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Biomedical subjects

B Sedgwick

Publications and source records attributed to B Sedgwick.

At least 37 records · Page 2Linked to original sources
PubMed1985

Active site and complete sequence of the suicidal methyltransferase that counters alkylation mutagenesis.

The inducible resistance to alkylation mutagenesis and killing in Escherichia coli (the adaptive response) is controlled by the ada gene. The Ada protein acts both as a positive regulator of the response and as a DNA repair enzyme, correcting premutagenic O6-alkylguanine in DNA by suicidal transfer of the alkyl group to one of its own cysteine residues. We have determined the DNA sequence of the cloned ada+ gene and its regulatory region. The data reveal potential sites of ada autoregulation. Amino acid sequence determinations show that the active center for the O6-methylguanine-DNA methyltransferase is located close to the polypeptide COOH terminus and has the unusual sequence -Pro-Cys-His-, preceded by a very hydrophobic region. These same structural features are present at the active site of thymidylate synthase, suggesting a common chemical mechanism for activation of the cysteine.

Alkylation↗
PubMed1984

Induction of resistance to alkylating agents in E. coli: the ada+ gene product serves both as a regulatory protein and as an enzyme for repair of mutagenic damage.

The expression of several inducible enzymes for repair of alkylated DNA in Escherichia coli is controlled by the ada+ gene. This regulatory gene has been cloned into a multicopy plasmid and shown to code for a 37-kd protein. Antibodies raised against homogeneous O6-methylguanine-DNA methyltransferase (the main repair activity for mutagenic damage in alkylated DNA) were found to cross-react with this 37-kd protein. Cell extracts from several independently derived ada mutants contain variable amounts of an altered 37-kd protein after an inducing alkylation treatment. In addition, an 18-kd protein identical with the previously isolated O6-methyl-guanine-DNA methyltransferase has been identified as a product of the ada+ gene. The smaller polypeptide is derived from the 37-kd protein by proteolytic processing.

Alkylating Agents↗
PubMed1983

Molecular cloning of a gene which regulates the adaptive response to alkylating agents in Escherichia coli.

The ada+ gene of E. coli is a regulatory gene of the adaptive response to simple alkylating agents. ada mutants are sensitive to both the mutagenicity and toxicity of alkylating agents, and are unable to induce O6-methylguanine DNA methyltransferase and 3-methyladenine DNA glycosylase II. The ada+ gene was cloned from wild type E. coli B by ligating bacterial DNA partially digested with Sau3A into the cosmid vector pJB8. The hybrid cosmid, pCS33, conveyed N-methyl-N'-nitro-N-nitrosoguanidine resistance to ada mutants of E. coli B and E. coli K12, and resulted in the constitutive synthesis of the two DNA repair enzymes at high levels. An alk mutation, which results in a deficiency of only the DNA glycosylase, was not complemented by this cosmid. It was concluded that the product of the ada+ gene is a positive regulator of the adaptive response. The cosmid insert DNA was subcloned into the plasmid vector pAT153, and the ada+ plasmids pCS42 and pCS58 selected. The ada+ gene was located in pCS58 by transposon mutagenesis and subcloning. Two polypeptides of Mr 37,000 and 27,000, were identified in 'maxi-cells' as products of the ada+ gene(s). It is as yet unclear whether they represent different forms of the same gene product, or are encoded by separate ada+ genes within the same operon.

Alkylating Agents↗
PubMed1982

The free coenzyme A requirement of animal fatty acid synthetase. Participation in the continuous exchange of acetyl and malonyl moieties between coenzyme a thioester and enzyme.

A hypothesis that the existence of common binding sites for acetyl and malonyl moieties on the animal fatty acid synthetase necessitates that free CoA be available continuously to facilitate unloading of inappropriately bound acetyl or malonyl moieties, allowing initial access of an acetyl moiety and subsequent access by malonyl moieties to the site of chain elongation, was formulated and tested. The unloading of acetyl or malonyl moieties from the enzyme was blocked by a CoA-scavenging system and the enzyme was unable to reload with the other substrate; the inhibition was relieved by the addition of CoA or pantetheine. The freely reversible nature of the loading/unloading reaction was established as follows. CoA or pantetheine, but not S-acetyl-N-acetylcysteamine, could act as donor or acceptor for acetyl moieties in the loading or unloading reactions; incubation of fatty acid synthetase, acetyl-CoA, and [G-3H]CoA resulted in the formation of acetyl-[G-3H]CoA in an amount consistent with the predicted equilibrium; and addition of a high concentration of CoA shifted the equilibrium toward unloading, leaving most of the substrate-binding sites vacant. These results support our hypothesis and provide a plausible explanation both for the requirement of free CoA by the fatty acid synthetase and for the observed inhibition of fatty acid synthesis by high concentrations of CoA.

