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PubMed1985

The effect of pH on the interaction of substrates and effector to yeast and rabbit muscle pyruvate kinase.

The interaction of fructose 1,6-bisphosphate, phosphoenolpyruvate and ADP with pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from yeast and rabbit muscle has been studied as a function of pH utilizing the quenching of protein fluorescence at 330 nm by these ligands. Both the muscle and the yeast pyruvate kinase interact with either ADP or phosphoenolpyruvate with similar affinity, indicating that the substrate-binding sites for these two isozymes are similar. The major difference between the yeast and muscle isozymes is their affinity with fructose 1,6-bisphosphate. Fructose 1,6-bisphosphate interacts with the yeast isozyme in orders of magnitude more strongly than with the muscle isozyme. Moreover, the affinity of fructose 1,6-bisphosphate to the yeast isozyme is strongly pH-dependent, while the interaction of fructose 1,6-bisphosphate with the muscle isozyme is independent of pH. The data indicate that yeast pyruvate kinase undergoes a conformational change as the pH is increased from 6.0 to 8.5.

Adenosine Diphosphate↗
PubMed1992

Characterization of the interaction of yeast enolase with polynucleotides.

Yeast enolase is inhibited under certain conditions by DNA. The enzyme binds to single-stranded DNA-cellulose. Inhibition was used for routine characterization of the interaction. The presence of the substrate 2-phospho-D-glycerate reduces inhibition and binding. Both yeast enolase isozymes behave similarly. Impure yeast enolase was purified by adsorption onto a single-stranded DNA-cellulose column followed by elution with substrate. Interaction with RNA, double-stranded DNA, or degraded DNA results in less inhibition, suggesting that yeast enolase preferentially binds single-stranded DNA. However, yeast enolase is not a DNA-unwinding protein. The enzyme is inhibited by the short synthetic oligodeoxynucleotides G6, G8 and G10 but not T8 or T6, suggesting some base specificity in the interaction. The interaction is stronger at more acid pH values, with an apparent pK of 5.6. The interaction is prevented by 0.3 M KCl, suggesting that electrostatic factors are important. Histidine or lysine reverse the inhibition at lower concentrations, while phosphate is still more effective. Binding of single-stranded DNA to enolase reduces the reaction of protein histidyl residues with diethylpyrocarbonate. The inhibition of yeast enolase by single-stranded DNA is not total, and suggests the active site is not directly involved in the interaction. Binding of substrate may induce a conformational change in the enzyme that interferes with DNA binding and vice versa.

Animals↗
PubMed1992

Glucose-induced activation of plasma membrane H(+)-ATPase in mutants of the yeast Saccharomyces cerevisiae affected in cAMP metabolism, cAMP-dependent protein phosphorylation and the initiation of glycolysis.

Addition of glucose-related fermentable sugars or protonophores to derepressed cells of the yeast Saccharomyces cerevisiae causes a 3- to 4-fold activation of the plasma membrane H(+)-ATPase within a few minutes. These conditions are known to cause rapid increases in the cAMP level. In yeast strains carrying temperature-sensitive mutations in genes required for cAMP synthesis, incubation at the restrictive temperature reduced the extent of H(+)-ATPase activation. Incubation of non-temperature-sensitive strains, however, at such temperatures also caused reduction of H(+)-ATPase activation. Yeast strains which are specifically deficient in the glucose-induced cAMP increase (and not in basal cAMP synthesis) still showed plasma membrane H(+)-ATPase activation. Yeast mutants with widely divergent activity levels of cAMP-dependent protein kinase displayed very similar levels of activation of the plasma membrane H(+)-ATPase. This was also true for a yeast mutant carrying a deletion in the CDC25 gene. These results show that the cAMP-protein kinase A signaling pathway is not required for glucose activation of the H(+)-ATPase. They also contradict the specific requirement of the CDC25 gene product. Experiments with yeast strains carrying point or deletion mutations in the genes coding for the sugar phosphorylating enzymes hexokinase PI and PII and glucokinase showed that activation of the H(+)-ATPase with glucose or fructose was completely dependent on the presence of a kinase able to phosphorylate the sugar. These and other data concerning the role of initial sugar metabolism in triggering activation are consistent with the idea that the glucose-induced activation pathways of cAMP-synthesis and H(+)-ATPase have a common initiation point.

