PubMed Health⌕ Search

Biomedical subjects

P W Robbins

Publications and source records attributed to P W Robbins.

At least 37 records · Page 2Linked to original sources
PubMed1997

Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae.

The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.

Cell Membrane↗
PubMed1996

Gene cloning and production of active recombinant Brugia malayi microfilarial chitinase.

Canlas and coworkers [Canlas et al. (1984) Am. J. Trop. Med. Hyg. 33, 420-424] isolated a monoclonal antibody (MF1) which, upon passive transfer, led to the clearance of Brugia malayi (Bm) microfilariae (mf) from infected jirds. The target of MF1 is a developmentally regulated mf chitinase (Cht) (Fuhrman et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1548-1552). This paper describes the production of enzymatically active Bm Cht in Escherichia coli. Standard expression conditions resulted in production of an insoluble maltose-binding protein (MBP)::Cht fusion protein, but by optimizing expression conditions, the amount of soluble MBP::Cht was increased 25-fold. The specific activity of the soluble MBP::Cht isolated from the E. coli cytoplasm was low. Exporting MBP::Cht into the E. coli periplasmic space increased the specific activity by 12-fold. This suggests that secretion through the membrane and/or the environment of the periplasmic space results in improved folding of recombinant Bm Cht.

ATP-Binding Cassette Transporters↗
PubMed1996

Cloning and characterization of chsD, a chitin synthase-like gene of Aspergillus fumigatus.

A chitin synthase-like gene (chsD) was isolated from an Aspergillus fumigatus genomic DNA library. Comparisons with the predicted amino acid sequence from chsD reveals low but significant similarity to chitin synthases, to other N-acetylglucosaminyltransferases (NodC from Rhizopus spp., HasA from Streptococcus spp. and DG42 from vertebrates. A chsD- mutant strain constructed by gene disruption has a 20% reduction in total mycelial chitin content; however, no differences between the wild-type strain and the chsD- strain were found with respect to morphology, chitin synthase activity or virulence in a neutropenic murine model of aspergillosis. The results show that the chsD product has an important but inessential role in the synthesis of chitin in A. fumigatus.

Amino Acid Sequence↗
PubMed1996

The isolation of a Dol-P-Man synthase from Ustilago maydis that functions in Saccharomyces cerevisiae.

Genomic DNAs from several fungi were screened for a homologous sequence to Saccharomyces cerevisiae DPM1, an essential gene which encodes dolichyl phosphoryl mannose synthase. The fungi examined included Aspergillus nidulans, Neurospora crassa, Schizophyllum commune and Ustilago maydis. Only U. maydis gave a significant signal after Southern hybridization using DPM1 as a probe. The Ustilago homolog was subsequently cloned and sequenced. The predicted protein of 294 amino acids has 60% identity to the S. cerevisiae protein, but lacks the putative "dolichol recognition sequence'. RNA of ca. 900 bp is transcribed in both yeast and filamentous cells of Ustilago. In Escherichia coli, the U. maydis sequence expressed a 35 kDa protein exhibiting dolichyl phosphoryl mannose synthase activity. The sequence was also shown to complement a haploid strain of S. cerevisiae containing a deletion of the DPM1 gene. The U. maydis sequence therefore, encodes a dolichyl phosphoryl mannose synthase that can support normal vegetative growth in S. cerevisiae.

Amino Acid Sequence↗
PubMed1996

Molecular cloning of the Golgi apparatus uridine diphosphate-N-acetylglucosamine transporter from Kluyveromyces lactis.

The mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they lack mannose phosphate and have terminal alpha1-->2-linked N-acetylglucosamine. The biosynthesis of these chains probably occurs in the lumen of the Golgi apparatus, by analogy to S. cerevisiae. The sugar donors, GDP-mannose and UDP-GlcNAc, must first be transported from the cytosol, their site of synthesis, via specific Golgi membrane transporters into the lumen where they are substrates in the biosynthesis of these mannoproteins. A mutant of K. lactis, mnn2-2, that lacks terminal N-acetylglucosamine in its mannan chains in vivo, has recently been characterized and shown to have a specific defect in transport of UDP-GlcNAc into the lumen of Golgi vesicles in vitro. We have now cloned the gene encoding the K. lactis Golgi membrane UDP-GlcNAc transporter by complementation of the mnn2-2 mutation. The mnn2-2 mutant was transformed with a genomic library from wild-type K. lactis in a pKD1-derived vector; transformants were isolated and phenotypic correction was monitored following cell surface labeling with fluorescein isothiocyanate conjugated to Griffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine, and a fluorescent activated cell sorter. A 2.4-kb DNA fragment was found to restore the wild-type lectin binding phenotype. Upon loss of the plasmid containing this fragment, reversion to the mutant phenotype occurred. The above fragment contained an open reading frame for a multitransmembrane spanning protein of 328 amino acids. The protein contains a leucine zipper motif and has high homology to predicted proteins from S. cerevisiae and C. elegans. In an assay in vitro, Golgi vesicles isolated from the transformant had regained their ability to transport UDP-GlcNAc. Taken together, the above results strongly suggest that the cloned gene encodes the Golgi UDP-GlcNAc transporter of K. lactis.

Amino Acid Sequence↗
PubMed1996

Homologs of the Xenopus developmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis of Nod-like chitin oligosaccharides during early embryogenesis.

The Xenopus developmental gene DG42 is expressed during early embryonic development, between the midblastula and neurulation stages. The deduced protein sequence of Xenopus DG42 shows similarity to Rhizobium Nod C, Streptococcus Has A, and fungal chitin synthases. Previously, we found that the DG42 protein made in an in vitro transcription/translation system catalyzed synthesis of an array of chitin oligosaccharides. Here we show that cell extracts from early Xenopus and zebrafish embryos also synthesize chitooligosaccharides. cDNA fragments homologous to DG42 from zebrafish and mouse were also cloned and sequenced. Expression of these homologs was similar to that described for Xenopus based on Northern and Western blot analysis. The Xenopus anti-DG42 antibody recognized a 63-kDa protein in extracts from zebrafish embryos that followed a similar developmental expression pattern to that previously described for Xenopus. The chitin oligosaccharide synthase activity found in extracts was inactivated by a specific DG42 antibody; synthesis of hyaluronic acid (HA) was not affected under the conditions tested. Other experiments demonstrate that expression of DG42 under plasmid control in mouse 3T3 cells gives rise to chitooligosaccharide synthase activity without an increase in HA synthase level. A possible relationship between our results and those of other investigators, which show stimulation of HA synthesis by DG42 in mammalian cell culture systems, is provided by structural analyses to be published elsewhere that suggest that chitin oligosaccharides are present at the reducing ends of HA chains. Since in at least one vertebrate system hyaluronic acid formation can be inhibited by a pure chitinase, it seems possible that chitin oligosaccharides serve as primers for hyaluronic acid synthesis.

3T3 Cells↗
PubMed1996

A mutant yeast deficient in Golgi transport of uridine diphosphate N-acetylglucosamine.

Mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they have terminal alpha1-->2-linked N-acetylglucosamine and lack mannose phosphate. In a previous study, Douglas and Ballou (Douglas, R. K., and Ballou, C. E. (1982) Biochemistry 21, 1561-1570) characterized a mutant, mnn2-2, which lacked terminal N-acetylglucosamine in its mannoproteins. The mutant had normal levels of N-acetylglucosaminyltransferase activity, and the partially purified enzyme from wild-type and mutant cells had the same apparent size, heat stability, affinity for substrates, metal requirement, and subcellular location. No qualitative or quantitative differences were found between mutant and wild-type cells in endogenous mannan acceptors and pools of UDP-GlcNAc. Chitin was synthesized at similar rates in wild-type and mutant cells, and the latter did not have a soluble inhibitor of the N-acetylglucosaminyltransferase or a hexosaminidase that could remove N-acetylglucosamine from mannoproteins. Together, the above observations led Douglas and Ballou ((1982) Biochemistry 21, 1561-1570) to postulate that the mutant might have a defect in compartmentation of substrates involved in the biosynthesis of mannoproteins. We determined whether the above mutant phenotype is the result of defective transport of UDP-GlcNAc into Golgi vesicles from K. lactis. Golgi vesicles which were sealed and of the same membrane topographical orientation as in vivo were isolated from wild-type and mnn2-2 mutant cells and incubated with UDP-GlcNAc in an assay in vitro. The initial rate of transport of UDP-GlcNAc into Golgi vesicles from wild-type cells was temperature dependent, saturable with an apparent Km of 5.5 microM and a Vmax of 8.2 pmol/mg of protein/3 min. No transport of UDP-GlcNAc was detected into Golgi vesicles from mutant cells. However, Golgi vesicles from both cells translocated GDP-mannose at comparable velocities, indicating that the above transport defect is specific. In addition to the above defect in mannoproteins, mutant cells were also deficient in the biosynthesis of glucosamine containing lipids.

