AU715449B2 - Human SMCY cDNA and related products - Google Patents

Description

WO 97/10267 PCTIUS96/1 4547 -1- HUMAN SMCY cDNA AND RELATED PRODUCTS This application claims the benefit of U.S. Provisional Application No. 60/003,744, filed 09/14/95, and U.S. Provisional Application No. 60/012,973, filed 03/07/96.

FIELD OF THE INVENTION The present invention relates to the fields of molecular, clinical and evolutionary biology. Specifically, the present invention relates to SMCY cDNA, genomic DNA, and proteins and their use in the analysis of SMCY derived histocompatibility antigens, and in the detection of evolutionary conservation and early embryonic expression of the SMCY gene.

BACKGROUND OF THE INVENTION The Y chromosome of mammals is known to play a crucial role in male development. Numerous genetic factors and/or functions have been localized to the Y chromosome, including the expression and regulation of male specific minor histocompatibility antigens such as H-Y antigen, the serologically detected male Sdma antigen, spermatogenesis factors such as Spy, and the primary testis determinant (Sry) (Tiepolo et al. (1976) Hum. Genet 34:119; McLaren et al. (1988) Proc. Nat/.

Acad. Sci. 85:6442; Roberts et al. (1988) Proc. Nat/. Acad. Sci.

85:6446; Koopman et al. (1991) Nature 351:117; Ma et al. (1992) Hum. Mol. Genet. 1(1):29).

Although many of the factors listed above have been known for some time, the identification and isolation of the genes encoding these proteins has remained elusive. One particular example is that of the male specific minor histocompatibility antigen H-Y. The H-Y antigen IWO 97/10267 PCT/US96/14547 -2was first identified in 1960 when it was observed that within an inbred mouse strain, male-to-female skin transplants were rejected but male-tomale or female-to-male skin grafts usually succeeded (Eichwald et al.

(1955) Transplant. Bull. 2:148; Billingham et al. (1960 J. Immunol.

85:14). A similar human antigen was identified in 1977 (Goulmy et al.

(1977) Nature 266:544), and was eventually found to be ubiquitously expressed in human tissues (de Bueger et al. (1992) J. Immunol.

149:1788; van der Harst et al. (1994) Blood 83:1060; Voogt et al.

(1990) Lancet 335:131). The same H-Y related human antigen was also found to be involved in immune responses during various organ transplantations, blood transfusions, and pregnancy (for review see E.

Goulmy (1988) Transplantation Reviews, Volume 2, pg. 29).

One group of researchers recently isolated a cDNA sequence encoding a mouse gene (mouse Smcy) which maps to the Sxr region of the mouse Y chromosome. This region of the mouse chromosome is known to be involved in the expression and/or regulation of the H-Y antigen and spermatogenesis factors. (Agulnik et al. (1994) Hum. Mol.

Genet. 3:873). Further evidence of the importance of this gene was shown by analysis of its expression. The mouse Smcy gene was found to be transcribed in all male mouse tissue, as well as in mouse preimplantation embryos, indicating a possible "housekeeping role" for the protein. (Id.) The X chromosome homolog of the SMCY gene has also been isolated recently from mouse (Smcx) and human (SMCX or XE169) (Agulnik et al., supra; Agulnik et al. (1994) Hum. Mo. Genet. 3:879; Wu et al. (1994) Hum. Mol. Genet. 3:153). These X homologs show a high degree of homology with the mouse Smcy gene at the amino acid level. (Id.) The mouse Smcy cDNA sequence has been used to construct primers, which in turn have been used to amplify short stretches of homologous genomic DNA from human, mouse, and horse subjects, WO 97/10267 PCT/US96/14547 -3using the polymerase chain reaction (PCR). (Agulnik et al. (1994) Hum.

Mol. Genet. 3:873.) Comparison of the sequences of the resulting amplified fragments of genomic DNA to the known sequence of the corresponding regions of mouse Smcy cDNA showed greater than 93% homology at the amino acid level. Further, analysis of the expression of the SMCY homolog in humans showed that, as in mouse, the gene is widely expressed in male tissues. This evidence of conservation with a high degree of homology in several species indicate that the SMCY protein likely performs important functions in a variety of different organisms, including in humans. Id. at 876.

Although, as noted above, the sequences or partial sequences of the Smcy and Smcx genes of several different species have been reported in the literature over the last several years, it has not been clear how useful any of that sequence information would be in evaluating the function, control and expression of other SMC homologs. One recent study, in particular, indicated the utility of such information is very limited. In that study, an evolutionary conservation study, a mouse Smcx probe failed to detect an Smcy homolog in cattle and rabbit (Agulnik et al. (1994) Hum. Mo. Genet. 3:879). The authors of that study concluded that the Y copy of the gene was probably no longer required and that the X copy, which escapes X inactivation in all species studied, had taken over its function. This last study strongly indicates that mouse Smcx sequence information, in particular, would not be suitable for use in studying Smcx or Smcy homologs in other organisms. Graves, J.A.M. (1995) BioEssays 17(4):311; Jones et al.

(1995) Zygote 3:133.

Although the importance of the SMCY gene in humans and in many other organisms was appreciated even before the fragment composition comparison study cited immediately above, the human SMCY gene has not been previously isolated and sequenced or otherwise characterized. Without such characterization studies, human WO 97/10267 PCT/US96/14547 -4- SMCYgene expression, mutation, and control can only be accomplished with great difficulty. Therefore, there is a need to isolate the human SMCY gene and to determine the sequence of that gene, as well as to construct probes and primers complementary to that gene for use in embryonic development and other research studies. Once known, the human SMCY gene sequence could be used to study how alterations in the sequence of that gene affect male fertility, to produce antibodies to human SMCY protein, to design possible male contraceptives, to evaluate evolutionary conservation, and to determine the sex of tissues.

SUMMARY OF THE INVENTION One aspect of the present invention is cDNA encoding a human SMCY protein. This first aspect of the present invention is, specifically, human SMCYcDNA, comprising a translation region encoding the amino acid sequence of a human SMCY protein. The translation region of the human SMCY cDNA of the present invention most preferably consists of nucleotides 276 to 4893 of SEQ ID NO:1.

A second aspect of the present invention is an oligonucleotide primer, consisting of a sequence of nucleotides homologous or complementary to the most preferred human SMCY cDNA of this invention, having the complete nucleotide sequence of SEQ ID NO:1.

A third aspect of the present invention is a recombinant human SMCY protein produced by, and isolated from, a host organism containing the human SMCYcDNA of this invention. The human SMCY cDNA is preferably introduced into the host cell by transfection or transformation. The recombinant protein of this invention is most preferably a protein consisting of the amino acid sequence of SEQ ID NO:2.

A fourth aspect of the present invention is an oligonucleotide probe to human SMCY cDNA consisting of a sequence of nucleotides homologous or complementary to SEQ ID NO:1.

WO 97/10267 PCT/US96/14547 A fifth aspect of the present invention is a method of isolating an oligonucleotide probe to human SMCY genomic DNA, comprising: a. providing at least one primer consisting of a sequence of nucleotides homologous or complementary to SEQ ID NO:1; b. providing isolated human genomic DNA; and c. using at least one primer to amplify the isolated genomic DNA, thereby producing an amplified oligonucleotide probe.

A fifth aspect of the present invention, related to the fourth aspect above, is an oligonucleotide probe to human SMCYgenomic DNA produced according to that last method.

In an additional aspect, the present invention is a method of detecting SMCY homologs comprising: a. providing a nucleic acid sample; b. providing a pair of oligonucleotide primers designed to flank a region of interest in the nucleic acid sample, wherein at least one of the primers consists of a sequence of nucleotides homologous or complementary to SEQ ID NO: 1; c. amplifying the nucleic acid sample with said primer pair, resulting in an amplified oligonucleotide; and d. detecting the amplified primers complementary to regions of human SMCY genomic or cDNA which are upstream and/or downstream of regions of interest.

It is anticipated that the SMCY cDNA, isolated gene, probes, primers, protein, and methods of this invention could be used for a wide variety of different purposes, including, but not limited to, the analysis of SMCY derived histocompatibility antigens, the detection of evolutionary conservation, the study of early embryonic expression of the SMCY gene, and studies related to mitosis such as studies of spermatogenesis and tumorgenesis.

The present invention has specific use in the fields of evolutionary and developmental biology. Evolutionary biologists look for ways in WO 97/10267 PCT/US96/14547 -6which to examine the evolutionary relationship between different species of animals. The SMCY sequence of the present invention provides a new tool for such studies both by allowing more detailed and accurate comparison of the sequences of SMC homologs, both presently known and those that will be isolated in the future, as well as providing a superior probe to those currently available for assaying the DNA of diverse species for SMC homologs. Developmental biologists are interested in determining the mechanism by which cellular growth and differentiation occur during embryogenesis. This involves, in part, the elucidation of which genes are active at the various stages of embryonic development and what impact the expression of the genes has on the overall development of the embryo. The human SMCY cDNA sequence and/or isolated genomic DNA of the present invention can be used to ascertain the developmental stage at which SMC homologs are activated in embryos of diverse species.

Further objects, features, and advantages of the invention will be apparent from the following detailed description of the invention and the illustrative figures.

BRIEF DESCRIPTION OF THE FIGURES Figure 1: A map showing the overlap of the human SMCY cDNA clones that were isolated and sequenced are depicted in relation to one another and to the SMCY mRNA.

Figure 2: A representative map of a AgtlO clone containing an SMCYcDNA insert, including the location and orientation of primers Al, A2, C1, and C2.

Figure 3A: Southern blot of human DNA digested with Eco RI and probed with human SMCY. The arrow indicates the male specific SMCY DNA bands.

Figure 3B: Southern blot of cattle DNA digested with Sac I and WO 97/10267 PCT/US96/14547 -7- Pst I and probed with human SMCY. The arrow indicates the male specific Smcy DNA bands.

Figure 3C: Southern blot of rabbit, horse and monodelphus

DNA

digested with Sac I and Pst I and probed with human SMCY. The arrow indcates the male specific Smcy DNA bands.

Figure 4A: Comparison of partial rhesus Smcy cDNA and human SMCY cDNA sequences, with homologous sequences shaded.

Figure 4B: Comparison of partial Rhesus and Human SMCY Amino Acid Sequences, with homologous sequences shaded.

Figure 5: Photograph of an ethidium bromide stained electrophoresis gel of DNA from Rhesus 4, 8, and 16 cell-stage embros amplified with a human SMCY primer pair and a rehsus Smcx primer pair.

Figure 6: Elution profiles of HPLC fractionated peptides extracted from HLA-B7 molecules showing which fractions contained reconstituted H-Y epitope: after a single HPLC elution, or after a second HPLC elution.

Figure 7: Plot of results of mass spectrometry and 5 Cr release assay of the peak eluent fraction from the second HPLC column shown in Figure 6B.

Figure 8: Collision-activated dissociation (CAD) spectrum of peptide 1171 from fraction 14 (Figure 7B) after converting the R residue to ornithine.

Figure 9: Plot of the results of a "'Cr release study of reconstitution of the H-Y epitope on incubation of T2-B7 cells with a synthetic peptide of SMCY, or of SMCX.

Figure 10: Plot of percent inhibition of the binding of an iodinated endogenous B7 peptide to purified HLA-B7 by various synthetic peptides, including an SMCY synthetic peptide.

WO 97/10267 PCT/US96/14547 -8- DETAILED DESCRIPTION The following definitions are intended to assist in providing a clear and consistent understanding of the scope and detail of the terms used.

The term "gene" as used herein refers to a DNA sequence that codes for a bioactive protein or its precursor. The protein or precursor can be encoded by the full length gene sequence or any portion thereof so long as the bioactivity of the protein product is maintained.

The term "SMC homolog" as used herein refers to Smcx and Smcy genes and their corresponding mRNAs from any species of animal.

The term "SMC locus" as used herein refers to a particular segment of an SMC homolog.

The term "upstream" is used herein refers to a nucleic acid sequence proceeding in the opposite direction from expression.

The term "downstream" is used herein to refer to a nucleic acid sequence proceeding in the same direction as expression.

The term "oligonucleotide" as used herein refers to a molecule composed of two or more deoxyribonucleotides or ribonucleotides which is either chemically synthesized or generated by restriction digestion of a DNA molecule.

