Identification and expression of a novel human testis-specific gene by digital differential display.
BACKGROUND
Evidence for the importance of genetic factors in male infertility is accumulating. This study was designed to identify a novel testis-specific gene related to spermatogenesis by a new strategy of digital differential display (DDD).


METHODS
Based on the generation of expressed sequenced tags (ESTs), comparing the testis libraries with other tissue or cell line libraries by the DDD program, we identified a new contig of the ESTs which were derived from testis libraries and represented a novel gene. Multi-tissue RT-PCR was performed to analyse its tissue-specific expression. The full-length cDNA of the new gene was obtained using the BLAST program. Sequencing was performed and the result was analysed. Semi-quantitative RT-PCR and Northern blot analyses of mRNA from differential normal tissues were performed to clarify the expression pattern of the new gene. The sequence of the opening reading frame was integrated into the pQE-30 vector expressed in Escherichia coil strain M15 (pREP4). With IPTG induction, the target protein was detected.


RESULTS
A full-length cDNA sequence of the new gene named SPATA12 (GeneBank accession number AY221117) in human testis was identified. SPATA12 was 2430 bp in length, located in chromosome 3p21.1-3p21.2. The sequence of the opening reading frame was 676-1248 bp, as was confirmed by RT-PCR and sequencing. The cDNA encodes a novel protein of 190 amino acids with a theoretical molecular weight of 20417.8 and isoelectric point of 5.23. The sequence has no significant homology with any known protein in databases. Semi-quantitative RT-PCR and Northern-blot analyses of multiple tissues showed that SPATA12 was expressed significantly in normal human testis. The expression recombinant of SPATA12 was constructed and a high level of the histidine-tagged fusion protein was obtained.


CONCLUSIONS
DDD can be confirmed by SPATA12 as a novel computational biology-based approach for identification of the testis-specific expression genes. SPATA12 may function as a testicular germ cell associated gene that plays some roles in spermatogenesis. Moreover, a great amount of SPATA12 protein could be obtained by the gene recombination technique, thus providing a reliable foundation for investigating the biological function of this new protein.
Li
Dan
D
Institue of Reproductive and Stem Cell Engineering, Central South University, Changsha 410078, China. leedanie@163.net
Lu
Guang-xiu
GX
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D020224:Expressed Sequence Tags; D015723:Gene Library; D005796:Genes; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013091:Spermatogenesis / Q000235:genetics*; D013737:Testis / Q000378:metabolism*