Cloning and characterization of a novel spermiogenesis-related gene, T6441, in rat testis.
We report in the present study the cloning and characterization of a novel gene, named T6441, initially derived by the suppressive subtracted hybridization (SSH) cDNA library. The full-length T6441cDNA was 664 bp long, containing a complete open-reading frame for a protein of 149 amino acids (aa). The protein bears no homology to any reported genes. It is predicted that the molecular mass was about 16.7 kDa. Northern blot analysis showed that the T6441 gene had about 4 transcripts in adult rat testis and was temporally regulated in a stage-dependent manner in the testis. In situ hybridization showed that T6441 mRNA was specifically localized in spermatids, and its expression level varied in the cells at different stages of the testicular development, with the highest level at steps 7-14. RT-PCR results showed that the T6441 mRNA was transcribed in most of the tested tissues with its strongest signal in the testis. Recombinant T6441 protein was prepared, purified, and was used to raise rabbit. Western blot analysis using the antiserum revealed four possible testicular specific proteins with their molecular weights being about 22, 25, 50 and 55 kDa respectively. The T6441 protein was expressed mainly in the cytoplasm of spermatids with the maximal levels at steps 12-19. At step 19 spermatid, the T6441 was mainly localized in the residual bodies. The cytoplasm localization of T6441 protein was supported by transient over expression of GFP-fusion protein in Hela cells. Interestingly, the expression of T6441 caused death of transfected cells within 48 h. Our preliminary experimental results suggest that the T6441 gene may play a role in cytoplasm movement and removal during spermiogenesis.
Song
Xin-Xin
XX
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.
Li
Yin-Chuan
YC
Shi
Yu-Qiang
YQ
Hu
Xiao-Qian
XQ
Hu
Zhao-Yuan
ZY
Han
Chun-Sheng
CS
Liu
Yi-Xun
YX
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008297:Male; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017474:Receptors, Laminin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013087:Spermatids / Q000378:metabolism; D013091:Spermatogenesis; D013737:Testis / Q000378:metabolism*; D013997:Time Factors; D014018:Tissue Distribution
D018076:DNA, Complementary; D012333:RNA, Messenger; C510624:RPSA protein, human; D017474:Receptors, Laminin; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; D049452:Green Fluorescent Proteins