PubMed Health⌕ Search

Biomedical subjects

B J Bowman

Publications and source records attributed to B J Bowman.

At least 19 recordsLinked to original sources
PubMed1997

Mutations of pma-1, the gene encoding the plasma membrane H+-ATPase of Neurospora crassa, suppress inhibition of growth by concanamycin A, a specific inhibitor of vacuolar ATPases.

Concanamycin A (CCA), a specific inhibitor of vacuolar ATPases, inhibited growth of Neurospora crassa in medium adjusted to pH 7 or above. Mutant strains were selected for growth on medium containing 1.0 microM CCA. Sixty-four (of 66) mutations mapped in the region of the pma1 locus, which encodes the plasma membrane H+-ATPase. Analysis of V-ATPase activity in isolated vacuolar membranes from the mutant strains showed wild-type activity and sensitivity to CCA. In contrast, plasma membrane H+-ATPase activity in isolated plasma membranes from the mutants was reduced as compared with wild-type, and in four strains the activity showed increased resistance to vanadate. The most interesting change in the plasma membrane H+-ATPase was in kinetic behavior. The wild-type enzyme showed sigmoid dependence on MgATP concentration with a Hill number of 2.0, while the seven mutants tested exhibited hyperbolic kinetics with a Hill number of 1.0. One interpretation of these data was that the enzyme had changed from a functional dimer to a functional monomer. Mutation of the plasma membrane H+-ATPase did not confer resistance by preventing uptake of CCA. In the presence of CCA both wild-type and mutant strains were unable to accumulate arginine, failed to concentrate chloroquine in acidic vesicles, and exhibited gross alterations in hyphal morphology, indicating that the CCA had entered the cells and inactivated the V-ATPase. Instead, we hypothesize that the mutations conferred resistance because the altered plasma membrane H+-ATPase could more efficiently rid the cell of toxic levels of Ca2+ or protons or other ions accumulated in the cytoplasm following inactivation of the V-ATPase by CCA.

Anti-Bacterial Agents↗
PubMed1997

Cultural pathways toward Antonovsky's sense of coherence.

This study empirically validated Antonovsky's suggestion that differing cultural pathways exist in the development of a Sense of Coherence Differences were found in the ways in which essentially identical levels of a Sense of Coherence were developed in Native Americans and Anglo-Americans. Native Americans' families appear to emphasize moral and religious values while Anglo-American families appear to emphasize achievement and independence.

Achievement↗
PubMed1997

The intriguing evolution of the "b" and "G" subunits in F-type and V-type ATPases: isolation of the vma-10 gene from Neurospora crassa.

We have characterized the vma-10 gene which encodes the G subunit of the vacuolar ATPase in Neurospora crassa. The gene is somewhat unusual in filamentous fungi because it contains five introns, comprising 71% of the region between the translation start and stop codons. The 5' untranslated region of the gene contains several elements that have been identified in other genes that encode subunits of the vacuolar ATPase in N. crassa. A comparison of G subunits from N. crassa, S. cerevisiae, and animal cells showed that the N-terminal half of the polypeptide shows the highest degree of sequence conservation. Most striking is the observation that this region could form an alpha helix in which all of the conserved residues are clustered on one face. Subunit G appears to be homologous to the b subunit found in F-type ATPases. The major difference between the b and G subunits is the lack of a membrane-spanning region in the G subunit. We have also identified homologous subunits in the operons which encode V-type ATPases in a eubacterium, Enterrococcus hirae, and an archaebacterium, Methanococcus jannaschii. As in eukaryotic vacuolar ATPases the G subunit homologs lack a membrane-spanning region. Although the b and G subunits appear to be derived from a common ancestor, significant changes have evolved. In F-type and V-type ATPases these subunits can have zero, one, or two membrane-spanning regions and can also differ significantly in the number of copies per enzyme.

Amino Acid Sequence↗
PubMed1996

Characterization of a vacuolar protease in Neurospora crassa and the use of gene RIPing to generate protease-deficient strains.

We have isolated a gene from Neurospora crassa that appears to encode a pepstatin-sensitive protease found both in membranes and in soluble contents of vacuoles. The gene contains two introns and encodes a 396-residue protein with a molecular mass of 42,900 Da. Because of the similarity of the protein to proteinase A in Saccharomyces cerevisiae the gene has been named pep-4. Strains with mutations in the pep-4 gene were generated in vivo by the gene RIPing procedure described by Selker and Garrett (Selker, E. U., and Garrett, P. W. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6870-6874). The mutant strains were deficient in pepstatin-sensitive protease activity and did not appear to produce a major 42-kDa polypeptide in the vacuole. The mutant strains grew at the same rate as the wild type and had no other observable phenotype. When compared with inactivation of the PEP4 gene of S. cerevisiae, inactivation of the pep-4 gene in N. crassa produced a phenotype that was different in several ways. In N. crassa the mutant strains did not exhibit reduced sporulation or reduced viability after nitrogen starvation, and they had elevated levels of proteinase B and carboxypeptidase activities. The pep-4 gene appears to encode the N. crassa, homolog of proteinase A, but the maturation of vacuolar hydrolases appeared to be less dependent on this protease than has been observed in S. cerevisiae.

