Cloning and characterization of the promoter of baboon XRCC1, a gene involved in DNA strand-break repair.

Z Q Zhou, C A Walter
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引用次数: 9

Abstract

The DNA repair gene XRCC1 was the first cloned human DNA repair gene involved in resistance to ionizing radiation. Previous studies have shown that rodent and baboon homologs of XRCC1 are expressed in all tested tissues with significantly higher levels in testis. Furthermore, expression of murine XRCC1 is most abundant in pachytene spermatocytes and round spermatids. To begin to study regulation of XRCC1 expression, the 5' region of baboon XRCC1 was cloned and characterized. 400 bp of 5'-flanking region showed the greatest promoter activity, while -194 to -8 bp of the 5'-flanking region displayed core promoter activity in transient transfection assays. A comparison between baboon and human 5'-flanking sequences in the core promoter region revealed a potential CAAT-box, an imperfect CREB-binding site and two putative Sp1-binding sites. Results from transient transfection assays in which each putative binding site was individually mutated, indicated that the distal Sp1-binding site has a functional role in transcription. In comparison, both putative Sp1-binding sites bound protein(s) from HeLa cell nuclear extracts in vitro. In vitro binding was lost when mutated Sp1 sites were used in gel mobility shift assays. Finally, anti-Sp1 antibodies produced mobility supershifts, thereby indicating Sp1 or an Sp1-like protein bound to the DNA fragment in vitro.

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狒狒参与DNA断链修复的基因XRCC1启动子的克隆与表征。
DNA修复基因XRCC1是第一个克隆的参与抗电离辐射的人类DNA修复基因。先前的研究表明,啮齿动物和狒狒的XRCC1同源物在所有测试组织中均有表达,且在睾丸中的表达水平明显较高。此外,小鼠XRCC1在粗粒精母细胞和圆形精母细胞中表达最为丰富。为了开始研究XRCC1的表达调控,我们克隆了狒狒XRCC1的5′区并对其进行了表征。在瞬时转染实验中,5′-翼区400 bp的启动子活性最高,而5′-翼区-194 ~ -8 bp的启动子活性最高。通过对狒狒和人类核心启动子区域5'侧序列的比较,发现了一个潜在的CAAT-box、一个不完美的creb结合位点和两个假定的sp1结合位点。瞬时转染试验的结果表明,每个假定的结合位点都单独突变,表明远端sp1结合位点在转录中具有功能性作用。相比之下,两种推测的sp1结合位点在体外都与HeLa细胞核提取物中的蛋白结合。当突变Sp1位点用于凝胶迁移转移试验时,体外结合丢失。最后,抗Sp1抗体产生迁移超移,从而表明Sp1或Sp1样蛋白在体外与DNA片段结合。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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