The term "primer" as used herein refers to an oligonucleotide that, when annealled to a complementary DNA or RNA under appropriate conditions, can serve as an initiation point for synthesis of a copy (primer extension product) of the DNA or RNA. Appropriate conditions include the presence of all four nucleotide triphosphates, a DNA polymerase, and buffer conditions suited to the performance of the particular polymerase chosen for the synthesis.

It will be understood that the term "primer," as used herein below, may refer to more than one primer. For example, in the case where the exact sequence of the end(s) of a locus to be amplified is not known, a plurality of primers will be synthesized and used that correspond to WO 97/10267 PCT/US96/14547 -9some or all of the possible sequences at the priming site of the fragment.

The term "primer pair" as used herein refers to primers which hybridize to opposing strands at opposite ends, and upstream, of an SMC locus.

The term "PCR" as used herein refers to the polymerase chain reaction technique in which cycles of denaturation, primer pair annealing, and extension with DNA polymerase are used to amplify the number of copies of a target nucleic acid. The polymerase chain reaction process for amplifying nucleic acids is described in U.S. Patent Nos. 4,683,195 and 4,683,202, which are incorporated herein by reference for a detailed description of the process.

Once the sequence of the human SMCY cDNA of the present invention is disclosed herein, it will be possible to obtain the gene, primers, and probes of the present invention by standard methods used in genetic engineering. For examples of methods suitable for use in making the products of the present invention, see J. Sambrook, E.F.

Frisch, T. Maniatis, Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory (1989), incorporated herein by reference. Below is a summary of the preferred methods for making the human SMCY cDNA and the other products of this invention.

The sequence of the human SMCY cDNA of the present invention is preferably assembled from a set of clones selected and isolated from at least one human male cDNA library, preferably from a human male lymphocyte and/or testis cDNA library. The library used to assemble the human SMCY cDNA of this invention is preferably produced as follows.

RNA is extracted from an appropriate biological material, preferably from human male lymphocyte or testis tissue, by any one of several methods well known in the art. (See for example Chirgwin et al. Biochemistry (1979) 18:5294 and Chomczynski et al. Anal Biochem. (1987) 162:156). RNA suitable for use in constructing the libraries used to WO 97/10267 PCT/US96/14547 make the cDNA of this invention can also be extracted using any one of a number of commerically available kits for this purpose, including the RNAgents® Total RNA Isolation System (Promega Corporation, Madison, Wisconsin).

The most preferred methods of extracting RNA for use in constructing the present libraries involve the steps of homogenizing the tissue in a denaturing solution containing guanidine, extracting the homogenate with phenol/chloroform, and then precipitating the total RNA from the resulting aqueous phase with isopropanol. Isolation of the mRNA fraction of the total RNA pool is then accomplished by passing the RNA over an oligo dT cellulose column or by binding to other such dT or dU containing material (Aviv et al. Proc. Nat/. Acad. Sci. (1972) 69:1408). Other oligo dT support material suitable for use in isolating the mRNA fraction include the streptavidin paramagnetic particles provided in commercial mRNA isolation kits, such as the PolyATract® System (Promega Corp., Madison, Wisconsin, USA). The mRNA retained on the dT or dU support is then eluted and concentrated by ethanol precipitation prior to use.

Any one of a number of known methods for generating double stranded cDNA equivalents of the mRNA isolated as described above are suitable for use in producing the library used to assemble the full human SMCYcDNA of the present invention. The most preferred such method is that of Gubler and Hoffman, in which an oligo-dT containing primer is annealed to the mRNA and used to prime synthesis of the first strand of the cDNA by reverse transcriptase. Gubler, U. and Hoffman,

B.J.

(1983) Gene 25:283. The second strand is subsequently synthesized by strand replacement using: RNase H to partially digest the mRNA thereby leaving gaps, a DNA polymerase to synthesize the second strand using the first cDNA strand as template and the remaining mRNA fragments as primers, and DNA ligase to ligate all the newly WO 97/10267 PCT/US96/14547 -11synthesized second strand cDNA fragments together resulting in double stranded cDNA molecules. Id.

A cDNA library is produced by inserting the double stranded cDNA molecules into a suitable cloning vector, such as Agt10, by any one of a number of known cloning methods. Suitable cloning methods for use in making the present cDNA library include direct blunt end ligation or ligation using linkers, for example EcoRI linkers attached to the ends of the cDNA inserts to facilitate ligation of each insert into the EcoRI restriction enzyme cut site of the vector. Packaging of the recombinant vectors into bacteriophage can be accomplished by on of several methods described in Sambrook et al. at sections 2.95-2.107.

The cDNA library thus produced is then screened for clones containing human SMCY cDNA. Any one of a number of known methods is suitable for such screening. However, either of the following two methods is most preferred. The first such preferred method uses plaque hybridization to screen the cDNA library. In this method aliquots of the cDNA library are mixed with an appropriate bacteria, plated at low density, and grown on agar plates. Once the bacteria are confluent, the plaques are lifted onto a nitrocellulose or a similar membrane filter and the filters are hybridized with one or more SMCY or SMCX DNA probes that have been labeled with a radioisotope or fluorescent reagent (Sambrook et al., sections 2.108-2.119). Plaques that hybridize with the probe are then serially isolated to single plaque purity and their cDNA inserts are analyzed. The inserts of AgtlO clones containing cDNAs of interest are then subcloned into plasmid vectors well known in the art such as pGEM® (Promega Corp.) or Bluescript® (Stratagene, La Jolla, California, USA).

The other preferred method for obtaining specific cDNAs of interest from a cDNA library involves amplification of the desired inserts by PCR using one or more sequence specific primers and reverse transcriptase. See, Kawasaki, "Amplification of RNA," PCR WO 97/10267 PCT/US96/14547 -12- Protocols: A Guide to Methods Applications (Acad. Press, pub.

1990), pp. 21-27; Rappolee et al. (5 Aug. 1988) Science 241:708; Chelly et al. (30 June 1988) Nature 33:858; Brenner et al. (1989) Biotechniques 7(10):1096; Block, Will (19 March 1991) Biochem.

30(11):2735, all of which are incorporated herein by reference. In this method, known as RT-PCR, a primer pair is constructed that will hybridize to opposite strands of the cDNA insert of interest such that their 3' ends are in closest proximity to each other. Alternatively, one of the primers is constructed to hybridize to the cDNA insert of interest and the other is constructed to hybridize to the vector DNA flanking the cDNA insert of interest. The primer pair is used to PCR amplify the selected cDNA inserts thus generating a subset of cDNAs specifically containing the inserts of interest. As above, these amplified cDNAs can then be subcloned into plasmid vectors by the methods described above or by use of commercially available "T-vectors" such as the pGEM®T-Vector System (Promega Corp.) or the original TA Cloning® Kit (Invitrogen®, San Diego, California, USA).

The DNA sequence of the cDNA clones selected in the library screening procedures described above can readily be determined by isolating DNA from the selected clones, and analyzing the insert sequence using any standard DNA sequencing method. Suitable standard sequencing methods for use in determining the cDNA insert sequences to assemble the sequence of the human SMCY cDNA of the present invention include the Sanger method of dideoxy sequencing (Sanger et al., J. Mol. Biol. (1975) 94:441 and Sanger et al., Proc. Nat/.

Acad. Sci. (1977) 74:5463), and the Maxam and Gilbert chemical cleavage method (Maxam et al., Proc. Nat/. Acad. Sci. (1977) 74:560).

See also Sambrook et al., Section 13.

Once the sequence of the insert portion of each cDNA clone is determined, the sequences are aligned with one another at regions of overlap to generate a composite cDNA sequence. This assembly WO 97/10267 PCT/US96/14547 -13process is preferably done by comparing the individual candidate clones to one another. Any missing cDNA sequences in the resulting composite map are resolved by additional library screening and sequencing until one obtains a contiguous sequence which includes at least the entire translation region of the protein.

Another aspect of the present invention is probes to the human SMCY gene sequence. The probes of this invention include any single or double stranded human SMCY DNA or RNA sequence of sufficient size to hybridize to a human SMCY cDNA sequence. The probes may be obtained by any of a number of methods well known in the art including, but not limited to the isolation of restriction enzyme fragments, synthesis of oligonucleotides, and the synthesis of single stranded DNA or RNA (Sambrook et al., Sections 5, 11, and respectively). The probes of this invention can be labeled and used to probe DNA or RNA samples from various sources to determine whether homologs of human SMCY are present.

The probes of this invention can be labeled by any of a number of procedures well known in the art so that on hybridization to a DNA or RNA sequence having homology with the probe, they can be detected.

Labeling procedures, including the use of 32 P or biotin, as well as hybridization techniques and protocols are described in detail in Sambrook et al., Sections 1 and 7-11, and Ausubel et al., Current Protocols in Molecular Biology (1 988), among others.

Yet another aspect of the present invention is an oligonucleotide primer having a human SMCY cDNA sequence which is complementary to a region of DNA or RNA adjacent to and upstream of a corresponding SMC locus of interest. Pairs of human SMCY primers of this invention which are complementary to opposing strands of DNA or RNA flanking a locus of interest can be used to amplify the DNA or RNA of that locus in an amplification reaction, such as the polymerase chain reaction (PCR). Thus, the primers of this invention provide a means to identify WO 97/10267 PCT/US96/14547 -14the presence or absence of an SMC homolog. The primer pairs of this invention are particularly useful in instances where one has only extremely small quantities of starting material (RNA or DNA) rendering standard probing techniques such as that described above impractical or impossible.

The primers of this invention are preferably single stranded, but may also be double stranded. The size of any particular primer of this invention depends on many factors, including the method being used and the source of the primer. However, in all instances the primer must be long enough to form a stable hybrid with the DNA or RNA substrate of interest. The primer must also have sufficient complementarity with the DNA or RNA being primed to form a stable hybrid. Exact complementarity is not required.

The primers of this invention may be prepared by any of several methods well known in the art, most preferably by automated synthesis.

See, Mullis, K.B. and Faloona, F.A. "Specific Synthesis of DNA in vitro via a Polymerase-Catalyzed Chain Reaction," Methods in Enzymology (Acad. Press, publ. 1987), pp. 335-350; Thein et al. "The use of synthetic oligonucleotides as specific hybridization probes in the diagnosis of genetic disorders," Human Genetic Disease Analysis: A Practical Approach (IRL Press, Herndon, VA, 1993), pp. 21-33, both of which are incorporated herein by reference.

Another aspect of the present invention is the isolated gene encoding for SMCY protein. The isolated gene of this invention is preferably obtained using the full length SMCY cDNA sequence, the SMCY cDNA fragments used to assemble that full length sequence, or probes or primers complementary to one or more region of the full length cDNA sequence. This aspect of the present invention uses known techniques, to isolate the gene of this invention, such as the genomic DNA isolation techniques described in Sambrook et al., supra, incorporated by reference herein.

WO 97/10267 PCT/US96/14547 An additional aspect of the present invention is a recombinant form of human SMCYprotein. The protein of this invention is preferably produced by cloning the human SMCY cDNA of this invention into an appropriate protein expression vector. The sequence of the recombinant human SMCY protein of this invention is preferably determined by analyzing the sequence of the human SMCY cDNA for potential open reading frames and comparing the possible amino acid sequences of those open reading frames with any specific peptide sequence known.

The open reading frame comtaining matches to the known peptide sequence is likely the correct amino acid sequence. See, Wu, J.

et al. (1994) Hum. Mol. Genetics 3(a):153, incorporated herein by reference.

The examples below illustrate the isolation and characterization of AgtlO clones with SMCY cDNA inserts, and the use of the resulting insert sequence information to assemble a contiguous human SMCY cDNA sequence encoding a human SMCY protein. The examples also illustrate the use of the sequence information to produce human SMCY DNA or RNA primers or probes to regions of interest in SMCY genes.

Finally, the examples below illustrate the use of the human SMCY cDNA sequence to identify and analyze human histocampatibility antigens such as the H-Y antigen.

EXAMPLE 1 ISOLATION OF HUMAN SMCY cDNA CLONES Two cDNA libraries were used for the isolation of human SMCY cDNA. The first was constructed by oligo-dT priming/reverse transcribing mRNA from male lymphocytes and cloning the products into AgtlO. The second, an oligo-dT/random primed human testis AgtlO cDNA library, was obtained from Clonetech Labs (Cat. #HL1162a).

