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R G Lloyd

Publications and source records attributed to R G Lloyd.

At least 91 records · Page 5Linked to original sources
PubMed1985

Analysis of the ruv locus of Escherichia coli K-12 and identification of the gene product.

The ruv gene of Escherichia coli, which is associated with inducible mechanisms of DNA repair and recombination, has been cloned into the low-copy plasmid vector pHSG415. The recombinant plasmid pPVA101 fully complements the DNA repair-deficient phenotype of ruv mutants. Restriction endonuclease analysis of this plasmid revealed a 10.6-kilobase (kb) HindIII DNA insert which contained a 7.7-kb PstI fragment identified as being from the chromosomal ruv region. Deletion analysis and Tn1000 insertional inactivation of ruv function located the ruv coding region to a 2.2-kb section of the cloned DNA fragment. A comparison of the proteins encoded by ruv wild-type and mutant plasmids identified the gene product as a protein of molecular weight 41,000.

Bacterial Proteins↗
PubMed1985

Identification and genetic analysis of sbcC mutations in commonly used recBC sbcB strains of Escherichia coli K-12.

Evidence is presented to show that Escherichia coli JC7618, JC7621, and JC7623, previously regarded as having a recB recC sbcB genotype, carry an additional mutation in a new gene designated sbcC at minute 9 on the standard genetic map. In the absence of the sbcC mutation these strains are sensitive to mitomycin C and have a reduced efficiency of recombination. Cultures of recBC sbcB (sbcC+) strains grow slowly, contain many inviable cells, and rapidly accumulate fast-growing variants due to mutation of sbcC. sbcC has been identified on recombinant plasmids and tentatively located by Tn1000 mutagenesis to a 0.9-kilobase DNA section between proC and phoR.

Chromosome Deletion↗
PubMed1984

A molecular model for conjugational recombination in Escherichia coli K12.

Conjugational recombination in Escherichia coli was investigated by measuring lacZ+ product, beta-galactosidase, in crosses between lacZ mutants. Enzyme production in both Hfr and F-prime crosses was detected very soon after transfer of the donor lacZ allele. The level of enzyme activity was reduced by no more than two-fold when the recipient carried a recB mutation. With an F-prime donor, recombination appeared to be restricted largely to a short period immediately after transfer, with little evidence of recombination during subsequent exponential growth of the transconjugant cells. These observations are interpreted to suggest that recA dependent recombination is able to initiate with high efficiency at gaps present in the donor DNA before synthesis of a complementary strand is completed, and independently of recB function. A molecular model for conjugational recombination based on this idea is presented in terms of the known activities of recA and recBC products. Some of the predictions of the model are tested by analysing the recombinant genotypes produced in Hfr crosses with multiply marked strains.

Bacterial Proteins↗
PubMed1984

Repair of DNA double-strand breaks in Escherichia coli K12 requires a functional recN product.

Mutation of the recN gene of Escherichia coli in a recBC sbcB genetic background blocks conjugational recombination and confers increased sensitivity to UV light and mitomycin C. The basis for this phenotype was investigated by monitoring the properties associated with recN mutations in otherwise wild-type strains. It was established that recN single mutants are almost fully resistant to UV irradiation, and that there is no detectable defect in repair of UV lesions by excision, error-prone, or recombinational mechanisms. However, recN mutations confer sensitivity to mitomycin C and ionizing radiation both in wild-type and recB sbcB strains. The sensitivity to ionizing radiation is correlated with a deficiency in the capacity to repair DNA double-strand breaks by a UV inducible mechanism. Recombinant lambda phages that complement the recombination and repair defects of recN recBC sbcB mutants have been identified, and the recN gene has been cloned from these phages into a low copy-number plasmid.

Bacterial Proteins↗
PubMed1984

Effect of ruv mutations on recombination and DNA repair in Escherichia coli K12.

