Cole1, cer site-specific recombination

Stewart, Gillian (1986) Cole1, cer site-specific recombination. PhD thesis, University of Glasgow.

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The multicopy, naturally occurring plasmid ColE1 is maintained stably under normal growth conditions, whereas plasmid cloning vectors related to it are relatively unstable. Summers and Sherratt (1984) presented evidence suggesting that ColE1 is partitioned randomly at cell division and that plasmid stability is inversely correlated with plasmid multimerization. Wild type ColE1 is stable as it utilizes a site-specific recombination system to breakdown multimers formed by homologous recombination. This increases the stability of ColE1 by maximising the number of independently segregating units. The ColE1 site-specific recombination system utilizes a site cer, contained in a 238bp fragment (3731-3969bp) and a recombinase xer, which is probably host encoded. To investigate if the recombinase acting at cer' was host encoded, mutants affecting cer-specific recombination were sought. A total of ten Tn5::xer mutants have been isolated, only three of which have been studied in any detail. These three host mutants are called DSX. 1, DSX. 2 and DSX. 300. DSX. 2 and probably DSX. 1 are spontaneous mutants, whereas DSX. 330 is a Tn5::xer mutant. These xer mutants have been used to demonstate: (i) a correlation between multimerization and instability for ColE1, (ii) that ColE1 cannot supply in trans any of the functions absent in the xer mutants and (iii) that the xer functions are used in the multimer resolution systems of the high copy number plasmids ColK, CloDF13, and perhaps ColE2 and CoIE3. The xerA gene mutant in DSX. 330 has been cloned and sequenced. Sequence analysis of the minimum complementing clone reveals an ORF, which would encode for a polypeptide of 17kd, whose presence has been confirmed using whole cell protein extracts and minicells. The xerA plasmid clone complements the Xer- phenotype of DSX. 300 but not that of DSX. 1 and 2, defining at least two host complementation groups necessary for cer-specific recombination. Classical genetic techniques were then used to determine the genetic map position of the xer genes. The data indicates that xerA mutant in DSX. 300 lies between 82 and 87mins, whereas the xer 1and 2 gene(s) mutant in DSX. 1 and 2 lie between 87 and 91mins. One of these xer genes is probably the recombinase, whereas the other may be a DNA binding protein, involved in the formation of higher order protein-DNA structures or in DNA bending.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Genetics
Date of Award: 1986
Depositing User: Enlighten Team
Unique ID: glathesis:1986-76627
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 14:01
Last Modified: 19 Nov 2019 14:01

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