Ser Leu Asp Pro Pro Giu Arg Ala 1825 1830 1835 1840 Val Thr Tyr Phe Pro Glu Asn Ser Cys Asp Ala Arg Ala Ala Ile Cys 1845 1850 1855 Gly Ile Gin Asp Thr Gin Gly Lys Trp Leu Ser Gly Met Phe Asp Arg 1860 1865 1870 Glu Ser Phe Vai Glu Thr Leu Glu Gly Trp Ala Lys Thr Val Ile Thr 1875 1880 1885 Gly Arg Ala Lys Leu Gly Gly Ile Pro Vai Gly Ile Ile Ala Val Glu 1890 1895 1900 Thr Glu Thr Val Met Gin Val Ile Pro Ala Asp Pro Gly Gin Leu Asp 1905 1910 1915 1920 Ser Ala Giu Arg Val Val Pro Gin Ala Gly Gin Val Trp Phe Pro Asp 1925 1930 1935 Ser Ala Ala Lys Thr Ala Gin Ala Leu Leu Asp Phe Asn Arg Glu Glu 1940 1945 1950 Leu Pro Leu Phe Ile Leu Ala Asn Trp Arg Gly Phe Ser Gly Gly Gin 1955 1551960 19G5 Arg Asp Leu Phe Glu Gly Ile Leu Gin Ala Gly Xaa Met Ile Val Glu 1970 1975 1980 Asn Leu Arg Thr Tyr Lys Gin Pro Ala Phe Val Tyr Ile Pro Lys Ala 1985 1990 1995 2000 Gly Glu Leu Arg Gly Gly Ala Trp Val Val Val Asp Ser Lys Ile Asn 2005 2010 2015 Pro Glu His Ile Glu Met Tyr Ala Glu Arg Thr Ala Arg Gly Asn Val 2020 2025 2030

I

Leu Glu Ala Pro Gly Leu Ile Glu Ile Lys Phe Lys Pro Asn Glu Leu 2035 2040 2045 Glu Glu Ser Met Leu Gly Leu Asp Pro Glu Leu Ile Ser Leu Asn Ala 2050 2055 2060 Lys Leu Leu Lys Glu Thr Ser Ala Ser Pro Ser Pro Trp Glu Thr Ala 2065 2070 2075 2080 Ala Ala Ala Glu Thr Ile Arg Arg Ser Met Ala Ala Arg Arg Lys Gin 2085 2090 2095 Leu Met Pro Ile Tyr Thr Gin Val Ala Thr Arg Phe Ala Glu Leu His 2100 2105 2110 Asp Thr Ser Ala Arg Met Ala Ala Lys Gly Val Ile Ser Lys Val Val 2115 2120 2125 Asp Trp Glu Glu Ser Arg Ala Phe Phe Tyr Arg Arg Leu Arg Arg Arg 2130 2135 2140 Leu Ala Glu Asp Ser Leu Ala Lys Gin Val Arg Glu Ala Ala Gly Glu 2145 2150 2155 2160 Gin Gln Met Pro Thr His Arg Ser Ala Leu Glu Cys Ile Arg Lys Trp 2165 2170 2175 Tyr Leu Ala Ser Gin Gly Gly Asp Gly Glu Lys Trp Gly Asp Asp Glu 2180 2185 2190 Ala Phe Phe Thr Trp Lys Asp Asp Pro Asp Lys Tyr Gly Lys Tyr Leu 2195 2200 2205 Glu Glu Leu Lys Ala Glu Arg Ala Ser Thr Leu Leu Ser His Leu Ala 2210 2215 2220 Glu Thr Ser Asp Ala Lys Ala Leu Pro Asn Gly Leu Ser Leu Leu Leu 2225 2230 2235 2240 Ser Lys Met Asp Pro Ala Lys Arg Glu Gin Val Met Asp Gly Leu Arg 2245 2250 2255 Gin Leu Leu Gly 2260 INFORMATION FOR SEQ ID NO: 32: t'J SEQUENCE CHARACTERISTICS: LENGTH: 3319 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: GGATCCTCTT GAGCTTCTTC AGCAGAGATA CAGTTGACAT GGCCACGTGC AGTGGTGGCT GGCTTGGCGT AGAACACTTT CCCTGTCGGC TTGCCACGGC CAACAGCTTT TCCAGATTGG 120

TTGGGGTTGG

GTCACCGAGC

GTAAACTGTT

AGGGCAGTGT

ATGTCACGGIC

GCTTTGTTAA

TCAGGACTAA

GTTGCATATC

TGAGTGAAAC

TCACGCAAAG

CCATAGTTCT

TCTGGGGACA

TGGTACGTGG

GGGCGGGGCA

TAGGCACCCA

CATGCTTGCG

CAAGCTTCTG

GTCCCCTATG

GTGCAAGGTT

TGCAGAACTC

CCTCGCTGAA

CCCACCATAG

CTCGCGCAGA

AGGAGCATTG

AGGCGCCTGA

CACACGGCGC

TGTTGCTTCA

AAAAGATGTG

GAACCTTGCT

ACCGAACTCC

CTCATGTTTA

TTCAGCCCAG

ACTGGCGGGG

TAGTGGCCCG

TTGGTTGGAC

AAGGATGGCC

TTCTGAGGAC

TAATCAGTCT

CACTCATGCA

TGCTTCTTGG

CTACTGTAAG

CGGTCCATGA

GTAGTGACAT

CCTTCAAGAT

GTTCCCCACA

CACCATAGGG

TCGGTGTGAA

CAGCACCGGA

GACCAGACTC

GACGGAGGTC

CTTCAGTAGA

CATCCACAGA

GACCCTTCGG

GGCCCGCTGG

GATATGCAGC

TTTAAAGCAA

CTTGGCTGGT

CCTGGGTGGC

TGAGGAACCC

AGCATTGATG

GCGGCGAAGC

GACTTCCTCA

AAGTCGACCT

AATGTGATGT

GCGCATAGCT

AAAGGTGACC

180 240 300 360 420 480 540 600 660 720 780 840 TTATCAGCCT CAGOTACTAG CGCAGAATGC AGTTTGTGAG TAATACTGCG AAGCCAGTCA TCGGCGTCGA GAGGCTCGAC CTGAGTGACA CCAAGTTATT AAACGGTTAG TCTCCCGCTT GGGTGAGGCA GGTTTGTTCC TTGGTGCGGG TGTTAACCAT ATTCTGACAT AGAACGAAGA GACATGGTAG ATACAGAAAC TAGTACAGAA CCAAACAAAG ATTCCGAGCT CCTATACATT ATCCCACGAG TCACGCAGGA ACTAACTCAG ACAACTCCGT AACGTGATCG TGATCCTTCT

GGAGTGGTGG

CCTCTGATGG

GTTTCTCTCG

CCCAACTCTG

CCTAGGAAAA

ATGTAATGGA

TGCTTCTTTT

TACTACTACG

ATTTTTCTAC

CGGTGGATGA

AGTAGTCTTC

TTTTCGTTCG

AAAGTGGGTG

TGTGCCATGT

GCTTCCAGCA

CTGCCTTCGG

CAAAACAATA

TAACTTGGAA

ATTCCATCGT

GTGAAAAGAG

ACCTCCGGAA

TACAGCTAGT

ATATAAGTCA

TCGTAGGGGC

GATGTAACTT GATGAAATCA TTTGCTCGAT GCGCTCCAAC TAACTTCGGC CAGAGAGGGA CCTGCTCTGG GGGAGCAGGG GTTTAGTCCA GGATGATAGG TGTAAGATGA CCATCCGTAT CATACACACC ATACAAGGTT GATTACATCT CATCGGAGGC GGCGGTACAA GCTAAGTCAT AGGATACTAG TGATACTACT CCTCCATAGC CTGGAAGCTC TGTTGGGAGG GATTAAATCA 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560

