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Biomedical subjects

V Stewart

Publications and source records attributed to V Stewart.

At least 73 records · Page 4Linked to original sources
PubMed1990

Nitrate regulation of anaerobic respiratory gene expression in narX deletion mutants of Escherichia coli K-12.

Previous studies have shown that narL+ is required for nitrate regulation of anaerobic respiratory enzyme synthesis, including formate dehydrogenase-N, nitrate reductase, and fumarate reductase. Insertions in the closely linked narX gene decrease, but do not abolish, nitrate regulation of anaerobic enzyme synthesis. Analysis of sequence similarities suggests that NarX and NarL comprise a two-component regulatory pair. We constructed lacZ operon and gene fusions to investigate the operon structure of narXL. We found evidence for a complex operon with at least two promoters; PXL-narX-PL-narL. We also investigated the role of NarX in nitrate regulation of anaerobic respiratory enzyme synthesis by constructing nonpolar loss of function narX alleles. These deletions were studied on narL+ lambda specialized transducing bacteriophage. The narX deletions had no effect on nitrate regulation in delta (narXL) strains. This finding suggest that the subtle effects of previously studied narX insertions are due to decreased expression of narL and that narX+ is not essential for normal nitrate regulation. The role of NarX in nitrate regulation remains to be determined.

Alleles↗
PubMed1990

Structure and expression of germ line immunoglobulin heavy-chain epsilon transcripts: interleukin-4 plus lipopolysaccharide-directed switching to C epsilon.

We have isolated cDNA clones complementary to a truncated immunoglobulin heavy-chain C epsilon RNA transcript previously found to be induced in B lymphoid cells by treatment with lipopolysaccharide (LPS) combined with interleukin-4 (IL-4). We demonstrate that this transcript initiates from a promoter upstream of the germ line epsilon class-switch recombination region (S epsilon region). The major germ line C epsilon transcript contains a small 5' exon contributed by sequences upstream of the S epsilon region spliced to the normal C epsilon exons. Treatment of splenic B lymphoid cells with LPS plus IL-4 induces the expression of transcripts from the germ line epsilon transcription unit followed by expression of normal immunoglobulin epsilon heavy-chain mRNA. Furthermore, we demonstrate that similar treatment of transformed precursor B cell lines induces the expression of germ line epsilon transcripts followed by class switching to epsilon expression in these lines. This is the first demonstration of switching to epsilon in cells of the pre-B stage. The general structure of the germ line epsilon transcript and transcription unit is similar to that previously characterized for germ line gamma 2b transcripts. However, expression of these two germ line transcription units in B-lineage cells is inversely regulated by IL-4 (plus LPS) treatment, correlating with the effects of these treatments on switching to these loci.

Animals↗
PubMed1990

Differential regulation of the N-myc gene in transfected cells and transgenic mice.

The N-myc gene is expressed specifically in the early developmental stages of numerous cell lineages. To assay for sequences that could potentially regulate N-myc expression, we transfected constructs that contained murine N-myc genomic sequences linked to a reporter gene and genomic clones that contained the complete human or murine N-myc genes into cell lines that either express or do not express the endogenous N-myc gene. Following either transient or stable transfection, the introduced N-myc sequences were expressed regardless of the expression status of the endogenous gene. In contrast, when the clones containing the complete human N-myc gene were introduced into the germline of transgenic mice, expression in some transgenic lines paralleled the tissue- and stage-specific expression of the endogenous murine gene. These findings demonstrate differences in the regulation of N-myc genes in recipient cells following in vitro versus in vivo introduction, suggesting that early developmental events may play a role in the regulation of N-myc expression.

Age Factors↗
PubMed1989

Role of glutathione and dependent enzymes in anthracycline-resistant HL60/AR cells.

