Amplification of an IS1-Flanked Unit in Escherichia coli K12

Clugston, Carol Keir (1986) Amplification of an IS1-Flanked Unit in Escherichia coli K12. PhD thesis, University of Glasgow.

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The rearrangement associated with US Cut mutants of E. coli K12 was shown to be the tandem amplification of an IS1-flanked unit, Tn2901, which contains the argF gene. The copy number of Tn2901 in Cut1, a US Cut mutant, was estimated to be 45. A single IS1 element was shown to be present at the novel joint between each amplified unit, indicating that IS1-IS1 recombination is responsible at least for the initial duplication of Tn2901. The amplified unit was cloned as two overlapping restriction fragments: an 11.7kb EcoR1 fragment and a 9.4kb Bg1ll fragment in pUC8/9. The latter fragment expresses the argF gene; the former does not. Protein analysis indicated that the argF gene product is greatly over-expressed in US Cut mutants. Preliminary northern analysis detected only one transcript (of approximately 2.4kb) from the 11 kb chromosomal DNA carried by Tn2901 other than a possible argF transcript. Therefore, at least 6kb of Tn2901 is not accounted for in terms of RNA transcripts. The F factor is required in cis for the initial IS1-IS1 recombination event. Two possible functions of the F factor were examined: an F factor IS-mediated rearrangement, and conjugal transfer. Southern hybridization of chromosomal digests to radiolabelled plasmids containing IS3, IS2 and gamma delta indicated that a major rearrangement involving the F factor IS elements had not taken place. The possibility of a minor rearrangement was not ruled out. Conjugal transfer was examined by the use of sodium dodecyl sulphate which depolymerises F pili filaments, and by the construction of a transfer-deficient mutant. The results indicated that conjugal transfer is not required for the amplification of Tn2901. A technique was developed for the directed integration of a plasmid vector into the bacterial chromosome. Using this technique, the CAT gene was inserted in Tn2901, producing a strain with which unamplified and amplified colonies can be distinguished by plating on minimal agar containing crystal violet. In the future, the directed integration technique will be of immense value in the replacement of wild-type chromosomal DNA segments with in vitro mutagenized sequences. This should allow the localization of the function provided by the F factor and the determination of whether an IS1 gene product is involved in the initial IS1-IS1 recombination event. A chromosomal gene bank was constructed from which a clone was isolated containing the right hand IS1 element of Tn2901 and approximately 2kb of adjacent chromosomal DNA. This clone will allow the in vitro construction of mutants of the right hand IS1 element of Tn2901 and their insertion into the bacterial chromosome in place of the wild-type IS1 sequence. The clone will also permit the isolation of overlapping clones from the gene bank extending up to the F factor, and these clones will be of value in the directed integration technique.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Genetics
Date of Award: 1986
Depositing User: Enlighten Team
Unique ID: glathesis:1986-77474
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jan 2020 09:07
Last Modified: 14 Jan 2020 09:07

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