ANALYSIS OF THE V-MYB STRUCTURAL COMPONENTS IMPORTANT FOR TRANSACTIVATION OF GENE-EXPRESSION
ANALYSIS OF THE V-MYB STRUCTURAL COMPONENTS IMPORTANT FOR TRANSACTIVATION OF GENE-EXPRESSION
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DOI：
10.1093/nar/19.7.1533
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发表时间：
1991-04-11
影响因子：
14.9
通讯作者：
OSTROWSKI, MC
中科院分区：
文献类型：
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作者：
BORTNER, DM;OSTROWSKI, MC
为了定义对基因表达反式激活很重要的 v-myb 蛋白结构域，我们在 NIH 3T3 细胞中使用瞬时转染测定研究了 v-myb 基因和一组 v-myb 缺失突变体的反式激活。 对 v-myb 缺失产物组的分析表明，蛋白质羧基末端部分中先前未识别的区域是反式激活所必需的。 该区域位于 v-myb 基因 5' 端的氨基酸 295-356 之间。 将 v-myb DNA 结合域与大鼠糖皮质激素受体 (rGR) 的 DNA 结合域交换，改变了 v-myb 作用的顺式元件要求：只有含有糖皮质激素反应元件的报告基因才会被 myb-rGR 融合蛋白激活。 完整v-myb基因反式激活所必需的羧基末端区域也是rGR融合基因反式激活所必需的。 当在瞬时共表达测定中进行测试时，包含新反式激活区域的羧基末端缺失突变能够阻断野生型 v-myb 反式激活。 在我们的研究中出乎意料的是，我们可以证明原核载体序列中存在的 lacZ 基因包含一个 DNA 元件，该元件偶然可以充当 v-myb 依赖性增强子元件，并且 v-myb 蛋白可以在体外与该元件结合。 lacZ 增强子包含 myb 共有 DNA 结合位点 YAAC(G/T)G。
In order to define the domains of the v-myb protein that are important for transactivation of gene expression, we have studied transactivation by the v-myb gene and a set of v-myb deletion mutants using transient transfection assays in NIH 3T3 cells. Analysis of the set of v-myb deletion products demonstrated that a previously unidentified region in the carboxyl-terminal portion of the protein is required for transactivation. This region lies between amino acids 295-356 with respect to the 5' end of the v-myb gene. Switching the v-myb DNA binding domain with the DNA binding domain of the rat glucocorticoid receptor (rGR) switched the cis-element requirement for v-myb action: only reporters containing glucocorticoid response elements were activated by myb-rGR fusion proteins. The carboxyl terminal region essential for transactivation by the intact v-myb gene was also necessary for transactivation by the rGR-fusion gene. Carboxyl-terminal deletion mutations that encompassed the novel transactivation region were able to block wild-type v-myb transactivation when tested in transient co-expression assays. In an unexpected sidelight to our studies, we could demonstrate that the lacZ gene present in the prokaryotic vector sequences contained a DNA element that fortuitously can act as a v-myb-dependent enhancer element, and that v-myb protein can bind to this element in vitro. The lacZ enhancer contains the myb consensus DNA binding site YAAC(G/T)G.