[Molecular cloning and expression analysis of a novel human testis-specific gene]

Abstract

Digital Differential Display (DDD) of the National Center for Biotechnology Information (NCBI) is a quantitative method that enables the user to determine the fold differences between the libraries being compaired, using a statistical method to quantitate the transcript levels. In this study, DDD program was performed between nine testis libraries ('tester') and seventy-six libraries derived from other tissues ('driver'). We identified a new contig of expression sequence tags (ESTs) HS. 129794 which were from testis libraries. To validate the use of bioinformatics approaches in gene discovery, the ESTs HS. 129794, which was predicted to be testis -specific, was chosen for further study. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of mRNA from different normal tissues indicated that HS. 129794 was specifically expressed in human testis. By querying EST and Unigene datagases, a full-length cDNA sequence of novel gene in human were identified, it was 2 430 bp in length, located in chromosome 3p21.1. The sequence of the open reading frame was 676 approximately 1 248 bp, as was confirmed by RT-PCR and sequencing in human testis. The cDNA encodes a novel protein of 190 amino acids with a theoretical molecular weight of 20 417.8 and isoelectric point of 5.23. The sequence shares no significant homology with any known protein in databases. Semi-quantitative RT-PCR analysis of multiple tissues further showed that the novel gene is expressed significantly in different stage of human testis and sperm. We hypothensize that its functions as a testis-specific and spermatogenesis related gene that plays some roles in spermatogenesis, and named it SRG5 (Testis Spermatogenesis Related Gene 5, SRG5) (GeneBank accession number: AY221117). Identification of SRG5 using DDD approaches validates gene discovery using computational approaches.