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D Y Thomas

Publications and source records attributed to D Y Thomas.

At least 127 records · Page 7Linked to original sources
PubMed1991

Expression in yeast of a cDNA copy of the K2 killer toxin gene.

A cDNA copy of the M2 dsRNA encoding the K2 killer toxin of Saccharomyces cerevisiae was expressed in yeast using the yeast ADH1 promoter. This construct produced K2-specific killing and immunity functions. Efficient K2-specific killing was dependent on the action of the KEX2 endopeptidase and the KEX1 carboxypeptidase, while K2-specific immunity was independent of these proteases. Comparison of the K2 toxin sequence with that of the K1 toxin sequence shows that although they share a common processing pathway and are both encoded by cytoplasmic dsRNAs of similar basic structure, the two toxins are very different at the primary sequence level. Site-specific mutagenesis of the cDNA gene establishes that one of the two potential KEX2 cleavage sites is critical for toxin action but not for immunity. Immunity was reduced by an insertion of two amino acids in the hydrophobic amino-terminal region which left toxin activity intact, indicating an independence of toxin action and immunity.

Amino Acid Sequence↗
PubMed1991

Ubiquitin gene expression: response to environmental changes.

It has previously been shown that the yeast ubiquitin genes UBI1, 2 and 3 are strongly expressed during the log-phase of batch culture growth, whereas the UBI4 gene is weakly expressed. We found that heat shock, treatment with DNA-damaging agents, starvation, and the feeding of starved cells all transiently induced UBI4. These results suggest that UBI4 is induced whenever a change in culture conditions dictates a dramatic shift in cellular metabolism, and that UBI4 expression returns to lower levels once cellular metabolism has adapted to the new conditions. In contrast, all of the treatments tested, except starvation, transiently repressed the UBI1, 2 and 3 genes. Although starvation also repressed UBI1, 2 and 3 its effect was not transient, and expression only recovered upon the addition of fresh media. These results, together with others presented here, suggest that high levels of UBI1, 2 and 3 expression are dependant upon ongoing cell growth, and that treatments which slow or stop growth repress their expression.

4-Nitroquinoline-1-oxide↗
PubMed1991

Removal of an inter-domain hydrogen bond through site-directed mutagenesis: role of serine 176 in the mechanism of papain.

A mutant of papain, where an inter-domain hydrogen bond between the side chain hydroxyl group of a serine residue at position 176 and the side chain carbonyl oxygen of a glutamine residue at position 19 has been removed by site-directed mutagenesis, has been produced and characterized kinetically. The mutation of Ser176 to an alanine has only a small effect on the kinetic parameters, the kcat/Km for hydrolysis of CBZ-Phe-Arg-MCA by the Ser176Ala enzyme being of 8.1 x 10(4) /M/s compared with 1.2 x 10(5) /M/s for papain. Serine 176 is therefore not essential for the catalytic functioning of papain, even though this residue is conserved in all cysteine proteases sequenced. The pH-activity profiles were shown to be narrower in the mutant enzyme by up to 1 pH unit at high ionic strength. This result is interpreted to indicate that replacing Ser176 by an alanine destabilizes the thiolate-imidazolium form of the catalytic site Cys25-His159 residues of papain. Possible explanations for that effect are given and the role of a serine residue at position 176 in papain is discussed.

Base Sequence↗
PubMed1991

Prohormone processing by yeast proteases.

Investigations of the precursors of alpha-pheromone and killer toxin in the yeast Saccharomyces cerevisiae have defined the genes coding (KEX1 and KEX2) for the proteases which are responsible for their processing. In addition to processing at pairs of basic residues it is evident that yeast can also process at monobasic sites. We present data on the Kex1p and Kex2p enzymes, their cellular localization, and their post-translational modification. In addition initial characterisation of the monobasic specific protease and the isolation of mutants defective in this activity are presented. The use of the yeast system as a model for the processing of mammalian prohormones is discussed.

Amino Acid Sequence↗
PubMed1990

Secretion of functional papain precursor from insect cells. Requirement for N-glycosylation of the pro-region.