Isolation of human SMCY cDNA was initiated by screening the lymphocyte cDNA library by plaque hybridization with a 190 bp human SMCY genomic DNA fragment, a fragment previously described in WO 97/10267 PCT/US96/14547 -16- Agulnik et al. (1994) Hum. Mol. Genet. 3:873. Library filters containing a total of 2 x 106 clones were hybridized with the random primed probe overnight at 60 0 C followed by washing with 0.1X SSC/0.1% SDS for minutes. Filters were exposed to Kodak XAR film overnight at -80 0

C

and positive colonies were isolated.

The plaque hybridization screening described above yielded two partial SMCY cDNA clones, H1 and H2 (see Figure the inserts of which were subcloned into pBluescript KSII (Stratagene, La Jolla, California, USA). H2 was found to contain 1410 bp of the SMCYcDNA sequence, including 44 bp of the 5' untranslated region, and 1366 bp of the protein coding region of the mRNA. Further, the sequence of H2 was determined to be chimeric, containing 1400 bases of unrelated DNA at its 3' end. H1, which overlaps H2, contains SMCY cDNA sequence more 3' than H2 but lacks the full coding sequence for the SMCY protein. H1 also contains a deletion and several insertions in the middle of the SMCY sequence as determined by comparison with the human SMCX sequence. Wu, J. et al., supra.

The insert regions of additional SMCY cDNA clones with sequences in the 3'direction of the target mRNA sequence of those contained in H1 and H2 were obtained by amplifying the lymphocyte and testis cDNA libraries prepared as described above, with RT-PCR and with primers complementary to SMCY cDNA and to Agt10 DNA, as follows. The insert region of the first such clones (clones 8 and 9) were determined by amplifying phage DNA from the libraries using RT-PCR, with primers complementary the downstream region of the SMCY cDNA sequence region of the H1 clone (primers S100 and S101) and with primers complementary to Agt10 vector DNA adjacent to and flanking the insert region of each clone. The insert region of the next clone (clone 1) was similarly amplified from the same libraries using RT-PCR and primers complementary to the downstream region of the SMCY cDNA sequence from clone 8 (primers S102 and S103), and WO 97/10267 PCT/US96/14547 -17primers. The insert region of the last clone containing SMCY cDNA sequence (clone A17) downstream of the the SMCY sequence of clone H1 was similarly amplified from the libraries, using the same Agtl 0 primers used above, and primers complementary to the downstream region of the SMCY cDNA insert sequence from clone 1 (primers S104 and S105). The above procedure resulted in the isolation of clone 1 from the lymphocyte library, clone 8 from the testis library, clone 9 from the testis library, and clone A17 from the lymphocyte library. Below is a representative detailed example of how the insert sequence of each such clone described above was first amplified and analyzed.

The sequence of clone 8 was used to screen for clone 1, as follows. SMCY primers S102 (5'-CCTAACATCCAGGCTCTCAA-3') [SEQ ID NO:3] and S103 (5'-AGAAGCTCTGACTAAGGCACAA-3')

[SEQ

ID NO:4], complementary to the sequence near the 3' end of clone 8, were synthesized. Primer S102 (nucleotides 3285-3304 of the insert region of clone 8) sits just 5' of primer S103 (nucleotides 3305-3326) in the SMCY sequence. In addition, two sets of AgtlO vector primers were synthesized: external primers C 1 (5'-CCACCTTTTGAGCAAGTTCAG-3') and C2 (5'-GAGGTGGCTTATGAGTATTTG-3') and internal primers Al (5'-AGCCTGGTTAAGTCCAAGCTG-3') and A2 (5'-CTTCCAGGGTAAAAAGCAAAAG-3'). After a first round of amplification using PCR with primers S102 and C1 or C2, the amplified products obtained were reamplified with primers S103 and Al or A2.

PCR conditions were: 94°C for 2 minutes then 30 cycles of 94°C for seconds, 55 0 C for 1 minute, 72°C for 3 minutes, followed by a 5 minute extension at 72°C. the resulting PCR fragments were electrophoresed on a 2.5% agarose gel, purified using Wizard'" PCR Preps DNA Purification System (Promega) and subcloned into pGEM®-T vector (Promega) according to the manufacturers protocols.

WO 97/10267 PCTfUS96/1 4547 -18- SMCY cDNA sequences 5' upstream) of those contained in clone H2 were isolated by RT-PCR with SMCY primers S32 and S33 complementary to sequences at the 5' end of H2 yielding clone from the testis library. Clone S20 contains an additional 231 bp of untranslated SMCY sequence.

Figure 1 provides a composite map of the various human SMCY cDNA clones that were isolated and sequenced in this example. The maps of each individual clone described above are depicted in relation to one another and to the human SMCY mRNA. The solid blocks in each map represent the protein coding region of the mRNA while the open blocks represent 3' or 5' untranslated regions (UTRs). The slashed portion of clone H2 represents sequence unrelated to SMCY. The hatched portion of clone H1 represents positions of deletions and insertions in the human SMCY sequence. Arrows indicate the position of human SMCY cDNA specific primers used to amplify and subclone AgtlO human SMCY cDNA inserts with RT-PCR, as described above.

The arrows point in the 5' to 3' direction. Extension of each of the SMCY primers proceeded in the direction of the arrow used to describe the primer in Figure 1.

Figure 2 provides a representative map of AgtlO clones with human SMCY cDNA inserts. The map depicts two principal regions of a representative clone, the Agt10 vector sequence shown as a shaded region, and the SMCY cDNA insert shown as an unshaded region. The arrows labeled A1, A2, C1, and C2 represent primers to the AgtlO vector sequence flanking the cloning site of the cDNA inserts. The arrows point inthe 5' to 3' direction. These primers were paired with SMCY specific primers in RT-PCR reactions to amplify and subclone sections of AgtlO clones containing SMCY inserts as described above.

WO 97/10267 PCT/US96/14547 -19- EXAMPLE 2 HUMAN SMCY cDNA AND PROTEIN SEQUENCE

ANALYSIS

The contiguous 5466 bp sequence of the human SMCY cDNA was determined by combining the sequences of the individual clones at regions of overlap, as depicted in Figure 1. The 5466 bp of sequence is made up of: a 4617 bp protein translation region, 275 bp of untranslated sequence, and 574 bp of 3' untranslated sequence.

The 4617 bp translation region codes for a human SMCY protein 1539 amino acids in length.

The 5466 bp human SMCY cDNA sequence obtained (SEQ ID NO. 1) is shown in the Sequence Listing below with the decoded amino acid sequence of the human SMCY protein. All DNA sequencing was done by the dideoxy termination method of Sanger.

EXAMPLE 3 EVOLUTIONARY CONSERVATION STUDY USING HUMAN SMCY cDNA DNA was extracted from the blood and/or liver of male and female human, cattle, horse, rabbit and monodelphus (South American Opposum) using standard procedures as previously referenced. The DNA was then digested to completion with the indicated restriction enzymes, electrophoresed on a 0.8 agarose gel, Southern blotted, and hybridized with random primed 32 P labeled human SMCY cDNA fragments at 65°C overnight. The Southern blot was washed under stringent conditions (two 15 minute washes in 2X SSC/0.1% SDS at RT; two 15 minute washes in 2X SSC/0.1% SDS at 55 0 C; two minute washes in 2X SSC/0.1 SDS at RT for the cattle, rabbit, horse and monodelphus blots; two 15 minute washes in 0.1X SSC/0.1 SDS at 55 0 C for the human blot) and autoradiographed.

Referring to Figures 3A, 3B and 3C, the Southern Blot shows that SMCY is conserved in the male in all species screened. Previously, using mouse Smcx as a probe, Smcy was demonstrated to be conserved WO 97/10267 PCT/US96/14547 in methatheria (wallaby: 150 million years diverged from eutherians) and in some (human, horse, pig, dog and mouse) but not all (cattle and rabbit) eutherian species. Agulnik et al. (1994) Hum. Mol Gen.

3(6):879. In these latter two species it was proposed that the Y copy of the gene was no longer required and that the X copy, which escapes X inactivation in all species studied, had taken over its function. In contrast, our results show that SMCY is conserved in monodelphus, which is 80 million years diverged from Australian marsupials, and in all eutherians probed, including cattle (Figure 3B) and rabbit (Figure 3C).

This is not surprising given that mouse Smcx is only about homologous to SMCY in humans and seemingly diverse species such as horse. Similarly, SMCY in primates shows more homology at the nucleotide level to all domestic species amplified using human primers than to SMCY in rodents including Chinese hamster.

EXAMPLE 4 USE OF HUMAN SMCY cDNA PRIMERS TO AMPLIFY AND STUDY PRIMATE SMCY The human SMCY cDNA sequence of Example 2 (SEQ ID NO. 1) was used to construct a primer pair, Rhl/S104 (5'-CCTCCAGACCTGGACAGAATT-3') and Rh2/S109 (5'-GTGGTCTGTGGAAGGTGTCA-3') flanking an SMCYIocus that codes for the 3' carboxyl region of the human SMCY protein (See SEQ ID NO:1). This region was chosen for amplification because comparison of the SMCY cDNA sequence from different species has shown that the 3' end of the cDNAs are most divergent from one another.

Using primers Rh1/S104 and Rh2/S109 at 100 pM concentration, 100 ng of male and female Rhesus DNA was amplified by PCR at low stringency (95°C for 2 minutes, then 1 minute at 94 0 C, 1 minute at 61 0 C and 1 minute at 72 0 C for 35 cycles followed by holding at 4°C).

The 359 bp male specific genomic DNA amplified product was gel purified using Wizard'" PCR Preps DNA Purification System (Promega), WO 97/10267 PCT/US96/14547 -21cloned into pGEM-T and sequenced using techniques described above.

As a control and for comparison, human and Rhesus RNA were also amplified, cloned and sequenced. The 264 bp Rhesus SMCYcDNA sequence obtained is shown in Figure 4A along with the human SMCY cDNA sequence. The decoded amino acid sequences for the cDNA sequences in Figure 4A is shown in Figure 4B. At the nucleotide level, there is 92% homology. At the amino acid level there is 82% homology.

EXAMPLE 5 USE OF HUMAN SMCY cDNA PRIMERS IN AN EARLY EMBRYONIC DEVELOPMENT STUDY IN PRIMATES To establish the onset of expression of Smcy in primates, Rhesus embryos were produced by superovulation, oocyte collection, in vitro fertilization, and embryo culture, and then collected. A total of 16 embryos were arrested in development at the 4, 8, and 16 cell-stage.

A single cell was removed from each of the 16 embryos and sexed using human SRYprimers amplifying the conserved "box" region of the gene.

Sinclair et al. (1990) Nature 346:216. Poly (A) RNA was then isolated from the remaining embryo cells by a modification of the PolyATtract® protocol (Promega) and amplified by RT-PCR with the human SMCY primer pair Rhl/S104 and Rh2/S109, described in Example 4, above, and the SMCX primer pair PRH1 (5'-AGAGTGGGGGCAGGGGTTAGTGTA-3') and PRH2 (5'-GGCCATCACCATTCCAGAGACAA-3'). Poly(A) RNA from Rhesus blood was also amplified as a control. The amplified products were electrophoresed on a 2.5% agarose gel and stained with ethidium bromide.

As can be seen in Figure 5, female and male embryos showed expression of Smcx (120 bp amplified product) at all three stages, however, Smcy expression (264 bp amplified product) was only seen in the male 8 and 16 cell-stage embryos indicating that expression of WO 97/10267 PCT/US96/14547 -22- Smcy begins between the 4 and 8 cell-stage. Although there is little data on early embryonic messages in primates, models suggest that only essential genes are expressed coincident with maternal/embryonic transition. Our data in primates, combined with the observation that Smcy is expressed in two-cell stage mouse embryos, demonstrates that Smcylx are two of the few early transcripts in embryos. Since Smcx escapes X-inactivation, two copies of the Smc gene may be necessary for normal function.

EXAMPLE 6 USE OF HUMAN SMCY PROTEIN SEQUENCE TO ANALYZE HUMAN H-Y ANTIGEN Endogenously processed H-Y peptides were processed as follows.

Class I MHC HLA-B7 molecules were purified by affinity chromatography from the H-Y positive, B lymphoblastoid cell line, JY. (Turner et al., (1975) J. Bio. Chem. 250: 4512; Parham et al. (1977) J. Biol. Chem.

