周畅 1, 2 ，李麓芸 2，卢光琇 2
（1,湖南师范大学生命科学院，长沙 410081， 2 ,中南大学生殖与干细胞工程研究所，长沙 410078）
摘要 : 本研究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1972bp，定位在5q23.2。通过RT-PCR及测序验证，其开放阅读框的位置在377-1471 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，我们推测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。关键词: 数字差异展示方法 ；表达序列标签；ZNF474基因
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Identification of a Novel Human Zinc Finger Protein Gene ZNF474 by Digital Differential Display
ZHOU Chang 1，2, LI Lu-Yun 2, LU Guang-Xiu 2
( 1,College of Life Science ,Human Normal University, Changsha 410081 , China； 2, Institude of Reproductive &Stem Cell Engineering, Central South University, Changsha, 410078 , China )
Abstract : 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 377-1471 bp, as was confirmed by RT-PCR and sequencing. The cDNA encodes a novel protein of 364 amino 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.
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