The synthetic gene coding for the precursor of the cysteine protease papain (EC 3.4.22.2) has been expressed using the baculovirus/insect cell system. The prepropapain gene was cloned into the transfer vector IpDC125 behind the polyhedrin promoter. The recombinant construct was then incorporated by homologous recombination into the Autographa californiaca nuclear polyhedrosis virus genome. The host Spodoptera frugiperda Sf9 cells infected with the recombinant baculovirus secrete an enzymatically inactive N-glycosylated papain precursor. This zymogen could be activated in vitro to yield about 400 nmol of active papain per liter of culture. The recombinant active mature papain was enzymatically indistinguishable from natural papain but the precursor was not processed to the same amino acid residue. The insect cells also accumulated prepropapain and glycosylated propapain intracellularly. This accumulation was an indication that there are rate-limiting steps in the secretion of proteins from insect cells in this expression system. Characterization of mutants of the precursor has shown that entry into the secretory pathway and addition of carbohydrate are prerequisite conditions for the production and secretion of functional propapain.

Amino Acid Sequence↗
PubMed1990

A protein engineering study of the role of aspartate 158 in the catalytic mechanism of papain.

The controversy concerning the various suggested roles for the side chain of Asp158 in the active site of papain has been clarified by using site-directed mutagenesis. Both wild-type papain and an Asp158 Asn variant were produced in a baculovirus-insect cell expression system, purified to homogeneity from the culture, and characterized kinetically. With CBZ-Phe-Arg-MCA as substrate, the kcat/KM and kcat values obtained for the Asp158Asn papain are 20,000 M-1.s-1 and 34 s-1, respectively, as compared with values of 120,000 M-1.s-1 and 51 s-1 obtained for the wild-type papain. In addition, the pH-(kcat/KM) profile for the Asp158Asn enzyme is shifted relative to that for the wild-type enzyme to lower values by approximately 0.3 pH unit. This shows clearly that Asp158 is not, as previously postulated, an essential catalytic residue. In addition, the pH dependency data are interpreted to indicate that, contrary to earlier suggestions, the negatively charged side chain of Asp158 does not significantly stabilize the active-site thiolate-imidazolium ion pair. However, its presence does influence the pKa's associated with ion-pair formation in a manner compatible with electrostatic considerations.

Animals↗
PubMed1990

Structure and transcriptional control of the Saccharomyces cerevisiae POX1 gene encoding acyl-coenzyme A oxidase.

We have cloned the Saccharomyces cerevisiae gene coding for the peroxisomal enzyme: fatty acyl-CoA oxidase (POX). The gene (named POX1) is unique in S. cerevisiae and has been identified through homology with the POX4 and POX5 genes of Candida tropicalis. The POX1 gene encodes a 84-kDa POX protein composed of 748 amino acids. The identity between the S. cerevisiae and C. tropicalis enzymes is about 40%, and there is a greater degree of similarity between the N termini than the C termini. A disruption of the POX1 coding sequence diminishes the ability of yeast cells to grow on oleic acid as a sole carbon source. The expression of the POX1 gene is regulated at the level of transcription, and is induced more than 25-fold by the addition of oleic acid to the medium.

Acyl-CoA Oxidase↗
PubMed1990

Genetic and molecular approaches to synthesis and action of the yeast killer toxin.

The K1 killer toxin of Saccharomyces cerevisiae is a secreted, virally-coded protein lethal to sensitive yeasts. Killer yeasts are immune to the toxin they produce. This killer system has been extensively examined from genetic and molecular perspectives. Here we review the biology of killer yeasts, and examine the synthesis and action of the protein toxin and the immunity component. We summarise the structure of the toxin precursor gene and its protein products, outline the proteolytic processing of the toxin subunits from the precursor, and their passage through the yeast secretory pathway. We then discuss the mode of action of the toxin, its lectin-like interaction with a cell wall glucan, and its probable role in forming channels in the yeast plasma membrane. In addition we describe models of how a toxin precursor species functions as the immunity component, probably by interfering with channel formation. We conclude with a review of the functional domains of the toxin structural gene as determined by site-directed mutagenesis. This work has identified regions associated with glucan binding, toxin activity, and immunity.

Cell Membrane↗
PubMed1990

Overexpression of the STE4 gene leads to mating response in haploid Saccharomyces cerevisiae.

