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puc启动子上游DNA二元对称序列突变对Rhodobacter sphaeroides 氧调节的影响

吴永强, Kaplan S

中科院上海植物生理研究所,上海 200032；德克萨斯大学健康科学中心,休斯顿 77225

摘要

为了鉴定pucBA基因表达受氧调控的顺式调节位点,通过PCR和多聚核苷酸定点突变的体外操作,在puc转录子5^上游非编码区产生了7个不同突变和5个不同10bp缺失序列。构建了含有各种顺式突变的puc上游区。启动子和报告基因lacZ的转录融合子。通过融合子#beta#-半乳糖苷酶活性分析，发现位于puc启动子上游二元对称结构的突变使得puc基因在有氧条件下去阻遏表达。IHF束缚位点的突变可使#beta#-半乳糖苷酶活性提高。

关键词： 氧调节; puc操纵子; 二元对称序列; 突变

The mutation in DNA sequences with dyad symmetry upstream of the puc promoter affects oxygen regulation of the puc operon(s) in Rhodobacter sphaeroides

Wu Yongqiang, Kaplan S

Shanghai Institute of Plant Physiology, The Chinese Academy of Sciences, Shanghai 200032

Abstract

Seven mutations and five 10-bp deletions were constructed in the 5^ noncoding region of the puc operon by in vitro manipulation of cloned puc upstream sequence with a modified Polymerase Chain Reaction (PCR) and oligonucleotide-directed mutagenesis.Transcriptional fusions of the puc upstream region containing different cis-mutations or 10-bp deletions were constructed by using the reporter molecule encoded by lacZ.The role of the target site controlling puc expression was investigated by analysing the effects of the mutations and deletions on the #bata#-galactosidase (#bata#-gal) activity resulting from the fusions when present in wild type R. sphaeroides 241 grown chemoheterotrophically and fermentatively. #bata#-gal activity of PCF611 with A to G transition mutation at-12 within a region of dyad symmetry was about 7.8 foldthat of the wild type fusion construct in the presence of O_2.The puc upstream sequence of PCFK carrying three mutations at -197,-193 and 190 between positions positions-199 to -185 within another dyad symmetry GCAGCN_5GCTGC produced a #bata#-gal activity 6.8 times as high as wild type activity.Binding of IHF is believed to induce a DNA bend which was proposed to faciliated interation between the O_(2)-regulatory repressor and the two regions of dyad symmetry.All these fusions showed almost the same level of lacZ gene expression under anaerobic condition.

Key words: Oxygen regulation;Puc operon;Dyad symmetry;Mutation

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引用本文：

吴永强, Kaplan S. puc启动子上游DNA二元对称序列突变对Rhodobacter sphaeroides 氧调节的影响[J]. 生理学报 1996; 48 (2): .

Wu Yongqiang, Kaplan S. The mutation in DNA sequences with dyad symmetry upstream of the puc promoter affects oxygen regulation of the puc operon(s) in Rhodobacter sphaeroides. Acta Physiol Sin 1996; 48 (2): (in Chinese with English abstract).