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V Stewart

Publications and source records attributed to V Stewart.

At least 55 records · Page 3Linked to original sources
PubMed1993

Structural genes for thiamine biosynthetic enzymes (thiCEFGH) in Escherichia coli K-12.

Escherichia coli K-12 synthesizes thiamine pyrophosphate (vitamin B1) de novo. Two precursors [4-methyl-5-(beta-hydroxyethyl)thiazole monophosphate and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate] are coupled to form thiamine monophosphate, which is then phosphorylated to make thiamine pyrophosphate. Previous studies have identified two classes of thi mutations, clustered at 90 min on the genetic map, which result in requirements for the thiazole or the hydroxymethylpryimidine. We report here our initial molecular genetic analysis of the thi cluster. We cloned the thi cluster genes and examined their organization, structure, and function by a combination of phenotypic testing, complementation analysis, polypeptide expression, and DNA sequencing. We found five tightly linked genes, designated thiCEFGH. The thiC gene product is required for the synthesis of the hydroxymethylpyrimidine. The thiE, thiF, thiG, and thiH gene products are required for synthesis of the thiazole. These mutants did not respond to 1-deoxy-D-threo-2-pentulose, indicating that they are blocked in the conversion of this precursor compound to the thiazole itself.

Amino Acid Sequence↗
PubMed1993

Structures of genes nasA and nasB, encoding assimilatory nitrate and nitrite reductases in Klebsiella pneumoniae M5al.

Klebsiella pneumoniae can use nitrate and nitrite as sole nitrogen sources during aerobic growth. Assimilatory nitrate and nitrite reductases convert nitrate through nitrite to ammonium. We report here the molecular cloning of the nasA and nasB genes, which encode assimilatory nitrate and nitrite reductase, respectively. These genes are tightly linked and probably form a nasBA operon. In vivo protein expression and DNA sequence analysis revealed that the nasA and nasB genes encode 92- and 104-kDa proteins, respectively. The NASA polypeptide is homologous to other prokaryotic molybdoenzymes, and the NASB polypeptide is homologous to eukaryotic and prokaryotic NADH-nitrite reductases. The narL gene product positively regulates expression of the structural genes for respiratory nitrate reductase, narGHJI. Surprisingly, we found that the nasBA operon is tightly linked to the narL-narGHJI region in K. pneumoniae, even though the nitrate assimilatory and respiratory enzymes serve different physiological functions.

Amino Acid Sequence↗
PubMed1992

Either of two functionally redundant sensor proteins, NarX and NarQ, is sufficient for nitrate regulation in Escherichia coli K-12.

Nitrate acts through the response regulator NarL to activate and repress anaerobic respiratory gene expression in Escherichia coli. The narX gene product encodes a cognate sensor (histidine protein kinase). However, previous work discovered that NarL-mediated nitrate regulation is essentially normal in delta narX deletion mutants. In other two-component regulatory systems studied, the cognate sensor gene is essential for normal regulation. We suggested that NarX-mediated signal transduction reactions are also provided by a functionally redundant nitrate sensor, NarQ. We report here the identification and analysis of narQ insertion mutants. In narX+ strains, a narQ::Tn10 insertion had no perceptible effect on nitrate regulation. However, the same narQ::Tn10 insertion eliminated nitrate regulation when present in delta narX deletion strains. Thus, either narX+ or narQ+ was sufficient for essentially normal NarL-mediated nitrate regulation. The narQ gene mapped to 53 minutes on the E. coli genetic map, a location distinct from all known nitrate regulatory or target genes. The predicted NarQ sequence shares substantial similarity with NarX, particularly in the histidine protein kinase region and in a region of shared similarity with the methyl-accepting chemotaxis proteins. Both NarQ and NarX apparently have N-terminal periplasmic domains, but the primary structures of these regions are largely dissimilar in the two sequences. Analysis of narX* and narL missense alleles in narQ+ versus narQ::Tn10 backgrounds suggests that NarQ and NarX may have subtle functional differences.