Acetyl Coenzyme A↗
PubMed1982

Genetic mapping of ada and adc mutations affecting the adaptive response of Escherichia coli to alkylating agents.

The adaptive response to alkylating agents is an inducible repair system which protects Escherichia coli against the mutagenicity and toxicity of these agents. Four mutations, ada-3, ada-5, ada-6, and adc-1, which confer differing phenotypes as regards this response, were shown to be cotransducible with gyrA, and were located at 47 min on the E. coli genetic map. A mutation already shown on the map at 47 min as tag (B. J. Bachmann and K. B. Low, Microbiol. Rev. 44:1--56, 1980; Karran et al., J. Mol. Biol. 140:101--127, 1980) is now known to be an ada mutation (G. Evensen and E. Seeberg, personal communication).

Alkylating Agents↗
PubMed1980

Isolation of mutants of Escherichia coli with increased resistance to alkylating agents: mutants deficient in thiols and mutants constitutive for the adaptive response.

A search has been made for mutants of E. coli that are constitutive for the adaptive response to alkylating agents. When selection was for resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) the resulting strains all proved to be low in intracellular thiols. As MNNG has to be converted by thiols to the proximal mutagen methylnitrosamine, these strains were resistant simply because their low thiol levels made them less readily methylated by MNNG. The selection was therefore undertaken for strains resistant to N-methylnitrosourea (MNU) because this alkylating agent does not require activation by thiols. Four MNU resistant mutants proved to be constitutive for the adaptive response. Following exposure to (3H) MNNG, they showed the same low ratio of O6-methylguanine (O6MeG) to 7-methylguanine (7MeG) as adapted wild type bacteria. The ratio of 3-methyladenine (3MeA) to 7MeG was the same in the wild type, the adapted wild type, and the constitutive strains. When exposed to very high MNNG doses, the constitutive mutants were even more resistant to mutation and killing than the adapted wild type. They have an even greater ability than the adapted wild type to remove O6MeG from methylated DNA (Lindahl personal communication).

Alkylating Agents↗
PubMed1976

Single-stranded regions in transforming deoxyribonucleic acid after uptake by competent Haemophilus influenzae.

About 15% of donor deoxyribonucleic acid (DNA) is single stranded immediately after uptake into competent Haemophilus influenzae wild-type cells, as judged by its sensitivity to S1 endonuclease. This amount decreases to 4 to 5% by 30 min after uptake. Mutants which are defective in the covalent association of recipient and donor DNA form little or no S1 endonuclease-sensitive donor. At 17 C donor DNA taken up by the wild type contains single-stranded regions although there is no observable association, either covalent or noncovalent. The single-stranded regions are at the ends of donor DNA molecules, as judged by the unchanged sedimentation velocity after S1 endonuclease digestion. The amount of single-stranded donor remains constant at 17 C for more than 60 min after uptake, suggesting that the decrease observed at 37 C is the result of association of single-stranded ends with single-stranded regions of recipient cell DNA. Three sequential steps necessary for the integration of donor DNA into recipient DNA are proposed: the synthesis of single-stranded regions in recipient DNA, the interaction of donor DNA with recipient DNA resulting in the production of single-stranded ends on donor DNA, and the stable pairing of homologous single-stranded regions.

DNA, Bacterial↗
PubMed1975

Minicell production and bacteriophage superinducibility of thymidine-requiring strains of Haemophilus influenzae.

Aminopterin- or trimethoprin-resistant thymidine-requiring strains of Haemophilus influenzae produce minicells, and the ratio of minicells to cells increases during the stationary phase of growth. Strain LB11, isolated after mutagenesis of a thymidine-requiring strain (Rd thd), produces more minicells than the parent strain. The mutations involved in high frequency minicell production have been transferred into the wild type (strain Rd) by transformation. The thymidine requirement in the resulting strain, MCl, is essential for minicell production, since spontaneous revertants of MCl to prototrophy do not produce minicells. The ratio of minicells to cells was increased more than 10(3)-fold by differential centrifugation. The minicells contain little or no deoxyribonucleic acid (DNA). Phage HPlcl apparently cannot attach to minicells. Competent cells of LB11 and its thymidine-requiring parent strain produce defective phage as a result of exposure to transforming DNA, whereas only LB11 produces many defective phage in response to the competence regime alone. Competent HP1c1 and S2 lysogens of MC1 and Rd thd are also superinducible by transforming DNA, but competent LB11 lysogens produced about the same amount of HP1c1 or S2 phage with or without exposure to transforming DNA possibly because of competition between the induced defective phage and Hp1c1 or S2 phage.

Bacteriophages↗