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G C Walker

Publications and source records attributed to G C Walker.

At least 163 records · Page 9Linked to original sources
PubMed1985

Cellular responses to DNA damage.

For many years, the study of the regulation of the SOS network was complicated by both the complexities of the responses and the interrelationships of the key regulatory elements. However, recently the application of powerful genetic and molecular biological techniques has allowed us to gain a detailed picture of the regulation of this complex network. The network is now known to consist of more than 17 genes, each of which is repressed by the LexA protein. Induction of the genes in the SOS network occurs when the RecA protein becomes activated in response to a signal generated by DNA damage. Two of the genes in this network, umuD and umuC, are absolutely required for mutagenesis by UV and various carcinogens. The umuD and umuC genes have molecular weights of 16,000 and 45,000 daltons, respectively, and are organized in an operon repressed by LexA. The mutagenesis-enhancing plasmid pKM101 carries two genes mucA and mucB, which are analogs of the umuD and umuC genes, respectively.

DNA Repair↗
PubMed1984

Analysis of mRNA synthesis following induction of the Escherichia coli SOS system.

Escherichia coli responds to impairment of DNA synthesis by inducing a system of DNA repair known as the SOS response. Specific genes are derepressed through proteolytic cleavage of their repressor, the lexA gene product. Cleavage in vivo requires functional RecA protein in a role not yet understood. We used mRNA hybridization techniques to follow the rapid changes that occur with induction in cells with mutations in the recA operator or in the repressor cleavage site. These mutations allowed us to uncouple the induction of RecA protein synthesis from its role in inducing the other SOS functions. Following induction with ultraviolet light, we observed increased rates of mRNA synthesis from five SOS genes within five minutes, maximum expression ten to 20 minutes later and then a later decline to near the initial rates. The presence of a recA operator mutation did not significantly influence these kinetics, whereas induction was fully blocked by an additional mutation in the repressor cleavage site. These experiments are consistent with activation of RecA protein preceding repressor cleavage and derepression of SOS genes. The results also suggest that the timing and extent of induction of individual SOS genes may be different.

Bacterial Proteins↗
PubMed1984

groEL and dnaK genes of Escherichia coli are induced by UV irradiation and nalidixic acid in an htpR+-dependent fashion.

Two proteins with molecular weights of 61,000 and 73,000 were found to be induced by UV light in Escherichia coli mutants in which the SOS responses are constitutively expressed. The induction of these proteins by UV light and nalidixic acid was shown to be independent of the recA+ lexA+ regulatory system. Analysis of these proteins by two-dimensional gel electrophoresis and comparison with the "heat-shock" proteins of E. coli revealed that the Mr 61,000 protein comigrated with the groEL gene product, that the Mr 73,000 protein comigrated with the dnaK gene product, and that other heat-shock proteins were also induced. The induction of groEL and dnaK by UV light and nalidixic acid is controlled by the htpR locus. The results suggest that the regulatory response of E. coli to agents such as UV light and nalidixic acid is more complex than previously thought.

Alleles↗
PubMed1984

Anaerobiosis induces expression of ant, a new Escherichia coli locus with a role in anaerobic electron transport.

Escherichia coli has a formate hydrogenlyase system which allows it to maintain an electron balance during anaerobic growth by passing electrons from formate to H+ ions, thus generating H2. The Mu d1(Ap lac) bacteriophage was used to generate mutants that were defective in passing electrons from formate to benzyl viologen, an artificial electron acceptor. A subset of these mutants was studied in which beta-galactosidase was expressed at much higher levels under anaerobic conditions than under aerobic conditions. If nitrate was present during anaerobic growth, the same levels of beta-galactosidase were seen in these fusion strains as were seen under aerobic conditions. The Mu d1(Ap lac) insertions in these mutants were genetically mapped between mutS and srl and thus define a new locus we have termed ant (anaerobic electron transport). Recombinant lambda derivatives were isolated which complemented the deficiency of the ant mutants in anaerobic electron transport and also carried a trans-acting region of DNA which reduced expression of the ant-lac fusions under anaerobic conditions; a probe to the ant region was generated from one of these recombinant lambda derivatives. Southern hybridization analysis revealed that the four independent ant::Mu d1(Ap lac) fusions we isolated spanned an approximately 5-kilobase region and that all were transcribed in the same direction, counterclockwise on the E. coli genetic map.