Amino Acid Sequence↗
PubMed1992

In vivo requirement of integration host factor for nar (nitrate reductase) operon expression in Escherichia coli K-12.

The nitrate reductase operon (narGHJI) of Escherichia coli encodes an anaerobic respiratory enzyme. Previous work has identified two cis-acting sites in the nar operon control region: a proximal site required for anaerobic induction mediated by the activator Fnr and a remote upstream site required for nitrate induction mediated by the activator NarL [Li, S. & DeMoss, J. A. (1988) J. Biol. Chem. 263, 13700-13705]. Our search for nar regulatory mutants yielded one strain with a mutation in himD, the structural gene for one of the subunits of integration host factor (IHF). Strains carrying null alleles of the IHF structural genes, himD and himA, had severe defects in nitrate induction of the nar operon but were normal for nitrate induction of the coordinately regulated fdn operon. Anaerobic expression of both operons was normal in him mutants. Gel-mobility-shift and DNase I protection experiments revealed a single IHF binding site in the nar operon control region, located midway between the upstream activation site and the promoter. We conclude that an IHF-mediated DNA bend is essential for efficient nitrate induction of the sigma 70-dependent nar operon promoter. This requirement of IHF for transcriptional activation had been noted for several sigma 54-dependent promoters.

Bacterial Proteins↗
PubMed1992

RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement.

We have generated mice that carry a germline mutation in which a large portion of the RAG-2 coding region is deleted. Homozygous mutants are viable but fail to produce mature B or T lymphocytes. Very immature lymphoid cells were present in primary lymphoid organs of mutant animals as defined by surface marker analyses and Abelson murine leukemia virus (A-MuLV) transformation assays. However, these cells did not rearrange their immunoglobulin or T cell receptor loci. Lack of V(D)J recombination activity in mutant pre-B cell lines could be restored by introduction of a functional RAG-2 expression vector. Therefore, loss of RAG-2 function in vivo results in total inability to initiate V(D)J rearrangement, leading to a novel severe combined immune deficient (SCID) phenotype. Because the SCID phenotype was the only obvious abnormality detected in RAG-2 mutant mice, RAG-2 function and V(D)J recombinase activity, per se, are not required for development of cells other than lymphocytes.

Animals↗
PubMed1992

Embryonic lethality in mice homozygous for a targeted disruption of the N-myc gene.

The N-myc gene encodes a putative transcription factor that is thought to function in the regulation of gene expression during cell differentiation and/or growth. To examine the role of N-myc during development, we have used targeted mutagenesis in embryonic stem cells to produce a mouse line that carries an N-myc null allele. Mice homozygous for the mutation died between 10.5 and 12.5 days of gestation. Histological analysis of mutant embryos revealed that organs and tissues expected at these stages of development were present. However, multiple defects were observed, primarily in tissues and organs that normally express N-myc. In particular, mutant hearts were underdeveloped, often retaining the S-shape more typical of 9-day-old embryos. In addition, cranial and spinal ganglia were reduced in size and/or cellularity. Most of the noted defects were more consistent with a role of N-myc in proliferation of precursor populations than with a block in differentiation per se, at least at these early stages. These results demonstrate that N-myc plays an essential role during development and clearly confirm that N-myc has a physiological function that is distinct from that of the other myc-family genes.

Alleles↗
PubMed1992

Mutational analysis reveals functional similarity between NARX, a nitrate sensor in Escherichia coli K-12, and the methyl-accepting chemotaxis proteins.

During anaerobic growth, nitrate induces synthesis of the anaerobic respiratory enzymes formate dehydrogenase-N and nitrate reductase. This induction is mediated by a transcription activator, the narL gene product. The narX gene product may be involved in sensing nitrate and phosphorylating NARL. We isolated narX mutants, designated narX*, that caused nitrate-independent expression of the formate dehydrogenase-N and nitrate reductase structural genes. We used lambda narX specialized transducing phage to genetically analyze these lesions in single copy. Two previously isolated narX* mutations, narX32 and narX71, were also constructed by site-specific mutagenesis. We found that each of these alleles caused nitrate-independent synthesis of formate dehydrogenase-N and nitrate reductase, and each was recessive to narX+. The narX* mutations lie in a region of similarity with the methyl-accepting chemotaxis protein Tsr. We suggest that the narX* proteins have lost a transmembrane signalling function such that phosphoprotein phosphatase activity is reduced relative to protein kinase activity.

