Distinct sequence elements involved in the glucocorticoid regulation of the mouse mammary tumor virus promoter identified by linker scanning mutagenesis.
Distinct sequence elements involved in the glucocorticoid regulation of the mouse mammary tumor virus promoter identified by linker scanning mutagenesis.
复制标题
通过接头扫描诱变鉴定出参与小鼠乳腺肿瘤病毒启动子糖皮质激素调节的独特序列元件。
DOI：
10.1016/0022-2836(86)90009-4
复制
发表时间：
1986
影响因子：
5.6
通讯作者：
B. Kühnel
中科院分区：
文献类型：
--
作者：
E. Buetti;B. Kühnel
在小鼠乳腺肿瘤病毒（MMTV）的前病毒DNA中，需要从RNA起始位点起长达200个碱基对的序列来刺激糖皮质激素在培养细胞中的转录。共构建了26个在MMTV长末端重复序列的激素控制区具有成簇点突变或小缺失的突变质粒，将其连接到单纯疱疹病毒胸苷激酶基因的编码部分，并通过转染引入LTK−细胞。通过S1核酸酶保护试验定量测定在激素存在或不存在下从突变体DNA的转录。我们的分析揭示了至少三个控制元件的存在，影响糖皮质激素的转录刺激的程度：（1）远端元件，在RNA起始位点的-181和-172碱基对之间。该区段中的接头扫描突变体相对于野生型在激素应答方面具有高达20倍的降低。(2)-120位附近的一个元素，由-121和-117之间的4个碱基对突变定义，导致五倍减少。(3)一个从10 - 78到-70的元素，由一个突变体定义，其刺激性也大约低五倍。前两个包括在已被其他人证明与受体复合物体外相互作用的区域中;最后一个与核蛋白因子的体外结合位点重叠。缺乏所有三个元件（-193至-70）的突变体完全不能被糖皮质激素诱导。与早期5′缺失突变体的结果一起，数据表明，对刺激反应的最大贡献是由远端元件做出的，然而，这确实需要两个更近端元件的存在才能使反应最大。在缺少远端元件的情况下，两个近端元件一起产生约为野生型的5%至10%的残余刺激，而单独的-70元件是无效的。此外，我们表明，一个功能性的TATA同源性是需要最大的刺激。糖皮质激素对MMTV的转录调控是通过多个序列模块的协同作用实现的，但并非所有序列模块都与体外受体结合位点相对应。
In the proviral DNA of mouse mammary tumor virus (MMTV), sequences up to ≈ 200 base-pairs from the RNA start site are required for stimulation of transcription by glucocorticoid hormones in cultured cells. A total of 26 mutant plasmids with clustered point mutations or small deletions in the hormone control region of the MMTV long terminal repeat were constructed, linked to the coding portion of theHerpes simplexvirus thymidine kinase gene, and introduced by transfection into LTK−cells. Transcription from mutant DNA in the presence or absence of hormone was quantified by S1nuclease protection assays. Our analysis revealed the presence of at least three control elements that affect the extent of transcription stimulation by glucocorticoid hormones: (1) a distal element, between − 181 and − 172 base-pairs from the RNA initiation site. Linker scanning mutants in this segment have a reduction of up to 20-fold in the hormone response with respect to wild type. (2) An element around position − 120, defined by a mutation of 4 base-pairs between − 121 and − 117, which causes a fivefold reduction. (3) An element from ≈ − 78 to − 70, defined by a mutant with also a roughly fivefold lower stimulation. The first two are included in areas that have been shown by others to interactin vitrowith hormone-receptor complexes; the last one overlaps thein vitrobinding site of a nuclear protein factor. A mutant lacking all three elements (− 193 to − 70) is completely non-inducible by glucocorticoids. Together with earlier results obtained with 5′ deletion mutants, the data show that the largest contribution to the stimulatory response is made by the distal element, which however does require the presence of both more-proximal ones for the response to be maximal. In the absence of the distal one, the two proximal elements together produce a residual stimulation in the order of 5 to 10% of wild type, while the − 70 element alone is ineffective. In addition, we show that a functional TATA homology is required for maximum stimulation. It appears that transcriptional regulation of MMTV by glucocorticoid hormones is achieved by the concerted action of multiple sequence modules, not all of which correspond to receptor binding sitesin vitro.