Sequence-specific DNA binding of the proto-oncoprotein ets-1 defines a transcriptional activator sequence within the long terminal repeat of the Moloney murine sarcoma virus.
Sequence-specific DNA binding of the proto-oncoprotein ets-1 defines a transcriptional activator sequence within the long terminal repeat of the Moloney murine sarcoma virus.
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原癌蛋白 ets-1 的序列特异性 DNA 结合定义了莫洛尼鼠肉瘤病毒长末端重复序列内的转录激活序列。
DOI：
10.1101/gad.4.4.667
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发表时间：
1990
影响因子：
10.5
通讯作者：
Graves,BJ
中科院分区：
文献类型：
--
作者：
Gunther,CV;Nye,JA;Bryner,RS;Graves,BJ
ETS原癌基因家族是一组正常细胞功能未知的序列相关基因。在对T淋巴细胞中参与小鼠逆转录病毒转录调控的细胞蛋白的研究中，我们发现ETS基因家族的一个成员编码一种序列特异性DNA结合蛋白。用Moloney小鼠肉瘤病毒(MSV)长末端重复序列(LTR)的双链寡核苷酸探针筛选小鼠胸腺表达文库，获得了小鼠Ets-1基因克隆。该序列有一个813bp的开放阅读框(ORF)，其预测的氨基酸序列与人Ets-1蛋白的272个羧基末端氨基酸序列有97.6%的同源性。ORF在细菌中表达，通过迁移率改变分析、Southwest印迹分析和甲基化干扰，30-kD蛋白产物以序列特异性的方式与DNA结合。构建了一个在Ets-1结合部位含有4个碱基对替换的突变体LTR，体外实验表明其结合能力降低。将含有该Ets-1结合位点的MSV LTR启动子的转录效率与野生型启动子在小鼠T淋巴细胞中的转录效率进行了比较，观察到报告基因的表达减少了15-20倍。我们认为Ets-1是哺乳动物C型逆转录病毒的转录激活因子，推测Ets相关基因构成了一组新的真核DNA结合蛋白。
The ets proto-oncogene family is a group of sequence-related genes whose normal cellular function is unknown. In a study of cellular proteins involved in the transcriptional regulation of murine retroviruses in T lymphocytes, we have discovered that a member of the ets gene family encodes a sequence-specific DNA-binding protein. A mouse ets-1 cDNA clone was obtained by screening a mouse thymus cDNA expression library with a double-stranded oligonucleotide probe representing 20 bp of the Moloney murine sarcoma virus (MSV) long terminal repeat (LTR). The cDNA sequence has an 813-bp open reading frame (ORF) whose predicted amino acid sequence is 97.6% identical to the 272 carboxy-terminal amino acids of the human ets-1 protein. The ORF was expressed in bacteria, and the 30-kD protein product was shown to bind DNA in a sequence-specific manner by mobility-shift assays, Southwestern blot analysis, and methylation interference. A mutant LTR containing four base pair substitutions in the ets-1 binding site was constructed and was shown to have reduced binding in vitro. Transcriptional efficiency of the MSV LTR promoter containing this disrupted ets-1 binding site was compared to the activity of a wild-type promoter in mouse T lymphocytes in culture, and 15- to 20-fold reduction in expression of a reporter gene was observed. We propose that ets-1 functions as a transcriptional activator of mammalian type-C retroviruses and speculate that ets-related genes constitute a new group of eukaryotic DNA-binding proteins.