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D Y Thomas

Publications and source records attributed to D Y Thomas.

At least 145 records · Page 8Linked to original sources
PubMed1988

Purification and characterization of a chloride-stimulated cellobiosidase from Bacteroides succinogenes S85.

A cellobiosidase with unique characteristics from the extracellular culture fluid of the anaerobic gram-negative cellulolytic rumen bacterium Bacteroides succinogenes grown on microcrystalline cellulose (Avicel) in a continuous culture system was purified to homogeneity by column chromatography. The enzyme was a glycoprotein with a molecular weight of approximately 75,000 and an isoelectric point of 6.7. When assayed at 39 degrees C and pH 6.5, the activity of the enzyme with p-nitrophenyl-beta-D-cellobioside as the substrate was stimulated by chloride, bromide, fluoride, iodide, nitrate, and nitrite, with maximum activation (approximately sevenfold) occurring at concentrations ranging from 1.0 mM (Cl-) to greater than 0.75 M (F-). The presence of chloride (0.2 M) did not affect the Km but doubled the Vmax. In the presence of chloride (0.2 M), the pH optimum of the enzyme was broadened, and the temperature optimum was increased from 39 to 45 degrees C. The enzyme released terminal cellobiose from cellotriose and cellobiose and cellotriose from longer-chain-length cellooligosaccharrides and acid-swollen cellulose, but it had no activity on cellobiose. The enzyme showed affinity for cellulose (Avicel) but did not hydrolyze it. It also had a low activity on carboxymethyl cellulose.

Bacteroides↗
PubMed1987

Yeast KEX1 gene encodes a putative protease with a carboxypeptidase B-like function involved in killer toxin and alpha-factor precursor processing.

The yeast KEX1 gene product has homology to yeast carboxypeptidase Y. A mutant replacing serine at the putative active site of the KEX1 protein abolished activity in vivo. A probable site of processing by the KEX1 product is the C-terminus of the alpha-subunit of killer toxin, where toxin is followed in the precursor by 2 basic residues. Processing involves endoproteolysis following these basic residues and trimming of their C-terminal by a carboxypeptidase. Consistent with the KEX1 product being this carboxypeptidase is its role in alpha-factor pheromone production. In wild-type yeast, KEX1 is not essential for alpha-factor production, as the final pheromone repeat needs no C-terminal processing. However, in a mutant in which alpha-factor production requires a carboxypeptidase, pheromone production is KEX1-dependent.

Amino Acid Sequence↗
PubMed1987

Determination of the carboxyl termini of the alpha and beta subunits of yeast K1 killer toxin. Requirement of a carboxypeptidase B-like activity for maturation.

The carboxyl-terminal sequences of the two polypeptide chains of the Saccharomyces cerevisiae K1 killer toxin were determined by protein sequencing and amino acid analysis of peptide fragments generated from the mature, secreted toxin. The COOH-terminal amino acid of the beta chain is histidine 316, the final residue encoded by the precursor gene. The COOH terminus of the alpha chain is at alanine 147 of the preprotoxin. Amino acid composition data for the purified toxin are consistent with that predicted from the gene sequence of the preprotoxin where the alpha and beta subunits consist of amino acid residues 45-147 and 234-316, respectively. The molecular weight of the mature alpha beta dimer is about 20,658. The COOH-terminal sequence determination completes the location of the toxin subunits in the precursor, and its configuration may be represented as prepropeptide-Pro-Arg-alpha-Arg-Arg-gamma-Lys-Arg-beta, where gamma represents the interstitial glycosylated peptide. The COOH terminal side of the paired basic residues (Arg-148 Arg-149 and Lys-232 Arg-233 of preprotoxin) are endoproteolytic processing sites for the product of the KEX2 gene (Julius, D., Brake, A., Blair, L., Kunisawa, R., and Thorner, J. (1984) Cell 37, 1075-1089), and thus maturation of the alpha subunit of killer toxin apparently requires a carboxypeptidase B-like activity. A possible candidate for this activity is the product of the KEX1 gene (Dmochowska, A., Dignard, D., Henning, D., Thomas, D.Y., and Bussey, H. (1987) Cell, in press).

Amino Acid Sequence↗
PubMed1987

A family of yeast expression vectors containing the phage f1 intergenic region.

