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At least 91 records · Page 5Linked to original sources
PubMed1990

Expression of Torpedo nicotinic acetylcholine receptor subunits in yeast is enhanced by use of yeast signal sequences.

We have produced the four subunits of the nicotinic acetylcholine receptor of Torpedo californica, an integral membrane protein, in the yeast Saccharomyces cerevisiae. Two of the subunits (alpha and delta) were readily produced from their cDNAs after simply subcloning them into a yeast shuttle vector adjacent to a yeast promoter. The other two protein subunits (beta and gamma) were not produced by this strategy, although the amounts of mRNA produced from these expression constructs are similar to those for alpha and delta. Replacing the DNA coding for the normal N-terminal signal sequences for the beta and gamma subunits with DNA coding for the signal sequence of yeast invertase results in successful protein synthesis. The yeast signal sequence allows these subunits to be translocated across the membrane of the endoplasmic reticulum and to be glycosylated. The appropriate final size of the subunit proteins suggests that the yeast signal sequence has been properly cleaved after translocation.

Animals↗
PubMed2006

A marine killer yeast against the pathogenic yeast strain in crab (Portunus trituberculatus) and an optimization of the toxin production.

A pathogenic yeast strain WCY which could cause milky disease in Portunus trituberculatus was identified to be Metschnikowia bicuspidate according to the results of routine yeast identification and 18S rDNA and ITS sequences. After screening of more than 300 yeast strains from different sources in marine environments, it was found that strain YF07b had the highest ability to produce killer toxin against the pathogenic yeast. Strain YF07b was identified to be Pichia anomala according to the results of routine yeast identification and 18S rDNA and ITS sequences. The optimal conditions for killer toxin production by strain YF07b were the production medium with 2.0% NaCl, pH 4.5, cultivation temperature of 20 degrees C and the optimal conditions for action of the crude killer toxin against the pathogenic yeast were the assay medium with 6.0% NaCl, pH 4.5 and temperature 15 degrees C.

Animals↗
PubMed1999

Yeast culture volatiles as attractants for Rhodnius prolixus: electroantennogram responses and captures in yeast-baited traps.

Responses to air currents carrying volatiles from yeast cultures were measured by means of electroantennograms (EAGs) in the haematophagous bug Rhodnius prolixus and we tested yeast-baited traps in the laboratory. The volatiles liberated by yeast cultures generated a clear electrophysiological response, much higher than that obtained during stimulation with clean air. The addition of yeast cultures to the traps dramatically increased overnight capture. A modification performed on the traps was effective for this species, as the bugs were immobilised once they fell into the device. Our results demonstrate that yeast culture volatiles are attractive for R. prolixus. The results obtained by means of electroantennogram techniques represent the first electrophysiological evidence of the perception of yeast volatiles by the antennae of a triatomine bug. We discuss the relevance of our results in view of currently used sampling techniques and control strategies.

Animals↗
PubMed1997

Incorporation of copper into the yeast Saccharomyces cerevisiae. Identification of Cu(I)--metallothionein in intact yeast cells.

Copper is an essential metal ion to many living organisms, including mammals, as it mediates a wide variety of important biochemical processes. At elevated concentrations, copper is extremely toxic to host cells. This paradoxical nature of copper has necessitated a highly regulated procedure for its cellular accumulation, transport, and excretion. One important group of proteins involved in eukaryotic copper speciation is the protein metallothionein. Luminescence microscopy data, emission, and circular dichroism spectral data are reported as copper is incorporated into metallothionein by the yeast Saccharomyces cerevisiae. These techniques provide information on the mechanism of copper uptake by S. cerevisiae. A two-stage kinetic mechanism for the uptake of copper from the growth medium by the yeast cells is observed. The first stage displays an uptake rate that is dependent on the initial copper concentration of the growth medium, and lasts for approximately 6 h. The second stage has a slower rate of copper uptake than the first, but the kinetics are independent of the initial copper concentration. Emission spectra recorded directly from the intact yeast cells (at 77 K) show that the cellular incorporation of copper proceeds via several species, eventually leading to storage of the copper in the form of Cu-metallothionein. The photomicrographs of yeast cells grown in a copper-containing medium clearly show an orange luminescence, indicating the formation of a Cu(I)-thiolate species. The identification of this species as copper-metallothionein was confirmed by measurement of the circular dichroism and emission properties following excretion and isolation of the copper-containing protein from the yeast cells. Analysis of the emission spectrum from S. cerevisiae Cu-metallothionein at 77 K reveals two emission bands, centered at 570 and 700 nm. The high-energy emission band exhibits a two-component decay, with excited state lifetimes of 4.70 and 48.5 microseconds. The low-energy emission exhibits one major decay component with a lifetime of 1.13 microseconds. A high-molecular-weight, copper-containing species is also isolated from the yeast cells and is characterized spectroscopically.

