在大肠杆菌中筛选Tn10四环素阻遏物与四环素操纵子的结合：温度敏感突变体的分离及DNA结合基序中的组合诱变
Selection for Tn10 tet repressor binding to tet operator in Escherichia coli: isolation of temperature-sensitive mutants and combinatorial mutagenesis in the DNA binding motif.
作者信息
Wissmann A, Wray L V, Somaggio U, Baumeister R, Geissendörfer M, Hillen W
机构信息
Lehrstuhl für Mikrobiologie, Friedrich Alexander Universität Erlangen/Nürnberg, Federal Republic of Germany.
出版信息
Genetics. 1991 Jun;128(2):225-32. doi: 10.1093/genetics/128.2.225.
Abstract
We have constructed a genetic assay which selects positively for a functional interaction between Tet repressor and its cognate operator in Escherichia coli. In this strain Tet repressor blocks expression of lacI and lacZ. This leads to derepression of a lacPO controlled galK gene. The strain can be selected by growth on galactose as the sole carbon source and screened for the beta-galactosidase phenotype. These features allow the identification of one candidate among 10(8) false clones on a single plate. The assay was applied to select mutants with a ts DNA binding phenotype and to screen oligonucleotide generated Tet repressor mutants. Analysis of these mutations revealed that they affect DNA and inducer binding and possibly the dimerization domains. These mutations are located at residues 21, 48, 49, 89 and at the C terminus of the protein (193), respectively.
摘要
我们构建了一种遗传检测方法，该方法能在大肠杆菌中对四环素阻遏物与其同源操纵基因之间的功能性相互作用进行正向选择。在这个菌株中，四环素阻遏物会阻断lacI和lacZ的表达。这会导致由lacPO控制的galK基因去阻遏。该菌株可以通过在以半乳糖作为唯一碳源的培养基上生长来进行选择，并针对β - 半乳糖苷酶表型进行筛选。这些特性使得在单个平板上能够从10⁸个假克隆中鉴定出一个候选克隆。该检测方法被用于筛选具有温度敏感型DNA结合表型的突变体以及对寡核苷酸产生的四环素阻遏物突变体进行筛选。对这些突变的分析表明，它们影响DNA和诱导剂结合，并且可能影响二聚化结构域。这些突变分别位于蛋白质的第21、48、49、89位残基以及蛋白质的C末端（193位）。
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本文引用的文献
1
High-frequency generalised transduction by bacteriophage T4.噬菌体T4介导的高频广义转导
Nature. 1979 Jul 5;280(5717):80-2. doi: 10.1038/280080a0.
2
THE PROPERTIES OF REPRESSOR AND THE KINETICS OF ITS ACTION.阻遏物的特性及其作用动力学
J Mol Biol. 1965 Jun;12:305-27. doi: 10.1016/s0022-2836(65)80255-8.
3
Nucleotide sequence of the repressor gene of the TN10 tetracycline resistance determinant.TN10四环素抗性决定因子阻遏基因的核苷酸序列。
Nucleic Acids Res. 1984 Jun 25;12(12):4849-63. doi: 10.1093/nar/12.12.4849.
4
Construction of a single-copy promoter vector and its use in analysis of regulation of the transposon Tn10 tetracycline resistance determinant.单拷贝启动子载体的构建及其在转座子Tn10四环素抗性决定因子调控分析中的应用。
J Bacteriol. 1984 Jun;158(3):910-9. doi: 10.1128/jb.158.3.910-919.1984.
5
A perfectly symmetric lac operator binds the lac repressor very tightly.一个完全对称的乳糖操纵子紧密结合乳糖阻遏物。
Proc Natl Acad Sci U S A. 1983 Nov;80(22):6785-9. doi: 10.1073/pnas.80.22.6785.
6
Identification of repressor binding sites controlling expression of tetracycline resistance encoded by Tn10.鉴定控制Tn10编码的四环素抗性表达的阻遏物结合位点。
J Bacteriol. 1983 Dec;156(3):1188-91. doi: 10.1128/jb.156.3.1188-1191.1983.
7
Control of expression of the Tn10-encoded tetracycline resistance genes. Equilibrium and kinetic investigation of the regulatory reactions.Tn10编码的四环素抗性基因表达的调控。调控反应的平衡和动力学研究。
J Mol Biol. 1983 Sep 25;169(3):707-21. doi: 10.1016/s0022-2836(83)80166-1.
8
Overlapping divergent promoters control expression of Tn10 tetracycline resistance.重叠的反向启动子控制Tn10四环素抗性的表达。
Gene. 1983 Aug;23(2):149-56. doi: 10.1016/0378-1119(83)90046-x.
9
pUR 250 allows rapid chemical sequencing of both DNA strands of its inserts.pUR 250可对其插入片段的两条DNA链进行快速化学测序。
Nucleic Acids Res. 1982 Oct 11;10(19):5765-72. doi: 10.1093/nar/10.19.5765.
10
A multifunctional gene (tetR) controls Tn10-encoded tetracycline resistance.一个多功能基因（tetR）控制着Tn10编码的四环素抗性。
J Bacteriol. 1982 May;150(2):633-42. doi: 10.1128/jb.150.2.633-642.1982.
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