TTCGCTCCAG

TGAATAAAGT

ATCTCCCACA

AGGCATCAAA

AACTATTCAT

CTGCCGCGTG

CGATTTGTGA

TCTCCGTTAC

CAATTAGCTA

GGTGACGGGG

GAGCACATCG

AACTGATGAC

ATGATATGAC

CATAACACGA

GGCGTCGAGA

CTTCCACCAG

CGCGTGTGTC

CCGTCAACTA

AAGGCACGGA

GACAACACAC

ACGTGGACGA

AGTCCAGGGC

ATCGCGTTAT

GTTATGGCGC

AAACCAGAAC

CCTATGCCAA

CCCCGCCTCC

CGTTTGCCTC

AATTTGGAAT

CAATTAGCTA

CCACGGACAA

AGCCGAGCGG

AGACACGCCG

GCACGTCCTA

AACGGACAAC

AAAACACCCC

GCACACCATC

ATGAATGTTG

GGCGGAACAC

CGTTGGCGCA

GACAACACAC

TTAGCACCGA

AGCACGCCAC

GAGAGACAGG

TTTAAAATCA

ATGGGAACAT

GCGACTACGA

CATCTGTGAC

GACTAGAATG

CACCAGCCCG

TAATCATTGG

CCATGATGCA

CGACTACGGG

CGGAGCGGAG

TGCAACGACG

CAGACATGAG

GACATGTTTC

TTGGAGATGT

CATGAAGCAC

TGAATGTGTA

GTACAGCAAG

AATATGCATG

ATTAGCTAGA

ACGGCCGGAC

GGAGCGAGCT

CACCCATCCG

1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 TCCATCCGCC CGCCCAACCA GGGCCATGCG GCCCAACTAC CCGTCGTCCC CGTCTAGACC ACGCCCCCCA CCTGCCCCGC CCCACCCCAC CCCCAACTCC TCCATGAATG CACGCATTTC

ATCGCTCCAA

TTATACCACG

CCCCGCGCGG

CTCCCCCTGC

CTCCGCCTCC

TGACCAGTGA

CGTAGGCTGC

CTCTCAGCGC

GCCGGATTGC

CTGGCCGGGT

CCACAACGCA

CAATTC CAT C

CATTCCGTCG

CCTTGCTATG

GCCTATGCGT

TGTACTCGCT

CTCCAGGCCC

CAGGAGATCA

CGGGTTCGCC

TTTGGGCACA

GCAGCCCCAG

TGGATCTCCA

AACGGCTTGG

CCCCTGCTTC

GCGGGCGATT

CGCTAGCCTC

CAATCCAAGC

CGGGACCAGA

CCGTCTGGCA

CTTGTTGCTT

CACCAGICGGC

CCTGGCCGCA

CGGCGCGCCT

TGCACGCATC

GACTGGGCCG

TCCGCCCACG

AGCGCAGCGC

TACCACTGCT

TTACGTCGAG

ACTTCCTTCT

CTCGGCGACG

GCACGGGTTT

CCGGACGGAC

TTCCGATTTT

GACTTGCCAT

CCGGCCTCAA

AGGGCCTTCC

AGCAGTCGAC

CGGGTGGTGG

GCTGAATGCC

CGGCGCGCAT

CCTCCTCCCT

CCACGGTAAG

CGCTGGAGCG

GGACTCGTAC

ATCGAGCGCG

TGCTGATTCT

CCGTGCCGTC

GCGCGCGCGA

GGAATTCAAG

2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 TCCATTTCCC TCTTTGCTCC TGCTTGGACT AACCAGTCCC CTAGTGTGGA CTACAGCATT

TTTTTCGCGT

CTAGAATTCT

GTTAATTAGT

AAAGGGCACA

CGGACATAAA

AAGGTTGGAG

ATTTTTAATG TGATCTCTGG GCACTCTCCC ATGGCACTCT GCCACCATGT TGTTGTTTTC CACATGCCAA ACCGAAAAGA TGATTGATGC GCTAACGGAC

GGGGCAATA

TCTTGCTCTT CTGGTTCTGC TGGTTGTTGA TGCCGGAGGA ATTTCCCGAT TTAGCTAGCC TGTAGTACCA TTTTAGCATC TGGTACAGAA AATATCCCAG TGCTGCAATT CTACGCTAAT GGACTTGTTC TTTTGCTTTT CCCAGCGCTG 3060 3120 3180 3240 3300 3319 INFORMATION FOR SEQ ID NO: 33: SEQUENCE CHARACTERISTICS: LENGTH: 3368 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