252: 7555.) The associated peptides were extracted in acid and separated from high molecular weight material by ultrafiltration as previously described (Hunt et al. (1991) Science 255:1261; Huczko et al. (1993) J. Immunol. 151:2572.), and subsequently fractionated by reverse-phase high-performance liquid chromatography (HPLC) (Cox etal. (1994) Science 264: 716). Aliquots of each fraction were incubated with HLA-B7 positive, H-Y negative T2-B7 target cells in order to assay for the ability to reconstitute the epitope recognized by an HLA-B7 restricted, H-Y specific cytotoxic T lymphocyte (CTL) clone, 5W4 (Huczko et al. (1993) J. Immuno. 151:2572). A single peak of reconstituting activity was observed (Fig. 6A, fraction 28 and 29), which was rechromatographed using a different organic modifier.

Although a single active peak of reconstituting activity was also observed from this separation (Fig. 6B, fraction 14, 15 and 16), it still contained more than 100 distinct peptide species, as assessed by electrospray ionization tandem mass spectrometry.

WO 97/10267 PCT/US96/14547 -23- The specific conditions used to reconstitute the H-Y epitope with the HPLC fractionated peptides extracted from HLA-B7 molecules, described generally above were as follows. In the first HPLC fractionation step, HLA-B7 molecules were immunoaffinity purified from 2x10 1 0 H-Y positive JY cells. Peptides were eluted from B7 molecules with 10% acetic acid, pH 2.2, filtered through a 10 kD cut-off filter and fractionated on a C18 reverse phase column. Buffer A was 0.1% heptafluorobutyric acid (HFBA); buffer B was 0.1 HFBA in acetonitrile.

The gradient consisted of 100% buffer A (0-20 min), 0 to 12% buffer B (20 to 25 min), and 12 to 50% buffer B (25 to 80 min) at a flow rate of 200 pl/min. 60 fractions of 200 pl each were collected from 20 to min.

Fractions 28 and 29 from the separation shown in Figure 6A were rechromatographed with the same acetonitrile gradient, but using trifluoroacetic acid (TFA) instead of HFBA as the organic modifier. The elution profile from this second HPLC column is shown in Figure 6B. For both panels A and B of Figure 7, 3% of each peptide fractions were preincubated with 1,000 5 1 CR-labeled T2-B7 cells at room temperature for 2 hours. The CTL clones were then added at an effector to target ratio of 10 to 1, and further incubated at 37 0 C for 4 hours. Background lysis of T2-B7 by the CTL clones in the absence of any peptides was in and in positive control lysis of JY was 75% in (A) and 74% in To identify the active H-Y peptide in the resulting mixture eluted from the second HPLC purification above, fraction 14 was chromatographed with an on-line microcapillary column effluent splitter as previously described (Cox et al. (1994) Science 264: 716; den Haan et al. (1995) Science 255:1261). One-fifth of the effluent was deposited into 100 pl of culture media in microtiter plate wells for analysis with CTLs as in Fig. 6. The remaining four-fifths of the material were directed into the electrospray ionization source, and mass spectra WO 97/10267 PCT/US96/14547 -24of the peptides deposited in each well were recorded on a triplequadrupole mass spectrometer (Finnigan-MAT, San Jose, California).

The results obtained from each well number assayed were were plotted as either H-Y epitope reconstitution activity measured as percent specific lysis or as abundance of peptide 1171 measured as ion current at m/z 391 A copy of the plot of these results is reproduced in Figure 7. The amount of the H-Y sensitizing activity in each well was correlated to signals observed in the mass spectrum, and therefore to the abundance of different peptide species.

By comparing the profile of H-Y activity and the ion abundance data in Figure 7, we were able to identify a (M 3H) +3 ion at a mass-tocharge ratio of 391 (neutral molecular mass 1171), whose abundance correlated with the amount of H-Y epitope reconstituting activity. Further confirmation of the importance of peptide 1171 was provided by the demonstration that a peptide with an identical mass and collision-activated dissociation (CAD) spectrum was also present in HLA-B7 associated peptides extracted from a second H-Y positive B lymphoblastoid line, DM, but absent from a spontaneous H-Y antigen loss variant of this cell, Assignment of a complete amino acid sequence to the 1171 peptide from the CAD mass spectrum recorded at the 20 fmol level proved difficult due to the absence of high mass fragment ions containing the amine terminus (b-type ions). A series of single and/or doubly charged fragment ions containing the carboxyl terminus (y-type ions) identified the C-terminal residue as either L or I and the first six amino acids as SPSVDK. The difference in molecular mass between this partial sequence and that of the full length peptide suggested the presence of four additional residues, for a total length of 11. Since the candidate peptide existed exclusively in the gas phase as an (M 3H)+ 3 ion, and underwent mass shifts of 42 and 84 Daltons (Da) on conversion to the corresponding methyl ester and acetylated WO 97/10267 PCT/US96/1 4547 derivative, respectively, two of the remaining residues were assigned as R and either D or E. Only two combinations of four residues (AREA and GRDV) meet the above criteria and satisfy the missing mass of 427 Da.

CAD spectra recorded on synthetic peptides suggested that R could not be located at either position 7 or 10. Databases were searched for proteins containing peptides with these characteristics, and an amino acid sequence (SPAVDKAQAEL) consistent at 9 out of 11 positions was found in residues 963-973 of the protein encoded by XE169 or SMCX (Wu et al. (1994) Hum. Mol. Genet. 3:153). Subsequent analysis of the SMCY sequence (Fig. 3) revealed the amino acid sequence (SPSVDKARAEL: residues 950-960) that is consistent at 11 out of 11 positions and has the expected mass of 1171 Da.

Given these results, peptide 1171 was further analyzed after conversion of the R residue to ornithine, using a CAD mass spectrometer. Material from second dimension HPLC fraction 14 shown in Fig. 6B was treated with 70% hydrazine hydrate for 1 hour. The CAD mass spectrum was recorded on the (M+2H) 2 ion at m/z 566.

the results, shown in Figure 8, confirmed that the peptide sequence of peptide 1171 was identical to that found in the predicted SMCY amino acid sequence.

To confirm this finding, synthetic peptides to the SMCY (SPSVDKARAEL) and SMCX (SPAVDKAQAEL) sequences were sythesized and purified to homogeneity by reverse phase-HPLC on a Vydac C4 column. Purity was established on an analytical RP column and the quantity of each peptide was confirmed by comparing the area of the peak with that of a standard peptide. The identity of the peptides was confirmed by mass spectrometry. These peptides were then used is experiments testing for reconstitution of the H-Y epitope. 5'Cr release was assayed at an effector to target ratio of 10 to 1 on T2-B7 cells that had been incubated in the presence of differing concentrations of the SMCY sythetic peptide SPSVDKARAEL or the SMCX sythetic WO 97/10267 PCT/US96/14547 -26peptide SPAVDKAQAEL using the concentrations of each such peptide indicated in the plot of the results of this assay, in Figure The results show the 11 residue SMCY sequence sensitized T2-B7 cells for recognition by the H-Y specific CTL clone. Half-maximal lysis was achieved at a peptide concentration of 10 pM (Fig. The corresponding peptide derived from the SMCX sequence was also able to sensitize T2-B7 cells for recognition, however, comparable levels of killing were only achieved by using a 10,000-fold higher peptide concentration. Binding studies showed that the concentration of the SMCY peptide that inhibited the binding of an iodinated standard peptide to purified HLA-B7 by 50% (IC 5 o) was 34 nM, while the ICo 5 for the SMCX peptide was 140 nM (Fig. Thus, the significant difference in the ability of the SMCY and SMCX peptides to sensitize targets for T cell recognition is almost entirely due to the fine specificity of the T cell receptor, rather than to differences in MHC binding affinities.

Based on this information, we conclude that the peptide epitope representing the HLA-B7 restricted H-Y antigen is derived from the protein encoded by SMCY.

Finally, the identity of the 11 amino acid segment of human SMCY protein identified above was further confirmed in a functional assay comparing the binding inhibition properties of a synthetic peptide constructed with that same amino acid sequence to other peptides.

Specifically, HPLC-purified test peptides were assayed for the ability to inhibit the binding of the iodinated endogenous B7 peptide APRTYVLLL to purified HLA-B7 as previously described (Ruppert et al. (1993) Cell 74:929; Chen et al. (1994) J. Immunol. 152:2874; Sette et al. (1994) J. Immunol. 153:5586). Figure 10 shows the results of this assay, expressed as percent inhibition measured for each peptide assayed plotted against peptide concentration. The following peptides were assayed herein: the SMCYpeptide SPSVDKARAEL the SMCX peptide SPAVDKAQAEL APRTLVLLL an endogenous peptide WO 97/10267 PCT/US96/14547 -27bound to HLA-B7; and LLDVPTAAV an endogenous peptide bound to HLA-A2.1, included as a negative control.

The assay results summarized above all used the human SMCY protein sequence derived from the human SMCY cDNA sequence information obtained in Examples 1 and 2, above, as summarized in the protein and DNA sequence depicted in SEQ ID NO:1 below, to identify and confirm the sequence of human H-Y antigen. In confirming the common identity of the sequence and functional characteristics of human H-Y antigen and the'l 1 amino acid synthetic peptide sythesized using the human SMCY protein sequence from Figure 3 (SEQ ID NO: 1), this example also strongly indicates that we correctly deduced the human SMCY amino acid sequence from the human SMCY cDNA sequence of Figure 3.

EXAMPLE 7 (HYPOTHETICAL) PRODUCTION OF ANTIBODIES

TO

THE HUMAN SMCY PROTEIN SEQUENCE Human specific antibodies, some of which may be H-Y antigen specific, are raised against a human SMCY protein, such as a human SMCY protein identified by SEQ ID NO:2 as described in Example 1.

EXAMPLE 8 (HYPOTHETICAL) USE OF ANTIBODIES TO THE HUMAN SMCY PROTEIN SEQUENCE IN IMMUNOLOGICAL

TESTING

The antibodies identified in Example 7 are used in an immunological testing and screening system. Information is obtained which has applications in clinical research, diagnostics and therapeutics.

Specifically, an adult and prenatal male infertility diagnostic test is designed.

WO 97/10267 PCT/US96/14547 -28- EXAMPLE 9 (HYPOTHETICAL) USE OF ANTIBODIES TO THE HUMAN SMCY PROTEIN SEQUENCE IN SEX SELECTION

AND

SCREENING FOR HUMANS The antibodies identified in Example 7 are used in a sex selection and screening system for humans.

EXAMPLE 10 (HYPOTHETICAL) USE OF ANTIBODIES TO THE HUMAN SMCY PROTEIN SEQUENCE IN SEX SELECTION

AND

SCREENING FOR NON-HUMAN

SPECIES

The antibodies identified in Example 7 are used in a sex selection and screening system for non-human species.

EXAMPLE 11 (HYPOTHETICAL) USE OF PRIMERS OF HUMAN SMCY cDNA IN EMBRYONIC EXPRESSION

STUDIES

An SMCY primer pair is synthesized using an oligonucleotide synthesizer. Each primer in the pair consists of a sequence of nucleic acid included in or complementary to a human SMCY cDNA sequence, such as SEQ ID NO:1. The sequence of each oligonucleotide primer is chosen such that the two primers are complementary to and flank a region of interest of RNA from a subject organism.

Embryos from the subject organism are harvested at various stages of development, 2 cell stage, 4 cell stage, 8 cell stage, etc.

RNA is then isolated from the individual embryos and amplified by PCR with an SMCY primer pair. After a sufficient number of cycles, any amplified DNA product is electrophoresed on a polyacrylamide gel, the gel is stained, and the gel is photographed or autoradiographed to reveal the presence or absence of amplified product.

By analyzing the amplified product, one can determine the stage at which embryos of the subject organism begin expressing the SMC encoded for by the RNA region amplified and tested.

WO 97/10267 PCT/US96/14547 -29- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Promega Corporation (ii) TITLE OF INVENTION: Human SMCY cDNA and Related Products (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Promega Corporation STREET: 2800 Woods Hollow Road CITY: Madison STATE: Wisconsin COUNTRY: U.S.A.

