Phase variation of gonococcal protein II: Regulation of gene expression by slipped-strand mispairing of a repetitive DNA sequence

Expression of outer membrane protein II (P.II) of Neisseria gonorrhoeae is subject to reversible phase variation at a rate of 10-3-10-4/cell/generation. The signal peptide coding regions of P.II genes contain variable numbers of tandem repeats of the sequence CTCTT. Changes in the number of CTCTT units, leading to frameshifting within the gene, are responsible for changes in P.II expression. Phase variation mediated by the CTCTT repeat also occurred in E. coli, as assayed with a P.II-alkaline phosphatase (phoA) gene fusion. Phase variation in both the gonococcus and E. coli was recA-independent, occurred at similar rates, and involved insertions or deletions of one or more repeat units. The characteristics of the phase variation process were consistent with a model in which expression of P.II genes is regulated by slipped-strand mispairing of the DNA in the CTCTT repeat region.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28047/1/0000486.pd

Improving Salmonella vector with rec mutation to stabilize the DNA cargoes

<p>Abstract</p> <p>Background</p> <p><it>Salmonella </it>has been employed to deliver therapeutic molecules against cancer and infectious diseases. As the carrier for target gene(s), the cargo plasmid should be stable in the bacterial vector. Plasmid recombination has been reduced in <it>E. coli </it>by mutating several genes including the <it>recA</it>, <it>recE</it>, <it>recF </it>and <it>recJ</it>. However, to our knowledge, there have been no published studies of the effect of these or any other genes that play a role in plasmid recombination in <it>Salmonella enterica</it>.</p> <p>Results</p> <p>The effect of <it>recA</it>, <it>recF </it>and <it>recJ </it>deletions on DNA recombination was examined in three serotypes of <it>Salmonella enterica</it>. We found that (1) intraplasmid recombination between direct duplications was RecF-independent in Typhimurium and Paratyphi A, but could be significantly reduced in Typhi by a Δ<it>recA </it>or Δ<it>recF </it>mutation; (2) in all three <it>Salmonella </it>serotypes, both Δ<it>recA </it>and Δ<it>recF </it>mutations reduced intraplasmid recombination when a 1041 bp intervening sequence was present between the duplications; (3) Δ<it>recA </it>and Δ<it>recF </it>mutations resulted in lower frequencies of interplasmid recombination in Typhimurium and Paratyphi A, but not in Typhi; (4) in some cases, a Δ<it>recJ </it>mutation could reduce plasmid recombination but was less effective than Δ<it>recA </it>and Δ<it>recF </it>mutations. We also examined chromosome-related recombination. The frequencies of intrachromosomal recombination and plasmid integration into the chromosome were 2 and 3 logs lower than plasmid recombination frequencies in Rec⁺strains. A Δ<it>recA </it>mutation reduced both intrachromosomal recombination and plasmid integration frequencies.</p> <p>Conclusions</p> <p>The Δ<it>recA </it>and Δ<it>recF </it>mutations can reduce plasmid recombination frequencies in <it>Salmonella enterica</it>, but the effect can vary between serovars. This information will be useful for developing <it>Salmonella </it>delivery vectors able to stably maintain plasmid cargoes for vaccine development and gene therapy.</p

Novel lambdoid cloning vectors.

The design of the general-purpose lambda cloning vector is analysed to determine several specific areas of usefulness and ideas are developed to improve the usefulness of lambda vectors in four of these areas: ease of use, cloning capacity, selection of recombinants and recombinational stability of cloned sequences. To make a vector which is easier to use, a derivative of EMBL4 which can be used to make recombinant phage capable of forming lysogens in a special host is constructed and tested to determine if high titre phage lysates can be prepared by thermo-induction of such a lysogen. To increase the cloning capacity of lambda vectors a special host lysogen capable of providing all the lambda late gene products is constructed and tested. Growth on this special lysogen allows the replaceable region of the phage vector to be extended through the left arm to the B gene giving a vector with 35 kilobases of capacity. To improve the selection of recombinant phage, a novel central fragment counterselection using the Tro phenotype, and which will work in the absence of host recombination systems, is designed and tested. During investigations of recombination-deficient host strains a novel, rec-independent recombination system is defined in E. coli.

The nucleotide sequence of the amiE gene of Pseudomonas aeruginosa.

The nucleotide sequence of the amiE gene, encoding the aliphatic amidase of Pseudomonas aeruginosa, has been determined. The sequence of 1038 nucleotides shows a strong bias in favour of codons with G or C in the third position, and only 44 different codons are utilised