本研究利用生物資訊學 (bioinformatics) 與分子生物學的方法選殖在牛與豬的Y染色體上具有性別特異性 (sex-chromosome specific loci) 表現的基因SMCY (Selected Mouse cDNA on the Y chromosome , H-Y antigen)，俾作為進一步應用分子免疫法進行選性繁殖 ( sex pre-selection )，以利控制家畜後裔之性別。試驗首先以生物資訊學之方法，利用以指數增加的各種DNA與蛋白質資料庫 (databases)，計包括有genome sequences、expressed sequences tags (ESTs)、sequence tagged sites (STSs)、SWISSPROT等進行蛋白質序列層面的比對，繼而將所有牛、豬與人類SMCX/SMCY基因具有同源性的DNA片段擷取出來，進行contig的組合。序列組合所得到的contigs和人類SMCX/SMCY基因的cDNA全長相較之下，牛與豬的cDNA片段長分別為2,932bp與977bp，約為人類cDNA全長的63及21%。
為獲得牛與豬SMCX/SMCY基因全長cDNA序列，萃取牛與豬之白血球細胞中的total RNA作為模板進行聚合酶鏈反應 (polymerase chain reaction, PCR) 之方式進行選殖，再利用reverse transcriptase-PCR (RT-PCR)、5’or 3’ rapid amplification of cDNA ends (5’or 3’RACE) 的方式獲得全長cDNA序列。
牛與豬之SMCX核苷酸序列長度分別為5,607 bp與5,096 bp，其轉譯之蛋白質長度為1,528 aa與1,559 aa。所得到部分豬SMCY基因序列1,366核苷酸，轉譯之蛋白質為455個胺基酸。不同物種間的胺基酸序列之相似度都大於80%，且SMCX更是相似度達到90%以上。

The objective of this investigation is to clone and characterize the bovine and porcine sex chromosome specific locus SMCY ( Selected Mouse cDNA on the Y chromosome ) (H-Y antigen) that causes the antibody response from male individual when skin grafting even between inbred lines for further application on sex pre-selection system using immunological method in domestic mammals, cattle and pigs. Since SMCX (a SMCY homologous locus on the X chromosome) and SMCY are highly conserved within and across mammalian species. Databases searches (bioinformatics approaches) were first performed using human SMCY sequence and translated protein as DNA and protein probes to acquire any cattle or pig homologous cDNA , genomic DNA or amino acid fragments. Searched databases including genomic sequences [(e.g., GenBank), cDNA sequences]、expressed sequences [e.g., database for expressed sequence tags (dbEST)] and protein databases [e.g., SWISSPORT, Protein Information Resource (PIR)]. A total of fourteen cattle and six pig SMCX/SMCY homologous DNA sequences have been retrieved and further subjected to contigs assembly. Assembled bovine and porcine SMCX/SMCY contigs span 3,185 and 977 bp, comparing to human SMCX/SMCY cDNA. To obtain full-length bovine and porcine SMCX/SMCY cDNAs, we proposed further PCR-based cloning strategies. Thus, total RNAs from cattle and pig lymphocytes have been isolated and served as templates for reverse transcriptase-polymerase chain reaction (RT-PCR), 5'-rapid amplification cDNA ends (5'-RACE) and 3'-RACE. The full-length nucleotides of bovine and porcine SMCX are 5,607 bp and 5,096 bp, which encode 1,528 and 1,599 amino acids, respectively. Cloned partial porcine SMCY are 1,366 bp and encodes 455 amino acids.

1. Johnson, L. A. Sexing mammalian sperm for production of offspring: the state-of-the- art. Anim Reprod Sci 60-61 93-107 (2000).
2. Moruzzi, J. F. Selecting a mammalian species for the separation of X- and Y-chromosom bearing spermatozoa. J Reprod Fertil 57 319-23 (1979).
3. Hohenboken, W. D. Applications of sexed semen in cattle production. Theriogenology 52 1421-33 (1999).
4. Eicher, E. M. and Washburn, L. L. Genetic control of primary sex determination in mice. Annu Rev Genet 20 327-60 (1986).
5. de la Chapelle, A. The Y-chromosomal and autosomal testis-determining genes. Development 101 33-8 (1987).
6. Sinclair, A. H., Berta, P.,Palmer, M., SHawkins, J. R., Griffiths, B., L.Smith, M., J.Foster, J. W., Frischauf, A., M.Lovell-Badge, R. and Goodfellow, P. N. A gene from the human sex-determining region encodes a protein with omology to a conserved DNA-binding motif. Nature 346 240-4 (1990).
7. Gubby J., J. C., P. Koopman, B. Capel, A. Economou, A. Munsterberg, N. Vivian, P. Goodfellow and R. Lovell-Badge. A gene mapping to the sex-determining region of the mouse Y chromosome is a member of novel family of embryonically expressed genes. Nature 346 245-250 (1990).
8. Wolf, U., W. Schempp and G. Scherer. Molecular biology of the human Y chromosome. Rev. Physiol. Biochem. Pharmacol.121 145-213 (1992).
