Wright C F, Zitomer R S
Mol Cell Biol. 1985 Nov;5(11):2951-8. doi: 10.1128/mcb.5.11.2951-2958.1985.
The transcription of the CYC7 gene of Saccharomyces cerevisiae, encoding the iso-2-cytochrome c protein, is controlled by two upstream regulatory elements, a positive element and a negative element. The nature of the DNA sequences in the negative element were investigated in a two-part approach. The first involved the construction of a CYC7-galK fusion gene which placed the coding sequence of the Escherichia coli galactokinase gene under the regulation of the CYC7 upstream sequences. This fusion allowed the quantitation by galactokinase enzyme assays of the effects on gene expression of a variety of previously isolated deletion mutations within the negative site. The results suggested that the negative site contained three related sequences. This hypothesis was tested in the second part of these studies, the selection of point mutations within the region of the negative site which led to increased CYC7 expression. Point mutations were introduced by a technique which induced mutations within a localized region at high efficiency. All but one of the mutations involved more than a single base-pair change. The mutations followed the pattern that multiple base-pair changes occurred in one repeat or single base-pair changes occurred in two repeats, with the exception of one mutant, which had a single base-pair change in one repeat. This pattern of mutations and the base pairs that were altered strongly supported the hypothesis that the repeats are integral elements of the negative site.
酿酒酵母CYC7基因编码同工-2-细胞色素c蛋白，其转录受两个上游调控元件控制，一个正调控元件和一个负调控元件。采用两部分方法研究了负调控元件中DNA序列的性质。第一部分涉及构建CYC7-galK融合基因，该基因将大肠杆菌半乳糖激酶基因的编码序列置于CYC7上游序列的调控之下。这种融合使得通过半乳糖激酶酶活性测定法能够定量分析负调控位点内各种先前分离的缺失突变对基因表达的影响。结果表明，负调控位点包含三个相关序列。在这些研究的第二部分中对这一假设进行了检验，即在负调控位点区域内选择导致CYC7表达增加的点突变。通过一种能在局部区域高效诱导突变的技术引入点突变。除一个突变外，所有突变都涉及不止一个碱基对的改变。这些突变遵循这样一种模式：在一个重复序列中发生多个碱基对的改变，或者在两个重复序列中发生单个碱基对的改变，但有一个突变体除外，它在一个重复序列中有一个碱基对的改变。这种突变模式以及发生改变的碱基对有力地支持了这样一种假设，即这些重复序列是负调控位点的组成元件。