The construction and characterization of a family of yeast expression vectors is described. They have the following features: plasmid replication and selection (ApR) in Escherichia coli, packaging of single-stranded (ss) DNA upon infection of E. coli with a filamentous helper phage, replication in Saccharomyces cerevisiae based on the 2 mu plasmid origin of replication (ori), selection in yeast by complementation of LEU2 (pVT-L series, size 6.3 kb) or URA3 gene (pVT-U series, size 6.9 kb) and seven unique restriction sites for cloning within an 'expression cassette' which includes the promoter and 3' sequence of the ADH1 gene. The multiple cloning site as well as the ori and intergenic region of the phage f1 have been cloned in two orientations for convenient gene cloning and ssDNA strand selection. As a result any of these eight vectors can be chosen for cloning, expressing genes in yeast, sequencing and mutagenesis without the need for recloning into specialized vectors.

Base Sequence↗
PubMed1987

Characteristics of the endoglucanase encoded by a cel gene from Bacteroides succinogenes expressed in Escherichia coli.

A cel gene from Bacteroides succinogenes inserted into the vector pUC8 coded for an enzyme which exhibited high hydrolytic activity on carboxymethylcellulose, p-nitrophenylcellobioside, and lichenan and low activity on laminarin and xylan. The enzyme was not synthesized by the Escherichia coli host when cells were cultured in complex medium containing added glucose. In the absence of added glucose, the endoglucanase and cellobiosidase activities synthesized were partitioned into the periplasmic space during growth, and practically all enzyme was located in the periplasm when the stationary phase of growth was reached. The enzyme exhibited 17- and sixfold higher Km values for the hydrolysis of carboxymethylcellulose and lichenan, respectively, than did the extracellular endoglucanase complex from B. succinogenes. The Cel endoglucanase had a pH optimum similar to that of the B. succinogenes enzyme except that the range was narrower, and the Cel endoglucanase was more readily inactivated on exposure to high temperature, detergents, and certain metals. Its activity was stimulated by calcium and magnesium. Nondenaturing polyacrylamide gel electrophoresis at different acrylamide concentrations revealed the presence of three endoglucanase components, two with molecular weights of 43,000 and one with a molecular weight of 55,000.

Bacteroides↗
PubMed1986

Yeast killer toxin: site-directed mutations implicate the precursor protein as the immunity component.

Yeast killer toxin and a component giving immunity to it are both encoded by a gene specifying a single 35 kd precursor polypeptide. This precursor is composed of a leader peptide, the alpha and beta subunits of the secreted toxin, and a glycosylated gamma peptide separating the latter. The toxin subunits are proteolytically processed from the precursor during toxin secretion. Using site-directed mutagenesis, we have identified a region of the precursor gene necessary for expression of the immunity phenotype. This immunity-coding region extends through the C-terminal half of the alpha subunit into the N-terminal part of the gamma glycopeptide. Mutations in other parts of the gene allow full immunity but produce precursors that fail to be processed. The precursor can therefore confer immunity, and we propose that it does so in the wild type by competing with mature toxin for binding to a membrane receptor.

Fungal Proteins↗
PubMed1986

Potential for manipulation of the rumen fermentation through the use of recombinant DNA techniques.

Recombinant DNA techniques offer a new approach to the study and eventual genetic manipulation of rumen bacteria to modify the rate and extent of nutrient digestion within the rumen. The foreign genes cloned into a rumen bacterium undoubtedly will have to enhance its ability to compete; otherwise, the new trait will be lost from the highly competitive environment within the rumen. As a general rule, the types of metabolic activities amenable to gene cloning may be those that enhance substrate versatility or eliminate a dependence of a specific rumen bacteria on cross-feeding by other rumen microorganisms. Enhanced substrate diversity and bypassing cross-feeding probably will become more important as the use of highly refined feeds and waste materials from the food and other industries are used in greater quantities to meet the dietary needs of ruminants.

Animals↗
PubMed1984

Cloning and sequencing of the preprotoxin-coding region of the yeast M1 double-stranded RNA.

Complementary DNA (cDNA) copies of the M1-1, toxin-coding region of the yeast M1 double-stranded RNA (dsRNA) have been cloned and sequenced. These sequences, in combination with the known terminal sequence of M1-1 dsRNA, identify a translation reading frame for a 316 amino acid protein of 34.7 kd, similar in size to the preprotoxin produced from M1 dsRNA by in vitro translation. Potential glycosylation sites in the preprotoxin peptide are identified. Based on its methionine content the extracellular yeast toxin appears to be contained within the C-terminal region of the precursor.

Amino Acid Sequence↗
PubMed1984

The expression of cDNA clones of yeast M1 double-stranded RNA in yeast confers both killer and immunity phenotypes.

Two cDNA clones of the segment of Saccharomyces cerevisiae M1 double-stranded RNA, which codes for the yeast killer toxin, have been expressed in yeast using the expression vector pYT760. Toxin expression and secretion depended upon the presence of a yeast promoter. Transformants not only contain an authentic preprotoxin precursor, as determined by precipitation of intracellular proteins with antitoxin antisera, but also display an immunity phenotype. The evidence is that the immunity protein is part of the preprotoxin and may act by masking toxin binding sites. Neither cDNA clone had a complete 5' terminus and the preprotoxin translational start was missing. The promoter and the initiator ATG were supplied by the expression vector. One clone with a full-length preprotoxin but altered N-terminal amino acids gave a normal glycosylated intracellular precursor. A clone with an N-terminal nine amino acid deletion gave a precursor which was not glycosylated but toxin was still secreted.

Amino Acid Sequence↗
PubMed1983

Synthesis of a human insulin gene. VI. Expression of the synthetic proinsulin gene in yeast.

The construction of plasmid vectors for the controlled expression of a synthetic human proinsulin gene in the yeast Saccharomyces cerevisiae is described. Attempts to express the proinsulin gene using the yeast ADH1 promoter alone did not yield detectable levels of proinsulin. Successful expression was achieved when the proinsulin gene was fused with the promoter and protein leader sequence of the GAL1 gene (coding for yeast galactokinase) in the yeast-Escherichia coli plasmid vector pYT7810. Two different-length leader sequences were employed; the longer leader (about 280 amino acids, fusion plasmid pPS13) gave about five times greater expression than the shorter leader fusion (30 amino acids, plasmid pPS5). Both fusions gave soluble protein products, and the proinsulin could be cleaved by cyanogen bromide treatment from the leader polypeptide. Proinsulin was detected by radioimmunoassay for human C-peptide only in cells induced with galactose, and was not detected in the gene fusions that were out of phase with the GAL1 leader sequence. Methods of improving the level of expression of the proinsulin gene in yeast using this system are discussed.

Amino Acid Sequence↗
PubMed1982

A yeast transcription system for the 5S rRNA gene.

A cell-free extract of yeast nuclei that can specifically transcribe cloned yeast 5S rRNA genes has been developed. Optima for transcription of 5S rDNA were determined and conditions of extract preparation leading to reproducible activities and specificities established. The major in vitro product has the same size and oligonucleotide composition as in vivo 5S rRNA. The in vitro transcription extract does not transcribe yeast tRNA genes. The extract does increase the transcription of tRNA genes packaged in chromatin.

Cell Nucleus↗
PubMed1982

Escherichia coli plasmid vectors containing synthetic translational initiation sequences and ribosome binding sites fused with the lacZ gene.

The construction of a series of Escherichia coli plasmid vectors suitable for assaying the effects of gene control signals fused with the E. coli lacZ gene is reported. A synthetic deoxyoligonucleotide dodecamer 5'-CATGAATTCATG GTACTTAAGTAC-5' containing two translation initiation codons (ATG) separated by an EcoRI site was ligated with a lacZ gene derivative which lacks the codons for the first eight amino acids in plasmid pMC1403 (Casadaban et al., 1980). Two ribosome-binding sequences were synthesised and inserted into the EcoRI site before an ATG, and the effects of these sequences on lacZ gene expression in vivo measured by assaying beta-galactosidase activity. The E. coli ribosomal RNA gene (rrnB) promoter, the tetracycline resistance gene promoter, and a lambda phage promoter were cloned using these plasmids. The plasmids are 9.9 kb in size, have ampicillin resistance as a selectable marker and are generally useful for the detection and in vivo assay of gene control regions.

Base Sequence↗
PubMed1981

Molecular and phenotypic characterization of penicillinase-producing Neisseria gonorrhoeae from Canadian sources.

The incidence of penicillinase-producing Neisseria gonorrhoeae (PPNG) infections has increased in Canada during the past 2 years. Most of these cases were imported from abroad. The PPNG strains from these cases were characterized with respect to susceptibility to 11 antibiotics, auxotype, and plasmid content. Rosaramicin and cefuroxime proved to be the most potent of the antibiotics tested. The molecular characterization of the isolates indicated that all carried a 2.6-megadalton cryptic plasmid. Most of the PPNG isolates (87%) harbored a 4.5-megadalton penicillinase-producing plasmid, whereas only 13% harbored the 3.2-megadalton penicillinase-producing plasmid. In those cases where contact tracing was possible, the correlation linking strains of Far Eastern etiology with carriage of the 4.5-megadalton plasmid was upheld. The penicillinase-producing strains were typed auxanographically in either the proline-requiring (57%) or prototrophic groups (42%). Substrate hydrolysis profiles and analytical isoelectric focusing of crude beta-lactamase extracts of several isolates has reconfirmed that these strains elaborate a type TEM-1 enzyme. Several of the penicillinase-producing plasmids were also examined for plasmid stability.

