一个新的睾丸特异表达基因的克隆及功能的初步分析(英文)

Molecular Cloning and Primary Functional Analysis of a Novel Human Testis-specific Gene

运用'数据库消减杂交'(digital differential display )方法来筛选人类睾丸特异表达新基因,获得了有差异显示的代表新基因的克隆重叠群.挑选其中一个克隆重叠群HS.326528进行多组织RT-PCR,初步获得该重叠群在人睾丸中有高表达.从该重叠群的IMAGE出发,采用生物信息学的方法快速克隆了一个人类新基因的全长cDNA序列,其全长1 044 bp,开放阅读框为214～529 bp,定位于15q26.2,编码由105个氨基酸组成、分子量为11.7 kD、等电点为10.09的一个碱性蛋白,该蛋白与已知蛋白无明显的同源性,克隆实验证明该基因的阅读框完全正确,RT-PCR和Northern blot显示该基因在人类睾丸中特异表达,实时PCR结果表明:该基因在成人睾丸中高表达,在精子中有中度表达,在胚胎睾丸中低表达,推测该基因与精子的生成有关,命名为SRG8(homo sapiens spermatogenesis -related gene 8)(GenBank登录号:AY489187),该基因编码的蛋白定位于细胞核.流式结果分析表明,SRG8基因能够促使HeLa细胞由S期向G2期的转变,从而加速细胞的分裂.这些结果表明SRG8基因可能在睾丸的发育及精子的形成过程中起重要的作用.

In this study,a new data mining tool called Digital Differential Display (DDD) from the NCBI was used to predict testis-specific expressed genes from the expressed sequence tag (EST) database.DDD(digital differential display) was performed between nine testis libraries and seventy libraries derived from other tissues.We identified a new contig of ESTs (HS.326528) which was from testis libraries.To validate the use of bioinformatic approaches in gene discovery,the ESTs (HS.326528),which were predicted to be testis-specific,were chosen for further study.Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis of matched sets of cDNAs from testis and other tissues indicated that the ESTs were specifically expressed in testis.This (result) was further validated by multi-tissue Northern blot.The full-length cDNA encompassing the entire open reading frame was cloned and,in view of its apparent specificity to testis,the gene was termed homo sapiens spermatogenesis-related gene 8——SRG8 (GenBank accession number: AY489187).The gene whose full (cDNA) length is 1 044 bp containing 3 exons and 2 introns is located in human chromosome 15q26.2,the cDNA (encodes) a novel protein of 105 amino acides with a theoretical molecular weight of 11.7 kD and isoelectric point of 10.09 which shares no significant homology with any known proteins in database.Real time PCR analysis of testis of different developmental periods revealed that SRG8 gene is significantly expressed in adult testis .The green fluorescent protein produced by pEGFP-C3/SRG8 was detected in the nucleus of HeLa cells after 24 h post-transfection.Cell cycle analysis showed that SRG8 can accelerate HeLa cells to traverse the S-phase and enter the G_2-phase compared with the control without transfection of SRG8,which suggested that this gene plays an important role in the development of testis.The discovery of SRG8 showed that DDD combined with (experiments) is a feasible,time-saving strategy to identify new candidate genes for testis-specific development