ZIP: 53711-5399 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette 3.5 inch, 1.44 Mb COMPUTER: IBM compatible PC OPERATING SYSTEM: DOS, version SOFTWARE: WordPerfect 5.1 (DOS text format) (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 06/003,744 FILING DATE: 9/14/95 APPLICATION NUMBER: 06/012,973 FILING DATE: 03/07/96 INFORMATION FOR SEQ ID NO:1 SEQUENCE CHARACTERISTICS: LENGTH: 5,476 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIEN TISSUE TYPE: TESTIS AND LYMPHOCYTE (vii) IMMEDIATE SOURCE: LIBRARY: cDNA I WO97/10267PCIS6147 PCTIUS96/14547 (ix) FEATURE: NAME/KEY: Human SMCY cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GGACGGCCAT ACTATTTTTA TCTTGCTTTT TCGTTCTGTC GCAGTACTGT TTAATATGAG TCCAGCGACG GCTCTGTGAC TGTTTTCCTC TGGTAAAATC GCTCTTGCGT CCTCAGCGTT TATCTCAGGT GCGGAAGGTC TCACAGGTTT GGAAATAGCG CCGGAAAAAT CGATCCGCGG AGTGAGACGG CTCGTACCAC ACTGCAGGGC CCGGAGGTCA AGATGGTGGC TGTAAAACTA GGATCCCTGA CGATTGCTTA GCATTAAGGC CCGAC 120 160 200 240 275 293 ATG GAA Met Glu 1

GAG

Giu

GAA

Giu

GAG

Giu

CCT

Pro

AGG,

Arg

GAT

Asp

CCC

Pro

GTG

Val

GCT

Ala 135

TTC

Phe

TTC

Phe

AAG

Lys

TTT

Phe

CTA

Leu

CAG

Gin

AAT

Asn

ATT

Ile 120

CGA

Arg CCG GGG TGT GAC Pro Gly Cys Asp CTG CCG CCA CCG Leu Pro Pro Pro CAA GAC CCG CTT Gin Asp Pro Leu TCT GGC ATC TGC Ser Gly Ile Cys GCA GTA GAA GTT Ala Val Giu Val 60 AAT GAA CTG GAG Asn Glu Leu Glu ATT GCA AAA TTC Ile Ala Lys Phe GTG GAG CGG AAG Vai Glu Arg Lys 105 GAG GAA GGT GGC Giu Giu Gly Giy GTT GCC CAG CGT Vai Ala Gin Arg 140

GAG

Giu

GGC

Gly

AAA

Lys 45

GAC

Asp

GCC

Ala

TGG

Trp

ATC

Ile

TAT

Tyr 125

CTC

Leu TGC CCG GTT TTT GAG CCT AGC TGG Cys Pro Val Phe Glu Pro Ser Trp 15

TAC

Tyr 30

ATC

Ile

AAT

Asn

CAA

Gin

GAA

Giu

TTG

Leu 110

GAA

Glu

CAC

His

ATT

Ile

CGC

Arg

TTC

Phe

ACT

Thr

ATT

Ile 95

GAC

Asp

CC

Ala

TAC

Tyr

GCG

Ala

CCA

Pro

AGA

Arg

AGA

Arg 80

CAA

Gin

CTC

Leu

ATC

Ile

CCA

Pro

AAA

Lys

CCC

Pro

TTT

Phe 65

GTG

Val1

GGC

Gly

TAC

Tyr

TGC

Cys

CCA

Pro 145

ATA

Ile

GCG

Ala

ACT

Thr

AAA

Lys

TCC

Ser

AGC

Ser

AAG

Lys 130

GGC

Gly

AGG

Arg

GAT

Asp

CCT

Pro

TTG

Leu

TCT

Ser

CTT

Leu 115

GAT

Asp

AAA

Lys

CCC

Pro

TG

Trp

CGC

Arg

AAC

Asn

TTA

Leu 100

AGT

Ser

COT

Arg

AAC

Asn

ATA

Ile

CAG

Gin

GTC

Val1

TAT

Tyr

AAG

Lys

AAG

Lys

CGG

Arg

ATT

Ile

OCT

Ala

GCA

Ala

CCT

Pro

CAA

Gin

TTO

Leu

ATT

I le

ATT

Ile

TGG

Trp

GGC

Gly 150 341 389 437 485 533 581 629 677 725 WO 97/10267 PCT/tJS96/14547 -31- CGC ATT ATT TAC CCC TAT GAA ATG TCC CTG CTA CGA Ser Leu Leu Arg TCA CAT TAC GAA Ser His Tyr Glu 155

TTT

Phe

AAT

Asn

CAG

Gin

AGG

Arg 215

CCA

Pro

GGC

Gly

GTC

Val

GGG

Gly

TGC

Cys 295

TTT

Phe

AAT

Asn

TGC

Cys

CCA

Pro

CAG

Gin

GAG

Glu

TCT

Ser 200

CTA

Leu

GAG

Glu

TTG

Leu

ACA

Thr

AAC

Asn 280

ACT

Thr

ATT

Ile

AAG

Lys

TTG

Leu

AAA

Lys 360

TCT

Ser

GTA

Val 185

GTG

Val

CAG

Gin

CTA

Leu

GGC

Gly

TGC

Cys 265

GTG

Val

AAG

Lys

GAC

Asp

CTT

Leu

TTA

Leu 345

TGT

Cys

GGA

Gly 170

AAA

Lys

CAG

Gin

CCT

Pro

AAG

Lys

CTT

Leu 250

CCC

Pro

TCA

Ser

ACA

Thr

TCA

Ser

CTT

Leu 330

CCA

Pro

ATC

Ile

GCC

Ala

GAT

Asp

CCT

Pro

GAT

Asp

AAG

Lys 235

ATG

Met

CCA

Pro

TCA

Ser

ACC

Thr

TAT

Tyr 315

TTC

Phe

CCC

Pro

TTG

Leu

AAC

Asn

AAG

Lys

TCA

Ser

CCA

Pro 220

TTA

Leu

GCT

Ala

ACT

Thr

ACA

Thr

ATG

Met 300

ATT

Ile

TGT

Cys

CTT

Leu

GCG

Ala

CAT

His

GAA

Glu

AAG

Lys 205

GAG

Giu

CAG

Gin

AAG

Lys

GTT

Val

TTG

Leu 285

CAA

Gin

TGC

Cys

GAT

Asp

CCT

Pro

GAG

Glu 365

GTG

Val

TAG

Tyr 190

TTC

Phe

CCT

Pro

ATA

Ile

GAT

Asp

ACG

Thr 270

CTC

Leu

CTT

Leu

CAA

Gin

GGC

Gly

GAA

Glu 350

TGT

Cys Arg

CAA

Gin 175

AAG

Lys

AGC

Ser

ACA

Thr

TAT

Tyr

AAG

Lys 255

GTG

Val

AAG

Lys

CGA

Arg

GTA

Vai

TGT

Cys 335

ATC

Ile

AAA

Lys Ile 160

TGT

Cys

CCC

Pro

AGC

Ser

GAG

Glu

GGG

Gly 240

GAT

Asp

AAG

Lys

CAG

Gin

AAG

Lys

TGC

Cys 320

GAT

Asp

CCC

Pro

CAG

Gin Ile Tyr Pro Tyr Glu Met 165

AAC

Asn

CAC

His

TAC

Tyr

GAG

Glu 225

CCA

Pro

AAG

Lys

GAT

Asp

CAC

His

AAT

Asn 305

TCC

Ser

GAC

Asp

AGA

Arg

CCT

Pro

ACA

Thr

AGC

Ser

AGT

Ser 210

GAC

Asp

GGT

Gly

ACT

Thr

GAG

Glu

TTG

Leu 290

CAC

His

CGT

Arg

AAT

Asn

GGC

Gly

CCT

Pro 370

CAC

His

ATC

Ile 195

CGA

Arg

ATT

Ile

CCC

Pro

GTG

Val

CAA

Gin 275

AGC

Ser

AGC

Ser

GGG

Gly

TAC

Tyr

ATC

Ile 355

GAA

Glu CCG TTT Pro Phe 180 CCC CTT Pro Leu CGG GCA Arg Ala GAG AAG Glu Lys AAA ATG Lys Met 245 CAT AAG His Lys 260 AGT GGA Ser Gly CTA GAG Leu Glu AGT GCC Ser Ala GAT GAA Asp Glu 325 CAC ATC His Ile 340 TGG AGG Trp Arg GCT TTT Ala Phe GAC 821 Asp AGA 869 Arg AAA 917 Lys CAT 965 His 230 ATG 1013 Met AAA 1061 Lys GGT i109 Gly CCC 1157 Pro CAG 1205 Gin 310 GAT 1253 Asp TTC 1301 Phe TGC 1349 Cys GGA 1397 Gly WO 97/10267 PCT[US96/14547 -32- TTT GAA CAG GCT ACC Phe Giu Gin Ala Thr CAG GAG TAC ACT TTG CAG AGT TTT GGT Gin Giu Tyr Ser Leu 375

GCT

Ala

CCT

Pro

GAG

Glu

TTT

Phe

GAG

Glu 455

GTG

Val

ATG

Met

TGG

Trp GGT 4 Gly

TTG

Leu 535

CCT

Pro I ATG 'I Met E

GAT

Asp

ACA

Thr

GAA

Glu

GGC

Gly 440

GAG

Glu

CTA

Leu

AAG

Lys

CAT

His

GAG

Glu 520

;AG

;AT

ksp

CC

er

TCC

Ser

GAA

Glu

GAC

Asp 425

AGT

Ser

AAG

Lys

GAT

Asp

GTG

Val

ATT

Ile 4 505

CCG,

Pro i

GAG

Glu

CTC

Leu CAT C His C

TTC

Phe

CTT

Leu 410

GTG

Val

GGC

Gly

GAG

Glu

CAG

Gin

CCC

Pro 490

GAG

Glu

AAG

Lys

.TG

Val =TA 4 eu I

IGT

AAC

Lye 395

GTI

Val

ACA

Thr

TTT

Phe

TAT

Tyr

TCT

Ser 475

TGG

Trp

GAT

Asp

ACC

Thr

ATG

Met

CAC

His 555 aTG Jal 380

TCC

Ser

GAG

Glu

GTT

Val

CCT

Pro

GCG

Ala 460

GTT

Val

CTG

Leu

CAC

His

TGG

Trp

AAG

Lys 540

CAG

Gin CCA 4 Pro

GAC

Asp

AAG

Lye

GAA

Glu

GTC

Val 445

ACC

Thr

CTC

Leu

TAC

Tyr

TGG

Trp

TAT

Tyr 525

ATG

Met

CTT

Leu

GTT

Val

TAC

I Tyr

CAA

Glu

TAT

Tyr 430

AGC

Ser

ACT

Ser

TGT

Cys

GTG

Vai

ACT

Ser 510

GGT

Gly

CTG

Leu

GTC

Val

GTC

Val

TTC

Phe

TTC

Phe 415

GGA

Cly

AAT

Asn

GGT

Gly

CAC

His

GGC

Gly 495

TAC

Tyr

OTA

Val

ACA

Thr I ACT C Thr I CGC I Arg 1 575

AAC

Asn 400

TGG

Trp

GCT

Ala

AGC

Ser

TGG

Trp

ATC

Ile 480

ATG

Met

TCT

Ser

=CC

Pro

,CT

?ro

:TC

.eu i60

LCA

hr Gin 385

ATG

Met

AGG

Arg

CAT

Asp

AAA

Lys

AAC

Asn 465

AAT

Asn

GTT

Val

ATT

Ile

TCC

Ser I GAG C Glu I 545 ATG I Met I AAC C Asn G Ser Phe Gly

CC]

Prc

CTC

Leu

ATT

Ile

CAA

Gin 450

CTG

Leu

GCA

Ala rTC Phe

NAC

ksn

,TG

,eu 530

:TG

.eu

LAT

~sn

'AG

lIn r GTA Val

GTG

I Val

CAT

His 435

AAC

Asn

AAT

Asn

GAC

Asp TCA I Ser

TAT

Tyr 515

CCA

Aia l TTT C Phe I CCC I Pro TGT C Cys P 5 TTT I Phe A 595

CAT

His

AGC

Ser 420

TCC

Ser

TTA

Leu

GTG

Val

ATC

Ile

GCA

Ala 500

CTG

Leu 3CA kIa 4

;AT

ksp IAC I ~sn rCA C la C

GAI

Ci

ATC

Met 405

AGC

Ser

AAA

Lys

TCT

Ser

ATG

Met

TCA

Ser 485

TTT

Phe

CAT

His

GAG

3iu kGC Ser kCT rhr 565

;GG

;ly ATG 1445 Met 390 G CTG 1493 Vai ATT 1541 Ile GAA 1589 Glu CCT 1637 Pro CCA 1685 Pro 470 GGC 1733 Gly TGT 1781 Cys TGC 1829 Trp CAT 1877 His CAG 1925 Gin 550 TTG 1973 Leu GAG 2021 Glu TTT GTC ATC ACT TTT CCT CGT GCT Phe Val Ile Thr Phe Pro Arg Ala 585 590 TAO CAC ACT GGT Tyr His Ser Gly LAC CAA GGC 2069 Lsn Gin Gly IWO 97/10267 PCTIUS96/1 4547 -33.