Amino Acid Sequence↗
PubMed1992

Localization of upstream sequence elements required for nitrate and anaerobic induction of fdn (formate dehydrogenase-N) operon expression in Escherichia coli K-12.

Two transcriptional activators, the FNR and NARL proteins, are required for induction of the fdnGHI operon, encoding Escherichia coli formate dehydrogenase-N. The FNR protein is required for anaerobic expression, while the NARL protein mediates nitrate induction. We used primer extension to locate the transcription initiation site 29 nucleotides upstream of the fdnG translation initiation codon. Expression assays with single-copy phi (fdnG-lacZ) gene fusions containing various deletions in the fdn 5'-regulatory region delimited three distinct cis-acting elements. One site, which is located at approximately -110, was required for nitrate induction. Two other sites share sequence similarity with the FNR protein binding site core consensus. The first site, centered at -42.5, was required for anaerobic induction. We used site-specific mutagenesis to change this putative FNR protein binding site into the CRP protein binding site core consensus. This change caused the fdn operon to be expressed aerobically, subject to CRP protein control. On the other hand, converting this putative FNR protein binding site into the FNR protein binding site core consensus resulted in elevated anaerobic induction of the fdn operon and also caused weak aerobic expression. The other putative FNR protein binding site, centered at -97.5, was not involved in anaerobic induction. It might play a negative role in fdn operon expression during anaerobic growth in the absence of nitrate.

Anaerobiosis↗
PubMed1992

Molybdenum cofactor (chlorate-resistant) mutants of Klebsiella pneumoniae M5al can use hypoxanthine as the sole nitrogen source.

Selection for chlorate resistance yields mol (formerly chl) mutants with defects in molybdenum cofactor synthesis. Complementation and genetic mapping analyses indicated that the Klebsiella pneumoniae mol genes are functionally homologous to those of Escherichia coli and occupy analogous genetic map positions. Hypoxanthine utilization in other organisms requires molybdenum cofactor as a component of xanthine dehydrogenase, and thus most chlorate-resistant mutants cannot use hypoxanthine as a sole source of nitrogen. Surprisingly, the K. pneumoniae mol mutants and the mol+ parent grew equally well with hypoxanthine as the sole nitrogen source, suggesting that K. pneumoniae has a molybdenum cofactor-independent pathway for hypoxanthine utilization.

Chromosome Mapping↗
PubMed1992

Comparison of two commonly used standard IVGTTs.

OBJECTIVE: To compare the magnitude and reproducibility of the FPIR measured during two different IVGTT protocols in nondiabetic subjects. RESEARCH DESIGN AND METHODS: Nine control subjects each had two pairs of IVGTTs with either a 4-min infusion of 0.5 g/kg glucose or a 1-min infusion of 0.3 g/kg glucose. Blood glucose and serum insulin were measured before and 1, 2, 3, 5, and 10 min after completion of the glucose infusion. The FPIR was measured with either 1 + 3-, 2 + 3 + 5-, or 1 + 3 + 5-min serum insulins, areas under the insulin curve (0-5 or 0-10 min), or the ratio of serum insulin to blood glucose area. RESULTS: The FPIR was higher in eight of nine subjects with the short-infusion test, but the within-subject variation of the two methods was identical. Reproducibility was not significantly improved with an integrated insulin area or insulin-to-glucose ratio measurement. CONCLUSIONS: Reproducibility of the FPIR measured during IVGTT is not significantly affected by the duration of the glucose infusion. However, the magnitude of the difference in FPIR observed between the two protocols highlights the need for standardization of the methodology if the IVGTT is to be used in studies of the preclinical stage of IDDM.

