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Biomedical subjects

R M Blumenthal

Publications and source records attributed to R M Blumenthal.

At least 19 recordsLinked to original sources
PubMed1992

Analysis of macromolecular biosynthesis to define the quinolone-induced postantibiotic effect in Escherichia coli.

Quinolones inhibit DNA gyrase, and the major effects of this inhibition are on replication and transcription of DNA. The postantibiotic effect (PAE) refers to continued inhibition of cell division, in terms of the viable count, following transient exposure to an antibiotic. Previous work has shown that quinolone-treated cells have not fully recovered by the time the classically defined PAE has ended. We describe the PAE of the quinolones CI-960, enoxacin, and ciprofloxacin on macromolecular biosynthesis in the clinical isolate Escherichia coli J96 in an attempt to relate the PAE to the time that it actually takes for the cells to recover fully. DNA synthesis was inhibited immediately upon exposure to these quinolones at 0.5x or 0.75x the MIC. This inhibition continued for several hours following quinolone removal. The effects of these quinolones on RNA and protein synthesis varied; enoxacin treatment at 0.5x the MIC resulted in an increase of over 60% in both RNA and protein synthesis per unit of cell mass, while ciprofloxacin and CI-960 at that level had no significant effects on either RNA or protein synthesis. The effects of enoxacin and ciprofloxacin on bacterial protein profiles were also distinguishable, and these changes corresponded to their PAE on DNA synthesis. Throughout the study, all measures of the physiological status of the cells returned to normal by the time DNA synthesis per unit of cell mass did so. These results suggest that DNA synthesis per unit of cell mass provides an accurate measure of the time required for quinolone-treated cells to recover fully.

Anti-Infective Agents↗
PubMed1992

Sequence and characterization of pvuIIR, the PvuII endonuclease gene, and of pvuIIC, its regulatory gene.

An open reading frame partially overlaps pvuIIR, and genetic evidence implies that this open reading frame, named pvuIIC, specifies a positive regulator of pvuIIR (T. Tao, J. C. Bourne, and R. M. Blumenthal, J. Bacteriol. 173:1367-1375, 1991). Inducible constructs of pvuIIC produced a protein of the expected size. The site of C.PvuII action appears to lie within pvuIIC itself; thus, pvuIIC may be a self-contained regulatory cassette.

Amino Acid Sequence↗
PubMed1991

A family of regulatory genes associated with type II restriction-modification systems.

Restriction-modification systems must be regulated to avoid autorestriction and death of the host cell. An open reading frame (ORF) in the PvuII restriction-modification system appears to code for a regulatory protein from a previously unrecognized family. First, interruptions of this ORF result in a nonrestricting phenotype. Second, this ORF can restore restriction competence to such interrupted mutants in trans. Third, the predicted amino acid sequence of this ORF resembles those of known DNA-binding proteins and includes a probable helix-turn-helix motif. A survey of unattributed ORFs in 15 other type II restriction-modification systems revealed three that closely resemble the PvuII ORF. All four members of this putative regulatory gene family have a common position relative to the endonuclease genes, suggesting a common regulatory mechanism.

Amino Acid Sequence↗
PubMed1989

Sequence, internal homology and high-level expression of the gene for a DNA-(cytosine N4)-methyltransferase, M.Pvu II.

The base sequence of the pvuIIM gene has been determined. This gene codes for a DNA-(cytosine N4)-methyltransferase, M.Pvu II. The base sequence contains a single large open reading frame that predicts a 38.3kDa polypeptide, consistent with experimental data. The pvuIIM gene contains some sequences common to DNA methyltransferases in general, but includes none of the sequences specifically conserved among DNA-(cytosine 5)-methyltransferases. The pvuIIM sequence also reveals an internal homology at the amino acid level, each half of which spans over 100 amino acids and is itself homologous to the sequences of some DNA-(adenine N6)-methyltransferases. A derivative of the pvuIIM plasmid was constructed to allow high-level production of M.Pvu II. Specifically, the composite Ptac promoter was inserted 5' to pvuIIM, intervening DNA was deleted, and the resulting construct was used to transform an mcrB laclq strain of Escherichia coli. When this transformant was induced with isopropyl-B-D-galactopyranoside (IPTG), growth rapidly ceased and M.Pvu II accumulated to the point of comprising over 10% of the total soluble protein.

Amino Acid Sequence↗
PubMed1988

Isolation of mutants in a DNA methyltransferase through mcrB-mediated restriction.