TAC AAT TTT GCT GAA GOT GTC AAC TGT ACT GCT GAO TGG CTA COT 2117 Cys Thr Ala Asp Trp Leu Pro 610 Tyr Asn 600 Phe Ala Glu Ala Val Asri Phe 605

GOT

Ala 615

GTC

Val

AOG

Thr

ATG

Met

GTC

Val

CGO

Arg 695

TGC

CyB

CTO

Leu

TTG

Leu

TOT

Ser

GAT

Asp 775

GOT

Ala2

GG)

Gl)

TTC

Phe

TTG

Leu

GTT

Val

AOG

Thr 680

CAG

Gln

TAO

Tyr

TGC

Cys

GAT

A~sp

~TTT

Phe 760 3G0 krg

OGC

Arg

TOO

Ser

GAT

Asp

CAG

Gin 665

GAG

Glu

TGC

Cys

GAO

Asp

AAG

Lys

GAG

Glu 745

GAO

Asp

CGT

Arg GAG2 Glu2

CA(

Gir

CAC

His

CTC

Leu 650

GAG

Glu

GOT

Ala

ATC

I le

TGO

Cys

TGO

Cys 730

CTC

Leu

AC

rhr kAA ys kGG krg

TGC

Cys

GAG

Glu 635

AAT

Asn

GAG

Glu

GAG

Glu

AAG

Lys

CCA

Pro 715

TOT

Ser 000 Pro

TGG

Trp

OGO

Arg

AGG

Arg 795

AT

I le 62 C

GAC

Glu

CTA

Leu

CGIA

Arg

OGA

Arg

TGC

Cys 700

GAT

Asp

AGT

Ser

ACC

Thr

GCC

Ala

RAGC

Ser 780 rTT he r' GAA SGlu

OTO

Leu

GCA

Ala

CGT

Arg

GAG

Glu 685

AAG

Lys

GGC

Gly

AGO

Ser

ATG

Met AAC2 Asn 1 765

TTTC

PheC

CT

Pro

CAC

Hi

AT(

I le

GTIZ

Val

CTIA

Leu 670

GOT

Ala

ACC

Thr

CTT

Leu

CGA

Arg

:TG

eu 750 .ys

~AA

LAT

snf tAO 5Tyr

TGC

Cys

GOT

Ala 655

OGA

Arg

TTT

Phe

AOG

Thr

GTA

Val

OAG

Gin 735

CAT

HisI

GTG

Val I GAG Glu I AGT G Ser G

CG(

Arc

AAC,

Lys 640

GTG

Val

AAG

Lys

GAG

Giu

TGC

Cys

TGC

Dys 720 rAC Tyr -,ys

,GA

~rg

~TA

~eu iAG ~lu ~00

CGG

SArg 625

ATG

Met

CAC

His

CO

Ala

OTG

Leu

TTC

Phe 705

OTT

Leu

OTO

Leu

CTG

Leu

GTG

Val

AGG

Arg 785

CTG

Leu

CT(

Let

GO]

Ala

AAG

Lys

OTT

Leu

OTO

Leu 690

TTG

Leu

TOO

Ser

OGG

Arg

P.AG

Lys

GCC

Ala 770

"CA

kla

,TT

.eu

CGG

1Arg

GC

Ala

GAG

Glu *TtG Leu 675

OCA

Pro

TOA

Ser

CAC

His

TAT

Tyr

ATT

Ile 755

TTG

Leu

OTG

Leu

CAG

Gin

CG(

Arc

TT(

Phe

ATG

met 660

GAG

Glu

GAT

Asp

CO

Ala

ATO

Ile

CGG

Arg 740

CGG

Arg

GAG

Glu

GAG

3Au

GA

krg

TAI

;Tyr

COIA

Pro 645

TTO

Phe

AAG

Lys

CAT

Asp

OTG

Leu

AAT

Asn 725

TAO

Tyr

GOT

Ala

GTG

Val

TOT

Ser

CTG

Leu 805 TGT 2165 Cys 630 GAG 2213 Glu ATT 2261 Ile GCC 2309 Gly GAA 2357 Glu GOC 2405 Ala 710 GAO 2453 Asp ACC 2501 Thr GAG 2549 Glu GAG 2597 Glu GAG 2645 Glu 790 AAG 2693 Lys AAC TGC CTG AGT GAG GTG GAG Asn Cys Leu Ser Glu Val Glu 810

GOT

Ala TGT ATT GOT CAA GTO CTG Cys 815 Ile Ala Gin Val Leu 820 GGG OTG 2741 Cly Leu WO 97/10267 PCTIUS96/1 4547 -34- GTC AGT GGT CAG GTG GCC Val Ser Gly Gin Val Ala 825 AGG ATG GAC ACT CCA CAG CTG ACT TTG Arg Met Asp Thr Pro Gin Leu Thr Leu ACT 2789 Thr 830 GAA CTC CGC Glu Leu Arg 840 CAT CAG ATT His Gin Ile 855 CAA GCT GAG Gin Ala Glu CTA TTC CGG Leu Leu Arg CCT GAA GCC Pro Glu Ala 905 GAT CAA GTC Asp Giu Val 920 GTC ATC ATG Val Ile Met 935 CCT TCT GTG Pro Ser Val CCA GAG CGC Ala Giu Arg AAG CAT CCA Lys His Pro 985 ATC CCT CTT Ile Pro Val I 1000 AAG GCA CAA C Lys Ala Gin 1 1015

GTC

Val

GGC

Gly

GCT

Ala

TCC

Ser 890

CAT

His

AAG

Lys CAG 4 Gin 4

GAC

Asp I

TGG

rrp C 970 CCA C Pro I ,AC C Us L ,CT lia T CT1 Let

GAIJ

Asy

CGT

Arg 875

CTG

Leu

CAC

Gin CAg Gin

GG

ly kAG Eys 955

;AA

:lu

;CC

la

:TG

leu 1GG rp r CTT GAG CAG a Leu Glu Gin 845 CTC AAG CAT Val Lys Asp 860 GAG GCT CTG Glu Ala Leu TTG GAG AGG Leu Glu Arg CTT CAG CAG Leu Gin Gin 910 gCC CTG GCC Ala Leu Ala 925 CTT TTG CTT Leu Leu Val 940 CCC CGG GCT Ala Arg Ala GAA AAG GCT Glu Lys Ala ACA TTG CAA 4 Thr Leu Glu 990 CCT AAC ATC Pro Asn Ile 1005 ATT GCT CAT Ile Ala Asp 1 1020

AT(

Met

GTC

Val

GCC

Ala

GGG

Cly 895

CAG

Gin

CCT

Pro

ATG

Met

GAG

Glu

CAT

His 975 3CC Ala

"AG

3mn

;TG

lal

GGC

Gly

CTG

Leu

ACA

Thr 880

CAC

Gin

GTG

Val

TCT

Ser

CGT

Gly

CTG

Leu 960

TTC

Phe

ATA

Ile

CCT

Ala

CAT

Asp 835 AGC CTC CCC Ser Leu Pro 850 GAA CAG GTG Glu Gin Val 865 CTG CCC TCT Leu Pro Ser CAG CTG GGT Gin Leu Cly GAG CAG GCG Glu Gin Ala 915 GCT CAC AGG Ala His Arg I 930 CCC AAG ATA 4 Ala Lys Ile 945 CAA GAA CTA Gin Giu Leu TGC CTG GAG Cys Leu Giu I ATT CCT GAG I Ile Arg Giu '3 995 CTC AAA GAA C Leu Lys Giu P 1010 GAG ATC CAA A Glu Ile Gin A 1025

TG(

Cy

GAC

Cit

AGI

Ser

GTA

Val 900

CAA

Gin

GGC

Gly

GCC

Ala

CTG

Leu 3CC kla

ICA

hr

;CT

la

AT

Lsn C GCC ATG 2837 a Ala Met G CCC TAT 2885 a Ala Tyr 870 CCA GGG 2933 Pro Gly 885 GAG GTG 2981 Glu Vai TGG CTA 3029 Trp Leu TCT CTC 3077 Ser Leu TCC AGC 3125 Ser Ser 950 ACC ATT 3173 Thr Ile 965 AGG CAG 3221 Arg Gin GAA AAC 3269 Glu Asn CTG ACT 3317 Leu Thr GGT GAC 3365 Cly Asp 1030 CAC TAC His Tyr CCC TCT CTA CAT GAC TTC GAG GGC Pro Cys Leu Asp Asp Leu Glu CTG GTG GCT CTG Leu Val Ala Val 1035 Gly 1040 GGC CGG 4313 Cly Arg 1045 WO 97/10267 PCT/US96/14547

GAC

Asp CTG CCT GTG GGG Leu Pro Val Gly 1050 TTG ACA GCA CAT TCC Leu Thr Ala His Ser 1065 AAG AAT TCT TGC TAC Lys Asn Ser Cys Tyr 1080 GCT GGC TCA GAC AGC Ala Gly Ser Asp Ser 1095 GGG TTG TAC CAG TGT Gly Leu Tyr Gin Cys 1115 CTC AGA GAC CCA GGC Leu Arg Asp Pro Gly 1130 AAG GAG AAG GAG GGT Lys Giu Lys Giu Gly 1145 CCC AGT CCA CTG GCA Pro Ser Pro Leu Ala 1160 TGT GTG TGT GGG CAG Cys Val Cys Gly Gin 1175 CTG TGT CAG GAC TGG Leu Cys Gin Asp Trp 1195 CTC ACC TCT CCA AAG Leu Thr Ser Pro Lys 1210 TGG GAA TGG GAC ACA Trp Giu Trp Asp Thr 1225 CGG CCA CGC CTA GAG Arg Pro Arg Leu Glu 1240 CTG GAA GAG CTG AGA CAG CTA GAG CTG CAG GTA 3461 Leu Glu Giu Leu Arg Gin Leu Giu Leu Gin Val 1055 1060 TGG AGA GAG AAG GCC TCC AAG ACC TTT CTC AAG 3509 Trp Arg Giu Lys Ala Ser Lys Thr Phe Leu Lye 1070 1075 ACA CTG CTT GAG GTG CTT TGC CCG TGT GCA GAC 3557 Thr Leu Leu Glu Val Leu Cys Pro Cys Ala Asp 1085 1090 ACC AAG CGT AGC CGG TGG ATG GAG AAG GCG CTG 3605 Thr Lye Arg Ser Arg Trp Met Giu Lys Ala Leu 1100 1105 1110 GAC ACA GAG CTG CTG GGG CTG TCT GCA CAG GAC 3653 Asp Thr Giu Leu Leu Gly Leu Ser Ala Gin Asp 1120 1125 TCT GTG ATT GTG GCC TTC AAG GAA GGG GAA CAG 3701 Ser Val Ile Val Ala Phe Lye Glu Gly Giu Gin 1135 1140 ATC CTG CAG CTG CGT CGC ACC AAC TCA GCC AAG 3749 Ile Leu Gin Leu Arg Arg Thr Asn Ser Ala Lye 1150 1155 CCA TCC CTC ATG GCC TCT TCT CCA ACT TCT ATC 3797 Pro Ser Leu Met Ala Ser Ser Pro Thr Ser Ile 1165 1170 GTG CCA GCT GGG GTG GGA CTT CTG CAG TGT GAC 3845 Val Pro Ala Gly Val Gly Leu Leu Gin Cys Asp 1180 1185 1190 TTC CAT GGG CAG TGT GTG TCA GTG CCC CAT CTC 3893 Phe His Gly Gin Cys Val Ser Val Pro His Leu 1200 1205 CCC AGT CTC ACT TCA TCT CCA CTG CTA GCC TGG 3941 Pro Ser Leu Thr Ser Ser Pro Leu Leu Ala Trp 1215 1220 kAA TTC CTG TGT CCA CTG TGT ATG CGC TCA CGA 3989 Lys Phe Leu Cys Pro Leu Cys Met Arg Ser Arg 1230 1235 kCA ATC CTA GCC TTG CTG GTT GCC CTG CAG AGG 4037 rhr Ile Leu Ala Leu Leu Val Ala Leu Gin Arg 1245 1250 CTG CCC GTG CGG CTG CCT GAG Leu Pro Val Arg Leu Pro Glu 1255 1260

