MECHANISM OF INSERTION SEQUENCE TRANSLOCATION

插入序列易位机制

• 批准号: 3275752
• 负责人: NIGEL David GRINDLEY
• 金额: $ 20.5万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-05-01 至 1986-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3275752
• 关键词: Escherichia coli; bacterial genetics; chromosome translocation; gene expression; genetic manipulation; genetic regulation; mutant

Transposable DNA elements have the ability to move from one chromosal site to another independent of normal homologous recombinational mechanisms. They play an important role in bacterial evolution and may be representative of a more universal class of elements involved in site-specific gene rearrangements and ammplification and perhaps control of gene expression in both prokaryotes and eukaryotes. I propose to study the mechanism of site-specific and recA-independent recombination events mediated by the kanamycin resistance transposon, Tn903, and its associated insertion element, IS903. Tn903 consists of a 1000 b.p. unique sequence (that contains the kanamycin resistance gene) flanked by two copies of a 1050 b.p. sequence in inverted orientation. We have shown that the 1050 b.p. sequence has the properties of an insertion sequence and propose that it be called IS903. By inserting a 10 b.p. DNA fragemtn into specific restriction endonuclease cleavage sites within IS903 we have constructed mutants that no longer mediate transposition. We have also determined the nucleotide sequence of IS903. Is propose to identify the protein(s) encoded by IS903 that is required for its transpostition (a) by examining proteins made from the wild type and mutant IS903 using the maxi-cell technique of Sancar et al., (J.Bact. 137, 692, (1979)) and (b) by programming a DNA-dependent protein synthesizing system with DNA of plasmids containing wild type or mutant IS903. Once the protein(s) is identified I intend to purify and characterize it. To do this it will probably be necessary to amplify its production by fusing the gene to an efficient promoter such as lac or PL. Initial characterization will include examination for sequence specific DNA binding or endonuclease activies.

转座的DNA元件具有从一个铬合成部位移动到 另一个独立于正常同源重组机制。 他们玩 在细菌进化中的重要作用，可以代表更多 参与特定地点基因重排的通用元素和 在原核生物和 真核生物。 我建议研究现场特异性和无依赖的机制 卡纳米霉素抗性转座子介导的重组事件，TN903和 它相关的插入元素，IS903。 TN903由公元前1000年组成。独特的 序列（包含卡纳霉素抗性基因），两侧是两个副本 公元前1050年倒置方向的序列。 我们已经证明了公元1050年。 序列具有插入序列的特性，并提出了它是 称为IS903。 通过插入10 B.P. DNA Fragemtn陷入特定限制 IS903内的核酸内切酶切割位点我们已经构造了突变体 较长的介导转位。 我们还确定了核苷酸序列 IS903。 建议识别由IS903编码的蛋白质所需的蛋白质 通过检查由野生型和突变体制成的蛋白质的转换（a） IS903使用Sancar等人的Maxi-Cell技术（J.Bact。137，692， （1979））和（b）通过对DNA依赖性蛋白质合成系统进行编程 含有野生型或突变体IS903的质粒的DNA。 一旦蛋白质是 确定我打算净化和表征它。 为此，可能会 通过将基因融合到有效的效率来扩增其产量 启动子，例如lac或pl。 最初的表征将包括检查 用于序列特异性DNA结合或核酸内切酶的激活。