9. Thompson, F. H. H-Y antigen gene loci. Science 201 842 (1978).
10. Hasen, J., Ross, H. and Boyar, R. M. H-Y antigen: localization of the H-Y gene. Horm Res 9 102-6 (1978).
11. Shelton, J. N. Reproductive technology in animal production. Rev Sci Tech 9 825-45 (1990).
12. Welch, G. R. and Johnson, L. A. Sex preselection: laboratory validation of the sperm sex ratio of flow sorted X- and Y-sperm by sort reanalysis for DNA. Theriogenology 52 1343-52 (1999).
13. Johnson, L. A., Flook, J. P. and Hawk, H. W. Sex preselection in rabbits: live births from X and Y sperm separated by DNA and cell sorting. Biol Reprod 41 199-203 (1989).
14. Johnson, L. A. and Welch, G. R. Sex preselection: high-speed flow cytometric sorting of X and Y sperm for maximum efficiency. Theriogenology 52 1323-41 (1999).
15. Morrell, J. M., Keeler, K. D., Noakes, D. E., Mackenzie, N. M. and Dresser, D. W. Sexing of sperm by flow cytometry. Vet Rec 122 322-4 (1988).
16. Ericsson, R. J. Sex selection. Fertil Steril 51 368-9 (1989).
17. Erickson, R. P. Does sex determination start at conception Bioessays 19 1027-32 (1997).
18. Ericsson, R. J. Sex selection via albumin columns: 20 years of results. Hum Reprod 9 1787-8 (1994).
19. Cartwright, E. J., Harrington, P. M., Cowin, A. and Sharpe, P. T. Separation of bovine X and Y sperm based on surface differences. Mol Reprod Dev 34 323-8 (1993).
20. Hendriksen, P. J., Welch, G. R., Grootegoed, J. A., Van der Lende, T. and Johnson, L. A. Comparison of detergent-solubilized membrane and soluble proteins from flow cytometrically sorted X- and Y-chromosome bearing porcine spermatozoa by high resolution 2-D electrophoresis. Mol Reprod Dev 45 342-50 (1996).
21. Blecher, S. R., Howie, R., Li, S., Detmar, J. and Blahut, L. M. A new approach to immunological sexing of sperm. Theriogenology 52 1309-21 (1999).
22. Simpson, E. The H-Y antigen and sex reversal. Cell 44 813-4 (1986).
23. Kent-First, M. G., Maffitt, M., Muallem, A., Brisco, P., Shultz, J., Ekenberg, S. Agulnik, A. I., Agulnik, I., Shramm, D., Bavister, B., Abdul-Mawgood, A. and VandeBerg, J. Gene sequence and evolutionary conservationof human SMCY. Nat Genet 14 128-9 (1996).
24. Greenfield, A. Scott, D., Pennisi, D., Ehrmann, I.. Ellis, P., Cooper, L. Simpson, E. and Koopman, P. An H-YDb epitope is encoded by a novel mouse Y chromosome gene. Nat Genet 14 474-8 (1996).
25. Muller, U. H-Y antigens. Hum Genet 97 701-4 (1996).
26. Agulnik, A. I., Longepied, G., Ty, M. T., Bishop, C. E. and Mitchell, M. Mouse H-Y encoding Smcy gene and its X chromosomal homolog Smcx. Mamm Genome 10 926-9 (1999).
27. Agulnik, A. I., Mitchell, M. J., Lerner, J. L., Woods, D. R. and Bishop, C. E. A mouse Y chromosome gene encoded by a region essential for spermatogenesis and expression of male-specific minor histocompatibility antigens. Hum Mol Genet 3 873-8 (1994).
28. Wang, W. Meadows, L. R., den Haan, J. M., Sherman, N. E., Chen, Y. Blokland, E., Shabanowitz, J., Agulnik, A. I., Hendrickson, R. C. and Bishop, C. E. Human H-Y: a male-specific histocompatibility antigen derived from the SMCY protein. Science 269 1588-90 (1995).
29. Anderson, G. B. Manipulation of the mammalian embryo. J Anim Sci 61 1-13 (1985).
30. Utsumi k, H. M., Takakura R, Utaka K, Iritain A. Embryo sex selection by a rat male-specific antibody and the cytogenetic and developmental confirmation in cattle embryos. Mol Reprod Dev 34 25-32 (1993).
31. Hendriksen, P. J., Tieman, M., Van der Lende, T. and Johnson, L. A. Binding of anti-H-Y monoclonal antibodies to separated X and Y chromosome-bearing porcine and bovine sperm. Mol Reprod Dev 35 189-96 (1993).
32. Sills, E. S., Kirman, I., Colombero, L. T., Hariprashad, J., Rosenwaks, Z. and Palermo, G. D.H-Y antigen expression patterns in human X- and Y-chromosome-bearing spermatozoa. Am J Reprod Immunol 40 43-7 (1998).