GGT

Gly GAG GCC CTT CAG TGT CTC ACA GAG 4085 Glu Ala Leu Gin Cys Leu Thr Glu 1265 1270 vud-1 017/inqCl rv PCTJUS96/1 4547 -36- AGG GCC ATT GGC TGG CAA GAO CGT GCC AGA AAG GCT CTG GCC TTT GAA 4133 Arg Ala Ile Gly Trp Gin Asp Arg Ala Arg Lys Ala Leu Ala Phe Glu 1275 1280 1285 GAT GTG ACT GCT CTG TTG OGA CAG CTG GCT GAG CTT CGC CAA CAG CTA 4181 Asp Val Thr Ala Leu Leu Arg Gln Leu Ala Giu Leu Arg Gln Gin Leu 1290 1295 1300 CAG GCC AAA CCC AGA CCA GAG GAG GCC TCA GTC TAO ACT TCA GCC ACT 4229 Gin Ala Lys Pro Arg Pro Giu Giu Ala Ser Val Tyr Thr Ser Ala Thr 1305 1310 1315 GCC TGT GAC CCT ATC AGA GAA GGC AGT GGC AAC AAT ATT TCT AAG GTO 4277 Ala Cys Asp Pro Ile Arg Glu Gly Ser Gly Asn Asn Ile Ser Lys Val 1320 1325 1330 CAA GGG CTG CTG GAG AAT GGA GAC AGT GTG ACC AGT COT GAG AAO ATG 4325 Gin Gly Leu Leu Glu Asn Giy Asp Ser Val Thr Ser Pro Giu Asn Met 1335 1340 1345 1350 GCT OCA GGA AAG GGC TOT GAC CTG GAG CTA CTG TCC TOG OTG TTG COG 4373 Ala Pro Gly Lys Gly Ser Asp Leu Giu Leu Leu Ser Ser Leu Leu Pro 1355 1360 1365 CAG TTG ACT GGC COT GTG TTG GAG CTG COT GAG GOA ATO CGG GOT CCC 4421 Gin Leu Thr Gly Pro Val Leu Giu Leu Pro Giu Ala Ile Arg Ala Pro 1370 1375 1380 CTG GAG GAG CTC ATG ATG GAA GGG ggC CTG OTT GAG GTG ACC CTG GAT 4469 Leu Giu Glu Leu Met Met Glu Gly Gly Leu Leu Giu Val Thr Leu Asp 1385 1390 1395 GAG AAO CAC AGO ATO TGG CAG CTG OTG CAG GOT GGA CAG COT OCA GAO 4517 Glu Asn His Ser Ile Trp Gin Leu Leu Gin Ala Gly Gin Pro Pro Asp 1400 1405 1410 CTG GAO AGA ATT CGC ACA OTT OTG GAG CTG GAA AAA TTT GAA CAT CAA 4565 Leu Asp Arg Ile Arg Thr Leu Leu Giu Leu Giu Lys Phe Giu His Gin 1415 1420 1425 1430 GGG AGT OGG ACA AGG AGO CGG GOT OTG GAG AGG OGA OGG CGG CGG CAG 4613 Gly Ser Arg Thr Arg Ser Arg Ala Leu Glu Arg Arg Arg Arg Arg Gin 1435 1440 1445 AAG GTG GAT CAG GGT AGA AAO GTT GAG AAT OTT GTT CAA CAG GAG OTT 4661 Lys Val Asp Gin Gly Arg Asn Val Giu Asn Leu Val Gin Gin Giu Leu 1450 1455 1460 CAG TCA AAA AGG GOT OGG AGO TOA GGG ATT ATG TOT CAG GTG GGC OGA 4709 Gin Ser Lys Arg Ala Arg Ser Ser Gly Ile Met Ser Gin Vai Gly Arg 1465 1470 1475 GAA GAA GAA OAT TAT CAG GAG AAA GCA GAO CGT GAA AAT ATG TTO CTG 4757 Glu Giu Glu His Tyr Gin Glu Lys Ala Asp Arg Glu Asn Met Phe Leu 1480 1485 1490 WO 97/10267 PCT/US96/1 4547 -37- ACA CCT TCO ACA GAC CAC AGO COT TTC TTG AAA GGA AAC CAA AAT AGO 4805 Thr Pro Ser Thr Asp His Ser Pro Phe Leu Lys Gly Asn Gin Asn Ser 1495 1500 1505 1510 TTA CAA CAC AAG GAT TCA GGO TOT TCA GOT GOT TGT COT TOT TTA ATG 4853 Leu Gin His Lys Asp Ser Gly Ser Ser Ala Ala Cys Pro Ser Leu Met 1515 1520 1525 CCT TTG OTA CAA CTC TCC TAO TOT GAT GAG CAA CAG TTG TGAOAGTGGO 4902 Pro Leu Leu Gin Leu Ser Tyr Ser Asp Glu Gin Gin Leu 1530 1535 ACCAAAGGTO ATTTGTGGTT GTTTTTGTTT GTTTGTTTOT TAAATCCTAO TATOTCCTGG 4962 CCTGGACCTC AGAAGGAGOT TTTTGCOTAT CTATAATTTT TCACTGOCAA TTTTTGATAT 5022 OCTCTCTCCT AGAGTTAOTG TTAAAAGGTT GGTTOGTAAA GTCCACACCC CGATGCTCAG 5082 AAGTGTCTTG CCAGCAAOAT TCCTGOTAGO ATAOAGGAGT GATTTCCTAA ACCAGTTTCA 5142 TTOTAGTOTG AATAGGGACA AACAAATCTT GAGGAAGCOC AAGTGOGTAC CTTTATTTTT 5202 GCCCCCACCA CCCTOTTTOT GTACTTOAAT TTTTGTTTGT TTTTTGTTTT TTTGTCCCTG 5262 TCATAAAATA TTTTGGTGC'T TCAAAACTTG TACCTTOATT GTAOATCCTT TTCtTTTOTC 5322 CCCTTGGGTO TTATTATAAA AGAAGACAAT GTAOGTTGTA ATTACCAAAA AGAATAGGGA 5382 AAAAOAAGAA TTTCATGACT OTACCTGTGG TOTATOTTTA ATTTOATTTC TTTTGTTAAA 5442 AATAAAACAA TGAGTATGTT TGGgaaaaaa aaaa 5476 INFORMATION FOR SEQ ID NO:2 SEQUENCE CHARACTERISTICS: LENGTH: 1539 amino acid residues TYPE: Amino Aoid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: Protein (iii) HYPOTHETICAL: yes (ix) FEATURE: NAME/KEY: Human SMCY Protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Giu Pro Gly Cys Asp 1 Glu Phe Leu Pro Pro Pro Giu Cys Pro Val Phe Giu Pro Ser Trp Ala 15 Glu Phe Gin Asp Pro Leu Gly Tyr Ile Ala Lys Ile Arg Pro Ile Ala 30 Glu Lys Ser Gly Ile Cys Lys Ile Arg Pro Pro Ala Asp Trp Gin Pro 45 I WO 97/10267 W097/0267PCT/US96/I 4547 -38- Pro Arg Asp Pro Vai Ala 135 Ser Phe Asn Gin Arg 215 Pro Gly Val Gly Cys 295 Phe Asn Cys Pro Phe 375 Ala Pro Phe Leu Gin Aen Ile 120 Arg Leu Gin Giu Ser 200 Leu Giu Leu Thr Asn 280 Thr Ile Lys Leu Lye 360 Giu Asp Thr Ala Asn Ile Vai 105 Giu Vai Leu Ser Val1 185 Val Gin Leu Giy Cys 265 Vai Lys Asp Leu Leu 345 Cys Gin Ser Giu Val Giu Ala Giu Giu Ala Arg Gly 170 Lys Gin Pro Lys Leu 250 Pro Ser Thr Ser Leu 330 Pro Ile Ala Phe Leu 410 Glu Leu Lys Arg Giy Gin Ser 155 Aia Asp Pro Asp Lye 235 Met Pro Ser Thr Tyr 315 Phe Pro Leu Thr Lye 395 Val Val 60 Giu Phe Lys Gly Arg 140 His Asn Lys Ser Pro 220 Leu Ala Thr Thr Met 300 Ile Cys Leu Ala Gin 380 Ser Giu Asp Aen Ala Gin Trp Giu Ile Leu 110 Tyr Giu 125 Leu His Tyr Giu His Val Giu Tyr 190 Lys Phe 205 Giu Pro Gin Ile Lys Asp Val Thr 270 Leu Leu 285 Gin Leu Cys Gin Asp Gly Pro Giu 350 Giu Cys 365 Giu Tyr Asp Tyr Lys Giu Phe Arg Thr Arg 80 Ile Gin 95 Asp Leu Ala Ile Tyr Pro Arg Ile 160 Gin Cys 175 Lye Pro Ser Ser Thr Giu Tyr Gly 240 Lye Asp 255 Val Lye Lye Gin Arg Lys Val Cys 320 Cys Asp 335 Ile Pro Lye Gin Ser Leu Phe Asn 400 Phe Trp 415 Phe 65 Val Gly Tyr Cys Pro 145 Ile Asn His Tyr Giu 225 Pro Lye Asp His Asn 305 Ser Asp Arg Pro Gin 385 Met Arg Thr Lye Ser Ser Lys 130 Gly Tyr Thr Ser Ser 210 Asp Gly Thr Giu Leu 290 His Arg Asn Gly Pro 370 Ser Pro Leu Pro Leu Ser Leu 115 Asp Lys Pro His Ile 195 Arg Ile Pro Val Gin 275 Ser Ser Gly Tyr Ile 355 Giu Phe Vai Val Arg Val Aen Tyr Leu Lye 100 Ser Lye Arg Arg Aen Ile Tyr Giu 165 Pro Phe 180 Pro Leu Arg Ala Giu Lye Lys Met 245 His Lye 260 Ser Gly Leu Giu Ser Ala Asp Giu 325 His Ile 340 Trp Arg Ala Phe Gly Giu His Met 405 Ser Ser 420 Gin Leu Ile Ile Trp Gly 150 Met Asp Arg Lys His 230 Met Lys Gly Pro Gin 310 Asp Phe Cys Gly Met 390 Val Ile WO 97/10267 PCT/US96/14547 -39.

