Molecular cloning and primary functional analysis of a novel human testis-specific gene.
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 G2-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
Nie
Dong-Song
DS
Human Reproductive and Stem Cell Engineering Institute, Central South University, Changsha 410078, China. nds200346@hotmail.com
Zhu
Wen-Bing
WB
Xiang
Yang
Y
Wang
Jian
J
Tan
Xiao-Jun
XJ
Deng
Yun
Y
Lu
Guang-Xiur
GX
D000328:Adult; D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002884:Chromosomes, Human, Pair 15; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013091:Spermatogenesis / Q000235:genetics*; D013737:Testis / Q000378:metabolism*; D014162:Transfection
D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C554273:SPATS1 protein, human; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins