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C F Thurston

Publications and source records attributed to C F Thurston.

At least 19 recordsLinked to original sources
PubMed1994

CEL1: a novel cellulose binding protein secreted by Agaricus bisporus during growth on crystalline cellulose.

The cel1 gene of Agaricus bisporus encodes a protein (CEL1) that has an architecture resembling the multi-domain fungal cellulases, although the sequence of its putative catalytic core is not matched by any other in the protein and nucleic acid data bases. The N-terminal half of the putative catalytic domain of CEL1 was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase. The fusion protein was used to raise a CEL1-specific antibody. CEL1 was detected as an extracellular 49.8 kDa protein in A. bisporus cellulose-grown cultures, where it bound strongly to cellulose. CEL1 was neither an endoglucanase, a cellobiohydrolase able to hydrolyze fluorogenic cellobiosides, a beta-glucosidase, a xylanase, nor a cellobiose: quinone oxidoreductase. CEL1 was present in some fractions of culture fluid separated by electrophoresis which released soluble sugars from crystalline cellulose.

Agaricus↗
PubMed1994

Regulation of transcription of the cel1 gene in Agaricus bisporus.

Northern analysis showed that accumulation of Agaricus bisporus cel1 mRNA was regulated by two independent mechanisms: (i) induction by cellulose; and (ii) repression by glucose and other sugars. Isolated A. bisporus nuclei were transcriptionally active. Nuclei isolated from cellulose-grown mycelium synthesized six times more cel1 mRNA than nuclei from glucose-grown mycelium. The start point of transcription (tsp) was identified by primer extension and S1 nuclease analysis. Putative glucose-, and cAMP-responsive elements as well as regions with homology to promoter regions of other fungal cellulase genes were detected both upstream and downstream from the tsp of the cel1 gene.

Agaricus↗
PubMed1994

The cel3 gene of Agaricus bisporus codes for a modular cellulase and is transcriptionally regulated by the carbon source.

A 52-kDa protein, CEL3, has been separated from the culture filtrate of Agaricus bisporus during growth on cellulose. A PCR-derived probe was made, with a degenerate oligodeoxynucleotide derived from the amino acid sequence of a CEL3 CNBr cleavage product and was used to select cel3 cDNA clones from an A. bisporus cDNA library. Two allelic cDNAs were isolated. They showed 98.8% identity of their nucleotide sequences. The deduced amino acid sequence and domain architecture of CEL3 showed a high degree of similarity to those of cellobiohydrolase II of Trichoderma reesei. Functional expression of cel3 cDNA in Saccharomyces cerevisiae was achieved by placing it under the control of a constitutive promoter and fusing it to the yeast invertase signal sequence. Recombinant CEL3 secreted by yeast showed enzymatic activity towards crystalline cellulose. At long reaction times, CEL3 was also able to degrade carboxymethyl cellulose. Northern (RNA) analysis showed that cel3 gene expression was induced by cellulose and repressed by glucose, fructose, 2-deoxyglucose, and lactose. Glycerol, mannitol, sorbitol, and maltose were neutral carbon sources. Nuclear run-on analysis showed that the rate of synthesis of cel3 mRNA in cellulose-grown cultures was 13 times higher than that in glucose-grown cultures. A low basal rate of cel3 mRNA synthesis was observed in the nuclei isolated from glucose-grown mycelia.

Agaricus↗
PubMed1993

The structure of laccase protein and its synthesis by the commercial mushroom Agaricus bisporus.

Agaricus bisporus secretes abundant laccase activity into the medium during mycelial growth. SDS-PAGE analysis of extracellular laccase protein, purified from compost extract, showed a predominant band of 65 kDa molecular mass, together with lesser amounts of smaller polypeptides. The main polypeptide was purified electrophoretically. Amino acid sequence analysis of the N-terminal region of the main polypeptide was used to specify the sequence of a 15-residue chemically synthesized peptide (N-terminal peptide). Rabbit antibodies were raised against pure laccase, electrophoretically purified main polypeptide and the synthetic N-terminal peptide. Electrophoretically purified main polypeptide antibody was further purified by affinity chromatography on laccase-CNBr-Sepharose. Western blot analysis showed that the antigenic behaviour of laccase in compost extract, culture filtrate from malt-extract culture, and the purified enzyme from both sources, differed. The patterns of bands revealed are most simply explained by generation of (proteolytically) partially cleaved enzyme molecules in the culture medium, possibly combined with differences in extent of glycosylation. [35S]Methionine incorporation and immunoprecipitation were used to follow laccase synthesis in cultures grown on malt extract. After short-term labelling, a single polypeptide of 68 kDa apparent molecular mass was immunoprecipitated from both mycelial extracts and the culture medium. When poly(A)-containing RNA from malt-extract-grown mycelium was translated in vitro in rabbit reticulocyte lysate, a single polypeptide of about 57 kDa molecular mass was immunoprecipitated, consistent with the previously measured carbohydrate content of 15% for the pure enzyme. After treatment with N-glycanase, the polypeptide showed an increase in mobility during SDS-PAGE consistent with a reduction in molecular mass of about 5 kDa, indicating about equal amounts of N- and O-linked carbohydrate. C-terminal labelling of pure laccase was attempted by transpeptidation with carboxypeptidase Y. Although some minor bands were labelled, the main polypeptide was not, indicating that the C-terminus of the enzyme may be blocked.