The STE4 gene of Saccharomyces cerevisiae encodes the beta subunit of the yeast pheromone receptor-coupled G protein. Overexpression of the STE4 protein led to cell cycle arrest of haploid cells. This arrest was like the arrest mediated by mating pheromones in that it led to similar morphological changes in the arrested cells. The arrest occurred in haploid cells of either mating type but not in MATa/MAT alpha diploids, and it was suppressed by defects in genes such as STE12 that are needed for pheromone response. Overexpression of the STE4 gene product also suppressed the sterility of cells defective in the mating pheromone receptors encoded by the STE2 and STE3 genes. Cell cycle arrest mediated by STE4 overexpression was prevented in cells that either were overexpressing the SCG1 gene product (the alpha subunit of the G protein) or lacked the STE18 gene product (the gamma subunit of the G protein). This finding suggests that in yeast cells, the beta subunit is the limiting component of the active beta gamma element and that a proper balance in the levels of the G-protein subunits is critical to a normal mating pheromone response.

Base Sequence↗
PubMed1990

Intracellular proteolytic processing of proopiomelanocortin in heterologous COS-1 cells by the yeast KEX2 endoprotease.

We have transiently expressed the yeast KEX2 gene together with the proopiomelanocortin (POMC) cDNA in COS-1 cells. Characterization of the POMC-related immunoreactive peptides by gel permeation and reversed-phase high pressure liquid chromatography showed that the KEX2 enzyme was active and capable of carrying out cleavage of POMC to release the authentic maturation product beta-endorphin(1-31). Peptides resembling beta-lipotropin, the amino terminal glycopeptide, and ACTH(1-39) were also detected as major products in the cell extracts. Our results indicate that the KEX2 enzyme can proteolytically release from POMC a set of peptides similar to that normally found in interior pituitary.

Adrenocorticotropic Hormone↗
PubMed1990

The yeast KEX-2-processing endoprotease is active in the Golgi apparatus of transfected NIH 3T3 fibroblasts.

Proteolytic processing of polyprotein precursors at pairs of basic amino acids is a prerequisite for the generation of bioactive peptide hormones. While the mammalian endoproteases responsible for these cleavages are yet to be identified, this function has been unequivocally assigned in yeast to the product of the KEX-2 gene. To study the molecular mechanisms involved in polyprotein processing, we have transfected the yeast KEX-2 gene into mouse NIH 3T3 fibroblasts and established a new cell line (called 2N-DK) where the KEX-2 endoprotease is permanently expressed. Immunofluorescence studies show that the KEX-2 enzyme is retained within the Golgi of the 2N-DK cells. The evidence for this cellular location is supported by measurement of intracellular and extracellular KEX-2 enzyme activity. In this permanently transfected cell line, KEX-2 activity is exclusively intracellular, in contrast to the situation previously described in transiently infected cell lines, where extracellular KEX-2 activity was detected. Furthermore, infection of 2N-DK cells with a recombinant retrovirus expressing a cDNA coding for porcine proopiomelanocortin (POMC) resulted in the synthesis of POMC and its efficient processing into beta-lipotropin and beta-endorphin, two of its physiologically authentic maturation products. These results suggest that in the fibroblast cell line 2N-DK, proteolytic processing of POMC by KEX-2 endoprotease occurs in the Golgi apparatus.

Amino Acid Sequence↗
PubMed1989

The expression in Escherichia coli of a synthetic gene coding for the precursor of papain is prevented by its own putative signal sequence.

A 1048-bp gene coding for prepropapain was assembled from chemically synthesized oligodeoxyribonucleotides and cloned into a variety of Escherichia coli expression plasmids. We observed loss of plasmid when the preproP gene was expressed in E. coli either as the native precursor or fused at the C terminus of the first 592 amino acids (aa) of beta-galactosidase (beta Gal). Deletion of the putative 26-aa signal peptide (pre-region) increased plasmid stability. The level of maintenance for the different plasmid constructs correlated with the level of expression detected by immunoblotting. Constitutive expression of the beta Gal-propapain fusion generated insoluble granules in a protease-deficient E. coli host. The fusion protein was easily purified to near homogeneity by differential solubilization of the granules.