Cell Membrane↗
PubMed1993

Flow cytometric detection of wild yeast in lager breweries.

A flow cytometric method for detection of wild yeast infections in breweries is reported. It is based on selective enrichment in Malt extract Yeast extract Glucose Peptone broth (MYGP) at 37 degrees C and in MYGP with 200 ppm CuSO4 at 25 degrees C, staining with a fluorochrome precursor and flow cytometry. In experiments with several types of wild yeast isolated from breweries and two different strains of lager yeast it has been possible to detect one wild yeast per 10(6) culture yeast after 48-72 h of incubation and, in some cases, after 24 h.

Beer↗
PubMed1995

An in vitro system for studying RNA-protein interaction: application to a study of yeast ribosomal protein L1 binding to 5S rRNA.

Previous attempts to study the binding of yeast ribosomal protein L1 with 5S rRNA in vitro have been impeded by the failure to form RNA-protein complexes with purified protein and RNA. To circumvent this difficulty, we have developed an in vitro system that allowed RNP formation. The system involved in vitro expression of the protein L1 from its cloned gene in the presence of exogenous yeast 5S rRNA. A protein of the expected size (34 kDa) was synthesized by in vitro transcription and translation. A specific 5S rRNA-protein L1 complex (RNP) was formed when the rRNA molecule was present during protein L1 synthesis. However, the full-length protein L1 failed to bind 5S rRNA. The extent of RNP formation was proportional to the concentration of the exogenous yeast 5S rRNA in the reaction. The RNP displayed properties identical to those isolated from mature 60S ribosome subunits. Addition of yeast 5.8S rRNA did not result in the formation of a specific RNP. Using this in vitro system, we examined the ability of several deletion mutant proteins to bind yeast 5S rRNA and concluded that protein L1 missing residues 261 to 295 from the C-terminus could not bind yeast 5S rRNA. This in vitro system should be useful for future studies on the molecular nature of 5S rRNA-protein L1 interaction.

Protein Binding↗
PubMed1989

Inhibition of yeast exoglucanases by glucosidase inhibitors.

Castanospermine, 1-deoxynojirimycin, and N-methyl-1-deoxynojirimycin, three well-characterized inhibitors of the glucosidases involved in the processing of N-linked oligosaccharides, did not affect the biosynthesis or the secretion of exoglucanases (EC 3.2.1.58) from Saccharomyces cerevisiae and Candida albicans but inhibited the activity itself. Regardless of the substrate used, laminarin or p-nitrophenyl beta-D-glucoside (pNPG), all three inhibitors proved to act in a competitive manner. Castanospermine was the most potent inhibitor, with Ki values ranging from 0.16 to 0.5 microM for three different purified yeast exoglucanases. The inhibition caused by 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin was poorer, but still significant. By contrast, the glucosidase inhibitors did not show any action on a partially purified endoglucanase (EC 3.2.1.39) Candida albicans. A purified exoglucanase from Basidiomycete QM 806, which was specific for laminarin, was unaffected by castanospermine but it was still inhibited in an uncompetitive manner by 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin. The presence of castanospermine in the culture medium of growing yeasts did not have any effect on yeast growth in spite of the fact that, under the conditions used, the external exoglucanase was fully inhibited. None of the yeast exoglucanases hydrolyzed the glucan synthesized in vitro by membrane preparations derived from either yeast. These results support the concept that yeast exoglucanases are glucosidases that also attack laminarin, rather than glucanases capable of attacking pNPG.

1-Deoxynojirimycin↗
PubMed1979

Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene.

We have constructed a plasmid, YEp13, which when used in conjunction with transformation in yeast is a suitable vector for isolating specific yeast genes. The plasmid consists of pBR322, the LEU2 gene of yeast, and a DNA fragment containing a yeast origin of replication from 2 mu circule. We have demonstrated the utility of this cloning system by isolating the yeast gene encoding the arginine permease, CAN1, from a pool of random yeast DNA fragments inserted into YEp13.

Arginine↗
PubMed1979

Expression of the yeast galactokinase gene in Escherichia coli.