Gly Ala Asp Ile Glu Phe Glu 455 Val Met Trp Gly Leu 535 Pro Met Phe Tyr Ala 615 Val Thr Met Val Arg 695 Cys Leu Leu I Ser 1 Asp C 775

GIL

Gly 440 Glu Leu Lys His Glu 520 Glu Asp Ser Val Asn 600 Gly Phe Leu Val rhr 680 ;In ryr 'ys ~sp 'he ly Asp 425 Ser Lys Asp Val Ile 505 Pro Glu Leu His Ile 585 Phe Arg Ser Asp Gin 665 Glu Cys Asp Lys Glu I 745 Asp Arg I Val Gly Glu Gin Pro 490 Glu Lys Vai Leu Gly 570 Thr Ala Qin His Leu 550 Glu kia Ile ,ys 'ys 130 .eu ~hr ,ys Thi Phe Tyr Ser 475 Trp Asp Thr Met His 555 Vai Phe Glu Cys Glu 635 Asn Glu Glu Lys Pro 715 Ser Pro Trp Arg Val Pro Ala 460 Val Leu His Trp Lye 540 Gin Pro Pro Ala Ile 620 Glu Leu Arg Arg Cys 700 Asp Ser Thr I Ala I Ser 1 780 Gl Val 445 Thr Leu Tyr Trp Tyr 525 Met Leu Vai Arg Val 605 Glu Leu Ala Arg Glu 585 Lys ;ly 5er 4et ksn 165 )he 1 Ty 43( Sei Sei Cys Val Ser 510 Gly Leu Va1 Val Ala 590 Asn His Ile Val Leu 670 Ala Thr Leu Arg Leu 750 Lys Glu His Ser Lys Glu 435 Asn Gly 5 His Gly 495 Tyr Val Thr Thr Arg 575 Tyr Phe Tyr Cys Ala 655 Arg i Phe Thr Val Gin 1I 735 His I Val I Glu L Set Tri Ile 48C Met Ser Pro Pro Leu 560 Thr His Cys Arg Lys 640 Val lys flu ,ys :ys 120 yr ~ys Lrg .eu Lys Asn 465 Asn Val Ile I Ser Glu 545 Met Asn Sen Thr Arg 625 Met His Ala Leu Phe 705 Leu Leu Leu Vai Arg I 785 Gl Let Ala Phe Asn Leu 530 Leu Asn Gin Gly Ala 610 Leu Ala Lys Leu Leu 690 Leu Ser Arg Lys kla 770 la i Asn I Asn Asp Sen Tyr 515 Ala Phe Pro Cys Phe 595 Asp Arg Ala Glu Leu 675 Pro Ser His Tyr Ile 755 Leu Leu Leu Val Ile Ala 500 Leu Ala Asp Asn Ala 580 Asn Trp Arg Phe Met 660 Glu Asp Ala Ile Arg 740 Rrg 3lu ;lu i Se Mel Set 48 Phe Hif Glu Sex Thr 565 Gly Gin Leu Tyr Pro 645 Phe Lys Asp Leu Asn 725 Tyr Ala Val Ser r Pro t Pro 470 c Gly Cys Trp His Gin 550 Leu Glu Gly Pro Cys 630 Glu Ile Gly Glu Ala 710 Asp Thr Glu Glu Glu 790 WO 97/10267 PTU9144 PCTIUS96/14547 Ala Arg Giu Arg Arg 795 Phe Pro Asn Ser Giu Leu Leu Gin 800 Arg Leu Lys 805 Asn Cys Leu Ser Giu 810 Val Ser Gly Gin Val 825 Giu Leu Arg Val Leu 840 His Gin Ile Gly Asp 855 Gin Ala Glu Ala Arg 875 Leu Leu Arg Ser Leu 890 Pro Giu Ala His Gin 905 Asp Giu Val Lys Gin 920 Val Ile Met Gin Gly 935 Pro Ser Vai Asp Lys 955 Ala Giu Arg Trp Giu 970 Lys His Pro Pro Ala 985 Ile Pro Val His Leu 1000 Lys Ala Gin Ala Trp 1015 His Tyr Pro Cys Leu 1035 Asp Leu Pro Val Gly2 1050 Leu Thr Ala His Ser 1065 Lys Asn Ser Cys Tyr 1080 Ala Gly Ser Asp Ser 1095 Gly Leu Tyr Gin Cys 1115 Leu Arg Asp Pro Gly 1130 Lys Giu Lys Giu Gly 1145 Val Glu Ala Cys Ile 815 Aia Arg Met Asp Thr 830 Leu Giu Gin Met Gly 845 Vai Lys Asp Val Leu 860 Giu Aia Leu Ala Thr 880 Leu Giu Arg Gly Gin 895 Leu Gin Gin Gin Vai 910 Ala Leu Ala Pro Ser 925 Leu Leu Val Met Gly 940 Ala Arg Ala Giu Leu 960 Giu Lys Ala His Phe 975 Thr Leu Glu Ala Ile 990 Pro Asn Ile Gin Ala 1005 Ile Ala Asp Val Asp 1020 Asp Asp Leu Giu Gly 1040 Leu Giu Giu Leu Arg 1055 rrp Arg Giu Lys Ala 1070 rhr Leu Leu Giu Val 1085 rhr Lys Arg Ser Arg 1100 ksp Thr Giu Leu Leu 1120 Ser Vai Ile Val Ala 1135 Ile Leu Gin Leu Arg 1150 Ala Gin Val Leu Gly Leu 820 Pro Gin Leu Thr Leu Thr 835 Ser Leu Pro Cys Ala Met 850 Glu Gin Val Giu Ala Tyr 865 870 Leu Pro Ser Ser Pro Gly 885 Gin Leu Gly Val Giu Val 900 Giu Gin Ala Gin Trp Leu 915 Ala His Arg Gly Ser Leu 930 Ala Lys Ile Ala Ser Ser 945 950 Gin Giu Leu Lpu Thr Ile 965 Cys Leu Giu Ala Arg Gin 980 Ile Arg Giu Thr Giu Aen 995 Leu Lys Giu Ala Leu Thr 1010 Giu Ile Gin Asn Gly Asp 1025 1030 Leu Val Ala Val Gly Arg 1045 Gin Leu Glu Leu Gin Val 1060 Ser Lys Thr Phe Leu Lys 1075 Leu Cys Pro Cys Ala Asp 1090 rrp Met Giu Lys Ala Leu 1105 1110 Gly Leu Ser Ala Gin Asp 1125 Phe Lys Giu Gly Giu Gin 1140 krg Thr Asn Ser Ala Lys 1155 WO 97/10267 WO 9710267PCT/US96/14547 -41- Pro Ser Pro Leu Ala Pro Ser Leu Met Ala Ser Ser Pro Thr Ser Ile 1160 1165 1170 Cys Val Cys Gly Gin Val Pro Ala Gly Val Gly Leu Leu Gin Cys Asp 1175 1180 1185 1190 Leu Cys Gin Asp Trp Phe His Gly Gin Cys Val Ser Val Pro His Leu 1195 1200 1205 Leu Thr Ser Pro Lys Pro Ser Leu Thr Ser Ser Pro Leu Leu Ala Trp 1210 1215 1220 Trp, Giu Trp Asp Thr Lys Phe Leu Cys Pro Leu Cys Met Arg Ser Arg 1225 1230 1235 Arg Pro Arg Leu Glu Thr Ile Leu Ala Leu Leu Val Ala Leu Gin Arg 1240 1245 1250 Leu Pro Val Arg Leu Pro Glu Gly Glu Ala Leu Gin Cys Leu Thr Giu 1255 1260 1265 1270 Arg Ala Ile Gly Trp Gin Asp Arg Ala Arg Lys Ala Leu Ala Phe Glu 1275 1280 1285 Asp Val Thr Ala Leu Leu Arg Gin Leu Ala Glu Leu Arg Gin Gin Leu 1290 1295 1300 Gin Ala Lys Pro Arg Pro Giu Giu Ala Ser Vai Tyr Thr Ser Ala Thr 1305 1310 1315 Ala Cys Asp Pro Ile Arg Glu Gly Ser Gly Asn Asn Ile Ser Lys Vai 1320 1325 1330 Gin Gly Leu Leu Glu Asn Gly Asp Ser Val Thr Ser Pro Glu Asn Met 1335 1340 1345 1350 Ala Pro Gly Lys Gly Ser Asp Leu Glu Leu Leu Ser Ser Leu Leu Pro 1355 1360 1365 Gin Leu Thr Gly Pro Val Leu Giu Leu Pro Giu Ala Ile Arg Ala Pro 1370 1375 1380 Leu Glu Giu Leu Met Met Giu Gly Gly Leu Leu Glu Val Thr Leu Asp 1385 1390 1395 Glu Asn His Ser Ile Trp Gin Leu Leu Gin Ala Gly Gin Pro Pro Asp 1400 1405 1410 Leu Asp Arg Ile Arg Thr Leu Leu Glu Leu Glu Lys Phe Glu His Gin 1415 1420 1425 1430 Gly Ser Arg Thr Arg Ser Arg Ala Leu Glu Arg Arg Arg Arg Arg Gin 1435 1440 1445 Lys Val Asp Gin Gly Arg Asn Vai Glu Asn Leu Val Gin Gin Giu Leu 1450 1455 1460 Gin Ser Lys Arg Ala Arg Ser Ser Gly Ile Met Ser Gin Val Gly Arg 1465 1470 1475 Giu Glu Giu His Tyr Gin Giu Lys Ala Asp Arg Glu Asn Met Phe Leu 1480 1485 1490 Thr Pro Ser Thr Asp His Ser Pro Phe Leu Lys Gly Asn Gin Asn Ser 1495 1500 1505 1510 Leu Gin His Lys Asp Ser Gly Ser Ser Ala Ala Cys Pro Ser Leu Met 1515 1520 11 WO 97/10267 PCT/US96/14547 -42- Pro Leu Leu Gin Leu Ser Tyr Ser Asp Glu Gin Gin Leu 1530 1535 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA primer to cDNA DESCRIPTION: Primer homologous to bases 3285-3304 of the human SMCY cDNA sequence identified by SEQ ID NO:1, above.

(iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (ix) FEATURE: NAME/KEY: Primer S102 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CCTAACATCC AGGCTCTCAA INFORMATION FOR SEQ ID NO:4 SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA primer to cDNA DESCRIPTION: Primer homologous to bases 3305-3326 of the human SMCY cDNA sequence identified by SEQ ID NO:1, above.

(iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (ix) FEATURE: NAME/KEY: Primer S103 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: AGAAGCTCTG ACTAAGGCAC AA The word 'comprising' or forms of the word 'comprising' as used in this description and in the claims do not limit the invention claimed to exclude any variants or additions.

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Claims (15)

1. A human SMCY cDNA, comprising a translation region encoding a human SMCY protein.

2. The human SMCYcDNA of claim 1, wherein the translation region consists of nucleotides 276 to 4893 of SEQ ID NO:1.

3. The human SMCY cDNA of claim 1, further comprising an untranslated sequence upstream of the translation region encoding the human SMCY protein.

4. The human SMCY cDNA of claim 3, wherein the untranslated sequence upstream of the translation region consists of nucleotides 1 to 275 of SEQ ID NO:1. The human SMCY cDNA of claim 1, further comprising an untranslated sequence downstream of the translation region encoding the human SMCY protein.

6. The human SMCY cDNA of claim 5, wherein the untranslated sequence downstream of the translation region consists of nucleotides 4893 to 5466 of SEQ ID NO:1.

7. An oligonucleotide primer to human SMCY cDNA, wherein the primer consists of a sequence of nucleotides homologous or complementary to SEQ ID NO:1.

8. The oligonucleotide primer of claim 7, wherein the sequence of nucleotides is selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4. WO 97/10267 PCT/US96/14547 -44-

9. A recombinant human SMCY protein having a molecular weight of approximately 1.5 kD, wherein the protein is produced by a host organism containing recombinant human SMCY cDNA. The recombinant human SMCY protein of claim 9, wherein the protein consists of an amino acid sequence as shown in SEQ ID NO:2.

11. An oligonucleotide probe to human SMCY cDNA, wherein the probe consists of a sequence of nucleotides homologous or complementary to SEQ ID NO:1.

12. A method of producing an oligonucleotide probe to human SMCY genomic DNA, comprising: a. providing at least one primer consisting of a sequence of nucleotides homologous or complementary to SEQ ID NO: 1; b. providing isolated human genomic DNA; c. using the primer to amplify the isolated genomic DNA, thereby producing an amplified oligonucleotide probe.

13. An oligonucleotide probe to human SMCY genomic DNA, produced by: a. providing at least one primer consisting of a sequence of nucleotides homologous or complementary to SEQ ID NO: 1; b. providing isolated human genomic DNA; c. using the primer to amplify the isolated genomic DNA.

14. A method of detecting SMCY homologs comprising: a. constructing a primer pair, wherein at least one primer of said primer pair consists of a sequence of nucleotides homologous or complementary to SEQ ID NO: 1; WO 97/10267 PCT/US96/14547 b. amplifying a nucleic acid sample with said primer pair, resulting in an oligonucleotide of an amplified fragment of SMCY DNA; and c. detecting the oligonucleotide. The method of claim 14, wherein the nucleic acid sample is DNA.

16. of DNA is The method of claim 15, wherein the nucleic acid sample obtained from a Agt10 cDNA library.

17. The method of claim 14, wherein the nucleic acid sample is RNA.

18. The method of claim 17, wherein the nucleic acid sample of RNA is poly(A)+RNA isolated from embryos.