Amino Acid Sequence↗
PubMed1996

Isolation of the vma-6 gene encoding a 41 kDa subunit of the Neurospora crassa vacuolar ATPase, and an adjoining gene encoding a ribosome-associated protein.

The vma-6 gene, encoding a membrane-associated subunit of the vacuolar H+-ATPase from Neurospora crassa, was cloned and sequenced. The gene contains three small introns and encodes a protein of 41 005 Da. When compared with homologous polypeptides from other species, the N. crassa protein contains a unique glycine-rich region. Three conserved cysteine residues, previously unrecognized, have been identified. An unrelated gene encoding a protein of 31 701 Da was found 2.1 kb downstream of vma-6. The second appears to encode the N. crassa homolog of a ribosome-associated protein identified previously in several plant and mammalian cells, and was named rap-1.

Adenosine Triphosphatases↗
PubMed1996

Cross-cultural validation of Antonovsky's Sense Of Coherence Scale.

This study empirically validated Antonovsky's suggestion that a Sense of Coherence is important in managing stress and remaining both physically and psychologically healthy. In both Anglo-American and Native American cultures, Sense of Coherence Measures were found to correlate negatively with measures of physical and mental distress. More interestingly, this study supported Antonovsky's prediction that people from various cultures may attain a similar level of a Sense of Coherence, despite great socioeconomic differences.

Adaptation, Psychological↗
PubMed1996

The vacuolar ATPase of Neurospora crassa is indispensable: inactivation of the vma-1 gene by repeat-induced point mutation.

To analyze the phenotype of cells lacking the vacuolar ATPase, we inactivated the vma-1 gene, which encodes the catalytic subunit of the enzyme. Because preliminary experiments suggested the vma-1 gene was essential, we developed a method of simultaneously inactivating the gene and complementing it with a functional copy. We call this method repeat-induced point mutation (RIP) & Rescue. Two strains, both of which contained an extra copy of the vma-1 gene, were mated. Progeny that had inherited a functional copy of the gene at an ectopic site in the genome were selected. In some of these progeny the endogenous vma-1 gene had been altered by the RIP process. Sequencing showed the endogenous vma-1 gene had been inactivated by multiple point mutations. Progeny from strains with an inactive endogenous vma-1 gene were inviable unless a functional copy of the gene cosegregated, indicating that the vacuolar ATPase is essential in Neurospora crassa.

Base Sequence↗
PubMed1995

Regulation of the expression of three housekeeping genes encoding subunits of the Neurospora crassa vacuolar ATPase.

The vacuolar ATPase is a complex enzyme and is encoded by at least nine genes, which appear to be scattered throughout the genome. We have examined the vma-1 vma-2, and vma-3 genes, which encode subunits present in multiple copies within the Neurospora crassa vacuolar ATPase. We wished to see if the expression of these genes is coordinately regulated and if these genes contain similar promoter elements. A region was sequenced of approximately 1 kb located upstream of the protein coding region for each gene. Several sequence elements were found in similar positions in each of the three genes. Each of the genes had several strong transcription initiation sites, clustered within 13-60 bp and located 112-193 bp upstream of the translation start site. The size and abundance of the RNA transcripts was also determined: the amount of RNA transcribed from each gene was roughly proportional to the numbers of each subunit present in the enzyme. A series of plasmids was constructed containing parts of the putative promoter region fused to beta-galactosidase. Analysis of these plasmids indicated that the essential region of the vma promoters lies within 370 bp of the protein coding region. Overall, the vma genes appear to have similar characteristics to "housekeeping" genes described in other organisms.

Adenosine Triphosphatases↗
PubMed1995

Isolation of the vma-4 gene encoding the 26 kDa subunit of the Neurospora crassa vacuolar ATPase.

We have isolated the vma-4 gene, which encodes a 25,746 Dalton subunit of the vacuolar ATPase, from Neurospora crassa. The gene contains two introns and was mapped to the left arm of linkage group I. Comparison of the predicted amino acid sequence with homologous proteins from Saccharomyces cerevisiae, Manduca sexta, and Bos taurus showed only 25% sequence identity. However, computer-assisted predictions of secondary structures gave similar results for all four proteins. Analysis of the sequence and the available biochemical data indicated that the vma-4 gene product may play the same structural role in the vacuolar ATPase as does the gamma-subunit in F-type ATPases.