We studied the cellular enzymatic defenses against anthracycline-induced free radical damage in the HL60 human myelogenous leukemia cell line and in its anthracycline-resistant subline, HL60/AR. Intracellular glutathione (GSH) levels and gamma-glutamyl transpeptidase activity were lower in HL60/AR than in HL60 cells. Glutathione-S-transferase (GST) and glutathione peroxidase activities were similar in both cell lines. The intracellular distribution of GSH/GST was visualized by digitized video fluorescence microscopy, utilizing the fluorescent probe monochlorobimane fluorescence microscopy, utilizing the fluorescent probe monochlorobimane (MBCl), which is specifically conjugated to GSH by GST. In HL60 cells stained with the MBCl probe, a bright diffuse cytoplasmic and nuclear fluorescence pattern was observed, whereas in HL60/AR cells, the fluorescence was mostly localized to the Golgi apparatus with a lesser component of diffuse cytoplasmic and nuclear fluorescence. Pretreatment of HL60/AR cells with buthionine sulfoximine (BSO) partially reversed resistance to daunorubicin. This effect of BSO on resistance was associated not only with the abolition of localized MBCl fluorescence to the Golgi apparatus but also with increased intracellular accumulation and retention of daunorubicin. The results of our studies demonstrate that inhibition of GSH synthesis in HL60/AR cells results in significant sensitization to daunorubicin and suggest that changes in the intracellular distribution of GSH/GST and/or increased drug retention may be involved in mediating this effect.

Antibiotics, Antineoplastic↗
PubMed1989

Genetic regulation of nitrate assimilation in Klebsiella pneumoniae M5al.

We isolated Mu dI1734 insertion mutants of Klebsiella pneumoniae that were unable to assimilate nitrate or nitrite as the sole nitrogen source during aerobic growth (Nas- phenotype). The mutants were not altered in respiratory (anaerobic) nitrate and nitrite reduction or in general nitrogen control. The mutations were linked and thus defined a single locus (nas) containing genes required for nitrate assimilation. beta-Galactosidase synthesis in nas+/phi(nas-lacZ) merodiploid strains was induced by nitrate or nitrite and was inhibited by exogenous ammonia or by anaerobiosis. beta-Galactosidase synthesis in phi(nas-lacZ) haploid (Nas-) strains was nearly constitutive during nitrogen-limited aerobic growth and uninducible during anaerobic growth. A general nitrogen control regulatory mutation (ntrB4) allowed nitrate induction of phi(nas-lacZ) expression during anaerobic growth. This and other results suggest that the apparent anaerobic inhibition of phi(nas-lacZ) expression was due to general nitrogen control, exerted in response to ammonia generated by anaerobic (respiratory) nitrate reduction.

Anaerobiosis↗
PubMed1988

Influence of nar (nitrate reductase) genes on nitrate inhibition of formate-hydrogen lyase and fumarate reductase gene expression in Escherichia coli K-12.

In Escherichia coli, aerobiosis inhibits the synthesis of enzymes for anaerobic respiration (e.g., nitrate reductase and fumarate reductase) and for fermentation (e.g., formate-hydrogen lyase). Anaerobically, nitrate induces nitrate reductase synthesis and inhibits the formation of both fumarate reductase and formate-hydrogen lyase. Previous work has shown that narL+ is required for the effects of nitrate on synthesis of both nitrate reductase and fumarate reductase. Another gene, narK (whose function is unknown), has no observable effect on formation of these enzymes. We report here our studies on the role of nar genes in fumarate reductase and formate-hydrogen lyase gene expression. We observed that insertions in narX (also of unknown function) significantly relieved nitrate inhibition of fumarate reductase gene expression. This phenotype was distinct from that of narL insertions, which abolished this nitrate effect under certain growth conditions. In contrast, insertion mutations in narK and narGHJI (the structural genes for the nitrate reductase enzyme complex) significantly relieved nitrate inhibition of formate-hydrogen lyase gene expression. Insertions in narL had a lesser effect, and insertions in narX had no effect. We conclude that nitrate affects formate-hydrogen lyase synthesis by a pathway distinct from that for nitrate reductase and fumarate reductase.

Aldehyde Oxidoreductases↗
PubMed1988

Identification and expression of genes narL and narX of the nar (nitrate reductase) locus in Escherichia coli K-12.

Previous studies have shown that narL+ is required for nitrate induction of nitrate reductase synthesis and for nitrate inhibition of fumarate reductase synthesis in Escherichia coli. We cloned narL on a 5.1-kilobase HindIII fragment. Our clone also contained a previously unidentified gene, which we propose to designate as narX, as well as a portion of narK. Maxicell experiments indicated that narL and narX encode proteins with approximate MrS of 28,000 and 66,000, respectively. narX insertion mutations reduced nitrate reductase structural gene expression by less than twofold. Expression of phi (narL-lacZ) operon fusions was weakly induced by nitrate but was indifferent to aerobiosis and independent of fnr. Expression of phi (narX-lacZ) operon fusions was induced by nitrate and was decreased by narL and fnr mutations. A phi (narK-lacZ) operon fusion was induced by nitrate, and its expression was fully dependent on narL+ and fnr+. Analysis of these operon fusions indicated that narL and narX are transcribed counterclockwise with respect to the E. coli genetic map and that narK is transcribed clockwise.

