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PubMed1995

A new human gene located in the PKD1 region of chromosome 16 is a functional homologue to ERV1 of yeast.

A new human gene has been identified on chromosome 16 in the interval containing the locus for polycystic kidney disease (PKD1) by analysis of a genomic cosmid clone and cDNAs. The gene contains at least one intron and is actively transcribed in tissues from kidney and brain. The putative gene product is predicted to be homologous to the yeast scERV1 protein by virtue of the high degree of identity (42%) over the entire length of the polypeptides. In former studies the yeast scERV1 gene was found to be essential for oxidative phosphorylation, the maintenance of mitochondrial genomes, and the cell-division cycle. In this study a yeast expression vector with a chimeric reading frame coding for the first 21 amino acids of the yeast protein and the terminal 100 amino acid residues of the human factor was transformed into yeast mutants with two different defects for scERV1. The chimeric human gene product was able to complement the yeast mutants and restored near normal viability. This identifies the human gene as a structural and functional homologue of the scERV1 gene.

Amino Acid Sequence↗
PubMed2001

Expression of protein tyrosine phosphatase-like molecule ICA512/IA-2 induces growth arrest in yeast cells and transfected mammalian cell lines.

The ICA512/IA-2 molecule, a protein with similarity to receptor-type protein tyrosine phosphatases, was discovered during studies to identify autoantigens in Type 1 diabetes. The biological function of ICA512/IA-2 is unknown. We describe striking effects of ICA512/IA-2 on viability and growth of both yeast cells and cultured mammalian cells. In transformed yeast Saccharomyces cerevisiae cells, expression of ICA512/IA-2 induced growth retardation as judged by measurements of optical density and counts of colony-forming units. In contrast, expression of the intracellular domain (amino acids 600-979) of ICA512/IA-2 in yeast or mammalian cells had no such effects. In investigations on apoptosis, expression of ICA512/IA-2 in yeast cells caused loss of plasma membrane asymmetry, but not release of cytochrome c from mitochondria which did occur in a control system after expression of the pro-apoptotic molecule Bax. Possible interactions between ICA512/IA-2 and components of the cytoskeleton were not supported by studies on staining of fixed yeast cells with phalloidin-Texas Red. With transfected mammalian cell lines COS-7 and NIH3T3, expression of ICA512/IA-2 likewise induced growth arrest, with some of the morphological features of apoptosis. Thus obligatory expression of ICA512/IA-2 in eukaryotic cells causes disruption of cellular activities, with growth arrest in yeast and nuclear pycnosis/fragmentation in mammalian cells. A possible explanation is that growth inhibition reflects a part of the presently unknown function of ICA512/IA-2.

3T3 Cells↗
PubMed1996

Mechanism, specificity and general properties of the yeast enzyme catalysing the formation of inosine 34 in the anticodon of transfer RNA.

In yeast, inosine is found at the first position of the anticodon (position 34) of seven different isoacceptor tRNA species, while in Escherichia coli it is present only in tRNAArg. The corresponding tRNA genes all have adenosine at position 34. Using as substrates in vitro T7-runoff transcripts of 31 plasmids carrying each natural of synthetic tRNA gene harbouring an anticodon with adenosine 34, we have characterised a yeast enzyme that catalyses the conversion of adenosine 34 to inosine 34. The homologous E. coli enzyme modifies adenosine 34 only in tRNAs with an arginine anticodon ACG. The base conversion occurs by a hydrolytic deamination-type reaction. This was determined by reversed phase high-pressure liquid chromatography/electrospray mass spectrometry analysis of the reaction product after in vitro modification in [18O]water. This newly characterised tRNA:adenosine 34 deaminase was partially purified from yeast. It has a molecular mass of approximately 75 kDa, and it does not require any cofactor, except magnesium ions, to deaminate adenosine 34 efficiently in tRNA. The observed dependence of the enzymatic reaction on magnesium ions probably reflects the need for a correct tRNA architecture. Enzymatic recognition of tRNA does not depend on the presence of any "identify" nucleoside other than adenosine 34. Likewise, the presence of pseudouridine 32 or 1-methyl-guanosine 37 in the anticodon loop does not interfere with inosine 34 biosynthesis. However, the efficacy of adenosine 34 to inosine 34 conversion depends on the nucleotide sequence of the anticodon loop and its proximal stem, the best tRNA substrates being those with a purine at position 35. Mutations that affect the size of the anticodon loop or one of several three-dimensional base-pairs abolish the capacity of the tRNA to be substrate for the yeast tRNA:adenosine 34 deaminase. Evidently, the activity of yeast tRNA:adenosine 34 deaminase depends more on the global structural feature (conformational stability/flexibility) of the L-shaped tRNA substrates than on the identity of any particular nucleotide other than adenosine 34. An apparent K(m) of 2.3 nM for its natural substrate tRNASer (anticodon AGA) was measured. Altogether, these results suggest that a single enzyme can account for the presence of inosine 34 in all seven cytoplasmic A34-containing precursor tRNAs in yeast.

