| In humans, infertility affects about 10% of couples worldwide, and in roughly half of these cases the defect can be traced to the man and about 20% of male infertility display a severe or complete loss of mature spermatozoa, which are called oligozoospermia or azoospermia. Spermatogenesis is a complex process of cell development and differentiation during which extensive changes in cell morphology and intracellular organization occur. This process requires the highly regulated expression of a network of genes located on autosomes and sex chromosomes. Further studies on these genes and their functions would help us tounderstand the molecular mechanism of spermatogenesis, as well as to clarify the genetic pathology of azoospermia.As an excellent animal model, mouse has been widely used in research on the genes related to spermatogenesis. To better understanding the function of our newly cloned human spermatogenesis related gene ZNF230, we cloned its mouse homolog by combining the rapid amplification of cDNA ends (RACE) technique and in silico cloning method. As the result, a 982bp full-length cDNA was obtained and then it was deposited in the GenBank under the accession number AF353167.The cDNA of znf230 contains a 690 nt open reading frame (ORF), with the start codon ATG at position 125 and stop codon TGA at position 815, encoding a polypeptide of 230 aa. A canonical polyadenylation signal AATAAA is found 18bp upstream of the poly (A) tail. When searched against InterPro database with the deduced aa sequence, a ring finger domain of C3HC4 type was found at the C-terminus (from aa residue 155 to 191). By comparing its nt and aa sequences with those of human homolog, 92% and 98% identity was observed respectively. Furthermore, it is remarkable that the aa of ring finger domain are completely identical. Aligned aa sequence of ring finger domain of znf230 with those of other species indicates that this domain is highly conserved. The conserved primary structure suggests, at least in some extent,that its function may also be conserved and important. After searching the human-mouse homology map, the mouse znf230 was mapped on chromosome 7. This result was also confirmed by chromosomal localization of its genomic sequence. Mouse znf230 gene spans at least 28 kb genomic sequences and the cDNA is split into six exons. The splicing sites of intron-exon boundaries are conformed to the standard ag/gt rule.To further understanding the expression profile of mouse znf230 gene, multiple-tissue Northern blot and RT-PCR were performed. As revealed by Northern blot, the expected 1 kb band was detected only in testis. Meanwhile, the 4.4 kb band was detected not only in heart, brain, skeletal muscle and kidney as its human homolog, but also in lung, liver and testis. In mouse, another 2.4 kb transcript was detected in heart, liver and kidney. This result was also confirmed by RT-PCR.We also investigated the expression of the znf230 gene during prenatal and postnatal mouse testicular tissues by RT-PCR. The expression of znf230 was first detected at day 6 postnatal (pn) and reached the adult level between day 14 to 21 pn. In mice, the seminiferous epithelium is basically organized by Sertoli cells and type-A spermatogonia cells at day 6 pn, and from day 18 pn until day 25 pn, round spermatids increase in number and progressively differentiateto condensing spermatids. Taken together the expression pattern of mouse znf230 and the fact that human ZNF230 is not expressed in idiopathic azoospermic patient whose spermatogenesis was blocked at spermatid phase, it can be deduced that the gene may play an important role in formation of spermatids.Analyzed with both PSORT program and GFP-labbeled iv-vivo method, the mouse znf230 was proposed to be a protein located in the nucleus, even though no nuclear location consensus signal was found in its sequence. The result of yeast one-hybrid analysis indicated that znf230 has not only transcriptional activity, but also that its intact ring finger domain is important for the activity. Although the n...
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