In Saccharomyces cerevisiae the genes for three of the enzymes involved in galactose metabolism are tightly linked near the centromere of chromosome II (Douglas and Hawthorne, 1964). However, the molecular mechanisms which control the expression of these genes are not well understood. A DNA fragment containing at least one of these yeast genes, the galactokinase gene (gal1), has been joined to the bacterial plasmid pBR322 and maintained in an Escherichia coli strain that carries a deletion in its own galactokinase gene, galK. The presence of the yeast gene was demonstrated by (i) complementation of the E. coli galactokinase deletion, (ii) by hybridization of the cloned DNA fragment to restriction enzyme digests of total yeast DNA and (iii) by assaying for yeast galactokinase activity in bacterial cell extracts. The yeast DNA fragment is 4700 base pairs long, and enables the host E. coli K-12 strain to grow in minimal medium containing galactose as the sole carbon source with a generation time of 14.3 h. The yeast galactokinase activity in the bacterial extracts is 0.7% of the bacterial galactokinase activity found in wild-type E. coli fully induced with fucose.

DNA↗
PubMed1981

Cloning of the HIS5 gene of Saccharomyces cerevisiae by yeast transformation.

A yeast DNA fragment complementing a yeast his5 mutation was cloned on a shuttle vector, YRp7, by transformation of the yeast host with plasmid DNA prepared from a gene bank constructed in the Escherichia coli host. That the DNA fragment contains the yeast HIS5 gene was confirmed by the integration of the cloned plasmid into the his5 region of the yeast chromosome. The cloned yeast HIS5 gene weakly complemented the E. coli hisC mutation and gave rise to a slow-growing E. coli transformant without addition of histidine. From the slow-growing culture, rapidly growing variants of E. coli were easily obtained by mutations either in the bacterial chromosome or in the plasmid.

Cloning, Molecular↗
PubMed1983

Copy number control by a yeast centromere.

Plasmids containing a cloned yeast (Saccharomyces cerevisiae) centromere (CEN3) in combination with a suitable DNA replication system are maintained in yeast at the low copy number typical of a chromosome. In composite plasmids containing CEN3 plus the yeast 2 mu plasmid, the CEN3 copy number control is dominant over the amplification system that normally drives the 2 mu plasmids to high copy number. The CEN3-2 mu composite plasmids are relatively stably maintained in yeast at a copy number of about one per haploid genome, and segregate through meiosis in a typical Mendelian pattern. Some of the CEN3-2 mu composite plasmids isolated from yeast contain deletions of variable size that remove the functional centromere, resulting in loss of the CEN3 control and reversion to high copy number. Formation of the CEN3 deletions requires the specialized recombination system (inverted repeat sequences and FLP gene) of the yeast 2 mu plasmid.

Centromere↗
PubMed1984

Cloning and expression of a yeast copper metallothionein gene.

The induction of a copper-binding metallothionein (Cu-MT) was studied in yeast, Saccharomyces cerevisiae, and a relationship between copper resistance and intracellular levels of Cu-MT in these eukaryotes was established. Poly(A)-containing RNA from a copper-resistant (Cur) yeast strain, which synthesized abundant quantities of Cu-MT and in which Cu-MT gene transcription was enhanced 50-fold upon exposure to CuSO4, was used to screen yeast genomic DNA clones. Restriction analysis revealed common XbaI and KpnI sites in five genomic clones isolated. The transcription of these clones was regulated by copper. Transformation of a copper-sensitive (Cus) yeast strain by one of these clones confers copper resistance in yeast. The results suggest that the expression of the Cu-MT gene is, in part, responsible for mediating copper resistance in yeast.

Cloning, Molecular↗
PubMed1985

Expression of a nitrogen-fixation gene encoding a nitrogenase subunit in yeast.

