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R C Greene

Publications and source records attributed to R C Greene.

At least 37 records · Page 2Linked to original sources
PubMed1982

Localization of the metJBLF gene cluster of Escherichia coli in lambda met transducing phage.

The position of the metJBLF gene cluster in the transducing phage lambda met102 was determined by ligation of its leftmost EcoRI fragment (102-1) to the lambda BCDEF (nin5) EcoRI fragment of lambda gtl (lambda BC) and characterization of the resultant recombinant phage. The new transducing phage carries about 6kb of bacterial DNA which contains the entire met gene cluster including the promoter of its rightmost member metF. Reasonable estimates of the coding capacity required for the four genes indicate that most of the bacterial DNA of the recombinant phage is occupied by the met gene cluster.

Bacteriophage lambda↗
PubMed1982

Construction and physical mapping of plasmids containing the metJBLF gene cluster of E. coli K12.

In vitro recombination techniques were used to clone the E. coli metJBLF gene cluster in a plasmid vector. Several chimeric plasmids were obtained, analyzed by restriction mapping and characterized genetically. The combined results establish that the met gene cluster is contained on an approximately 5.6 kilobase segment of bacterial DNA with metL between metB and metF. The origin of metL was localized precisely by its DNA sequence and its transcription direction was established.

Bacteriophage lambda↗
PubMed1981

Structural studies of lambda transducing bacteriophage carrying bacterial deoxyribonucleic acid from the metBJLF region of the Escherichia coli chromosome.

The structures of several lambda dmet and related lambda darg transducing phage were studied by restriction fragment mapping and electron microscopic measurements of homoduplexes and heteroduplexes. A new transducing phage (lambda dmet141), in which metF is the only functional gene of the cluster, was isolated. In contrast, lambda dmet117, which expresses the entire metBJLF cluster, has only 3 kilobases more bacterial deoxyribonucleic acid (DNA) than lambda dmet141. An EcoRI restriction fragment of lambda dmet117, which carries the leftmost 6 kilobases of the bacterial DNA insert, was isolated and shown to contain a functional copy of metB. Small structural differences at the attachment sites of some of the phage were shown to result from different sites of lambda integration in the two parent insertion lysogens.

Attachment Sites, Microbiological↗
PubMed1978

In vitro synthesis of cystathionine gamma-synthetase in Escherichia coli K-12.

Synthesis of cystathionine gamma-synthetase directed by DNA from a lambdadmet transducing phage has been achieved in cell extracts from Escherichia coli K-12. Enzyme synthesis was stimulated two- to threefold by the addition of guanosine 3'-diphosphate 5'-diphosphate to the incubation mixtures. Kinetic studies showed a 1.5- to 2.0-min lag between initiation of transcription and completion of a translatable message. This lag is shorter than that observed for beta-galactosidase synthesis with DNA from a lac transducing phage known to initiate transcription at the lac promoter. This result, together with information on the structure of the transducing phage, shows that pL is not used for initiation of in vitro metB transcription. Attempts to demonstrate repression were not successful, and unexpectedly, extracts from metJ+ strains were found to be more effective at enzyme synthesis than those from their metJ derivatives.

Coliphages↗
PubMed1977

Isolation and characterization of specialized lambda transducing bacteriophage carrying the metBJF methionine gene cluster.

Secondary attachment site lysogens of Deltaatt(lambda)Deltappc-argECBH strains of Escherichia coli with lambdacI857 integrated into the bfe gene (88 min) were isolated. Of 20 such lysogens examined, 2 produce lysates with transducing phage containing the metBJF gene cluster (87 min). Reintroduction of the ppc-argECBH chromosome segment (which lies between the bfe and met genes) into these strains virtually abolishes the production of met transducing phage. All of the phage examined have lost essential genes from the left arm of the lambda chromosome. Approximately 85% of the phage appear to have the same genetic composition, containing the metBJF gene cluster, but not the closely linked gene cytR, and having lost phage genes G and J. Analytical CsCl density gradient centrifugation of five representatives of this major class of phage shows four of them to have identical densities (lighter than lambda), while the fifth cannot be resolved from lambda. The four apparently identical phage were isolated from three separate lysates, which suggests the existence of preferred sites for illegitimate recombination on the bacterial and phage chromosomes. Three specialized transducing phage that carry cytR in addition to metB, metJ, and metF have also been studied. Each of these viruses has a different amount of phage deoxyribonucleic acid. Two of them have less deoxyribonucleic acid than lambda, whereas the third has about the same amount. The metB, metF, and cytR genes of the transducing phage have been shown to function in vivo. The phage-borne metB and metF genes are subject to metJ-mediated repression.