Agaricus↗
PubMed1993

Identification of two laccase genes in the cultivated mushroom Agaricus bisporus.

A cDNA library was constructed in lambda gt11 using mRNA from 11-d-old mycelium of Agaricus bisporus. Three clones containing laccase sequence were identified using an affinity-purified anti-laccase antibody. From one of these clones, a 333 bp sequence was used to identify further cDNA clones (including one which is close to full length) and a genomic clone. The coding sequences found were of two similar but not identical versions with differences at 36 out of 520 residues of deduced amino acid sequence. The laccase genes each encode a sequence expressed as a 2.3 kb mRNA, specifying a 520 residue polypeptide including a 19 amino acid residue signal peptide that is absent from the N terminus of the mature (extracellular) protein. The coding sequence of lcc1 is interrupted by 14 short introns. The lcc1 and lcc2 genes are not allelic as they do not segregate in uninucleate spores derived from a four-spored basidium. Comparison of the deduced amino acid sequences with that of the other fungal laccases that have been cloned, and with the very similar ascorbate oxidases from higher plants shows that whilst some sequence is absolutely conserved at and around the amino acid residues involved in copper binding, the overall sequence similarities are low.

Agaricus↗
PubMed1993

Purification and characterization of a serine proteinase from senescent sporophores of the commercial mushroom Agaricus bisporus.

A proteinase has been purified from the stipes of senescent sporophores of the mushroom Agaricus bisporus. The proteinase was inhibited by PMSF. It has a broad pH optimum, 6.5-11.5, and a narrow substrate specificity, requiring both a hydrophobic amino acid in the P1 position and a minimum peptide chain length. The apparent molecular mass of the proteinase was 27 kDa when determined by SDS-PAGE and 14.1 kDa when measured by gel filtration. The isoelectric point of the proteinase was 9.0. Polyclonal antibodies have been raised to the proteinase. The proteinase from A. bisporus has similar properties to, and 60% N-terminal sequence identity with, proteinase K from the fungus Tritirachium album.

Agaricus↗
PubMed1992

Isolation and characterization of a cellulose-growth-specific gene from Agaricus bisporus.

The edible basidiomycete, Agaricus bisporus, produces extracellular endoglucanase. Endoglucanase production is induced by cellulose and repressed by fructose in A. bisporus grown on minimal medium, and is regulated in activity during fruiting body development. An anti-endoglucanase antibody was used to isolate cellulase-related genes. Three main polypeptides of 38, 58, and 60 kDa were immunoprecipitated by the antibody from products of in vitro cell-free translation of mRNAs isolated from cellulose-grown mycelium. No cross-reaction was detected with the translated products from fructose-grown mycelium. This antibody was used to immunoscreen a lambda ZAPII-cDNA expression library made from mRNA isolated from cellulose-grown mycelium. Two cDNA cross-reacting clones, pSRc110 and pSRc200, were isolated. Clones pSRc110 and pSRc200 cross-hybridized and had the same restriction map. Clone pSRc200 hybrid selected an mRNA that on cell-free translation produced a 38-kDa polypeptide. The cDNA fragment from pSRc200 hybridized to a 1.3-kb mRNA from cellulose-grown mycelium. No hybridization was observed when using fructose-grown mycelium mRNA. Thus, the gene (cel1) expressing the 1.3-kb mRNA, is differentially regulated by the carbon source of the culture medium. The cell gene was isolated in a 8.9-kb EcoRI genomic fragment after hybridization to pSRc200. Sequences similar to those in the egl1 and cbh2 genes from Trichoderma reesi were found upstream from the ATG start codon in cel1. Nine short intervening sequences disrupt the cel1 coding sequence, and a strong bias against codons ending with G and A was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Agaricus↗
PubMed1992

An investigation of dye reduction by food-borne bacteria.