Anaerobiosis↗
PubMed1984

General transduction in Rhizobium meliloti.

General transduction by phage phi M12 in Rhizobium meliloti SU47 and its derivatives is described. Cotransduction and selection for Tn5 insertions which are closely linked to specific loci were demonstrated. A derivative of SU47 carrying the recA::Tn5 allele of R. meliloti 102F34 could be transduced for plasmid R68.45 but not for chromosomally located alleles. Phage phi M12 is morphologically similar to Escherichia coli phage T4, and restriction endonuclease analysis indicated that the phage DNA was ca. 160 kilobases in size.

Bacteriophages↗
PubMed1984

Host-dependent transposon Tn5-mediated streptomycin resistance.

Transposon Tn5 encodes streptomycin resistance in addition to kanamycin-neomycin resistance. This resistance was not detectable in Escherichia coli but was efficiently expressed in Rhizobium meliloti and certain other strains. By analysis of cloned Tn5 restriction endonuclease fragments, the streptomycin resistance (str) gene was located in the right-hand side of the central region as the transposon is conventionally drawn. Transcription of str appeared to originate at pL, the promoter for the neo gene (neomycin phosphotransferase type II). Expression of streptomycin resistance in E. coli was obtained after cloning of the neo-str region downstream of a strong E. coli promoter. A construct in which PL was deleted also showed differential expression of streptomycin resistance.

Cloning, Molecular↗
PubMed1983

Proteins required for ultraviolet light and chemical mutagenesis. Identification of the products of the umuC locus of Escherichia coli.

umuC is the genetic locus showing the greatest specificity for the "error-prone repair" process that is responsible for u.v. and chemical mutagenesis in Escherichia coli. By generating a probe specific to umuC DNA, we have been able to clone the umuC locus. Through a combination of subcloning and Tn1000 mutagenesis, we have identified a region of 2.2 X 10(3) bases which contains the information necessary to complement umuC mutations. This region of DNA codes for two polypeptides with molecular weights of 16,000 and 45,000. The genes for these proteins are organized in an operon that is repressed by the lexA protein. Complementation of previously isolated umuC mutations revealed that these proteins correspond to two complementation groups, umuC, which codes for the 45,000 Mr protein, and umuD, which codes for the 16,000 Mr protein, and that therefore both proteins are essential for "error-prone repair" in E. coli.

Bacterial Proteins↗
PubMed1983

Mutagenesis, by methylating and ethylating agents, in mutH, mutL, mutS, and uvrD mutants of Salmonella typhimurium LT2.

Salmonella typhimurium LT2 mutH, mutL, mutS, and uvrD mutants were especially sensitive to mutagenesis by both the recA+-dependent mutagen methyl methane sulfonate and the recA+-independent mutagen ethyl methane sulfonate, but not to mutagenesis by agents such as 4-nitroquinoline-1-oxide and UV irradiation. Similarly, these mutator strains were very sensitive to mutagenesis by the methylating agents N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea. The increased susceptibility to mutagenesis by small alkylating agents due to mutH, mutL, mutS, and uvrD mutations was not accompanied by an increased sensitivity to killing by these agents. Various models are discussed in an effort to explain why strains thought to be deficient in methyl-instructed mismatch repair are sensitive to mutagenesis by methylating and ethylating agents.

4-Nitroquinoline-1-oxide↗
PubMed1983

Identification of the uvrD gene product of Salmonella typhimurium LT2.

