A genetic analysis of the influence of Escherichia coli host factors on Tn5 transposition and excision.
Item
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Title
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A genetic analysis of the influence of Escherichia coli host factors on Tn5 transposition and excision.
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Identifier
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AAI8820863
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identifier
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8820863
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Creator
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Fuchs, Trudy.
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Contributor
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Adviser: Sharon D. Cosloy
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Date
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1988
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Microbiology
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Abstract
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The role of E. coli general recombination pathways and their components were examined for their possible influence on transposition and excision of Tn5. Strains were infected with a lysogeny defective derivative of {dollar}\lambda{dollar}, which harbors Tn5, and transposition of Tn5 was measured by selection with kanamycin. A RecF strain was observed to have 10-36 times higher insertion frequencies than strains utilizing other general recombination pathways. The effects of ExoV and ExoVIII were equivalent with respect to their individual impact on insertion, however a strain with both ExoV and ExoVIII (RecBC/E pathway), had significantly higher insertion frequencies than a strain in which both were absent (Rec{dollar}\sp-{dollar}). The frequency of insertion in a Rec{dollar}\sp-{dollar} strain was recF dependent. Although ExoV does not seem to be involved in insertion because RecBC and Rec{dollar}\sp-{dollar} strains had equivalent insertion frequencies, the presence of ExoV influenced insertion frequencies in recF mutants.;Precise excision was studied in strains utilizing different recombination pathways harboring Tn5 in the lacZ gene on an F{dollar}\sp\prime {lcub}lac{rcub}{dollar} plasmid. In addition, thyA and polA mutants with the same plasmid were studied. A precise excision was signalled by the formation of a red pigmented papilla on a colony grown on MacConkey agar. Papillae appeared only after 48 hours. The host factors found to affect excision were the RecF recombination pathway, DNA Polymerase I and the concentration of deoxythymidylate. A RecF strain was reduced in precise excision by two orders of magnitude and precise excision was found to be inhibited by five orders in thyA mutants. Excision was also reduced in polA mutants, and the degree of inhibition depended on the particular mutant allele. Excision frequencies were depressed at 30{dollar}\sp\circ{dollar} as compared to 37{dollar}\sp\circ{dollar} and 42{dollar}\sp\circ{dollar}. This was due to the fact that at 30{dollar}\sp\circ{dollar} the cells reached maturity at a later time than at other temperatures. We concluded that precise excisions occur in mature colonies during repair synthesis mediated by DNA Polymerase I.;The use of thyA hosts for insertional mutagenesis using transposable elements or the addition of fluorouracil to the medium can stabilize such mutations when excision can result in reversion or loss of the mutant. (Abstract shortened with permission of author.).
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
