The Human Hufp Gene Preliminary Study

Hybridizing human adult testis cDNA microarrays with human adult and 6 months embryonic testis cDNA probes, we identified a novel human testis gene, termed hUFP, of which mRNA expression level was 8.1-fold stronger in human adult testes than fetal testes. The result was confirmed by the means of RT-PCR in adult testis, fetal testis and sperm. hUFP cDNA contains 3256bp with a 218Ibp open reading frame encoding a protein of 727 amino acids with presumed molecular weight of 80.369kDa. hUFP gene was mapped to lOqll.l regions of chromosome 10. Predicted protein motif analysis of hUFP revealed that it had an ubiquitin-like N-terminus followed by a C-terminal Anl-like Zinc finger domain. This novel gene shows 82% nucleotide identity and 48.15% amino acid identity to the ubiquitin-like fusion protein involving An la and Anla in Xenopus laevis. hUFP has 51.18% amimo acid identity to rat RSD-7 and is highly similar to SUMO-1 and NEDD8 and therefore hUFP gene is one member of the ubiquitin-like modifiers (UBLs) family. Expression profile of hUFP with multiple-tissue RT-PCRs showed that hUFP also expressed ubiquitously in 16 adult human tissues but significantly expressed in testis. To examine hUFP special expression in adult human testis, hUFP protein was expressed and purified which we used to immunize mouse and obtain anti-hUFPantibodies. Then we did the immunohistochemistry test and found hUFP expression greatly strong in the Leydig cells of testis. To examine the role of ubiquitin domain of hUFP in vivo, in terms of hUFP high homology to gene An la and Anlb in Xenopus, we injected the three constructs of hUFP A ubiquitin which means ubiquitin domain was deleted from hUFP, Zn-hUFP which means only the Anl-like Zn-fmger domain was left and the full-length of hUFP into embryonic animal, dorsal and lateral at four cell stage of Xenopus laevis, respectively. Microinjection of hUFP A ubiquitin and Zn-hUFP into dorsal blastomeres caused obviously embryonic neural system defects. Whereas embryos that overexpressed full-length hUFP didn't show the anterior structure defects of smaller brain or other organ defects. We have also observed that microinjection of hUFPubiquitin and Zn-hUFP, both of which had no ubiquitin domains, could produce not only smaller anterior brain but larger ventral part than that of microinjection of hUFP. Taken together, these results suggested that hUFP expression was developmentally regulated and hUFP was potentially involved in testicular development and spermatogenesis. Especially, ubiquitin domain in hUFP is very important to regulate hUFP in playing some physiological roles in vivo.To primarily explore the function of hUFP, we constructed two retrovirus vectors to transfect 293T cells, including full-length hUFP and hUFPubiquitin. Cytoimmunohistochemistry was employed to confirmthe transfection results, on the basis of which we could research the function of hUFP further.