Mutation of the ruv gene of E. coli is associated with sensitivity to radiation, and filamentous growth after transient inhibition of DNA synthesis. The filamentation of ruv strains is abolished by mutations in sfiA or sfiB that prevent SOS induced inhibition of cell division, but this does not restore resistance to UV radiation. Double mutants carrying both ruv and uvr mutations are considerably more sensitive to UV radiation than the single mutants, but there is no additive effect of ruv with recA, recF, recB, or recC mutations. ruv mutations have little effect on conjugal recombination in wild-type strains but confer recombination deficiency and extreme sensitivity to ionizing radiation in recBC sbcB strains. These results, together with the fact that ruv strains are excision proficient and mutable by UV light, are interpreted to suggest that the ruv + product is involved in recombinational repair of damaged DNA rather than in cell division as suggested by Otsuji et al. (1974).

Bacterial Proteins↗
PubMed1984

Genetic analysis and molecular cloning of the Escherichia coli ruv gene.

The genetic organisation of the ruv gene, a component of the SOS system for DNA repair and recombination in Escherichia coli, was investigated. New point mutations as well as insertions and deletions were generated using transposon Tn10 inserted in eda as a linked marker for site specific mutagenesis, or directly as a mutagen. The mutations were ordered with respect to one another and previously isolated ruv alleles by means of transductional crosses. The direction of chromosome mobilization from ruv ::Mud( ApRlac ) strains carrying F42lac + established that ruv is transcribed in a counterclockwise direction. Recombinant lambda phages able to restore UV resistance to ruv mutants were identified,and the ruv + region was subcloned into a low copy number plasmid. The ruv + plasmid was able to correct the extreme radiation sensitivity and recombination deficiency of ruv recBC sbcB strains.

Chromosome Mapping↗
PubMed1983

lexA dependent recombination in uvrD strains of Escherichia coli.

Mutation of the uvrD gene of Escherichia coli is associated with an increased capacity for genetic recombination. The hyper-recombination effect is abolished by an additional mutation in lexA that limits synthesis of RecA protein and other gene products regulated by LexA repressor, and is not restored when increased synthesis of RecA protein is facilitated by a recAoc mutation. The viability of uvrD lexA strains is reduced and revertants selected on the basis of improved growth fall into three categories: those that are lexA+, or carry another mutation in lexA that directly suppresses the lexA defect; recA mutants that have lost the capacity for recombination altogether; and a third class which carry a mutation that is not in lexA or recA and which restores the hyper-rec phenotype but does not otherwise suppress the lexA defect. These results indicate that the hyper-recombination effect of a uvrD mutation is an induced response catalysed by RecA protein and at least one other lexA regulated activity.

Bacterial Proteins↗
PubMed1983

On the nature of the RecBC and RecF pathways of conjugal recombination in Escherichia coli.

The molecular mechanisms of the RecBC and RecF pathways for genetic recombination in E. coli were investigated by studying the kinetics of RecA protein function during conjugation. RecF recombination in recBC sbcB mutants is shown to be a much slower process than RecBC recombination in recBC+ sbcB+ strains, and is blocked by a mutation in lexA that prevents induction of RecA protein. Progress of the RecF pathway is greatly accelerated by a recAoc mutation which increases synthesis of RecA protein, but this does not restore recombination proficiency to a recBC sbcB lexA mutant. These results are interpreted to suggest that the RecF pathway directs integration of single-stranded Hfr DNA into the recipient chromosome whereas the RecBC pathway catalyses the exchange of largely double stranded DNA. This is consistent with the known stoichiometry of RecA protein catalysed heteroduplex DNA formation in vitro and with the delayed replication of RecF pathway recombinants which approximates to the time required for one round of DNA replication to generate homoduplex DNA. The regulation of the RecF pathway by lexA repressor is discussed in relation to the factors that govern the relative utilization of the two recombination pathways in wild-type cells.

Bacterial Proteins↗
PubMed1983

Inducible expression of a gene specific to the RecF pathway for recombination in Escherichia coli K12.