A procedure has been developed that permits the positive selection of mutants in a DNA methyltransferase (MTase) gene. The stringency of this selection can be varied so as to yield null mutants only, or a mixture of null and partially defective mutants. The procedure was developed with the PvuII MTase gene (pvuIIM), which was subcloned into a bacteriophage lambda vector. Growth of this lambda pvuIIM construct on an mcrB+ host selected for non-methylating mutants, and the stringency of selection was proportional to the number of consecutive lytic cycles. Many cytosine MTases have been found to generate substrates for mcrB-mediated restriction, and this procedure should be applicable to a number of cytosine MTase genes.

Bacterial Proteins↗
PubMed1988

McrA and McrB restriction phenotypes of some E. coli strains and implications for gene cloning.

The McrA and McrB (modified cytosine restriction) systems of E. coli interfere with incoming DNA containing methylcytosine. DNA from many organisms, including all mammalian and plant DNA, is expected to be sensitive, and this could interfere with cloning experiments. The McrA and B phenotypes of a few strains have been reported previously (1-4). The Mcr phenotypes of 94 strains, primarily derived from E. coli K12, are tabulated here. We briefly review some evidence suggesting that McrB restriction of mouse-modified DNA does occur in vivo and does in fact interfere with cloning of specific mouse sequences.

5-Methylcytosine↗
PubMed1985

Cloning of a restriction-modification system from Proteus vulgaris and its use in analyzing a methylase-sensitive phenotype in Escherichia coli.

A 4.84-kilobase-pair plasmid was isolated from Proteus vulgaris (ATCC 13315) and cloned into the plasmid vector pBR322. Plasmid pBR322 contains substrate sites for the restriction endonucleases PvuI and PvuII. The recombinant plasmids were resistant to in vitro cleavage by PvuII but not PvuI endonuclease and were found to cause production of PvuII endonuclease or methylase activity or both in Escherichia coli HB101. The approximate endonuclease and methylase gene boundaries were determined through subcloning, Bal 31 resection, insertional inactivation, DNA-dependent translation, and partial DNA sequencing. The two genes are adjacent and appear to be divergently transcribed. Most E. coli strains tested were poorly transformed by the recombinant plasmids, and this was shown by subcloning and insertional inactivation to be due to the PvuII methylase gene. At a low frequency, stable methylase-producing transformants of a methylase-sensitive strain were obtained, and efficiently transformed cell mutants were isolated from them.

Bacteriophage lambda↗
PubMed1983

The isolation and characterization of the Escherichia coli DNA adenine methylase (dam) gene.

The E. coli dam (DNA adenine methylase) enzyme is known to methylate the sequence GATC. A general method for cloning sequence-specific DNA methylase genes was used to isolate the dam gene on a 1.14 kb fragment, inserted in the plasmid vector pBR322. Subsequent restriction mapping and subcloning experiments established a set of approximate boundaries of the gene. The nucleotide sequence of the dam gene was determined, and analysis of that sequence revealed a unique open reading frame which corresponded in length to that necessary to code for a protein the size of dam. Amino acid composition derived from this sequence corresponds closely to the amino acid composition of the purified dam protein. Enzymatic and DNA:DNA hybridization methods were used to investigate the possible presence of dam genes in a variety of prokaryotic organisms.

Amino Acid Sequence↗
PubMed1983

Occurrence and expression of imipemide (N-formimidoyl thienamycin) resistance in clinical isolates of coagulase-negative staphylococci.

More than 500 clinical isolates were screened for resistance to a number of antibiotics, including imipemide (N-formimidoyl thienamycin [MK0787]). Of the 25 coagulase-negative staphylococcal isolates present in the screening sample, almost one-third showed one of two patterns of imipemide resistance. One pattern apparently involves constitutive expression of drug resistance, whereas the other pattern seems to result from an inducible resistance having an apparent induction threshold higher than the minimal inhibitory concentration of imipemide. The mechanism(s) responsible for this imipemide resistance is unclear, but may be distinct from the more common staphylococcal mechanisms of resistance to beta-lactam antibiotics. Only two of the patients from whom imipemide-resistant staphylococci were cultured had actually been treated with the antibiotic.

Coagulase↗
PubMed1981

Proteins Specified by bovine herpesvirus 1 (infectious bovine rhinotracheitis virus).