Adult↗
PubMed1991

Nitrate-inducible formate dehydrogenase in Escherichia coli K-12. I. Nucleotide sequence of the fdnGHI operon and evidence that opal (UGA) encodes selenocysteine.

The fdnGHI operon of Escherichia coli encodes nitrate-inducible formate dehydrogenase. We report here the entire nucleotide sequence of fdnGHI. The sequence contains three open reading frames of sizes appropriate to encode the three subunits of formate dehydrogenase-N. fdnG contains an in-frame UGA codon that specifies selenocysteine incorporation, and the predicted amino acid sequence of FdnG shows similarity to two other bacterial formate dehydrogenase enzymes. FdnH contains 4 cysteine clusters typical of those found in iron-sulfur proteins. FdnG also contains a cysteine cluster. Evidence from sequence and spectral analyses suggest that FdnI encodes cytochrome bFdn556. Implications for the membrane topology of formate dehydrogenase-N and its mechanism of proton translocation are discussed.

Amino Acid Sequence↗
PubMed1991

Nitrate-inducible formate dehydrogenase in Escherichia coli K-12. II. Evidence that a mRNA stem-loop structure is essential for decoding opal (UGA) as selenocysteine.

fdnG, encoding the selenopeptide of Escherichia coli formate dehydrogenase-N, contains an in-frame opal (UGA) codon at amino acid position 196 that directs selenocysteine incorporation. We have identified sequences that contribute to the mRNA context required for decoding this UGA as selenocysteine. We identified a potential stem-loop structure immediately downstream of UGA196 that is comparable in size and structure to a stem-loop predicted to form in fdhF, which encodes the selenopeptide of E. coli formate dehydrogenase-H. Mutational analysis of the fdnG stem-loop structure suggests that it is critical for decoding UGA196 as selenocysteine. Our data indicate that both stability and specific nucleotide sequences of the stem-loop likely contribute to the appropriate mRNA context for selenocysteine incorporation into the fdnG gene product.

Base Composition↗
PubMed1991

Genetic evidence that genes fdhD and fdhE do not control synthesis of formate dehydrogenase-N in Escherichia coli K-12.

Enterobacteria synthesize two formate dehydrogenases, formate dehydrogenase-N (encoded by fdnGHI) and formate dehydrogenase H (encoded by fdhF). Previous work has identified two rha-linked Salmonella typhimurium genes, fdnB and fdnC, which are required primarily for formate dehydrogenase-N activity. Analogous mutants, termed fdhD and fdhE, have been isolated in Escherichia coli. We used gene fusions between fdnG, the structural gene for the large subunit of formate dehydrogenase-N, and lacZ, the structural gene for beta-galactosidase, to examine E. coli fdnGHI operon expression in fdhD and fdhE insertion mutants. Expression of the phi (fdnG-lacZ) gene fusions was little affected by these insertions, suggesting that fdhD and fdhE do not control transcription or UGA decoding of the formate dehydrogenase-N structural genes. Our complementation tests, with cloned E. coli fdhD and fdhE genes, indicate that the S. typhimurium fdnC and fdnB genes are functionally homologous to the E. coli fdhD and fdhE genes, respectively.

Cloning, Molecular↗
PubMed1991

Mutational analysis of nitrate regulatory gene narL in Escherichia coli K-12.

The narL gene product, NarL, is the nitrate-responsive regulator of anaerobic respiratory gene expression. We used genetic analysis of narL mutants to better understand the mechanism of NarL-mediated gene regulation. We selected and analyzed seven nitrate-independent narL mutants. Each of three independent, strongly constitutive mutants had changes of Val-88 to Ala. The other four mutants were weakly constitutive. The narL505(V88A) allele was largely dominant to narL+, while narX+ had a negative influence on its constitutive phenotype, suggesting that NarX may play a negative role in nitrate regulation. We also constructed two narL mutations that are analogous to previously characterized constitutive degU alleles. The first, narL503(H15L), was a recessive null allele. The second, narL504(D110K), functioned essentially as wild type but was dependent on narX+ for full activity. We changed Asp-59 of NarL, which corresponds to the site of phosphorylation of other response regulators, to Asn. This change, narL502(D59N), was a recessive null allele, which is consistent with the hypothesis that NarL requires phosphorylation for activation. Finally, we tested the requirement for molybdate on regulation in a narL505(V88A) strain. Although narL505(V88A) conferred some nitrate-independent expression of fdnGHI (encoding formate dehydrogenase-N) in limiting molybdate, it required excess molybdate for full induction both in the absence and in the presence of nitrate. This finding suggests that narL505(V88A) did not confer molybdate-independent expression of fdnGHI.