Biological nitrogen fixation is catalyzed by nitrogenase, an enzyme complex exclusive to prokaryotes. We used the yeast Saccharomyces cerevisiae to study the synthesis, and subsequently the assembly, of nitrogenase components in a eukaryote. Here, the Klebsiella pneumoniae nifH gene, encoding the subunit of the Fe protein (Kp2) component of nitrogenase, was expressed in S. cerevisiae from the yeast ADHI promoter. The nifH gene product, detected in yeast by immunoblot analysis with anti-Kp2 antibodies, exhibited the same electrophoretic mobility in SDS-polyacrylamide gels as that of the Kp2 subunit synthesized in K. pneumoniae. Estimates of Kp2 antigen and assays of beta-galactosidase activity specified by nifH'-'lacZ fusions showed that the level of nifH product was similar in anaerobically and aerobically grown yeast, but varied with different transforming plasmids and in various haploid and diploid yeast strains. A cistron located downstream to nifH in a transcript resembling the polycistronic mRNA of the nifHDKY operon in K. pneumoniae is not translated in yeast.

Gene Expression Regulation↗
PubMed1986

Construction of stable laboratory and industrial yeast strains expressing a foreign gene by integrative transformation using a dominant selection system.

An expression cassette of mouse dihydrofolate reductase (Mdhfr) cDNA under control of the yeast cytochrome c promoter was inserted in a yeast plasmid containing the ARS1 sequence. The ARS replicating function was destroyed by BglII treatment prior to yeast transformation. Using this linearized plasmid, genomic transformants could be obtained from either laboratory or industrial strains of bakers' yeast based on direct methotrexate (MTX)-resistance selection. The entire sequence of the linearized plasmid was integrated by homologous recombination at the ARS region of the host chromosome. The results indicate that repetitive and homologous recombination occurs readily in such transformations. The stability of the constructed integrants was more than 99.95% per generation in non-selective medium, and tandem repeats of up to six copies (i.e., about 44 kb) were not changed even after 30 generations in rich medium. Expression in rich medium of cointegrated, human interleukin 2 cDNA under control of the triose phosphate isomerase promoter was shown by Western blot experiments in both laboratory and industrial yeast strains. Furthermore, a comparison of the transcription efficiency of the Mdhfr gene in the chromosome with that in the plasmid revealed that the efficiency was almost proportional to the number of gene copies, irrespective of the location of the transcription unit. These results show that by using the MTX/Mdhfr dominant selection-amplification system one can construct stable recombinant yeast strains suitable for heterologous gene expression in laboratory as well as in industrial fermentation conditions.

DNA, Fungal↗
PubMed1987

Glycosylation and secretion of human alpha-1-antitrypsin by yeast.

Human alpha-1-antitrypsin (alpha-AT) is a major serum protein with protease inhibitory activity. Three asparagine residues in alpha-AT are glycosylated with the mammalian 'complex' pattern of carbohydrate as the protein is secreted from cells in the liver. To examine the glycosylation and secretion of human alpha-AT by Saccharomyces cerevisiae, the yeast invertase secretion signal codons were substituted for the native secretion signal coding DNA of an alpha-AT cDNA, and the fusion gene was placed on an autonomously replicating yeast--Escherichia coli shuttle vector under control of the yeast triosephosphate isomerase (TPI) promoter. Yeast strains transformed with this plasmid produce human alpha-AT and secrete about one-fifth of it into the culture broth. Approximately 80% of the alpha-AT produced in yeast is not in the culture broth but is inside the cell within the secretory pathway. This internal alpha-AT is heterogeneous, consisting of molecules with core carbohydrate on either two or all three of the asparagine receptors. Human alpha-AT secreted into the culture broth contains, in addition to core carbohydrate, variable numbers of mannose outer chains, typical of secreted yeast proteins such as invertase. All carbohydrate is removed by endoglycosidase H treatment. Examination of alpha-AT secreted from an mnn9 mutant, which blocks addition of variable numbers of outer mannose chains, revealed a homogeneous alpha-AT product which, like alpha-AT isolated from human serum, bears carbohydrate on three asparagine residues per molecule.

Asparagine↗
PubMed1988

A series of shuttle vectors using chloramphenicol acetyltransferase as a reporter enzyme in yeast.