Biological Transport↗
PubMed1996

chs-4, a class IV chitin synthase gene from Neurospora crassa.

In Saccharomyces cerevisiae, most of the cellular chitin is produced by chitin synthase III, which requires the product encoded by the CSD2/CAL1/DIT101/KT12 gene. We have identified, isolated and structurally characterized as CSD2/CAL1/DIT101/KT12 homologue in the filamentous ascomycete Neurospora crassa and have used a "reverse genetics" approach to determine its role in vivo. The yeast gene was used as a heterologous probe for the isolation of a N. crassa gene(designated chs-4) encoding a polypeptide belonging to a class of chitin synthases which we have designated class IV. The predicted polypeptide encoded by this gene is highly similar to those of S. cerevisiae and Candida albicans. N. crassa strains in which chs-4 had been inactivated by the Repeat-Induced point mutation (RIP) process grew and developed in a normal manner under standard growth conditions. However, when grown in the presence of sorbose (a carbon source which induces morphological changes accompanied by elevated chitin content), chitin levels in the chs-4RIP strain were significantly lower than those observed in the wild type. We suggest that CHS4 may serve as an auxiliary enzyme in N. crassa and that, in contrast to yeasts, it is possible that filamentous fungi may have more than one class IV chitin synthase.

Acetylglucosamine↗
PubMed1996

Molecular cloning of a third chitinase gene (CHT1) from Candida albicans.

Here we report the complete nucleotide sequence of a third chitinase gene (CHT1) from the dimorphic human pathogen Candida albicans. The deduced amino acid (aa) sequence of Cht1 consists of 416 aa and displays 36% protein sequence similarity to chitinases Cht2 and Cht3, from C. albicans. Interestingly the domain structure of Cht1 is truncated when compared to the other chitinases of C. albicans and lacks a Ser/Thr-rich region.

Amino Acid Sequence↗
PubMed1996

The chsD and chsE genes of Aspergillus nidulans and their roles in chitin synthesis.

Two chitin synthase genes, chsD and chsE, were identified from the filamentous ascomycete Aspergillus nidulans. In a region that is conserved among chitin synthases, the deduced amino acid sequences of chsD and chsE have greater sequence identity to the polypeptides encoded by the Saccharomyces cerevisiae CHS3 gene (also named CSD2, CAL1, DIT101, and KTI1) and the Candida albicans CHSE gene than to other chitin synthases. chsE is more closely related to the CHS3 genes, and this group constitutes the class IV chitin synthases. chsD differs sufficiently from the other classes of fungal chitin synthase genes to constitute a new class, class V. Each of the wild-type A. nidulans genes was replaced by a copy that had a substantial fraction of its coding region replaced by the A. nidulans argB gene. Hyphae from both chsD and chsE disruptants contain about 60-70% of the chitin content of wild-type hyphae. The morphology and development of chsE disruptants are indistinguishable from those of wild type. Nearly all of the conidia of chsD disruption strains swell excessively and lyse when germinated in low osmotic strength medium. Conidia that do not lyse produce hyphae that initially have normal morphology but subsequently lyse at subapical locations and show ballooned walls along their length. The lysis of germinating conidia and hyphae of chsD disruptants is prevented by the presence of osmotic stabilizers in the medium. Conidiophore vesicles from chsD disruption strains frequently swell excessively and lyse, resulting in colonies that show reduced conidiation. These properties indicate that chitin synthesized by the chsD-encoded isozyme contributes to the rigidity of the walls of germinating conidia, of the subapical region of hyphae, and of conidiophore vesicles, but is not necessary for normal morphology of these cells. The phenotypes of chsD and chsE disruptants indicate that the chitin synthesized by each isozyme serves a distinct function. The propensity of a chsD disruptant for osmotically induced lysis was compared to that of strains carrying two other mutations (tsE6 and orlA::trpC) which also result in reduced chitin content vegetative cell lysis. The concentration of osmotic stabilizer necessary to remedy the lysis of strains carrying the three mutations is inversely related to the chitin content of each strain. This finding directly demonstrates the importance of chitin to the integrity of the cell wall and indicates that agents that inhibit the chsD-encoded chitin synthase could be useful anti-Aspergillus drugs.