33. Sills, E. S., Kirman, I., Thatcher, S. S., 3rd and Palermo, G. D. Sex-selection of human spermatozoa: evolution of current techniques and applications. Arch Gynecol Obstet 261 109-15 (1998).
34. Burgoyne, P. S., Levy, E. R. and McLaren, A. Spermatogenic failure in male mice lacking H-Y antigen. Nature 320 170-2 (1986).
35. Scott, D. M., Ehrmann, I. E., Ellis, P. S., Bishop, C. E.,Agulnik, A. I.,Simpson, E. and Mitchell, M. J. Identification of a mouse male-specific transplantation antigen, H-Y. Nature 376 695-8 (1995).
36. Alexander I. Agulink, M. J. M., Jody L. Lerner, Diane R.Woods and Colin E. Bishop. A mouse Y chromosome gene encoded by a region essential spermatogenesis and expression of male-specific minor histocompatibility. Human Molecular Genetics 3 873-878 (1994).
37. Pennisi, E. Long-sought H-Y antigen found. Science 269 1515-6 (1995).
38. Agulnik, A. I., Bishop, C. E., Lerner, J. L., Agulnik, S. I. and Solovyev, V. V. Analysis of mutation rates in the SMCY/SMCX genes shows that mammalian evolution is male driven. Mamm Genome 8 134-8 (1997).
39. Lingenfelter, P. A. Adler, D. A.,Poslinski, D.,Thomas, S.,Elliott, R. W., Chapman, V. M. and Disteche, C. M. Escape from X inactivation of Smcx is preceded by silencing during mouse development. Nat Genet 18 212-3 (1998).
40. Agulnik, A. I., Mitchell, M. J., Mattei, M. G., Borsani, G., Avner, P. A.,Lerner, J. L. and Bishop, C. E. A novel X gene with a widely transcribed Y-linked homologue escapes X- inactivation in mouse and human. Hum Mol Genet 3 879-84 (1994).
41. Thompson, F. H. H-Y antigen. N Engl J Med 301 387-8 (1979).
42. Fattaey, A. R., Helin, K., Dembski, M. S., Dyson, N., Harlow, E., Vuocolo, G. A., Hanobik, M. G., Haskell, K. M., Oliff, A. and Defeo-Jones, D. Characterization of the retinoblastoma binding proteins RBP1 and RBP2. Oncogene 8 3149-56 (1993).
43. Aasland, R., Gibson, T. J. and Stewart, A. F. The PHD finger: implications for chromatin-mediated transcriptional regulation. Trends Biochem Sci 20 56-9 (1995).
44. Pamilo P, B. N. Evolution of the Zfx and Zfy genes. Rates and interdependence between the genes. Mol Biol Evol 10 271-281 (1993).
45. Miyata T, H. H., Kuma, K., Mitsuyasy, K., and Yasunaga, T. Male-driven molecular ecolution: a model and nucleotide sequence analysis. Cold Spring Harbor Symp Quant Biol 52 863-867 (1987).
46. Banfi, S., Guffanti, A. and Borsani, G. How to get the best of dbEST. Trends Genet 14 80-1 (1998).
47. Boguski, M. S., Lowe, T. M. and Tolstoshev, C. M. dbEST--database for "expressed sequence tags". Nat Genet 4 332-3 (1993).
48. Rodriguez-Tome, P. Searching the dbEST database. Methods Mol Biol 69 269-83 (1997).
49. Hartl, D. L. Est! Est!! Est!!! Bioessays 18 1021-3 (1996).
50. Boguski, M. S. Bioinformatics. Curr Opin Genet Dev 4 383-8 (1994).
51. Wu, J. Salido, E. C., Yen, P. H., Mohandas, T. K., Heng, H. H., Tsui, L. C. Park, J., Chapman, V. M. and Shapiro, L. J. The murine Xe169 gene escapes X-inactivation like its human homologue. Nat Genet 7 491-6 (1994).
52. Wu, J., Ellison, J., Salido, E., Yen, P., Mohandas, T. and Shapiro, L. J. Isolation and characterization of XE169, a novel human gene that escapes X-inactivation. Hum Mol Genet 3 153-60 (1994).
53. Parker, K. C., Bednarek, M. A. and Coligan, J. E. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J Immunol 152 163-75 (1994).
54. Rammensee, H., Bachmann, J., Emmerich, N. P., Bachor, O. A. and Stevanovic, S. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50 213-9 (1999).
55. Sun, Y., Song, M., Stevanovic, S., Jankowiak, C., Paschen, A., Rammensee, H. G. and Schadendorf, D. Identification of a new HLA-A(*)0201-restricted T-cell epitope from the tyrosinase-related protein 2 (TRP2) melanoma antigen. Int J Cancer 87 399-404 (2000).
56. Vonderheide, R. H., Hahn, W. C., Schultze, J. L. and Nadler, L. M. The telomerase catalytic subunit is a widely expressed tumor-associated antigen recognized by cytotoxic T lymphocytes. Immunity 10 673-9 (1999).