The product of the uvrD gene of Salmonella typhimurium LT2 and Escherichia coli K-12 is thought to play a role in both the correction of mismatched bases and the repair of DNA damage, since insertion mutations in the uvrD gene increase the spontaneous mutation frequency and make the cells more sensitive to killing by UV irradiation. To clone the uvrD gene of S. typhimurium, we first generated a uvrD-specific probe by using DNA from an S. typhimurium uvrD421::Tn5 mutant. This probe was used to screen a lambda library of S. typhimurium DNA. Bacteriophage carrying intact uvrD+ genes were subsequently identified, and the uvrD+ gene was subcloned onto a low-copy-number vector. By using a combination of Tn1000 insertion mutagenesis and the maxicell technique, the product of the uvrD gene was shown to be a 75,000-dalton protein, and the relative direction of transcription of this protein was determined. Introduction of a low-copy-number plasmid carrying the S. typhimurium uvrD+ gene into uvrD insertion mutants of either S. typhimurium or E. coli restored the spontaneous mutation frequency and degree of UV sensitivity to the levels in the corresponding uvrD+ strains.

Bacterial Proteins↗
PubMed1983

Isolation and characterization of Tn5 insertion mutations in the lexA gene of Escherichia coli.

A Mu d(Ap lac)-generated fusion of lacZ to dinD, a gene induced by DNA damage, was used to isolate Tn5 insertion mutations that affect the regulation of the SOS responses. Three mutants were obtained that contained Tn5 insertions genetically linked to the lexA gene and had properties that suggested the mutants were deficient in lexA expression. The lexA protein has been shown to function as the repressor for genes involved in the SOS responses. By Southern blotting experiments, the three Tn5 insertions were physically mapped to distinct locations within the coding region of the lexA gene. The introduction of these mutations in six strains carrying lacZ fusions to different damage-inducible genes resulted in high expression of beta-galactosidase in all but one of the strains. In the dinF fusion strain, lacZ expression was reduced below that seen in a lexA+ background. Physical mapping studies of the dinF locus gave results consistent with the notion that dinF is part of the lexA transcription unit and that a lexA::Tn5 mutation has a polar effect on dinF expression. With certain din-lac fusion strains, a correlation was seen between the amount of beta-galactosidase production and the location of the particular Tn5 insertion within the lexA gene.

Alleles↗
PubMed1983

Genetic localization and characterization of a pKM101-coded endonuclease.

The genetic and biochemical properties of an endonuclease mediated by the mutagenesis-enhancing plasmid pKM101 have been investigated. Taking advantage of the observation that this endonuclease, unlike host-coded DNases, is active in the presence of EDTA, we have developed an assay with nondenaturing acrylamide gels containing DNA. We have localized the plasmid DNA sufficient for nuclease expression to a 0.8-kilobase sequence that is near regions of DNA necessary for conjugal transfer, and we have determined that this gene is transcribed clockwise on the pKM101 map. The pKM101 gene mediating this activity codes for a 16,000-dalton protein, which is the same molecular mass as the nuclease monomer, leading us to conclude that this gene codes for the nuclease itself rather than for an activator of some host-coded enzyme. Cellular fractionation experiments have shown that the enzyme is localized in the periplasm. We have not been able to demonstrate any physiological role for the enzyme, but we have ruled out a direct involvement of the nuclease in any of the following known plasmid-associated phenotypes: (i) mutagenesis enhancement, (ii) conjugal transfer, (iii) entry exclusion, (iv) fertility inhibition of coresident P-group plasmids, (v) killing of Klebsiella pneumoniae used as conjugal recipients, and (vi) plasmid curing induced by treatment of cells with fluorodeoxyuridine. In addition, we have shown that the enzyme does not restrict bacteriophage or affect the ability of the host to utilize DNA as a source of thymine. Finally, we have shown that 11 of the 26 other plasmids tested also elaborated EDTA-resistant DNases.

Conjugation, Genetic↗
PubMed1983

The Salmonella typhimurium LT2 uvrD gene is regulated by the lexA gene product.

The uvrD gene product apparently plays a role in the repair of UV damage, in mismatch repair, and in genetic recombination. A lower level of expression of the Salmonella typhimurium LT2 uvrD gene was observed in maxicells prepared from an Escherichia coli strain that contained a lexA+ plasmid than in maxicells prepared from an E. coli strain that lacked functional LexA protein. These results suggest that the uvrD+ gene is repressed by the LexA protein and is thus a member of the set of genes whose expression is increased by "SOS"-inducing treatments.

Bacterial Proteins↗