Adenosine↗
PubMed1999

Yeast polypeptide fusion surface display levels predict thermal stability and soluble secretion efficiency.

Efficiency of yeast cell surface display can serve as a proxy screening variable for enhanced thermal stability and soluble secretion efficiency of mutant proteins. Several single-chain T cell receptor (scTCR) single-site mutants that enable yeast surface display, along with their double and triple mutant combinations, were analyzed for soluble secretion from the yeast Saccharomyces cerevisiae. While secretion of the wild-type scTCR was not detected, each of the single, double, and triple mutants were produced in yeast supernatants, with increased expression resulting from the double and triple mutants. Soluble secretion levels were strongly correlated with the quantity of active scTCR displayed as a fusion to Aga2p on the surface of yeast. Thermal stability of the scTCR mutants correlated directly with the secreted and surface levels of scTCR, with evidence suggesting that intracellular proteolysis by the endoplasmic reticulum quality control apparatus dictates display efficiency. Thus, yeast display is a directed evolution scaffold that can be used for the identification of mutant eucaryotic proteins with significantly enhanced stability and secretion properties.

Culture Media, Conditioned↗
PubMed1998

Prothymosin alpha is not found in yeast.

According to published accounts, prothymosin alpha exhibits high evolutionary conservation from yeast to man (Makarova, T., Grebenshikov, N., Egorov, C., Vartapetian, A., and Bogdanov, A. FEBS Lett. 257, 247-250, 1989). We report here our failure to find evidence for prothymosin alpha in yeast using three biochemical approaches: hybridization of yeast mRNA and genomic DNA with human prothymosin alpha coding region probes, performance of the polymerase chain reaction with yeast genomic template DNA and three sets of primers recognizing human prothymosin alpha coding region sequences, and isolation of yeast proteins essentially as described in the publication above. A survey of the Saccharomyces cerevisiae complete genome database using the program BLASTp verified our findings: there is no prothymosin alpha-homologue in yeast. Furthermore, DNA representing organisms from bacteria to amphibians also failed to hybridize with the same probes. Therefore, the presence of a prothymosin alpha gene in animals other than mammals is highly unlikely.

Amino Acid Sequence↗
PubMed1997

Cell cycle inhibitory effects of HIV and SIV Vpr and Vpx in the yeast Schizosaccharomyces pombe.

The Vpr gene of human immunodeficiency virus type 1 and type 2 (HIV-1, HIV-2) and simian immunodeficiency virus (SIV) encodes a small nuclear protein which is virion-associated and assists nuclear transport of the preintegration complex. Expression of HIV-1 Vpr has been shown to induce differentiation and prevent proliferation of human cells. HIV-1 Vpr has also been shown to arrest cell growth and cause morphological defects in yeast. In contrast, the Vpx gene of HIV-2 and SIV, which shares sequence homology with Vpr, does not seem to inhibit proliferation of human cells. It has been suggested that the cell cycle arrest effect of Vpr and Vpx is species and cell-type dependent. In this study, we have taken advantage of a conditional expression system to characterize the growth inhibitory effects of Vpr and Vpx of HIV-1, HIV-2, and SIV in the fission yeast Schizosaccharomyces pombe. Our results show that both Vpr and/or Vpx of HIV-1, HIV-2, and SIV arrest cell growth in S. pombe, and HIV-1 Vpr is more cytotoxic than HIV-2 or SIV Vpr or Vpx. Flow cytometry analysis indicated that yeast cells cease proliferating with DNA contents indicative of arrest in G1 and G2, with some cells showing signs of overreplication of DNA. While the observed cell cycle arrest phenotype was not identical to that observed in mammalian cells, there were similarities of growth arrest phenotype caused by Vpr and Vpx in yeast and mammalian cells. Specifically, the observation that yeast and mammalians cell both arrest in G2 with reduced p34/cdc2 kinase activity indicates that Vpr and Vpx interact with conserved target(s) in yeast and mammalian cells. The ability to use genetic analysis to elucidate the mechanisms involved makes S. pombe an excellent model system in which to study the effects of Vpr and Vpx on cellular function.

Animals↗
PubMed2004

The role of phosphatases in TOR signaling in yeast.