The rates of reduction of seven redox dyes by 13 bacterial strains were measured and found to vary greatly between different bacterium/dye combinations. Phenazine ethosulphate and toluidine blue were the most rapidly reduced dyes by the majority of bacteria and resorufin and 2-hydroxy-1,4-naphthoquinone were reduced slowly, if at all. There was also considerable variation in the rates of reduction with any single dye/organism combination. Glucose stimulated the rates of endogenous dye reduction in about half of the organisms. For Bacillus cereus, Pseudomonas fluorescens and Escherichia coli, dye reduction was stimulated by a range of exogenous substrates but lactose, the primary available carbon and energy source in milk, had little effect. In Lactococcus lactis, dye reduction was stimulated by sugars but not by organic acids. Oxygen successfully competed with dye reduction in organisms containing respiratory chains, but with membrane fractions, dye reduction was more rapid than oxygen consumption. All the organisms showed little cytosolic dye reduction, except L. lactis which showed substantial rates of reduction of some dyes by this fraction. With the membrane fraction of E. coli and Ps. fluorescens, cyanide inhibited NADH and succinate-dependent dye reduction, Antimycin A inhibited lactate and succinate and rotenone had no significant effect, but inhibition was not always observed with membrane from both organisms.

Animals↗
PubMed1991

Glucose control of staphylococcal enterotoxin A synthesis and location is mediated by cyclic AMP.

The regulation of staphylococcal enterotoxin A (SEA) synthesis in a defined medium was studied using continuous culture techniques. SEA production was repressed by glucose and repression could be overcome by addition of exogenous cyclic AMP. As well as this classical catabolite repression control, addition of glucose to de-repressed steady-state cultures resulted in rapid disappearance of toxin from the medium (also mediated by loss of cyclic AMP). When the toxin dissappeared from the medium, it was taken up again by the bacteria without apparent modification.

Culture Media↗
PubMed1983

The complexity of poly(A)+ RNA in the alga Chlorella fusca.

RNA excess hybridization with radioactively labelled complementary DNA (cDNA) was used to reveal the complexity of poly(A)+ RNA from Chlorella fusca var. vacuolata. RNA was tested from cells during photosynthetic exponential growth and during adaptation to heterotrophic growth in the dark on acetate. Both RNA populations were resolved into abundant, intermediate and rare sequence classes. Abundant sequences (200-400 copies per cell) constituted a significantly larger proportion of total poly(A)+ RNA in acetate-adapting cells than in exponentially growing autotrophic cells. Both types of RNA contained a rare sequence class of complexity consistent with a composition of about 20 000 different sequences. This indicated a substantially greater complexity of genes expressed in this alga and Euglena than in fungi and slime moulds. Heterologous hybridization, and hybridization with fractionated cDNA, showed that the majority of differences between RNA populations from exponentially growing and adapting cells were changes in relative abundance of groups of sequences, rather than presence of different sets of sequences in the two populations.

Acetates↗
PubMed1981

Evidence for messenger ribonucleic acid of an ammonium-inducible glutamate dehydrogenase and synthesis, covalent modification, and degradation of enzyme subunits in uninduced Chlorella sorokiniana cells.

The cells of Chlorella sorokiniana cultured in nitrate medium contain no detectable catalytic activity of an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH). However, several lines of experimental evidence indicated that the NADP-GDH messenger ribonucleic acid was present at high levels and was being translated in uninduced cells. First, binding studies with 125I-labeled anti-NADP-GDH immunoglobulin G and total polysomes isolated from uninduced and induced cells showed that NADP-GDH subunits were being synthesized on polysomes from both types of cells. Second, when polyadenylic acid-containing ribonucleic acid was extracted from polysomes from uninduced and induced cells and placed into a messenger ribonucleic acid-dependent in vitro translation system, NADP-GDH subunits were synthesized from the ribonucleic acid from both sources. Third, when ammonia was added to uninduced cells, NADP-GDH antigen accumulated without an apparent induction lag. Fourth, by use of a specific immunoprecipitation procedure coupled to pulse-chase studies with [35S]sulfate, it was shown that the NADP-GDH subunits are rapidly synthesized, covalently modified, and then degraded in uninduced cells.

Ammonium Sulfate↗
PubMed1978

Control of isocitrate lyase synthesis in Chlorella fusca var. vacuolata. Rate of enzyme synthesis in the presence and absence of acetate measured by [35S]methionine labelling and immunoprecipitation.