Adenosine Triphosphatases↗
PubMed1995

The vacuolar ATPase: sulfite stabilization and the mechanism of nitrate inactivation.

Using vacuolar membranes from Neurospora crassa, we observed that sulfite prevented the loss of vacuolar ATPase activity that otherwise occurred during 36 h at room temperature. Sulfite neither activated nor changed the kinetic behavior of the enzyme. Further, in the presence of sulfite, the vacuolar ATPase was not inhibited by nitrate. We tested the hypothesis that sulfite acts as a reducing agent to stabilize the enzyme, while nitrate acts as an oxidizing agent, inhibiting the enzyme by promoting the formation of disulfide bonds. All reducing agents tested, dithionite, selenite, thiophosphate, dithiothreitol and glutathione, prevented the loss of ATPase activity. On the other hand, all oxidizing agents tested, bromate, iodate, arsenite, perchlorate, and hydrogen peroxide, were potent inhibitors of ATPase activity. The inhibitory effect of the oxidizing agents was specific for the vacuolar ATPase. The mitochondrial ATPase, assayed under identical conditions, was not inhibited by any of the oxidizing agents. Analysis of proteins with two-dimensional gel electrophoresis indicated that nitrate can promote the formation of disufide bonds between proteins in the vacuolar membrane. These data suggest a mechanism to explain why nitrate specifically inhibits vacuolar ATPases, and they support the proposal by Feng and Forgac (Feng, Y., and Forgac, M. (1994) J. Biol. Chem. 269, 13244-13230) that oxidation and reduction of critical cysteine residues may regulate the activity of vacuolar ATPases in vivo.

Adenosine Triphosphatases↗
PubMed1994

Characterization of the cit-1 gene from Neurospora crassa encoding the mitochondrial form of citrate synthase.

We have isolated the cDNA and corresponding genomic DNA encoding citrate synthase in Neurospora crassa. Analysis of the protein coding region of this gene, named cit-1, indicates that it specifies the mitochondrial form of citrate synthase. The predicted protein has 469 amino acids and a molecular mass of 52,002 Da. The gene is interrupted by four introns. Hybridization experiments show that a cit-1 probe binds to two different fragments of genomic DNA, which are located on different chromosomes. Neurospora crassa may have two isoforms of citrate synthase, one in the mitochondria and the other in microbodies.

Amino Acid Sequence↗
PubMed1994

The proteolipid subunit of the Neurospora crassa vacuolar ATPase: isolation of the protein and the vma-3 gene.

We have isolated the proteolipid subunit from the vacuolar ATPase of Neurospora crassa, using ion-exchange chromatography. We have also isolated several cDNA clones and the corresponding genomic DNA that encodes this subunit. The derived protein sequence indicates that the polypeptide is composed of 161 amino acid residues with an M(r) of 16,328 kDa. The gene encoding the proteolipid, named vma-3, is unusual in several respects. It contains four introns and, unlike other fungal genes, has non-coding regions that are as large as the coding regions. The 3' untranslated regions of the cDNAs were quite heterogeneous, with polyadenylation sites more than 300 bp apart. Analysis of the mRNA indicates that two size classes of transcripts are produced, differing in the length of the 3' untranslated region. Mapping of the vma-3 gene showed that it is closely linked, but not adjacent to, vma-1, the gene encoding the 67 kDa subunit of the vacuolar ATPase. This raises the possibility that in N. crassa some of the vacuolar ATPase genes may be clustered.

Adenosine Triphosphatases↗
PubMed1992

Structure of the vacuolar ATPase from Neurospora crassa as determined by electron microscopy.

We have examined the structure of the vacuolar ATPase of Neurospora crassa using negatively stained preparations of vacuolar membranes and of detergent-solubilized and gradient-purified ATPase complexes. We also examined the peripheral sector (V1) of the enzyme after it had been removed and purified. Using different stains, vacuolar membranes displayed ball-and-stalk structures similar to those of the intact mitochondrial ATPase. However, the vacuolar ATPase was clearly different from the mitochondrial ATPase in both size and structural features. The vacuolar enzyme had a much larger head domain with a distinct cleft down the middle of the complex. This domain was held above the membrane by a prominent stalk. Most intriguing was the presence of basal components. These structures appeared to project from the vacuolar membrane near the base of the stalks. Detergent-solubilized, gradient-purified ATPases displayed the same head, stalk, and basal features as those found with the intact enzyme on vacuolar membranes. The mitochondrial ATPase was significantly smaller, and no clefted head domains or basal components were observed. When V1 and F1 particles were directly compared, a significant difference in size and shape between these two soluble ATPase sectors was apparent. V1 retained all of the features seen in the globular head of the intact complex: V-shaped, triangular, and square forms around a stain-filled core.

