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Biomedical subjects

G C Walker

Publications and source records attributed to G C Walker.

At least 199 records · Page 11Linked to original sources
PubMed1978

Isolation and characterization of mutants of the plasmid pKM101 deficient in their ability to enhance mutagenesis and repair.

A screening procedure was developed for identifying mutants of the plasmid pKM101 no longer capable of enhancing mutagenesis. The test was based on the large pKM101-mediated increase in the number of Gal+ papillae observed on colonies of Salmonella typhimurium gal mutants plated on tetrazolium-galactose plates in the presence of a mutagen. The pKM101 mutant plasmids transferred normally, were stably maintained in cells, caused normal levels of ampicillin resistance, and still imparted sensitivity to phage Ike to their hosts. However, the pKM101 mutants had lost the ability to (i) enhance the reversion of both point and frameshift mutations, (ii) protect the cells against killing by UV irradiation, (iii) increase the spontaneous reversion rates of point mutations, (iv) enhance plasmid-mediated reactivation of UV-irradiated phage P22, (v) enhance Weigle reactivation. One pKM101 mutant with different properties from the others was identified by its increased spontaneous mutator effect. It is suggested that pKM101 amplifies the activity of the inducible error-prone repair systems in bacteria and that this is the function of pKM101 in the Ames Salmonella tester strains used for detection of carcinogens as mutagens.

Ampicillin↗
PubMed1978

Inducible reactivation and mutagenesis of UV-irradiated bacteriophage P22 in Salmonella typhimurium LT2 containing the plasmid pKM101.

The inducible (Weigle) reactivation of UV-irradiated bacteriophage P22 has been examined on strains of Salmonella typhimurium with and without the mutagenesis-enhancing plasmid pKM101. A large inducible reactivation was observed in the plasmid-containing strain, but only a small response was observed in the strain lacking the plasmid. An increased frequency of clear-plaque mutants was detected among the survivors. The efficiencies of the plasmid-mediated and cellular repair processes have been determined. The kinetics of induction of the phage reactivation have been investigated. The relationship of the observed results to the inducible reactivation of UV-irradiated lambda in Escherichia coli and to error-prone repair is discussed.

Genes, Viral↗
PubMed1977

Plasmid (pKM101)-mediated enhancement of repair and mutagenesis: dependence on chromosomal genes in Escherichia coli K-12.

The drug resistance plasmid pKM101 plays a mojor role in the Ames Salmonella/microsome carcinogen detecting system by enhancing chemical mutagenesis. It is shown that in Escherichia coli K-12 the plasmid pKM101 enhances both spontaneous and methyl methanesulfonate-caused reversion of an ochre mutation, bacterial survival after ultraviolet irradiation, and reactivation of ultraviolet-irradiated lambda in unirradiated cells. All these effects are shown to be dependent on the recA+ lexA+ genotype but not on the recB+ recC+ or recF+ genotypes. The recA lexA-dependence of the plasmid-mediated repair and mutagenesis suggests an interaction with the cell's inducible error-prone repair system. The presence of pKM101 is shown to cause an additional increase in methyl methanesulfonate mutagenesis in a tif mutant beyond that caused by growth at 42 degrees. The presence of the plasmid raises the level of the Weigle-reactivation curve for the raactivation of ultraviolet-irradiated lambda in E. coli and causes a shif of the maximum to a higher UV fluence. These observations suggest that pKM101 does not exert its effects by altering the regulation of the cell's error-prone repair system but rather by supplying a mechanistic component or components.

Chromosomes, Bacterial↗
PubMed1977

Characterization of an endonuclease associated with the drug resistance plasmid pKM101.

An endonuclease was detected in strains of Salmonella typhimurium containing the drug resistance plasmid pKM101. The enzyme was not detectable in strains lacking this plasmid, but it was present in strains containing mutants of pKM101 that were no longer able to enhance host cell mutagenesis. The endonuclease had a molecular weight of roughly 75,000 and, at pH 7.0, was equally active on single-stranded and duplex deoxyribonucleic acid (DNA). The reaction with single-stranded DNA was optimal at pH 5.5, whereas with duplex DNA the optimum was pH 6.8. The enzyme required a divalent cation for activity, and it had no detectable exonuclease activity with single-stranded or duplex DNA. The endonuclease extensively degraded DNA with no apparent base specificity, forming 5'-phosphomonoester termini. Although characterization of the endonuclease has not revealed its function, the enzyme does not appear to be a restriction endonuclease.