Amino Acid Sequence↗
PubMed1996

Isolation of the ALG6 locus of Saccharomyces cerevisiae required for glucosylation in the N-linked glycosylation pathway.

N-Linked protein glycosylation in most eukaryotic cells initiates with the transfer of the oligosaccharide Glc3Man9GlcNAc2 from the lipid carrier dolichyl pyrophosphate to selected asparagine residues. In the yeast Saccharomyces cerevisiae, alg mutations which affect the assembly of the lipid-linked oligosaccharide at the membrane of the endoplasmic reticulum result in the accumulation of lipid-linked oligosaccharide intermediates and a hypoglycosylation of proteins. Exploiting the synthetic growth defect of alg mutations in combination with mutations affecting oligosaccharyl transferase activity (Stagljar et al., 1994), we have isolated the ALG6 locus. alg6 mutants accumulate lipid-linked Man9GlcNAc2, suggesting that this locus encodes an endoplasmic glucosyltransferase. Alg6p has sequence similarity to Alg8p, a protein required for glucosylation of Glc1Man9GlcNAc2.

Amino Acid Sequence↗
PubMed1995

Synthesis of "Nod"-like chitin oligosaccharides by the Xenopus developmental protein DG42.

The Xenopus DG42 gene is expressed only between the late midblastula and neurulation stages of embryonic development. Recent database searches show that DG42 has striking sequence similarity to the Rhizobium NodC protein. NodC catalyzes the synthesis of chitin oligosaccharides which subsequently are transformed into bacterium-plant root signaling molecules. We find that the DG42 protein made in an in vitro coupled transcription-translation system catalyzes the synthesis of an array of chitin oligosaccharides. The result suggests the intriguing possibility that a bacterium-plant type of "Nod" signaling system may operate during early stages of vertebrate embryonic development and raises issues about the use of chitin synthase inhibitors as fungal-specific drugs.

Acetylglucosamine↗
PubMed1995

Molecular cloning and characterization of chitinase genes from Candida albicans.

Chitinase (EC 3.2.1.14) is an important enzyme for the remodeling of chitin in the cell wall of fungi. We have cloned three chitinase genes (CHT1, CHT2, and CHT3) from the dimorphic human pathogen Candida albicans. CHT2 and CHT3 have been sequenced in full and their primary structures have been analyzed: CHT2 encodes a protein of 583 aa with a predicted size of 60.8 kDa; CHT3 encodes a protein of 567 aa with a predicted size of 60 kDa. All three genes show striking similarity to other chitinase genes in the literature, especially in the proposed catalytic domain. Transcription of CHT2 and CHT3 was greater when C. albicans was grown in a yeast phase as compared to a mycelial phase. A transcript of CHT1 could not be detected in either growth condition.

Amino Acid Sequence↗
PubMed1995

Cloning and analysis of murine cDNA that encodes a fibrogenic lymphokine, fibrosin.

Tissue fibrosis that complicates chronic inflammation can be a cause of serious morbidity. The molecular links between inflammation and fibrosis appear to be a variety of proteins produced by activated chronic inflammatory cells. Collectively, these fibrogenic cytokines promote the growth of fibroblasts and the production of extracellular matrix that are the characteristic features of fibrotic tissue. In an attempt to clone cDNA for a fibrogenic lymphokine that we had isolated, we transfected COS-7 cells with a cDNA library derived from concanavalin A-stimulated lymphocyte line CDC25. Conditioned medium from the transfected COS-7 cells but not from sham-transfected cells stimulates fibroblast proliferation in vitro. We used heterologous expression in COS-7 cells of pools of CDC25 cDNA and screening for biological activity in conditioned medium to enrich for the cDNA clone(s) that encodes this activity. With this strategy of sib selection we isolated clone 2B3. The culture supernatants of 2B3-transfected COS-7 cells exert maximum growth-stimulating effects on fibroblasts at a dilution of 1:20,000. The isolated cDNA has one open reading frame (216 nucleotides) that has no significant homology with nucleotide sequences that encode other proteins. A synthetic peptide constructed from the deduced amino acid sequence is biologically active in picomolar concentrations, even though it may represent only a portion of the native fibrosin. This lymphokine, which we designate fibrosin, may play a role in regulating fibrogenesis in certain chronic inflammatory diseases.

