In Silico and Molecular Cloning of the Bovine and Porcine SMCX/SMCY Loci

• Main Authors: Pei-Shan Hsieh, 謝佩珊
• Other Authors: Yow-Ling Shiue
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/zx35v8

碩士 === 國立成功大學 === 生物科技研究所碩博士班 === 90 === 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.