The rate of increase of isocitrate lyase activity was measured in darkened Chlorella fusca var. vaculoata cultures in the presence and absence of acetate and compared with the rate of incorporation of [35S]methionine into isocitrate lyase enzyme protein under the same conditions. Isocitrate lyase enzyme protein was isolated for this purpose by specific immunoprecipitation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. After 4h in the dark, in the presence of acetate the rate of increase of isocitrate lyase activity was 75 times that in the absence of acetate. Incorporation of [35S]methionine into isocitrate lyase was 140 times greater in the presence of acetate. Incorporation of [35S]methionine into the trichloroacetic acid-insoluble fraction overall was about five times as fast in the presence of acetate. These data are not consistent with an increased turnover of isocitrate lyase enzyme molecules, sufficient to account for the low rate of increase of isocitrate lyase activity in the absence of acetate. The greater rate of enzyme synthesis in the presence of acetate must therefore be due to some effect of this metabolite on the processing or translation of isocitrate lyase mRNA.

Acetates↗
PubMed1977

Simultaneous operation of three catabolic pathways in the metabolism of glucose by Thiobacillus A2.

Enzymes essential to the operation of the Embden-Meyerhof glycolytic pathway, the Entner-Duodoroff pathway and oxidative pentose phosphate pathway were present in Thiobacillus A2 grown on glucose and other sugars. Radiorespirometry under various conditions with Thiobacillus A2 oxidising glucose specifically labelled with 14C in carbon atoms 1, 2, 3, 3 + 4, 6 or universally labelled demonstrated the simultaneous operation of the Embden-Meyerhof (48%), Entner-Doudoroff (28%), and pentose phosphate (24%) pathways in release of carbon dioxide from glucose. Growth on succinate, or autotrophically on formate or thiosulphate resulted in repression of most enzymes of the pathways, but high aldolase levels were retained indicating its role in gluconeogenesis and the Calvin cycle. Different fructose diphosphatase activities were found in succinate- and thiosulphate-grown organisms. The results indicate that all three major catabolic pathways for glucose function in Thiobacillus A2 grown on sugars. Thiobacillus acidophilus showed a different radiorespirometric pattern and apparently used the Entner-Duodoroff (64.5%) and pentose phosphate (35.5%) pathways, but showed unusually high release of carbon atom 6, as was also found for T. ferrooxidans.

Arsenic↗
PubMed1977

Control of isocitrate lyase synthesis in Chlorella fusca var. vacuolata. The basal activity of the enzyme and the kinetics of induction.

1. Isocitrate lyase activity was measured in non-induced Chlorella fusca var. vacuolata cells. 2. During exponential autotrophic growth about 1-2 molecules of the enzyme per cell were present. 3. In light-limited cultures the amount of the enzyme increased to 10-20 molecules/cell. 4. When autotrophic cultures were placed in the dark, the basal activity of isocitrate lyase increased after a 2h lag so that after 8h in the dark there was a 500-fold increase in activity. 5. When isocitrate lyase was induced (by addition of acetate and removal of illumination) in autotrophic cultures which had been growing exponentially, the full induced rate of enzyme synthesis was obtained after 70-80min. 6. When light-limited autotrophic cultures were induced, the rate of isocitrate lyase synthesis was maximal after only 40-50min. 7. These data are consistent with a catabolite-repression control co-ordinated with photosynthetic activity,which may be independent of the specific inducing effect of acetate.

Acetates↗
PubMed1975

The kinetic of degradation of chondroitin of sulphates and hyaluronic acid by chondroitinase form Proteus vulgaris.

Km and Vmax. were determined for the degradation by chondroitinase of chondroitin 4-sulphate, 4-sulphate-proteoglycna, chondroitin 6-sulphate, dermatan sulphate and hyaluronic acid. Degradation of chondroitin 4-sulphate was inhibited by hyaluronic acid but not by keratan sulphate. The results are discussed with regard to the use to the use of chondroitinase as a sleective reagent for the degradation of tissue glycosaminoglycans.

Chondroitin↗
PubMed1975

Characterization and messenger activity of poly(A)-containing RNA from Chlorella.

Poly(A)-containing RNA was isolated by cellulose column chromatography from total RNA extracted from Chlorella fusca var. vacuolata 211/8p. RNA retained by the column was identified as poly(A)-containing RNA because it contained ribonuclease-resistant tracts, 25 to 55 nucleotides in length, from which not less than 80% of base was found to be adenine after acid hydrolysis. The base composition of poly(A)-containing RNA differed from that of RNA (largely ribosomal) which did not adsorb to cellulose, having a higher adenine content and a lower guanine content. Poly(A)-containing RNA was polydisperse including molecules with mobilities from 10S to 40S with a mean of about 20S. In an in vitro system derived from wheat-germ, protein synthesis was stimulated by adding poly(A)-containing RNA from Chlorella. Optimum conditions were established in this system with respect to the amount of poly(A)-containing RNA added and the concentration of KCl and Mg-2+. It is proposed that, in Chlorella, poly(A)-containing RNA includes cytoplasmic mRNA as has been shown for some other eucaryotic organisms.

Adenine Nucleotides↗