MECHANISM OF INSERTION SEQUENCE TRANSLOCATION

点击翻译按钮获取中文摘要

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.