Human Testis-specific Expression Of The Gene Family, Vcx / Y Protein-coding Function Of A Preliminary Study

Variable Charge X/Y is a human testis-specific gene family that localized on sex chromosomes. VCX/Y family include VCY, which localized on Y chromosome, and VCX-2r, VCX-8r and VCX-10r that localized on X chromosome. All of the proteins, encoded by the gene family, have a putative bipartite nuclear localization signal near the N-terminus, suggesting that they are nuclear proteins. The main difference among VCX-2r, VCX-8r, VCX-10r and VCY is that the protein products of the X-linked loci have varied tandem iteration of an acidic ten-amino-acid motif present singly in the Y homologues. In addition, Lys and Arg are rich in the region outside of the tandem iteration. VCX/Y protein molecules are small size and high charge, which resemble those chromatin-associated proteins, such as histones or protamines, the latter mediate condensed DNA packaging in sperm. Because of the two features, VCX/Y proteins had been speculated as components of chromatin in previous study.VCY and VCXSr were selected as representatives of VCX/Y family in our study. VCY protein was expressed in E. coli in the form of glutathione-S-transferase (GST) fusion protein. With the purified fusion protein as antigen, the anti-GST-VCY antibody was generated and the localization of VCY protein in human testis was determined by immunohistochemistry. In the testis seminiferous epithelium, VCY proteins were localized in nuclei of germ cells. Intense staining of spermatocyte and round spermatid occurred while elongate spermatid were not stained. In addition, there was also some stronger staining region in some nuclei.With the strategy of PCR and subcloning, VCY was cloned into eukaryotic expression vectors pDsRed1-N1 and pEGFP-N1 by Xho I /BamH I directional insertion. With the same methods, VCX-8r was subcloned in-frame into pEGFP-N1. Using propidium iodide staining and green fluorescent protein (GFP) tag technologies, VCY and VCX-8r proteins were mainly localized in the nucleoli of COS7 cells. The colocalization for VCY and VCX-8r in COS7 cells was also observed. Based on the same strategy, a series of segmental deleted mutants of VCY were cloned into pEGFP-N1 vector. The localization of the mutants in COS7 cells gave us the verification to the bipartite nuclear localization signal. In addition, the tandem iteration of an acidic ten-amino-acid motif was definite to be the sequence, which aid VCX/Y proteins to be localized in nucleoli.With VCY cDNA (cloned into pAS2-1 plasmid) as bait, a cDNA fragment of acidic...