Adenosine Triphosphatases↗
PubMed1992

The vacuolar ATPase of Neurospora crassa.

The filamentous fungus Neurospora crassa has many small vacuoles which, like mammalian lysosomes, contain hydrolytic enzymes. They also store large amounts of phosphate and basic amino acids. To generate an acidic interior and to drive the transport of small molecules, the vacuolar membranes are densely studded with a proton-pumping ATPase. The vacuolar ATPase is a large enzyme, composed of 8-10 subunits. These subunits are arranged into two sectors, a complex of peripheral subunits called V1 and an integral membrane complex called V0. Genes encoding three of the subunits have been isolated. vma-1 and vma-2 encode polypeptides homologous to the alpha and beta subunits of F-type ATPases. These subunits appear to contain the sites of ATP binding and hydrolysis. vma-3 encodes a highly hydrophobic polypeptide homologous to the proteolipid subunit of vacuolar ATPases from other organisms. This subunit may form part of the proton-containing pathway through the membrane. We have examined the structures of the genes and attempted to inactivate them.

Amino Acid Sequence↗
PubMed1992

Vacuolar ATPase of Neurospora crassa: electron microscopy, gene characterization and gene inactivation/mutation.

We are using three approaches to investigate the vacuolar ATPase, V-ATPase, from Neurospora crassa. (1) Examination in the electron microscope shows the enzyme has a 'ball and stalk' structure like the F-type ATPases. However, the vacuolar ATPase is significantly larger, has a prominent cleft in the head sector, and has extra components associated with the stalk and membrane sectors. (2) Genes encoding three of the major subunits of the vacuolar ATPase and the homologous subunits of the mitochondrial F-ATPase have been isolated. The exon/intron structures of the genes have been analyzed and the chromosomal locations have been determined. Two of the vacuolar ATPase genes map very close to each other, suggesting the possibility of a cluster of ATPase genes. (3) The function of the ATPase is being investigated by isolating strains with altered or inactivated ATPase. We are characterizing strains that are resistant to bafilomycin A1, a potent and specific inhibitor of the vacuolar ATPase. Initial attempts to inactivate a vacuolar ATPase gene indicate that the enzyme may be essential for growth.

Adenosine Triphosphatases↗
PubMed1990

Mutations that affect circadian rhythms in Neurospora crassa can alter the reduction of cytochromes by blue light.

We have examined membrane fractions from mutant strains of Neurospora crassa that have altered responses to blue light or have altered circadian rhythms. Using an in vitro assay, we assessed whether the mutations affected the levels of photoreducible cytochromes. Three of the mutant strains, prd-1, rib-1, and wc-1, were not qualitatively different from the wild type. The poky strain was found to have high concentrations of photoreducible cytochrome c. After removal of this cytochrome, however, the photoreducible cytochromes in the plasma membrane and endoplasmic reticulum were also similar to those of the wild type. The most significant differences were found in strains mutated at the frq locus, which affects circadian rhythms. In the frq-9 strain, the cytochrome in the endoplasmic reticulum was not detectably reduced by blue light. The frq-1 mutation caused a significant shift in the spectrum of blue-light-reduced cytochrome in the endoplasmic reticulum.

Cell Membrane↗
PubMed1989

Nucleotide sequence of pho-4+, encoding a phosphate-repressible phosphate permease of Neurospora crassa.

The nucleotide (nt) sequence of the Neurospora crassa pho-4+ gene, which encodes a phosphate-repressible phosphate permease, has been determined. The gene specifies a protein of 590 amino acids (aa) and contains two introns. Two RNA transcripts of 3.3 and 2.4 kb have been identified, and transcription start points (tsp) and termination sites and/or processing sites have been located. The 3.3-kb message is initiated about 890 nt upstream from the tsp for the 2.4-kb transcript. A hydropathy profile of the aa sequence suggests ten to twelve membrane-spanning helices with a large hydrophilic domain between the eighth and ninth helices. This model for the predicted secondary structure of the protein is very similar to models proposed for other sequenced integral membrane proteins from both prokaryotes and eukaryotes. Since very few permease-encoding genes of eukaryotes have been examined in molecular detail, it will be of interest to compare the sequence of pho-4+ with those encoding other anion transport proteins, as they become available.

Amino Acid Sequence↗