Carrier Proteins↗
PubMed1994

Structural elements in yeast tRNAs required for homologous modification of guanosine-26 into dimethylguanosine-26 by the yeast Trm1 tRNA-modifying enzyme.

In eukaryotic tRNAs, guanosines in position 26 (G26), located at the junction between the D-stem and the anticodon stem of tRNA, are usually modified to N2,N2-dimethylguanosine (m2(2)G). Although G26 is a prerequisite for biosynthesis of m2(2)G26, it is not self-sufficient for the formation of the dimethylated G26, since in exceptional cases eukaryotic tRNAs have an unmodified G26. In the yeast Saccharomyces cerevisiae the only tRNA species with an unmodified G26 is tRNAAsp. Using in vitro transcripts of this tRNA, as well as of yeast tRNAPhe, a tRNA containing m2(2)G26 in vivo, we have investigated the requirements on tRNA sequences and structures for the formation of m2(2)G26 by the yeast enzyme, i.e. in a homologous in vitro system. We have now demonstrated that G26 was efficiently dimethylated in vitro also after deletion of the entire anticodon stem and loop. We conclude that the elements necessary for a productive interaction between G26 in nuclear coded yeast tRNAs and the yeast G26 modifying enzyme are located within the core of the tRNA. For modification of G26 to m2(2)G26 via monomethylated G26, important primary and secondary structural elements in the tRNAs are a size of at least five nucleotides in the variable loop together with two G-C base pairs in the D-stem. This is the first case reported where the minimal requirements on nuclear coded tRNAs for a yeast modifying enzyme has been elucidated.

Base Sequence↗
PubMed1983

Isolation of a yeast single-strand deoxyribonucleic acid binding protein that specifically stimulates yeast DNA polymerase I.

We sought a protein from yeast that would bind more strongly to single-stranded DNA than to duplex DNA and would stimulate the activity of the major yeast DNA polymerase, but not polymerases from other organisms. We isolated a protein that binds about 200 times more strongly to single-stranded DNA than duplex DNA and stimulates yeast DNA polymerase I activity 4-5-fold. It inhibits synthesis catalyzed by calf thymus DNA polymerase alpha and has little effect on T4 DNA polymerase. This yeast protein, SSB-1, has a molecular weight of approximately 40 000. At apparent saturation there is one protein molecule bound per 40 nucleotides. Protein binding causes the single-stranded DNA molecule to assume a relatively extended conformation. It binds to single-stranded RNA as strongly as to DNA. SSB-1 increases the initial rate of polymerization catalyzed by yeast DNA polymerase I apparently by increasing the processivity of the enzyme. We estimate there are 7500-30 000 molecules of SSB-1 per yeast cell, enough to bind at least 400-1600 nucleotides per replication fork. Thus it is present in sufficient abundance to participate in DNA replication in vivo in the manner suggested by these in vitro experiments.

DNA↗
PubMedDate not supplied

Evidence for yeast autophagy during simulation of sparkling wine aging: a reappraisal of the mechanism of yeast autolysis in wine.

Yeast autolysis is the source of several molecules responsible for the quality of wines aged in contact with yeast cells. However, the mechanisms of yeast autolysis during wine aging are not completely understood. All descriptions of yeast autolysis in enological conditions emphasize the disturbance of cell organization as the starting event in the internal digestion of the cell, while no reference to autophagy is found in wine-related literature. By using yeast mutants defective in the autophagic or the Cvt pathways we have demonstrated that autophagy does take place in wine production conditions. This finding has implications for the genetic improvement of yeasts for accelerated autolysis.