Reports from numerous laboratories have shown that the gene coding for the bacterial enzyme chloramphenicol-3-O-acetyltransferase can be used as a reporter gene (cat) in mammalian and plant systems to analyze gene activity at the transcriptional level when combined with endogenous regulatory signals; the enzyme activity can be quantified by a chromatographic or a photometric assay. To adapt this simple and highly sensitive test for the yeast system, we constructed a series of yeast vectors containing the cat gene together with selectable markers for Escherichia coli and yeast; integrating, autonomously replicating and centromere-carrying plasmids were used. We show that the cat gene lacking the endogenous promoter is expressed at low levels in yeast transformants. To demonstrate functional expression of the cat gene placed under the control of a yeast promoter, we chose the PHO5 regulatory region. We found that cat expression was induced via the PHO5 promoter in a manner as observed for the endogenous PHO5 gene, whereas in the repressed state cat expression remained low. Using these vectors, it should be feasible to analyze other sequences conferring promoter activity or other control functions in yeast.

Chloramphenicol O-Acetyltransferase↗
PubMed1989

Expression and activity of a gene encoding rat cytochrome c in the yeast Saccharomyces cerevisiae.

A rat-processed pseudogene, which encodes normal rat cytochrome c, has been expressed in the yeast, Saccharomyces cerevisiae. The translated region of the chromosomal CYC1+ locus, which encodes yeast iso-1-cytochrome c, was replaced by the translated region of the gene encoding rat cytochrome c (CYC1-RAT), thus preserving the proper CYC1 transcription initiation and termination signals. Although the levels of transcription of the normal CYC1+ gene and the CYC1-RAT gene in yeast were equivalent, rat cytochrome c was produced at approx. 40% of the level of iso-1-cytochrome c. In addition, the specific activity in vivo was estimated to be approx. 60% that of the yeast iso-1-cytochrome c. N-terminal processing of indigenous rat cytochrome c, in which the N-terminal methionine residue is cleaved and the penultimate glycine residue is acetylated, also occurred in yeast. Methionine cleavage was complete, while acetylation proceeded to only 70% completion. Lys-72 was trimethylated to 66% completion in the rat cytochrome c produced in yeast. The near normal expression (40%) and specific activity (60%) in vivo indicates that the 40% difference in amino acid sequence is not critical for mitochondrial import, heme attachment and interactions with redox partners.

Amino Acid Sequence↗
PubMed1989

Yeast cyclophilin: isolation and characterization of the protein, cDNA and gene.

Cyclophilin (CPH) has been isolated from the yeast Saccharomyces cerevisiae, purified to homogeneity and partially sequenced. Oligodeoxyribonucleotides deduced from this sequence were used to isolate the corresponding cDNA and gene. An open reading frame coding for a 162-amino acid (aa) protein with a calculated Mr of 17,392, was deduced from the nucleotide sequence. Comparison between yeast and human CPH shows a very high overall sequence conservation (65% aa homology). The binding of yeast CPH to cyclosporin A is identical to that of human and bovine CPH. Unlike in Neurospora crassa, a mitochondrial form of CPH could not be detected in yeast. Southern-blot analysis of yeast DNA shows that only one CPH-related sequence is present per haploid genome, whereas at least 20 genes or pseudogenes were detected in the human and rat genome. Purified yeast CPH exhibits peptidyl-prolyl cis-trans isomerase activity, albeit to a far lesser extent than the mammalian protein.

Amino Acid Sequence↗
PubMed1990

Disruption of the gene XRN1, coding for a 5'----3' exoribonuclease, restricts yeast cell growth.

As a step toward determining the metabolic role(s) of a 5'----3' exoribonuclease (XRN1), a yeast gene, XRN1, encoding XRN1, was first cloned, then disrupted to test its essentially or effect on yeast cell growth. Clones in the high-copy-number plasmid YEp24 cause overproduction (fivefold) of XRN1 in yeast cells, as measured by either poly(A) hydrolytic activity or immunoreactivity. Restriction mapping and deletion analysis showed that the XRN1 gene is located on a 6.7-kb XbaI-XhoI fragment of chromosome VII. The normal gene was disrupted in two haploid yeast strains by integrating a fragment with a BglII-deleted segment replaced with the yeast URA3 gene, and the disrupted strains lack XRN1. Successful transformation of haploid cells showed that the gene is not essential, but its absence markedly affected the cell growth rate. The growth defect is corrected by introduction of the XRN1 gene on a plasmid back into the disrupted yeast.

Blotting, Southern↗