Amino Acid Sequence↗
PubMed1989

The STE4 and STE18 genes of yeast encode potential beta and gamma subunits of the mating factor receptor-coupled G protein.

The STE4 and STE18 genes are required for haploid yeast cell mating. Sequencing of the cloned genes revealed that the STE4 polypeptide shows extensive homology to the beta subunits of mammalian G proteins, while the STE18 polypeptide shows weak similarity to the gamma subunit of transducin. Null mutations in either gene can suppress the haploid-specific cell-cycle arrest caused by mutations in the SCG1 gene (previously shown to encode a protein with similarity to the alpha subunit of G proteins). We propose that the products of the STE4 and STE18 genes comprise the beta and gamma subunits of a G protein complex coupled to the mating pheromone receptors. The genetic data suggest pheromone-receptor binding leads to the dissociation of the alpha subunit from beta gamma (as shown for mammalian G proteins), and the free beta gamma element initiates the pheromone response.

Amino Acid Sequence↗
PubMed1989

Cloning and characterization of the SKI3 gene of Saccharomyces cerevisiae demonstrates allelism to SKI5.

We have identified a mutant strain of the yeast Saccharomyces cerevisiae which overproduces killer toxin. This strain contains a single mutation which fails to complement defects in both the SKI3 and SKI5 genes, while a cloned copy of this gene complements both the ski3 and ski5 defects. The level of secreted toxin from a cDNA based plasmid is not increased in a ski3 strain, showing that the overproduction phenotype is dependent upon an increased level of M1 dsRNA.

Alleles↗
PubMed1989

Structure of the cel-3 gene from Fibrobacter succinogenes S85 and characteristics of the encoded gene product, endoglucanase 3.

The cel-3 gene cloned from Fibrobacter succinogenes into Escherichia coli coded for the enzyme EG3, which exhibited both endoglucanase and cellobiosidase activities. The gene had an open reading frame of 1,974 base pairs, coding for a protein of 73.4 kilodaltons (kDa). However, the enzyme purified from the osmotic shock fluid of E. coli was 43 kDa. The amino terminus of the 43-kDa protein matched amino acid residue 266 of the protein coded for by the open reading frame, indicating proteolysis in E. coli. In addition to the 43-kDa protein, Western immunoblotting revealed a 94-kDa membranous form of the enzyme in E. coli and a single protein of 118 kDa in F. succinogenes. Thus, the purified protein appears to be a proteolytic degradation product of a native protein which was 94 kDa in E. coli and 118 kDa in F. succinogenes. The discrepancy between the molecular weight expected on the basis of the DNA sequence and the in vivo form may be due to anomalous migration during electrophoresis, to glycosylation of the native enzyme, or to fatty acyl substitution at the N terminus. One of two putative signal peptide cleavage sites bore a strong resemblance to known lipoprotein leader sequences. The purified 43-kDa peptide exhibited a high Km (53 mg/ml) for carboxymethyl cellulose but a low Km (3 to 4 mg/ml) for lichenan and barley beta-glucan. The enzyme hydrolyzed amorphous cellulose, and cellobiose and cellotriose were the major products of hydrolysis. Cellotriose, but not cellobiose, was cleaved by the enzyme. EG3 exhibited significant amino acid sequence homology with endoglucanase CelC from Clostridium thermocellum, and as with both CelA and CelC of C. thermocellum, it had a putative active site which could be aligned with the active site of hen egg white lysozyme at the highly conserved amino acid residues Asn-44 and Asp-52.

Amino Acid Sequence↗
PubMed1988

Expression of MF alpha 1 in MATa cells supersensitive to alpha-factor leads to self-arrest.

MATa cells of Saccharomyces cerevisiae defective in both the SST1 and SST2 gene products exhibit self-arrest when they express the MF alpha 1 gene under the control of the GAL1 promoter. This response to endogenously produced pheromone can be alleviated by mutations which prevent the production of, or response to, alpha-factor. Suppressors of the self-arrest phenotype include a class of mutants which remain responsive to low levels of pheromone, but are resistant to high levels of alpha-factor. One of these mutants has been mapped to chromosome X, 31 cM distal to SUP4, and defines a new locus designated STE18.

Crosses, Genetic↗