Identification And Functional Study Of TSARG2, A Novel Human Testis Spermatogenesis Apoptosis-related Gene, And SRG2, The Homologous Gene In Mouse

Testis spermatogenic cell apoptosis is a complicated polygene-related process. At present, the research on testis spermatogenic cell apoptosis is in primitive stage. So far, specific testis spermatogenic cell apoptosis-related gene has not been cloned. Cloning new apoptosis related novel gene is a key to further understanding of apoptosis mechanism and the biology process of germ cell, and it is of momentous significance on clarifying physiology and pathology process of spermatogenesis.In previous study, Janghong have succeeded cloning 24 ESTs of mouse testis spermatogenic cell apoptosis-related gene by creating mouse cryptorchidism model and making use of suppression subtractive hybridization, and registered in Genbank.Using nested PCR and draft human genome searching, we successfully cloned the TSARG2 and mouse homeobox gene and made rudiment study on its functions. There were three parts in the study, and the following is its main experimental methods and results.Part 1: Identification of a novel gene, TSARG2, and its sequence characterCloning new apoptosis-related novel gene is a key to further understanding of apoptosis mechanism and the biological process of germ cell, and it is of momentous significance on clarifying physiology and pathology process of spermatogenesis. To rapidly attain human novel gene full-length cDNAsequence, the gene-specific primers and the vector-specific primers have been designed for successful performing nested PCR and draft human genome searching to rapidly identify the TSARG2 (Genebank accession number AY040204) 5' end from a human testis cDNA library by using a cDNA fragment (Genebank accession number BE644542) as a electronic probe, which was significantly changed in cryptorchidism and represents a novel gene. Furthermore, a mouse homologue of this gene was identified(Genebank accession number AF395083) by lab on-line. TSARG2 with 1 233 bp length was composed of 6 exons and spaned about 115 kb of genomic DNA, The putative protein encoded by this gene was 305 amino acid with a theoretical mass of 34 751 and with no significant homology with any known protein in databases. A kind of nucleoprotein was the most impossible. In this gene, a CpG island, whose GC content was 61.6 %, was located from -277 to +163. In this CpG island, from -191 to +16, we predicted that there was a 208 bp promotor in the above region. Northern blots indicated that the gene only had high expression in human testis. TSARG2 was mapped to chromosome 4q34.1-34.2 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.Part 2: Preliminary function study of TSARG2Collected the periphermal blood specimens of azoospermia, severeoligozoospermia and cryptorchidism, which were 122 samples, extracted the DNA and established DNA sample pool. But the mutation of this gene has not been found in those samples by PCR-SSCP and this indicated that this gene might carry out its function through up-regulation or down-regulation.The open reading frame of TSARG2 was obtained from human testis cDNA library by PCR. Using in situ hybridization on tissue section of human testis. We preliminarily studied the expression and function of TSARG2. The purple brown granules of hybridized signal were found in different stages of spermatogenesis cell. The results showed that this gene performs function by different expression in different stages of spermatogenesis cell.To observe TSARG2 fusion protein expression in mammalian cell, pEGFP-Nl/TSARG2 fusion plasmid was constructed and transiently introduced into COS7 cell by liposome transfection. Under the fluorescence microscope, the green fluorescence produced by pEGFP-Nl/rSARG2 was detected on the nucleus of COS7 cells after 24 hours post transfection, while the fluorescence produced by pEGFP-Nl was detected through the cells. Consistent with the prediction by Bioinformatics, this re...