Coliphages↗
PubMed1975

Genetic characterization of the metK locus in Escherichia coli K-12.

Three independently isolated metK mutants have been shown to have leisions lying between speB and glc near 57 min on the Escherichia coli chromosome. Two deletions result in a lack of the metC gene product but neither extends into the metK glc region. The three metK mutations are recessive to the wild-type allele carried on the KLF16 episome.

Arginine↗
PubMed1975

Role of methionine in the regulation of serine hydroxymethyltransferase in Eschericia coli.

Significant derepression of serine hydroxymethyltransferase is observed when metE or metF mutants of Escherichia coli K-12 are grown on D-methionine sulfoxide instead of L-methionine. The derepression is not prevented by addition of glycine, adenosine, guanosine, guanosine, and thymidine to the growth medium of methionine-limited metF cells showing that the effect is not due to a secondary deficiency of these nutrients. On the other hand, methionine-limited growth of a metA mutant leads to derepression of met regulon enzymes, but only a marginal increase in serine hydroxymethyltransferase activity. A prototrophic metJ strain grown on minimal medium has about the same serine hydroxymethyltransferase as the wild type. The enzyme activity of the metJ strain is not influenced by methionine, but it is partially repressed by glycine, adenosine, and thymidine. metK strains have about twice as much serine hydroxymethyltransferase activity as wild-type cells when grown on minimal medium; but when both types of cells are grown on medium supplemented with glycine, adenosine, guanosine, and thymidine, their enzyme activities are about the same. The results show that methionine limitation can lead to depression of serine hydroxymethyltransferase, but that the regulatory system is different from the one which controls the methionine regulon.

Adenosine↗
PubMed1973

Properties of metK mutants of Escherichia coli K-12.

Some of the properties of three metK mutants of Escherichia coli K-12 have been examined. All three strains have lower than normal levels of SAM (S-adenosyl-l-methionine) synthetase and elevated levels of cystathionine synthetase and cystathionase. One strain (RG73) appears to have an unstable SAM synthetase, suggesting that it carries a structural gene mutation. The two strains (RG62 and RG109) which have the lowest levels of SAM synthetase when grown on minimal medium have appreciably higher levels of enzyme when grown on complete medium. Growth on defined media supplemented with leucine or methionine causes a several-fold increase in the specific activity of SAM synthetase with associated decreases in cystathionine synthetase and cystathionase, but the changes are not as large as those seen in cells grown on LB broth. The SAM pools of strains RG62 and RG109 are markedly lower than normal while that of strain RG73 is slightly below normal. The methionine pools of all three strains are elevated several-fold. The metK strains are able to synthesize cyclopropane fatty acids, but the rate of their formation is slowed. Modification and restriction of phage 21 appears to be normal, suggesting that these strains are able to methylate DNA.

Carbon Isotopes↗
PubMed1973

Methionine limitation in Escherichia coli K-12 by growth on the sulfoxides of D-methionine.

The synthesis and resolution of the diastereoisomers of d-methionine sulfoxide in high yield is described. Growth of two methionine auxotrophs (strains HfrC and AB1932) on the d-methionine sulfoxides is slower than on l-methionine, and the resultant cells are markedly derepressed for three enzymes of the methionine regulon (cystathionine synthetase, cystathionase, and S-adenosyl-l-methionine synthetase). Strain HfrC grows more rapidly on the sulfoxides and shows less derepression than strain AB1932. Although growth on d-methionine-d-sulfoxide is much slower than on d-methionine-l-sulfoxide (two- to threefold increase in division times), cells grown on d-methionine-l-sulfoxide generally have higher enzyme activities. The sulfoxides of d-methionine appear to provide a useful supplement to obtain methionine-limited growth in Escherichia coli.

Adenosine Triphosphate↗
PubMed1971

Regulation of methionine biosynthesis in Escherichia coli: mapping of the metJ locus and properties of a metJ plus-metJ minus diploid.

MetJ(-) mutants of Escherichia coli have elevated nonrepressible levels of the enzymes of methionine biosynthesis and S-adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase, EC 2.5.1.6). In E. coli, as in Salmonella typhimurium, the metJ locus is close to metB (95% cotransduction of metB and metJ markers), but in E. coli the order is reversed, with metJ mapping clockwise to metB. A stable merodiploid, heterozygous for metJ, is subject to repression by methionine. Thus, metJ functions via a diffusible product. MetJ could either be a regulatory locus or could code for an enzyme required for the synthesis of a methionine metabolite that functions in the control system.

Journal Article↗