DNA, Bacterial↗
PubMed1975

Stepwise enzymatic oligoribonucleotide synthesis including modified nucleotides.

A method has been developed for the routine synthesis of 2'(3')-o-monoacyl ribonucleoside 5'-diphosphates for stepwise synthesis of oligoribonucleotides with Escherichia coli polynucleotide phosphorylase. The use of triethyl orthoisovalerate allows the facile preparation of 2'(3')-o-isovaleryl-UDP, -CDP, -ADP, -GDP, -IDP, -EPLISON-APD, eplison-CDP, and N6-isopentenyl-ADP. The synthesis of N6-isopentenyl-ADP from ADP by N1-alkylation and the Dimroth rearrangement to N6 is reported. The effects of several factors including the nature of the divalent cation, pH, SALT CONCENTRATION, AND TIME ON THE EFFICIENCY OF THE POLYNUCLEOTIDE PHPSPHORYLASE CATALYZED SINGLE ADDITIONS OF THE 2'(3')-O-ISOVALERYL RIBONUCLEOSIDE 5'-DIPHOSPHATES TO AN OLIGORIBONUCLEOTIDE PRIMER ARE REPORTED. The syntheses of many tetranucleoside triphosphates and two pentanucleoside tetraphosphates in yields of 20-75 per cent are reported. The 2'(3')-o-isovaleryl derivatives of IDP, eplison-ADP, eplison-CDP, and N6-isopentenyl-ADP were all accepted by polynucleotide phosphorylase as substrates for the monoaddition reaction. The extension of the method to include the syntheses of oligoribonucleotides containing modified nucleosides offers a means of studying the role s of these modification by the use of relatively simple model compounds.

Cytosine Nucleotides↗
PubMed1975

T4-induced RNA ligase joins single-stranded oligoribonucleotides.

RNA ligase isolated from Escherichia coli infected with bacteriophage T4 will catalyze the formation of an intermolecular 3' leads to 5' phosphodiester linkage between an oligoribonucleotide with a free 3'-hydroxyl and another oligoribonucleotide with a 5'-phosphate. Upon reaction with (Ap)5C, nearly quantitative conversion of the hexamer [5'-32P]p(Up)5U to the dodecamer (Ap)5C[3' leads to 5'-32P]p(Up)5U was observed. The product was identified by its mobility on RPC-5 column chromatography, its resistance to alkaline phosphatase, and the appearance of the expected radiolabeled products on hydrolysis with alkali, ribonuclease A, snake venom phosphodiesterase, and spleen phosphodiesterase. The coupling of other pairs of single-stranded oligoribonucleotides has also been demonstrated. The intermolecular joining reaction is probably mechanistically similar to the intramolecular cyclization activity previously reported for Tr RNA ligase. It is expected that this enzyme will be useful for the synthesis of RNA fragments of defined sequence.

Chromatography, Gel↗
PubMed1974

The mode of incorporation of 6-benzylaminopurine into tobacco callus transfer ribonucleic Acid: a double labeling determination.

Cytokinin-dependent tobacco (Nicotiana tabacum) tissue was grown in the presence of N(6)-benzyladenine (6-benzylaminopurine) labeled in the phenyl ring with (3)H and in the 8-position of the purine ring with (14)C. The (3)H/(14)C ratio of N(6)-benzyladenosine recovered from labeled tobacco callus transfer RNA preparations was compared with the corresponding ratio for N(6)-benzyladenine supplied in the medium. By this means, with suitable controls, the incorporation of the synthetic cytokinin, N(6)-benzyladenine, into the transfer RNA of tobacco callus tissue was shown to involve the intact moiety. The observed level of incorporation was up to one N(6)-benzyladenosine molecule per 10(4) transfer RNA molecules.

Journal Article↗
PubMed1966

Thermal resistance of Staphylococcus aureus in milk, whey, and phosphate buffer.

The thermal resistance of four strains of coagulase-positive Staphylococcus aureus was determined in phosphate buffer, whole milk, skim milk, and Cheddar cheese whey. The logarithmic order of death prevailed until about 99.99 to 99.999% of the organisms were destroyed, after which there was a decline in the rate of destruction. The organisms were more resistant in skim milk and Cheddar cheese whey than in phosphate buffer and whole milk. Thermal resistance varied among strains of S. aureus but was consistent with individual strains. As the age of cultures of strain B-120 increased from 12 to 228 hr, the D(55) values increased from 0.95 to 3.0. The thermal resistance of cultures obtained from survivors to partial thermal destruction was similar to that of the parent cultures.

Animals↗