人类生精相关基因TSARG4的cDNA克隆

为了探索精子生成的分子机制 ,从人精子外部致密纤维蛋白相关基因SPAG4(spermantigen 4)和小鼠精母细胞中表达的AK0 0 62 2 5基因出发 ,找到两个人类EST ,BG72 0 5 64和AI70 0 45 4,其中BG72 0 5 64在人睾丸中表达。运用“间隙填充法”填平这两个EST之间的间隙 ,从人睾丸文库中快速克隆了同源于SPAG4和AK0 0 62 2 5基因的人类TSARG4基因 (testisandspermatogenesisrelatedgene 4) (GenBank登录号为AF40 13 5 0 ) ,并用RT PCR对该基因阅读框进行验证。TSARG4基因全长 12 5 2bp ,开放阅读框为 94～ 12 3 3bp ,定位于 2 0q11.2 ,推定编码 3 79个氨基酸 ,预计分子量为 43 0 81.45 ,等电点为 8.61,该基因与小鼠精母细胞基因AK0 0 62 2 5编码的氨基酸序列同源性 74% ,与人类SPAG4基因编码的氨基酸序列同源性 45 %。RT PCR表明人类TSARG4基因在多个组织中均有表达 ,而同源的小鼠AK0 0 62 2 5基因仅在睾丸中表达

Cloning of cDNA of TSARG4, a Human Spermatogensis Related Gene

To understand molecular mechanism of spermatogenesis, two ESTs BG720564 and AI700454, were found from SPAG4 (sperm antigen 4), a gene related to dense fiber protein of outer membrane of the human sperm and mouse spermatocytes gene AK006225. The gap was filled by polymerase chain reaction, and a 1252 bp fragment was obtained. This 1252 bp fragment was named TSARG4 (testis and spermatogenesis related gene 4 (GenBank accession number AF401350). Its opening reading frame was 94-1233 bp, as was proved by RT-PCR in human testis. TSARG4 gene was located in 20q11.2, and the putative protein was 379 amino acid with a theoretical molecular weight of 43 081 and isoelectric point of 8.61. The homologies of amino acid sequences were 74% between TSARG4 and AK006225 gene and 45% between TSARG4 and SPAG4 gene, respectively. The human TSARG4 mRNA is expressed in a wide range of adult tissues, including testis, whereas the homologous mouse spermatocytes gene AK006225 is expressed specifically in the testis.