Autophagy↗
PubMed1994

A yeast system for expression of human cystathionine beta-synthase: structural and functional conservation of the human and yeast genes.

Human cystathionine beta-synthase (CBS; EC 4.2.1.22) deficiency results in a recessive genetic disorder whose clinical and biochemical manifestations vary greatly among affected individuals. In an effort to identify and analyze mutations in the human CBS gene, we have developed a yeast expression system for human CBS. We have cloned and sequenced a human cDNA that codes for CBS and have expressed the human CBS protein in yeast cells lacking endogenous CBS. The human enzyme produced in yeast is functional both in vitro and in vivo. We have also cloned and sequenced the yeast gene, CYS4, that codes for CBS. The predicted human and yeast CBS proteins are 38% identical and 72% similar to each other, as well as sharing significant similarity with bacterial cysteine synthase. These results demonstrate the evolutionary conservation of CBS and establish the utility of a yeast expression system for studying human CBS.

Animals↗
PubMed1999

Histidine-tagged wild-type yeast actin: its properties and use in an approach for obtaining yeast actin mutants.

Wild-type and an N-terminal 6-histidine-tagged actin have each been expressed by using a yeast strain that contains the actin gene on a plasmid and not on the chromosome. Yeast strains have also been constructed that use two plasmids, one expressing the wild-type protein and the other the 6-histidine-tagged protein. Yeast cells can be grown with either plasmid alone or with both plasmids together and appear to be normal in that the growth rates of all the yeast strains are quite similar, as is the morphology of the yeast cells. The polymerization properties of the 6-histidine-tagged actin appear almost identical to wild-type actin expressed from the chromosome. When the wild-type and 6-histidine-tagged actin are coexpressed, they can be purified by standard techniques and then separated using nickel-nitrilotriacetate chromatography. The method can be used to prepare actin mutants including those that are nonfunctional or might not support yeast growth for other reasons.

Actins↗
PubMed2000

Poly(A) tail-dependent exonuclease AtRrp41p from Arabidopsis thaliana rescues 5.8 S rRNA processing and mRNA decay defects of the yeast ski6 mutant and is found in an exosome-sized complex in plant and yeast cells.

Eukaryotic 3'-->5' exonucleolytic activities are essential for a wide variety of reactions of RNA maturation and metabolism, including processing of rRNA, small nuclear RNA, and small nucleolar RNA, and mRNA decay. Two related but distinct forms of a complex containing 10 3'-->5' exonucleases, the exosome, are found in yeast nucleus and cytoplasm, respectively, and related complexes exist in human cells. Here we report on the characterization of the AtRrp41p, an Arabidopsis thaliana homolog of the Saccharomyces cerevisiae exosome subunit Rrp41p (Ski6p). Purified recombinant AtRrp41p displays a processive phosphorolytic exonuclease activity and requires a single-stranded poly(A) tail on a substrate RNA as a "loading pad." The expression of the Arabidopsis RRP41 cDNA in yeast rescues the 5.8 S rRNA processing and 3'-->5' mRNA degradation defects of the yeast ski6-100 mutant. However, neither of these defects can explain the conditional lethal phenotype of the ski6-100 strain. Importantly, AtRrp41p shares additional function(s) with the yeast Rrp41p which are essential for cell viability because it also rescues the rrp41 (ski6) null mutant. AtRrp41p is found predominantly in a high molecular mass complex in Arabidopsis and in yeast cells, and it interacts in vitro with the yeast Rrp44p and Rrp4p exosome subunits, suggesting that it can participate in evolutionarily conserved interactions that could be essential for the integrity of the exosome complex.

Arabidopsis↗
PubMed2005

Cytokinesis depends on the motor domains of myosin-II in fission yeast but not in budding yeast.

Budding yeast possesses one myosin-II, Myo1p, whereas fission yeast has two, Myo2p and Myp2p, all of which contribute to cytokinesis. We find that chimeras consisting of Myo2p or Myp2p motor domains fused to the tail of Myo1p are fully functional in supporting budding yeast cytokinesis. Remarkably, the tail alone of budding yeast Myo1p localizes to the contractile ring, supporting both its constriction and cytokinesis. In contrast, fission yeast Myo2p and Myp2p require both the catalytic head domain as well as tail domains for function, with the tails providing distinct functions (Bezanilla and Pollard, 2000). Myo1p is the first example of a myosin whose cellular function does not require a catalytic motor domain revealing a novel mechanism of action for budding yeast myosin-II independent of actin binding and ATPase activity.