TACTCGCCGC

TACTCGCCGC

CAGCTGGACA

TTGCCGTGGT

TCGTAGACGT

GTCGCCATGG

GGCCACTTGC

TCACGGAAAA

TCAGGCAGCG

AGGTGGTGTG

GATATGTTTT

AGGGATGAAG

CGGCAGCGGC

CGGCAGCGGC

GGGACTCGGA

TGGCCGGCAC

CTCCTGCACG

CCTCCTGCCG

CGGTCCGCTG

CAGGACTGCC

CGTCGAATCA

TAGCGTATCC

CAGCCGAGAA

GGGCATGTTT

GTAGGCATGG

GTAGGCATGG

GCCCATGGAC

GATAGCCCCT

CGCCATCCAT

CCGTGCGCGC

AGTCCGGCCC

AGGCTGGTCG

GACGCCGCGG

TTGGCAGATG

AGAACAGGAA

GGATGCCGAT

GCGTATGCAT

GCGTGTGCGG

AACGTCAGCG

TCGACGTCCT

GGCCTCCTGC

TCCTCATGAA

GGTCTGGAGA

GATCGGCCCG

CAACTCCGAT

CAACGGCGGA

GGGATGACGA

AGCATGAGAT

CCTACTGTTT

GCGCCTGCAG

GGTGCAGGAG

CGTCGTCGCC

CGCCATGCGC

CTACTGCCGC

GCCGCCGGTC

GACAGTTTCC

GTCCCAGACG

TAGTAAGAGG

CGTAGATAGA

GCGGGGCGGG

CTGTCGGATC

CACGCTGACG

GGCACCAGGG

GGTGTCCACG

CGCCGCGACA

CGCTCGCCGT

TGGTCAGTGG

ACCCTGATGA

GCGGCGACAG

GATTAGAGAA

CGGCACGGGG

AAGAGATCAA

120 180 240 300 360 420 480 540 600 660 720

TTAGGTTGAG

TAGGAGTAAT

GATGCACTCT

GCCCAGGCGT

GCTAAGTGCA

CCTAATCGTG

TTATTTGATT

AAAAAATATT

CCGGTGACAC

ACACCCCGCA

ACATCCATCC

GCACCAACGG

TGGCTTCCTA

AAGTTTTTTA

GATTTAGTTT

GGAGTTTCAT

CACAGAAAAT

CGGTGGTATT

GTTTAATAGT

TGAATCACAA

GGGACTACAA

GTATGTTTGA

GTGACGGCGA

GAGCGGAGGG

TTTTAGCTGA

ATAGGATGGA

CATAGAAAAG

ATTTTCCTCC

GGTTTAGGTT

GGATCTGTGT

AGTATAGATA

ACAAGAGCTA

CATGTTTCAC

GGCGTTGGCA

AGCGCAACTA

AGTCTACATC

TAATAATAAT

TTTGTCTGAG

GATGCACCGC

ACAGTGGAGT

AAGGCTAACC

ACGCTTTGTG

AAAAAGGCAC

ACAAAAGCAT

CACCAACACG

TCAAAAGCGT

TTGTCTTCAA

ACACCCGTCA

TAGATGACAA

ATTAGTTTCC

GATTATATAG

ACGGCCAGTC

GTTAGATTGA

GGGTGTGCTT

GCCTTCGTTA

GATATGCCCT

TCACCCGAGA

TGGGACCTAT

GGGGAAATGG

CGTGTCCCCG

TTATATATGG

TAATAGGATG

TTTCCTAATT

AATGTAAATT

TTTTAGTGGG

AAAAAAGTTC

ACGCGCGTAG

TGTGGCAAAA

AACGGAATAA

GCTAGGCACA

AATCGACTCC

CCCCGTAAAT

7800 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 GCACGACTAG AAGGTGCACC ATTGCATCCT CAAAAAAGAA AAAAAAAAGC GAATCAACCT