Cloning, Molecular↗
PubMed1986

Molecular cloning of nit-2, a regulatory gene required for nitrogen metabolite repression in Neurospora crassa.

We used an efficient sib-selection procedure to isolate a cosmid clone that complemented a mutated nit-2 gene of Neurospora crassa. Restriction fragment length polymorphism mapping indicated that the cosmid DNA insert was derived from linkage group IL, between 5S rDNA locus 12 and mt, the region of the N. crassa genome that contains nit-2. We conclude that the cosmid carries the nit-2 gene.

Chromosome Mapping↗
PubMed1986

Rho-dependent transcription termination in the tryptophanase operon leader region of Escherichia coli K-12.

Recent studies have suggested that expression of the tryptophanase (tna) operon of Escherichia coli is subject to transcription termination-antitermination control (V. Stewart and C. Yanofsky, J. Bacteriol. 164:731-740, 1985). In vivo studies have indicated that the transcribed leader region, tnaL, contains a site or sites of rho-dependent transcription termination (rho is the polypeptide product of the gene rho). We now report direct in vitro evidence that tnaL contains rho-dependent termination sites. In vivo termination appeared to occur at the rho-dependent termination sites identified in vitro. Transcription pausing analyses correlated sites of pausing in tnaL with sites of rho-dependent termination.

Base Sequence↗
PubMed1985

Evidence for transcription antitermination control of tryptophanase operon expression in Escherichia coli K-12.

Tryptophanase, encoded by the gene tnaA, is a catabolic enzyme distinct from the enzymes of tryptophan biosynthesis. Tryptophanase synthesis is induced by tryptophan and is subject to catabolite repression. We studied the mechanism of tna operon induction. Mutants with altered rho factor were partially constitutive for tna expression, implicating rho-dependent transcription termination in the control of tna expression. Measurements of mRNA synthesis from the transcribed leader region preceeding the tna operon suggested that the tna promoter was constitutive and that in the absence of inducer, transcription terminated in the leader region. Upon induction, this transcription termination was relieved. Cis-acting constitutive mutants had genetic alterations in the tna leader region. These lesions defined a site that is homologous to the bacteriophage lambda boxA sequence, which is thought to play a role in antitermination control of lambda lytic gene expression. We propose that tna expression is subject to transcription antitermination control. We hypothesize that a tryptophan-activated antiterminator protein mediates induction by suppressing the rho-dependent termination sites in the leader region, thus allowing transcription to proceed into the tna operon structural gene region.

Base Sequence↗
PubMed1984

Periarticular calcifications in association with intra-articular corticosteroid injections.

The authors report on three patients who received intra-articular corticosteroid injections (IACI) in the small joints of their hands for the symptomatic relief of local inflammation. In these cases radiographs showed that among the complications of IACI are periarticular soft-tissue calcifications. Evidence suggests that these calcifications are composed of hydroxyapatite crystals, which may themselves produce inflammatory arthritis.

Adult↗
PubMed1982

Nitrate reductase in Escherichia coli K-12: involvement of chlC, chlE, and chlG loci.

We examined the properties of mutants of E. coli which are defective with respect to nitrate reductase activity. chlE::Mu cts and chlG::Mu cts mutants were all chlorate resistant, and the strains that we examined all synthesized nitrate reductase apoenzyme. We concluded that the chlE and chlG loci, like the chlA, chlB, and chlD loci, are involved in the synthesis of insertion of molybdenum cofactor. We identified at least four distinct phenotypic classes of chlC::Tn10 mutants, all of which were fully or partially sensitive to chlorate. Two of these classes may represent lesions in the structural genes for nitrate reductase subunits A and C. Two other classes may be altered in the regulation of the expression of nitrate reductase or other anaerobic enzymes. We propose the mnemonic nar for naming individual genes within the chlC locus.

Apoenzymes↗
PubMed1982

Requirement of Fnr and NarL functions for nitrate reductase expression in Escherichia coli K-12.

I used a chlC-lac operon fusion to study regulatory mutations which affect nitrate reductase expression in Escherichia coli. A NarL- mutant apparently lacks a nitrate-specific positive regulatory component. Furthermore, an fnr (nirR) mutation prevented enzyme induction under any conditions. These data are consistent with a two-step, positive control model for nitrate reductase regulation.

DNA, Recombinant↗