MECHANISM OF INSERTION SEQUENCE TRANSLOCATION

可转座的DNA元件能够从一个染色体位置移动到 另一种独立于正常同源重组机制。他们打球 在细菌进化中的重要作用，并可能代表更多 参与位点特异性基因重排的一类通用元件 原核生物中基因表达的扩增及其可能的调控 真核生物。 我建议研究位置特异性和区域非依赖性的机制。 卡那霉素抗性转座子Tn903和 与其关联的插入元素IS903。Tn903由1000 b.p。独一无二 序列(包含卡那霉素抗性基因)两侧有两个拷贝 1050桶。反方向的序列。我们已经证明了1050 b.p。 序列具有插入序列的性质，并提出它是 名为IS903。通过插入10 b.p。DNA片段进入特定的限制条件 我们已经构建了IS903内的核酸内切酶切割位点的突变体 更长的中间转位。我们还测定了核苷酸序列 IS903。 建议鉴定IS903编码的蛋白质(S)是ITS所必需的 转座(A)通过检测野生型和突变体产生的蛋白质 使用Sancar等人的最大细胞技术的IS903(J.Bact.137,692， (1979))和(B)通过编程依赖DNA的蛋白质合成系统 含有野生型或突变型IS903的质粒的DNA。一旦蛋白质(S) 我想对它进行提纯和定性。要做到这一点，它可能会 有必要通过将基因融合到有效的基因来扩大其产量 促进剂，如Lac或PL。最初的特征将包括检查 用于序列特异性DNA结合或核酸内切酶活性。

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.