GTGGTTGGTT

CTCGCATTTA

CATAGGGATG

AAGAAAAAAA

TAGAGAACAG

GCGGCACAAG

ACGTGAGCGA

GTTGGGGCCC

AACCAGGGCG

CACCCCCCAC

GGTTAGAGGG

TTTCTGGATT

AGTGTATACG

AAGATTATGT

GGTCATGATG

ACGCCGTGGT

AGCCGAAGGG

TACCTACACA

ATGAGGCCCA

CTGCCCCGCC

ACTGTGGTAT

TATTTTAGGA

CGTGTATATG

ACCATTGCGC

CAGTCCGAGT

GGCTTGGCCG

AGCACGCCAC

CACGCCGGAG

ACAACCTGTC

CCACCCCACC

CCCCAGCCCA

TTTCCGGCGA

AGCGCTTGCG

GTGTATGTCC

TACGGTAACG

ACGACTACGG

CGGAGCGGAA

AGACAGGTGC

GTCGACTCCT

CCACCCCCAA

CCATGGTTCA

TGCGCATTCA

TCTGTACTGT

ATACACTTGA

AACAAACGGG

GACGGCCGGA

GGAGCGAGCA

AACGACACAC

CCCCGTCTCC

CTCCTCCATG

AATCCTGGTG

GTGGGAGGTT

GTTAAAAAAA

GCCGATTAGC

AGTCAACAAG

CGGGTCGGGG

CATCGAAGGC

CAATCCGTCC

ACCTCCACCA

AATGCACGCA

1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 TTTCATCGCT CCTACCACAA CGCAGCAGCA CCAGCGGCCT CGGCGACGCG CCGCGCATTT ATAGCAAGCA ATTCCTCGTT CTGGCCGCAG CACGGCCTTC CGCGGCGCGG CTCCGGCCGA GTGCCCCTG'C TTCTGCCCCC GGTGATTGAG CGAGTCGGTC CGCGCGTAAG AACAGTAGTA CACGAGCCGG CTAGCTCCAG TTCTAGCGGC ACGAGATCAC TCGTGCGCGC GGAGTCCTGC GAATTGGCGG GCGGCTTCTT GTGATTGGCC GGGTTTTGGG

GCCTCCGCCT

TTCCTCCTTC

ACCGACGGTA

CTCTTCCGGT

TCGCTACTGG

CTACCACCAG

GCCCCCCAGT

GGAGCCGGAT

TGACGCGGGA

CGTGCCGTCT

TGCTTGCTGC

CCGCCGCCGC

CTCCCTCCGC

CGCGCCCTGC

TTTCGCTGGA

CTTCGGCCCG

CTTCTCCGTC

CCCGCAAGCG

ACTGCTCTCG

TCCTGCCGAC

GGCATTACGT

TTCCGTCCTT

TGCCTCTCCT

GGCATTCCGT

CCGTCCCCCC

GCACCGCGTG

AGCTGCCGTG

CCCGGGGCCT

GCGCGGGGCC

CGCGCGCGAT

GATCGACCCG

CGAGCGGGTG

GTGCTGAATG

GGATCTCCAT

CGAACGGCTT

TGCCCCCGCC

CGTGTGTGTA

TCCCGGCGCG

TCAAATCGAG

TTCCTGCTG

TCGAGCTAGT

CGCCGTCGCC

GTGGGCGTGC

TCGGAATTCA

2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 AGTCCCTTTT CCCCTTCCCT CCTGCTTGGA GTGGACTAAC CTTAGTGTGG ACTTCAACAT

TTTTTTCATG

ACTGTTGCGG

TAGTTATTAG

TCCAGTACAG

TTCGCTAAC!C

ACTACTATGT

ACATGCCAAA

GATTGATGCG

GGGCAATA

TGATCTAGGG

ATTGCGCACT

TGGTTAAATA

AAAAAAAGGA

AGACAGAATT

TTTTGTTCTG

CCGAAAAGAA

CTAACAGACG

TCTTGCTGTT CTGTTTCTGC TGGCTGTTGA CTATCAGCTT TTCCCCTGGC ACTGTTTCCG GAGGAATTTC CTGATTTTTT GTACCATTAT GTCTTTGTTT GCTTTGTGCC ATTTTTAGCA ATAAACGTGC AAAACTGAAA AATAATAACC CGGTGCTGTT GATTCCACCA TTTTCCTGAT TTAGTTAGTA GTTAAATAGG TTTGTACCAT TTTAGCATCT AGTACAGAAA AAGCGCACAC ATATCCCAAT GCTGCAATTC TACGCTAATC GGACATAAAT GATTTGTTCT TTTGCTTTTC CCAGTGCTGA AGGTTGGAGG 2940 3000 3060 3120 3180 3240 3300 3360 3368 INFORMATION FOR SEQ ID NO: 34: Wi SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs WO 96/32484 PCT/US96/05095 231 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: ATCGATCGGC CTCGGCTCCA ATTTCATT 28 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GTTCCCAAAG GTCTCCAAGG INFORMATION FOR SEQ ID NO: 36: SEQUENCE CHARACTERISTICS: LENGTH: 37 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: GCGGACTCGA GTCGACAAGC TTTTTTTTTT TTTTTTT 37 INFORMATION FOR SEQ ID NO: 37: WO 96/32484 PCT/US96/05095 232 SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: ACGCGTCGAC TAGTAGGTGC GGATGCTGCG CATG 34 INFORMATION FOR SEQ ID NO: 38: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: GCGGACTCGA GTCGACAAGC INFORMATION FOR SEQ ID NO: 39: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: ACGCGTCGAC CATCCCATTG TTGGCAACC 29 WO 96/32484 PCT/US96/05095 233 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GACTCATTGA GATCAAGTTC

Claims (36)

1. An isolated DNA segment that encodes canola or wheat acetyl-CoA carboxylase further defined as encoding the amino acid sequence of SEQ ID NO:20 or SEQ ID NO:31.