Amino Acid Sequence↗
PubMed1990

Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12.

Formate oxidation coupled to nitrate reduction constitutes a major anaerobic respiratory pathway in Escherichia coli. This respiratory chain consists of formate dehydrogenase-N, quinone, and nitrate reductase. We have isolated a recombinant DNA clone that likely contains the structural genes, fdnGHI, for the three subunits of formate dehydrogenase-N. The fdnGHI clone produced proteins of 110, 32 and 20 kDa which correspond to the subunit sizes of purified formate dehydrogenase-N. Our analysis indicates that fdnGHI is organized as an operon. We mapped the fdn operon to 32 min on the E. coli genetic map, close to the genes for cryptic nitrate reductase (encoded by the narZ operon). Expression of phi(fdnG-lacZ) operon fusions was induced by anaerobiosis and nitrate. This induction required fnr+ and narL+, two regulatory genes whose products are also required for the anaerobic, nitrate-inducible activation of the nitrate reductase structural gene operon, narGHJI. We conclude that regulation of fdnGHI and narGHJI expression is mediated through common pathways.

Aldehyde Oxidoreductases↗
PubMed1990

Structure and expression of germline immunoglobulin gamma 3 heavy chain gene transcripts: implications for mitogen and lymphokine directed class-switching.

We have characterized the structure and expression of transcripts synthesized from the murine germline immunoglobulin gamma 3 heavy chain gene in certain B-lineage cells. The transcripts initiate upstream of the switch gamma 3 region, generating a 5' exon that is spliced to C gamma 3 exons. Expression of this germline transcript is induced when splenic B cells or A-MuLV-transformed pre-B cell lines are cultured in the presence of lipopolysaccharide (LPS). Addition of interleukin-4 (IL-4) to these lipopolysaccharide (LPS) cultures dramatically inhibits induction of the germline gamma 3 transcript. Induction of germline gamma 3 transcripts occurs before the increased accumulation of gamma 3-producing cells and VDJ-gamma 3 mRNA in cultures of splenic B cells. These data provide further evidence that germline CH transcriptional units are important components in the regulation of heavy chain class-switching. In addition, the pre-B cell lines that we describe represent the first example of permanent cell lines that regulate expression of the germline gamma locus in response to LPS plus IL-4 treatment in a manner analogous to normal B cells; therefore these lines should represent an excellent model system to further study the molecular mechanisms by which germline expression is regulated by these agents.

Animals↗
PubMed1990

Genetic evidence that NarL function is not required for nitrate regulation of nitrate assimilation in Klebsiella pneumoniae M5al.

We cloned the narL gene, required for nitrate induction of respiratory nitrate reductase synthesis, from Klebsiella pneumoniae. The E. coli narL gene product shares sequence similarity with the response regulator proteins of two-component regulatory systems. We found that narL(+)-containing plasmids restored nitrate regulation of anaerobic respiratory gene expression in appropriate Escherichia coli hosts. The K. pneumoniae narL region encoded a protein whose migration in Laemmli gels was indistinguishable from that of the narL product of E. coli. We constructed a narL::Km mutant of K. pneumoniae. This mutation abolished nitrate induction of respiratory nitrate reductase synthesis but had no effect on nitrate induction of assimilatory nitrate and nitrite reductase synthesis. We conclude that K. pneumoniae has distinct nitrate-responsive regulators for controlling respiratory and assimilatory gene expression.

Cloning, Molecular↗