Anti-Bacterial Agents↗
PubMed1980

Saccharomyces cerevisiae plasmid, Scp or 2 mum: intracellular distribution, stability and nucleosomal-like packaging.

Cell fractions from yeast strains known to harbor the plasmic 2 mum or Scp were treated with nucleases used to probe eukaryotic chromosome structure. Scp and subfragments were identified by hybridization to natural or cloned Scp probes according to Southern (34). Specificity was confirmed with non-Scp probes. Copy/haploid nuclear genome(n) was estimated from reconstructions at a resolution of 0.5/n. About 43-67% of the total cellular copy exists as nucleoprotein complexes which separate from other debris on isokinetic sucrose gradients with s-values of 67-110. These complexes are totally degraded by DNAase I. Digestion with micrococcal nuclease produced integral-sized fragments; they are not generated by direct mixing of pure Scp with nuclear chromatin from a[cir] strain. Initial digests gave a repeat of 168 +/- 3 base pairs (bp) for both Scp and nuclear nucleoprotein; advanced digests reduced the nuclear repeat relative to Scp by 8 bp. Of a potential 37 repeat units/plasmid, 31-32 were directly measured. A strain difference in Scp autodegradation was found. A partial nuclease resistant form was also demonstrated whose abundance was cell strain and growth stage dependent. Both Scp isomers exist in these complexes which are structurally similar to simian viral 40 minichromosomes.

Deoxyribonuclease I↗
PubMed1980

Genetic analysis of Saccharomyces cerevisiae transformed by plasmid containing a supressor transfer ribonucleic acid gene.

The behavior in Saccharomyces cerevisiae of plasmid pYTE1, which contains yeast tyrosine-inserting ochre suppressor SUP4.o, a 4-kilobase EcoRI fragment of yeast 2muDNA, and the bacterial plasmid pBR322, has been studied. Selection of yeast transformants was by suppression of multiple ochre mutations. About 10(3) to 10(4) transformants per microgram of pYTE1 dfeoxyribonucleic acid were obtained. The majority of transformants contained both an integrated copy of the SUP4.o gene plus pBR322 deoxyribonucleic acid sequences and autonomously replicating forms of the plasmid. The integrated copy was extremely stable mitotically and meiotically, but the associated nonintegrated copies were lost at meiosis. The chromosomally integrated pBR322 sequences were linked to the SUP4.o gene. The integration site was at the SUP4+ locus. In transformants with only nonintegrated copies of pYTE1, the expression of suppression was reduced, and the plasmid was unstable in mitosis. Plasmid deoxyribonucleic acid preparations from both types of transformant could be used to retransform yeast cells. Plasmid pYTE1 has restriction enzyme sites useful for the high frequency and stable transformation of other genes into yeasts. The potential uses of this plasmid for transformation of other organisms is discussed.

Genetic Linkage↗
PubMed1977

Synthesis of cytochrome c oxidase polypeptides in an Escherichia coli cell-free system directed by Saccharomyces cerevisiae mitochondrial DNA.

Using purified yeast mitochondrial DNA as a template for E. coli RNA polymerase (holoenzyme) complementary mitochondrial RNA has been synthesized in vitro. This RNA has been used to direct a low background E. coli S-30 protein-synthesizing system. The synthesis of mitochondrial polypeptides has been detected by using antiserum raised against purified cytochrome c oxidase holoenzyme and shown to be specific for this antigen. The antiserum-antigen complex was dissociated and subject to SDS-polyacrylamide gel electrophoresis and the presence of 3 polypeptides of 39, 31, and 26 X 10(3) daltons molecular weight demonstrated, which correspond to the subunits synthesized by mitochondria in whole cells which are inhibited with cycloheximide.

Cell-Free System↗
PubMed1975

Synthesis of mitochondrial proteins in an Escherichia coli cell-free system directed by yeast mitochondrial DNA.

The optimum conditions for transcription in vitro of yeast mtDNA into biologically relevant RNA by Escherichia coli RNA polymerase holoenzyme and yeast mitochondrial RNA polymerase was found to critically depend on salt concentration. RNA was transcribed (at 0.25 M KCl concentration) from high-molecular-weight mtDNA which was then translated in an E. coli (S-30) cell-free protein synthesising system. Efficient translation of mitochondrial RNA was achieved using conditions which had also been determined to be optimal in other systems. Identification of the polypeptides produced in the translation system was made using antiserum raised against mitochondrial membranes. Electrophoresis of the completely dissociated antigen-antibody complexes using dodecylsulphate-polyacrylamide gels revealed that the system in vitro produced polypeptides of similar molecular weight to those synthesised in vivo by cycloheximide-inhibited whole cells.

Cell-Free System↗