2. The DNA segment of claim 1, further defined as comprising SEQ ID NO: 19 or SEQ ID

3. The DNA segment of claim 1, wherein said DNA segment encodes wheat acetyl- CoA carboxylase.

4. The DNA segment of claim 3, further defined as encoding the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:31.

5. The DNA segment of claim 4, further defined as SEQ ID NO:9 or SEQ ID NO:

6. The DNA segment according to any one of claims 1 to 5, wherein said DNA is operatively linked to a promotor, said promoter expressing the DNA segment.

7. The DNA segment of claim 6, defined further as a vector.

8. The DNA segment of according to any one of claims 1 to 5, wherein said DNA encodes a portion of a dicotyledonous acetyl-CoA carboxylase functionally linked to a portion of a monocotyledonous acetyl-CoA carboxylase.

9. A host cell comprising the DNA segment according to any one of claims 1 to The host cell of claim 9, defined further as being a prokaryotic cell.

11. The host cell of claim 10, further defined as a bacterial or cyanobacterial host cell. P:\OPER\JEH\1945381 r2.doc46/070) -235-

12. The host cell of claim 9, defined further as being a eukaryotic cell.

13. The host cell of claim 12, further defined as a yeast cell or a plant host cell.

14. The host cell of claim 13, wherein said cell is a monocotyledonous plant cell. The host cell of claim 11, wherein the bacterial host cell is E coli.

16. The host cell of claim 11, wherein the cyanobacterial host cell is Synechococcus or Anabaena.

17. The host cell of claim 9, wherein the host cell expresses the DNA segment to produce the encoded acetyl-CoA carboxylase protein or peptide.

18. The host cell of claim 9, wherein the expressed acetyl-CoA carboxylase protein or peptide includes a contiguous amino acid sequence 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; SEQ ID or SEQ ID NO:31.

19. A method of using the DNA segment of claim 1, comprising the steps of: preparing a recombinant vector in which the acetyl-CoA carboxylase- encoding DNA segment of claim 1 is positioned under the control of a promoter; introducing said vector into a host cell; culturing the host cell under conditions effective to allow expression of the encoded acetyl-CoA carboxylase protein or peptide; and collecting said expressed acetyl-CoA carboxylase protein or peptide. P:\OPERULEH\1945381 rs2.doc-06/07/( -236- An isolated nucleic acid segment comprising: a nucleic acid segment comprising a sequence region that consists of at least contiguous nucleotides that have the same sequence as, or are complementary to, 25 contiguous nucleotides 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; SEQ ID NO:19; or SEQ ID NO:30; or a nucleic acid segment of from 25 to about 13,000 nucleotides in length that hybridizes 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; SEQ ID NO:19; or SEQ ID NO:30; or the complements thereof, under hybridization I conditions including about 0.15M to about 0.9M salt at temperatures from about 20 0 C. to about 55 0 C.

21. The nucleic acid segment of claim 20, further defined as comprising a sequence region that consists of at least 25 contiguous nucleotides that have the same sequence as, or are complementary to, 25 contiguous nucleotides of SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ IDNO:7; SEQ IDNO:9; SEQ IDNO: 1; SEQ ID NO:19; or SEQ ID

22. The nucleic acid segment of claim 20, further defined as comprising a nucleic acid segment of from 25 to about 13,000 nucleotides in length that hybridizes 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; SEQ ID NO:19; or SEQ ID NO:30, or the complements thereof, under hybridization conditions including about 0.15M to about 0.9M salt at temperatures from about 20 0 C. to about 55 0 C.

23. The nucleic acid segment of claim 20, wherein the segment comprises a sequence region of at least 25 contiguous nucleotides from SEQ ID NO:19 or SEQ ID RT NO:30, or the complements thereof, or wherein the nucleic acid segment hybridizes to the nucleic acid segment of SEQ ID NO:19 or SEQ ID NO:30, or the P:\OPERUEH\1945381 rs2.doc-O6/07AX -237- complements thereof, under hybridization conditions including about 0.15M to about 0.9M salt at temperatures from about 20 0 C to about 55 0 C.