An electrophoretic analysis of radioactively labeled, purified, "empty" and DNA-containing infectious bovine rhinotracheitis virions revealed the presence of 25 to 33 structural (virion) polypeptides. A total of 11 of these polypeptides could be labeled with [3H]glucosamine and were identified as glycoproteins. In addition to the 25 structural polypeptides, infectious bovine rhinotracheitis virus infected cells also contained at least 15 nonstructural (nonvirion) polypeptides that were not present in purified virions. Expression of the viral polypeptides in infected cells was controlled temporally. Thus, most viral polypeptides could be categorized as "alpha" (immediate early), "beta" (early), or "gamma" (late) on the basis of their order of appearance in infected cells and whether their syntheses were dependent upon prior viral protein or DNA synthesis. None of the glycoproteins belongs to the alpha class, although at least one (GVP11) was synthesized in the absence of viral DNA synthesis. Serum from a cow in which infectious bovine rhinotracheitis virus lesions were reactivated by dexamethasone precipitated both structural and nonstructural polypeptides.

Animals↗
PubMed1980

Gene expression in Escherichia coli after amino acid, purine, or pyrimidine exhaustion.

Three strains of Escherichia coli B auxotrophic for leucine, guanine, or uracil were analyzed after exhaustion of the respective required nutrient from the growth medium. The pattern of transcription was analyzed by ribonucleic acid-deoxyribonucleic acid filter hybridization to specific deoxyribonucleic acid probes, and the pattern of translation was analyzed by autoradiography after the resolution of proteins on sodium dodecyl sulfate-polyacrylamide gels. The results obtained suggest the following conclusions. (i) Specific regulation of rpoBC transcription occurs at both the promoter (PL10) and the putative attenuator between rplL and rpoB. (ii) The stringent response of ribosomal protein gene expression to amino acid insufficiency is only partially mimicked by purine or pyrimidine insufficiency. (iii) Transcription initiation at PL10 decreases in response to guanine exhaustion, but in contrast increases significantly in response to uracil exhaustion. (iv) The expression of the induced lac operon is severely depressed during any of these exhaustions. These conclusions argue against simple models for regulation of ribonucleic acid polymerase production or promoter choice by the intracellular levels of its substrate nucleotides.

DNA-Directed RNA Polymerases↗
PubMed1980

Regulation of ribonucleic acid polymerase synthesis during restriction of an Escherichia coli mutant temperature sensitive for transcription factor sigma.

An Escherichia coli mutant temperature sensitive for the sigma subunit of ribonucleic acid polymerase was shifted to restrictive temperatures. In response to these restrictions the transcription of rpoBC increased markedly, and the synthesis rates of the beta and beta' subunits of ribonucleic acid polymerase increased. The ratio of the beta and beta' synthesis rates (beta/beta') decreased.

DNA-Directed RNA Polymerases↗
PubMed1978

Gene expression in Escherichia coli B/r during partial rifampicin-mediated restrictions of transcription initiation.

The antibiotic rifampicin inhibits transcription initiation, but not the elongation and completion of nascent RNA transcripts. Addition of low concentrations of rifampicin only partially blocks initiation but at the same time specifically alters the general pattern of transcription in the culture. The transcription of genes specifying the beta and beta' subunits of RNA polymerase, and to a lesser extent of the genes specifying the RNA and protein components of the ribosome, was specifically stimulated relative to total transcription. In contrast, the transcription of the lactose operon was selectively reduced. These results are consistent with the ideas that the level of expression of the genes specifying the beta and beta' subunits is sensitive to the general rate of RNA synthesis in the culture, and that the expression of the beta and beta' RNA polymerase genes is related to the expression of ribosome component genes.

DNA-Directed RNA Polymerases↗
PubMed1976

The effects of the relA gene on the synthesis of aminoacyl-tRNA synthetases and other transcription and translation proteins in Escherichia coli A.

The effects of a partial restriction of valyl-tRNA aminoacylation on the synthesis of aminoacyl-tRNA synthetases, ribosomal proteins, and other translation and transcription proteins were examined in otherwise isogenic stringent (relA+) and relaxed (relA1) derivatives of E. coli B. The synthesis of individual ribosomal proteins, elongation factor G, and to a lesser extent elongation factors Tu and Ts, and the valyl- and arginyl-tRNA synthetases was found to be subject to the influence of the stringent control system. The synthesis of the alpha and beta subunits of RNA polymerase and several of the aminoacyl-tRNA synthetases, in contrast, is either not subject to the influence of the stringent control system, or is subject to additional regulatory constraints.

Amino Acyl-tRNA Synthetases↗