Actins↗
PubMed1987

A single intronless action gene in the fission yeast Schizosaccharomyces pombe: nucleotide sequence and transcripts formed in homologous and heterologous yeast.

The actin gene of the fission yeast Schizosaccharomyces pombe has been isolated by using as a hybridization probe cloned actin DNA from the budding yeast Saccharomyces cerevisiae. In contrast to most actin genes studied from diverse eukaryotic species, the S. pombe gene is not interrupted by introns. The protein sequence deduced from the nucleotide sequence of the gene shows that the S. pombe actin is more closely related to the mammalian gamma-actin than to the actin of S. cerevisiae. Three transcripts of 1240, 1650 and 1850 nucleotides having the same 5' end but differing in the length of their 3' untranslated region are generated in the fission yeast. Only one messenger RNA of 1330 nucleotides is formed from the S. pombe actin gene in S. cerevisiae. Contrary to the observation made with other S. pombe genes transcribed in the budding yeast, the heterologous actin gene transcript is initiated 39 nucleotides upstream of the initiation start site used in the homologous yeast. The mRNA termination (or 3' processing) mechanism in the two ascomycetes also differs as the 3'end of the S. pombe actin gene transcript in S. cerevisiae does not coincide with either of the three 3'ends mapped in the fission yeast.

Actins↗
PubMed1992

Human and human-yeast chimeric U6 snRNA genes identify structural elements required for expression in yeast.

U6 is the most highly conserved spliceosomal snRNA. Previous mutational studies have shown that the majority of essential residues in U6 are located in a region of 35 nucleotides encompassing a conserved hexanucleotide and stem I and stem II of the U4-interaction domain. Although the yeast and human U6 RNAs are 80% identical in this region, the human U6 gene cannot functionally replace the yeast gene in vivo. The human gene is not transcribed when placed in the context of yeast flanking sequences. Transcription of the human gene, but not its function, can be stimulated by the introduction of an A block promoter element in the U6 coding region. Using a set of human-yeast chimeras, we show that the 5' domain and the 3' terminal region of the human U6 gene can each functionally replace the corresponding yeast domains. However, a combination of both domains in a single molecule is lethal. The basis of the inability of the human U6 snRNA to function in yeast cells is discussed.

Base Composition↗
PubMed1993

Human snRNP polypeptide D1 promotes pre-mRNA splicing in yeast and defines nonessential yeast Smd1p sequences.

Parallel investigations of yeast and metazoan pre-mRNA splicing have documented enormous complexity in the nucleic acid and protein components of the cellular splicing apparatus, the spliceosome. The degree to which yeast and metazoan spliceosomal proteins differ in composition and structure is currently unknown. In this report we demonstrate that the human small nuclear ribonucleoprotein (snRNP) polypeptide D1 complements the cell lethality, splicing deficiency, and snRNA instability phenotypes associated with a yeast smd1 null allele. Mutational analysis of yeast SMD1, guided by a comparison of the predicted yeast and human proteins, reveals that a large, nonconserved portion of Smd1p is dispensable for biological activity. These observations firmly establish D1 as an essential component of the cellular splicing apparatus and suggest that yeast and metazoa are remarkably similar in the polypeptides guiding early snRNP assembly.

Alleles↗
PubMed1997

Variations of the C2H2 zinc finger motif in the yeast genome and classification of yeast zinc finger proteins.

The PROSITE pattern Zinc_Finger_C2H2 was extended to permit the detection of all C2H2 zinc fingers and their parent proteins in the recently completed sequence of the yeast genome. Additionally, a new computer program was written that extracts other zinc binding motifs (non C2H2 'fingers'), overlapping with the classical zinc finger pattern, from the found set of yeast C2H2 fingers. The complete and correct detection of all fingers is a prerequisite for the classification of the yeast zinc finger proteins in functional terms. The detected 53 yeast C2H2 zinc finger proteins do not contain finger clusters with 10 or more repeats, as is frequently found in higher eukaryotes. Only three proteins contain four or more fingers in a cluster. Moreover, nearly all 27 yeast proteins with tandem arrays of two or three finger domains can be classified into nine subgroups with high sequence conservation in their finger clusters, in particular of their DNA recognition helices. These results and application of the recently elaborated finger/DNA recognition rules suggest that the yeast proteins belonging to the same subgroup may recognize identical or very similar DNA sites.

