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首页> 外文期刊>Nucleic Acids Research >IDENTIFICATION AND CHARACTERISATION OF TWO TRANSCRIPTIONAL REPRESSOR ELEMENTS WITHIN THE CODING SEQUENCE OF THE SACCHAROMYCES CEREVISIAE HXK2 GENE
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IDENTIFICATION AND CHARACTERISATION OF TWO TRANSCRIPTIONAL REPRESSOR ELEMENTS WITHIN THE CODING SEQUENCE OF THE SACCHAROMYCES CEREVISIAE HXK2 GENE

机译：酿酒酵母HXK2基因编码序列内两个转录阻遏元件的鉴定和表征

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摘要

A well-defined set of isogenic yeast strains has been constructed whereby each strain contains a different HXK2::lacZ gene fusion integrated at the URA3 locus. These HXK2::lacZ fusions differ in the amount of the HXK2 gene (encoding hexokinase 2 isoenzyme) that is fused to the lacZ reporter gene. Comparison of the P-galactosidase activities of each strain during growth on glucose or ethanol revealed that some part of the coding region between +39 and +404 bp is involved in repressing gene expression in a carbon source dependent manner. A series of deletions of this HXK2 coding region were constructed and fused upstream of a minimal CYC1::lacZ promoter. beta-Galactosidase activities on glucose or ethanol growth yeast cells revealed that two different regulatory elements are present in this DNA region. Gel mobility shift analysis and in vitro DNase I footprinting have shown that proteins bind specifically to two downstream repressor sequences (DRS1 located from +140 to +163 and DRS2 located between +231 and +251) that influence the rate of HXK2 transcription when ethanol is used as carbon source by Saccharomyces cerevisiae. We identified and partially purified a 18 kDa protein that binds specifically to synthetic double-stranded oligonucleotides containing the (A/C)(A/G)GAAAT box sequence. Our data suggest that p18 synthesis is under the control of genes involved in glucose repression (MIG1 = CAT4) and glucose derepression (SNF1 = CAT1).
机译：已经构建了一组定义明确的等基因酵母菌株，其中每个菌株都包含整合在URA3基因座上的不同HXK2 :: lacZ基因融合体。这些HXK2 :: lacZ融合体在与lacZ报告基因融合的HXK2基因（编码己糖激酶2同工酶）的数量上有所不同。比较每个菌株在葡萄糖或乙醇上生长期间的P-半乳糖苷酶活性，发现+39和+404 bp之间的编码区的某些部分以碳源依赖性方式参与了基因表达的抑制。该HXK2编码区的一系列缺失被构建并融合在最小CYC1 :: lacZ启动子的上游。葡萄糖或乙醇生长酵母细胞上的β-半乳糖苷酶活性表明，该DNA区域中存在两种不同的调控元件。凝胶迁移率迁移分析和体外DNase I足迹分析表明，当乙醇为乙醇时，蛋白质与两个下游阻遏物序列（位于+140至+163的DRS1和位于+231至+251的DRS2）特异性结合，从而影响HXK2转录的速率。被酿酒酵母用作碳源。我们鉴定并部分纯化了一种18 kDa蛋白，该蛋白与包含（A / C）（A / G）GAAAT盒序列的合成双链寡核苷酸特异性结合。我们的数据表明p18合成受葡萄糖抑制（MIG1 = CAT4）和葡萄糖抑制（SNF1 = CAT1）相关基因的控制。

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