Amino Acid Sequence↗
PubMed1995

A multigene family related to chitin synthase genes of yeast in the opportunistic pathogen Aspergillus fumigatus.

Two approaches were used to isolate fragments of chitin synthase genes from the opportunistic human pathogen Aspergillus fumigatus. Firstly, regions of amino acid conservation in chitin synthases of Saccharomyces cerevisiae were used to design degenerate primers for amplification of portions of related genes, and secondly, a segment of the S. cerevisiae CSD2 gene was used to screen an A. fumigatus lambda genomic DNA library. the polymerase chain reaction (PCR)-based approach led to the identification of five different genes, designated chsA, chsB, chsC, chsD and chsE. chsA, chsB, and chsC fall into Classes I, II and III of the 'zymogen type' chitin synthases, respectively. The chsD fragment has approximately 35% amino acid sequence identity to both the zymogen type genes and the non-zymogen type CSD2 gene. chsF appears to be a homologue of CSD2, being 80% identical to CSD2 over 100 amino acids. An unexpected finding was the isolation by heterologous hybridization of another gene (chsE), which also has strong sequence similarity (54% identity at the amino acid level over the same region as chsF) to CSD2. Reverse transcriptase-PCR was used to show that each gene is expressed during hyphal growth in submerged cultures.

Amino Acid Sequence↗
PubMed1995

Architecture of the yeast cell wall. The linkage between chitin and beta(1-->3)-glucan.

To isolate the putative linkage region between chitin and beta(1-->3)-glucan, Saccharomyces cerevisiae cell walls were digested with beta(1-->3)-endoglucanase and the reducing ends of the enzyme-resistant glucose chain stubs were labeled by reduction with borotritide. The radioactive material was further digested with exochitinase to remove the bulk of the chitin, and the liberated oligosaccharides were fractionated on a sizing column. A single peak (compound I) was found to consist of N-acetylglucosamine, glucose, and glucitol residues in the ratio 1:2:1. By digestion with beta-N-acetylglucosaminidase and by NMR spectroscopy, N-acetylglucosamine was identified as the nonreducing terminus, linked to laminaritriitol by a beta(1-->4) bond. Five additional oligosaccharides were recovered, two being analogs of compound I, with 1 or 3 glucose units, respectively; the remaining three were shown to be the reduced analogs of laminaribiose, laminaritriose, and laminaritetraose. The presence of N-acetylglucosamine-containing oligosaccharides arises from the activity of chitinase in cleaving 2 sugar units sequentially in those chains containing an odd number of N-acetylglucosamine residues; correspondingly, oligosaccharides containing only glucose and sorbitol derived from even-numbered chitin chains, a result implying that chitinase can hydrolyze the linkage between N-acetylglucosamine and glucose. It is concluded that the terminal reducing residue of a chitin chain is attached to the nonreducing end of a beta(1-->3)-glucan chain by a beta(1-->4) linkage. Experiments with appropriate mutants showed that synthesis of the chitin combined with glucan is catalyzed by chitin synthetase 3. The timing and possible mechanism of formation of the chitin-glucan linkage is discussed.

Acetylglucosaminidase↗
PubMed1995

Specific beta glucanases as tools for polysaccharide structure determination.

A number of interesting beta-glucanases with a variety of substrate specificities have recently been described. These enzymes are ideal tools for elucidating the structure of the wide array of glucose polymers found in nature. Since many of the enzymes can be easily produced in substantial quantities by cloning and 'overexpression' techniques, they are available for other purposes as well. For example, they may substantially increase the nutritional value of grain and other plant materials.

Amino Acid Sequence↗