24. The nucleic acid segment of claim 20, wherein the segment comprises a sequence region of at least 25 contiguous nucleotides from SEQ ID NO:11, or the complement thereof, or wherein the nucleic acid segment hybridizes to the nucleic acid segment of SEQ ID NO:11, or the complement thereof, under hybridization conditions including about 0.15M to about 0.9M salt at temperatures from about 0 C. to about 55 0 C. The nucleic acid segment of claim 20, wherein the segment comprises a sequence region of at least 25 contiguous nucleotides from SEQ ID NO:9, or the complement thereof, or wherein the nucleic acid segment hybridizes to the nucleic acid segment of SEQ ID NO:9 or SEQ ID NO:30, or the complements thereof, under hybridization conditions including about 0.15M to about 0.9M salt at temperatures from about 20 0 C. to about 55 0 C.

26. The nucleic acid segment of claim 20, wherein the segment comprises a sequence "region of at least 25 contiguous nucleotides from SEQ ID NO:7, or the complement a thereof, or wherein the nucleic acid segment hybridizes to the nucleic acid segment Sof SEQ ID NO:7, or the complement thereof, under hybridization conditions including about 0.15M to about 0.9M salt at temperatures from about 20°C. to about 55 0 C.

27. The nucleic acid segment of claim 20, wherein the segment comprises a sequence region of at least 25 contiguous nucleotides from SEQ ID NO:3, or the complement thereof, or wherein the nucleic acid segment hybridizes to the nucleic acid segment of SEQ ID NO:3, or the complement thereof, under hybridization conditions including about 0.15M to about 0.9M salt at temperatures from about 20°C. to about 55 0 C. P:\OPERVEH\1945381 rs2.doc4)6/A7/X) -238-

28. The nucleic acid segment of claim 20, wherein the segment comprises a sequence region of at least about 25 nucleotides; or wherein the segment is about nucleotides in length.

29. The nucleic acid segment of claim 20, wherein the segment comprises a sequence region of at least about 30 nucleotides; or wherein the segment is about nucleotides in length. The nucleic acid segment of claim 20, wherein the segment comprises a sequence region of at least about 50 nucleotides; or wherein the segment is about nucleotides in length.

31. The nucleic acid segment of claim 20, wherein the segment comprises a sequence region of at least about 100 nucleotides; or wherein the segment is about 100 nucleotides in length.

32. The nucleic acid segment of claim 20, wherein the segment comprises a sequence region of at least about 200 nucleotides; or wherein the segment is about 200 nucleotides in length. 0

33. The nucleic acid segment of claim 20, wherein the segment comprises a sequence region of at least about 500 nucleotides; or wherein the segment is about 500 nucleotides in length.

34. The nucleic acid segment of claim 20, wherein the segment comprises a sequence region of at least about 1000 nucleotides; or wherein the segment is about 1000 nucleotides in length. The nucleic acid segment of claim 20, wherein the segment comprises a sequence region of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7. P:\OPERUEH\!945381 rs2.doc-06/077A -239-

36. The nucleic acid segment of claim 20, wherein the segment comprises a sequence region of SEQ ID NO:9, SEQ ID NO: 1, SEQ ID NO:19, or SEQ ID

37. The nucleic acid segment of claim 20, wherein the segment is up to 13,000 basepairs in length.

38. The nucleic acid segment of claim 20, wherein the segment is up to 5,000 basepairs in length.

39. The nucleic acid segment of claim 20, wherein the segment is up to 3,000 basepairs in length.

40. The nucleic acid segment of claim 20, wherein the segment is up to 1,000 basepairs in length.

41. A nucleic acid detection kit comprising, in suitable container means, an isolated S: plant or acetyl-CoA carboxylase-encoding nucleic acid segment according to any one of claims 1 to 5 and a detection reagent. p

42. The nucleic acid detection kit of claim 41, wherein the detection reagent is a detectable label that is linked to said acetyl-CoA carboxylase nucleic acid segment. S43. The isolated nucleic acid according to any one of claims 1-8 or 20-40, the host cell according to any one of claims 9-18, the method according to claim 19 and the kit according to claim 41 and 42 substantially as herein before described with reference to the Figures and/or Examples. DATED this 7 h day of JULY, 2000 ARCH DEVELOPMENT CORPORATION by DAVIES COLLISON CAVE 4 Patent Attorneys for the applicant(s)