Rapid Identification of Human Testis Spermatocyte Apoptosis-related Gene, TSARG, by Nested PCR and Draft Human Genome Searching

LIU Shang-Feng, LI Lu-Yun, FU Jun-Jiang, LIU Gang, XING Xiao-Wei, LU Guang-Xiu^*
( Human Reproductive Engineering Institue, Central South University Xiangya Medical School, Changsha 410078, China )

Abstract    Cloning apoptosis-related novel genes is a key to further understanding of apoptosis mechanism and the biology process of germ cells, and is of momentous significance on clarifying physiological and pathological process of spermatogenesis.To rapidly attain human novel gene full-length cDNA sequence, the gene-specific primers and the vector-specific primers were designed for nested PCR, and draft human genome searching was performed to rapidly identify the TSARG2 (GenBank accession number AY040204) 5' end from a human testis cDNA library, by using a cDNA fragment (GenBank accession number BE644542) as an electronic probe,which was significantly changed in cryptorchidism and represented a novel gene. Furthermore, a mouse homologue of this gene was identified (GenBank accession number AF395083) by lab on-line.TSARG2 with a 1 233 bp length was composed of 6 exons and spanned about 115 kb of genomic DNA,The putative protein encoded by this gene was 305 amino acid with a theoretical molecular weight of 34 751 dalton and did not share significant homology with any known protein in databases.TSARG2 was expressed in many tissues and mapped to chromosome 4q33—34.1 by database analyses.Therefore, we propose that nested-PCR and draft human genome searching are rapid, sensitive, accurate and efficient method for isolating gene 5' end, even full-length gene from cDNA library.
Key words    gene cloning; nested PCR; draft human genome searching; testis; apoptosis

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