CAJ | 학술논문

运用数字差异展示方法,克隆一个与生精相关的睾丸高表达基因.借助公共ESTs数据库,利用DDD软件比较分析各种睾丸文库与其他组织或细胞系文库有差异表达的ESTs,成功克隆到一个在人类睾丸中高表达的新基因.结合实验获得新基因cDNA全长,该基因被国际人类基因命名委员会命名为ZNF474(GeneBank登陆号AY461732).ZNF474的cDNA全长为1 972 bp,定位在5q23.2.通过RT-PCR及测序验证,其开放阅读框的位置在377 bp～1 471 bp处,编码364个氨基酸,在氨基酸水平与小鼠同源基因有66%的一致性,而与其他已知蛋白质无明显同源性.Northern杂交分析显示ZNF474在成体睾丸组织特异高表达,卵巢组织弱表达,在多种其他组织中不表达,为单一转录本.原位杂交显示ZNF474基因在正常成人睾丸组织各级生精细胞、隐睾组织以及精原细胞癌组织中均有较高表达.综上考虑,推测ZNF474作为生殖细胞中特异的转录因子,对人类的精子发生和卵母细胞的发育可能起重要作用.
운용수자차이전시방법,극륭일개여생정상관적고환고표체기인.차조공공ESTs수거고,이용DDD연건비교분석각충고환문고여기타조직혹세포계문고유차이표체적ESTs,성공극륭도일개재인류고환중고표체적신기인.결합실험획득신기인cDNA전장,해기인피국제인류기인명명위원회명명위ZNF474(GeneBank등륙호AY461732).ZNF474적cDNA전장위1 972 bp,정위재5q23.2.통과RT-PCR급측서험증,기개방열독광적위치재377 bp～1 471 bp처,편마364개안기산,재안기산수평여소서동원기인유66%적일치성,이여기타이지단백질무명현동원성.Northern잡교분석현시ZNF474재성체고환조직특이고표체,란소조직약표체,재다충기타조직중불표체,위단일전록본.원위잡교현시ZNF474기인재정상성인고환조직각급생정세포、은고조직이급정원세포암조직중균유교고표체.종상고필,추측ZNF474작위생식세포중특이적전록인자,대인류적정자발생화란모세포적발육가능기중요작용.
This study was designed to identify a novel testis overexpressed gene related to spermatogenesis by a new strategy of digital differential display (DDD). 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. A full-length cDNA sequence of the new gene named ZNF474 ( GeneBank accession number AY461732) by the HUGO Gene Nomenclature Committee in human testis was identified. ZNF474 was 1972 bp in length, located in chromosome 5q23.2. The sequence of the opening reading frame was 377 bp ～ 1471 bp, as was confirmed by RT-PCR and sequencing. The cDNA encodes a novel protein of 364amino acids protein that was 66 % identical to a mouse homologue. Northern blot analyses revealed that ZNF474 mRNA was exclusively expressed in testis and ovary and has one transcript. In situ hybridization revealed that ZNF474 high expressed in adult testis and seminoma. The brown granules of hybridization signal were found in spermatocyte, spermatids and spermatozoa, no expression in sertoli's cells and leydig's cells. We hypothesize that ZNF474 functions as a germ cell-specific transcription factor that plays important roles in spermatid differentiation and oocyte development.