Cloning and Characterisation of E. coli xer Genes

Stirling, Colin John (1987) Cloning and Characterisation of E. coli xer Genes. PhD thesis, University of Glasgow.

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The heritable stability of the natural multicopy plasmid, ColE1, can be greatly reduced by the formation of plasmid multimers via homologous recombination. Wild-type ColE1 carries a 233bp site, cer. which when present in direct repeat (as is the case in a ColE1 dimer), acts as a substrate for a site-specific recombinase which efficiently resolves plasmid multimers to monomers. This monomerisation function is strongly correlated with an increase in plasmid stability. Current evidence suggests that ColE1 segregates randomly, and that cer-specific monomerisation serves to enhance stability by maximising the number of independently segregating plasmid copies (Sherratt et al, 1984; Summers & Sherratt, 1984). The 233bp cer site represents the only ColE1 sequence necessary for cer-specific recombination, however the available evidence makes it appear highly improbable that this locus also encodes the cer-specific recombinase; on this assumption the recombinase would require to be host encoded. A procedure was devised for the selection of E. coli K12 mutants defective in cer-specific recombination (Xer-). Using this procedure a total of 11 independent xer mutants have been isolated from Tn5-mutagenised populations; of the 11 xer lesions, six are genetically linked to an inserted copy of Tn5, whilst two appear to be unlinked spontaneous mutations, the remaining 3 mutants are uncharacterised. This collection of mutants has been employed to clone the wild-type xer genes by genetic complementation. This has revealed that the 11 mutants fall into two complementation groups; two mutants are complemented by the cloned xerA gene, whilst the remaining nine are complemented by a clone of the xerB gene. High resolution mapping of the xerA and xerB loci indicates that they are quite discrete, mapping to 70.5 and 96.5 minutes respectively on the E. coli K12 linkage map. The xerA gene was delineated to within a 920bp Sphl/AccI fragment, and the DNA sequence of this fragment determined. Sequence analyses revealed the presence of a 156 codon open reading frame (ORF) which would encode a polypeptide with a predicted molecular weight equivalent to 17.2KDa; the expression of this ORF has been confirmed in mini-cell analyses. This ORF has been shown to be disrupted by the insertion of Tn5 in the mutant allele xerA3 which, coupled with a deletion analysis of the locus, provides convincing evidence that this 156 codon ORF must represent the xerA gene. The xerB locus (a 1.92 Kb Hindlll fragment) has also been sequenced and the xerB gene identified as a 503 codon open reading frame which encodes a polypeptide with a predicted molecular weight equal to 55.3KDa. Interestingly there are several features of the DNA sequence flanking the xerB gene which indicate that it may be transcribed as part of a polycistronic mRNA, with at least one other gene immediately downstream of xerB: whether or not this downstream gene encodes an Xer-related function remains to be determined. The XerB protein has also been visualised in minicell expression studies, and has been over-expressed in whole cells to a level of 5% of total cell protein. The XerB protein has been purified to homogeneity and its identity confirmed by N-terminal peptide sequencing. This purified material has been used to raise anti-XerB antisera which might now be employed to investigate several features of XerB including its expression, cellular location(s), and function(s). The sequences of the xerA and xerB genes (and their products), coupled to the genetic map positions of both loci, indicate that we have identified two entirely novel E. coli functions involved in site-specific recombination at the ColE1 cer locus. The precise role(s) the two proteins play in cer-specific recombination, and in the biology of E. coli remain to be determined.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Genetics
Date of Award: 1987
Depositing User: Enlighten Team
Unique ID: glathesis:1987-76667
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 13:56
Last Modified: 19 Nov 2019 13:56

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