Amino Acid Sequence↗
PubMed1992

Antibody (IgG, IgA, and IgM) to baker's yeast (Saccharomyces cerevisiae), yeast mannan, gliadin, ovalbumin and betalactoglobulin in monozygotic twins with inflammatory bowel disease.

To assess whether dietary antigens play a role in inflammatory bowel disease, 26 monozygotic twin pairs with inflammatory bowel disease and 52 healthy controls were investigated for serum antibodies (IgA, IgG, IgM) against ovalbumin, betalactoglobulin, gliadin, whole yeast (Saccharomyces cerevisiae) and yeast cell wall mannan. The twins were made up of five pairs concordant and nine pairs discordant for Crohn's disease, and two pairs concordant and 10 pairs discordant for ulcerative colitis. Two patients with Crohn's disease had a slight increase in disease activity, the others were in clinical remission. Two striking observations were made: first, individuals with ulcerative colitis were indistinguishable from healthy twins, and controls except for the response to gliadin. Both healthy and diseased twins had higher IgA levels to gliadin than controls. Second, twins who had developed Crohn's disease displayed higher antibody titres towards yeast cell wall mannan in particular, but also to whole yeast (Saccharomyces cerevisiae) of all antibody types (IgA, IgG, and IgM). In contrast, the response to gliadin, ovalbumin, and betalactoglobulin did not differ from healthy twins and was even lower than in the controls. The results argue against an increased systemic antigen presentation caused by an impaired mucosal barrier in the inflammatory bowel disease. Rather, they suggest that yeast cell wall material--that is, mannan, or some antigen rich in mannose and cross reacting with mannan, may play an aetiological role in Crohn's disease, but not in ulcerative colitis. The increases in IgA and IgM, as well as IgG suggest that local and systemic immune systems are selectively activated by antigen(s) present in the cell wall of baker's yeast.

Adult↗
PubMed1987

The interaction of yeast citrate synthase with yeast mitochondrial inner membranes.

The specific interaction of yeast citrate synthase with yeast mitochondrial inner membranes was characterized with respect to saturability of binding, pH optimum, effect of ionic strength, temperature response, and inhibition by oxalacetate. The binding ability of the inner membranes is inhibited by proteolysis and heat treatment, which implies that the membrane component(s) responsible for binding is a protein. A protein fraction from inner membranes when added to liposomes will bind citrate synthase. In addition, the binding of yeast fumarase, mitochondrial malate dehydrogenase, and cytosolic malate dehydrogenase to yeast inner membranes was examined. For these studies the yeast mitochondrial matrix enzymes, citrate synthase (from two types of yeast), malate dehydrogenase, and fumarase, as well as cytosolic malate dehydrogenase, were purified using rapid new techniques.

Animals↗
PubMed1994

Proteolytic processing and secretion of human beta-amyloid precursor protein in yeast. Evidence for a yeast secretase activity.

Human beta-amyloid precursor protein (APP), the transmembrane precursor of the Alzheimer's disease beta-amyloid peptide, was expressed in the yeast Saccharomyces cerevisiae by fusion to prepro-alpha-factor. From analysis of protease-deficient yeast strains, the fusion protein underwent partial processing by Kex2 protease to generate full-length APP and a fraction of the molecules were degraded in the vacuole. A soluble APP ectodomain fragment bearing lumenal but not cytosolic epitopes was released into the media, indicating cleavage by a "membrane protein-solubilizing proteinase" or "secretase" activity. Yeast cells contained a C-terminal APP fragment that co-migrated with authentic C-terminal fragment derived from alpha-secretase cleavage of full-length APP in human cells. The N-terminal sequence of immunoaffinity purified C-terminal APP fragment from yeast was identical to that reported in mammalian and insect cells. These results demonstrate the existence of a secretase activity in yeast. Furthermore, this yeast secretase activity may be related to an APP processing activity present in metazoan cells.

Alzheimer Disease↗