The Mud(Aplac) operon fusion technique of Casadaban and Cohen (1979) was used to search for inducible functions specific to the RecF pathway of conjugal recombination. A fusion mutant of a recBC sbcB mutant which showed less than 1% of the normal level of recombination in Hfr crosses has been isolated and designated as rec-259. The mutation is shown to be closely linked to tyrA at approximately 57.5 min in relation to the standard genetic map, and is quite distinct from recA. Two point mutations within this gene have also been obtained. Mutation of this gene interferes specifically with the RecF pathway of recombination, and also causes increased sensitivity to mitomycin C and UV light. Expression of the lac genes in the rec-259 fusion strain is increased following damage to DNA, but not in lexA and recA derivatives. These observations demonstrate the existence of an inducible gene which is regulated by lexA and whose expression is required for RecF recombination and DNA repair.

DNA Repair↗
PubMed1983

Control of recA dependent activities in Escherichia coli: a possible role for the recF product.

DNA repair and genetic recombination have been studied in recF143 mutants of Escherichia coli in which expression of inducible, SOS repair activities was altered by additional mutations either in lexA or recA. recF143 and lexA3 appeared to act additively to increase sensitivity to UV irradiation and to reduce recombination proficiency. The recF defect was suppressed in strains carrying the tif-1 allele of recA but not in strains carrying mutations that increased synthesis of recA+ protein or which directly inactivated lexA repressor. These and other data are interpreted to suggest that the recF defect is related to a reduced activity of recA protein. The implications of these findings are discussed in relation to the control of SOS activities and cell division during normal growth.

Bacterial Proteins↗
PubMed1982

Damage to DNA induces expression of the ruv gene of Escherichia coli.

Regulation of the ruv gene of E. coli was studied using phage Mud (Ap lac) to obtain a fusion of the lac genes to the ruv promoter. Beta-galactosidase synthesis in the ruv-lac fusion strain was induced by mitomycin C and other agents that damage DNA. The induction of beta-galactosidase could be altered by mutations either in lexA or recA from which it is concluded that ruv is regulated by lexA repressor. A possible function of ruv in promoting cell recovery following damage to DNA is discussed.

Bacterial Proteins↗
PubMed1980

Hyper-recombination in uvrD mutants of Escherichia coli K-12.

A mutant strain of E. coli which was isolated initially because of its strong hyper-recombination phenotype was shown to carry a lesion in uvrD. The presence of this mutation, designated uvrD210, increased the frequency of recombination between chromosomal duplications in F-prime repliconant cells and reduced linkage between closely linked markers in crosses with Hfr donors. A comparable hyper-rec phenotype was demonstrated in strains carrying other alleles of uvrD previously referred to as mutU4, uvr502 and recL152. The recombination activity of a uvrD210 strain was abolished by mutation of recA but the mutator activity associated with this allele proved to be independent of recA. It is suggested that uvrD mutations reduce the fidelity of DNA replication and that the accumulation of lesions in the newly synthesized strand provides additional sites for initiating recombination.

DNA Replication↗
PubMed1980

Altered regulation of the recA gene in Escherichia coli strains carrying a recA-linked suppressor of lexA.

A mutant strain of E. coli displaying altered regulation of the recA gene was isolated as a revertant of a lexA3 recA200 double mutant which showed improved DNA repair and recombination functions. The mutant (alc-24) was located between srl and recA200 and caused synthesis of high levels of recA protein in both lexA+ and lexA3 strains. The possibility that alc-24 defines the regulatory site for recA is discussed.

Bacterial Proteins↗
PubMed1978

Hyper-recombination in Escherichia coli K-12 mutants constitutive for protein X synthesis.

Genetic recombination was studied in Escherichia coli F- strains in which synthesis of the recA gene product protein X is increased due to mutation in either recA (tif-1) or lexA (spr). When a single donor marker was selected, the recombination proficiency of these strains was not significantly altered in Hfr crosses. However, linkage of unselected, proximal Hfr markers was found to be much reduced among the progeny tested, and more of the progeny showed evidence of multiple exchanges between donor and recipient DNA. These effects were much more apparent when the recipient carried both tif-1 and spr mutations, but in this case recombination proficiency was reduced compared with those strains carrying either mutation alone, particularly in crosses with Hfr Cavalli. A lexA mutation was found to suppress the effect of tif-1 on the recombinant genotype.

Bacterial Proteins↗