The TOR pathway controls cellular functions necessary for cell growth and proliferation of yeast and larger eukaryotes. The search for members of the TOR signaling cascade in yeast led to the discovery of type 2A protein phosphatases (PP2A) as important players within the pathway. We describe the roles in yeast of PP2A and the closely related phosphatase, Sit4, and then focus on complexes formed between the catalytic subunit of these phosphatases and Tap42, a direct target of the Tor protein kinases in yeast. Recent results suggest that Tap42 mediates many of the Tor functions in yeast, especially those involved in transcriptional modulation. However, whether Tap42 executes its function by inhibiting phosphatase activity or by activating phosphatases is still uncertain. In addition, Tor affects some transcriptional and physiological processes through Tap42 independent pathways. Thus, Tor proteins use multiple mechanisms to regulate transcriptional and physiological processes in yeast.

Adaptor Proteins, Signal Transducing↗
PubMed1985

Mannose inhibition of Escherichia coli adherence to urinary bladder epithelium: comparison with yeast agglutination.

The adherence of piliated strains of Escherichia coli (E. coli) to mammalian epithelial cells has been reported by several investigators to be specifically inhibited by D(+)-mannose or its derivatives. Much of this work utilized mannose type compounds to inhibit agglutination of mannan containing yeast cells by E. coli to demonstrate mannose sensitivity. This report investigates the ability of the neotype strain of E. coli (which is sensitive to mannose inhibition of yeast cell agglutination) to bind and metabolize radiolabeled D(+)-mannose. In addition the relative efficacy of D(+)-mannose and heparin to inhibit the adherence of E. coli to rabbit bladder mucosa was compared. Results showed that although D(+)-mannose did block E. coli--yeast cell agglutination in a reversible manner, radiolabeled D(+)-mannose binding by E. coli could not be displaced by 1,000 fold excess unlabeled D(+)-mannose. This suggests uptake of the sugar as opposed to a surface binding phenomenon which was confirmed by the demonstration of significant metabolism of mannose by E. coli. The same concentration of D(+)-mannose which prevented E. coli--yeast cell agglutination was not particularly effective in preventing E. coli adherence to the acid denuded rabbit bladder. Heparin treatment of the acid denuded bladder was very effective in preventing E. coli adherence but was ineffective in preventing E. coli--yeast cell agglutination. This indicates that E. coli--yeast cell agglutination should not be correlated with E. coli adherence to mammalian epithelial tissue.

Agglutination↗
PubMed1993

Genetic analysis of human p34CDC2 function in fission yeast.

The p34cdc2 protein kinase plays a key role in the control of the mitotic cell cycle of fission yeast, being required for both entry into S-phase and for entry into mitosis in the mitotic cell cycle, as well as for the initiation of the second meiotic nuclear division. In recent years, structural and functional homologues of p34cdc2, as well as several of the proteins that interact with and regulate p34cdc2 function in fission yeast, have been identified in a wide range of higher eukaryotic cell types, suggesting that the control mechanisms uncovered in this simple eukaryote are likely to be well conserved across evolution. Here we describe the construction and characterisation of a fission yeast strain in which the endogenous p34cdc2 protein is entirely absent and is replaced by its human functional homologue p34CDC2. We have used this strain to analyse aspects of the function of the human p34CDC2 protein genetically. We show that the function of the human p34CDC2 protein in fission yeast cells is dependent upon the action of the protein tyrosine phosphatase p80cdc25, that it responds to altered levels of both the mitotic inhibitor p107wee1 and the p34cdc2-binding protein p13suc1, and is lethal in combination with the mutant B-type cyclin p56cdc13-117. In addition, we demonstrate that the human p34CDC2 protein is proficient for fission yeast meiosis, and examine the behaviour of two mutant p34CDC2 proteins in fission yeast.

Alleles↗
PubMed1995

Cloning and expression of a Xenopus gene that prevents mitotic catastrophe in fission yeast.

In fission yeast the Wee1 kinase and the functionally redundant Mik1 kinase provide a regulatory mechanism to ensure that mitosis is initiated only after the completion of DNA synthesis. Yeast in which both Wee1 and Mik1 kinases are defective exhibit a mitotic catastrophe phenotype, presumably due to premature entry into mitosis. Because of the functional conservation of cell cycle control elements, the expression of a vertebrate wee1 or mik1 homolog would be expected to rescue such lethal mutations in yeast. A Xenopus total ovary cDNA library was constructed in a fission yeast expression vector and used to transform a yeast temperature-dependent mitotic catastrophe mutant defective in both wee1 and mik1. Here we report the identification of a Xenopus cDNA clone that can rescue several different yeast mitotic catastrophe mutants defective in Wee1 kinase function. The expression of this clone in a wee1/mik1-deficient mutant causes an elongated cell phenotype under non-permissive growth conditions. The 2.0 kb cDNA clone contains an open reading frame of 1263 nucleotides, encoding a predicted 47 kDa protein. Bacterially expressed recombinant protein was used to raise a polyclonal antibody, which specifically recognizes a 47 kDa protein from Xenopus oocyte nuclei, suggesting the gene encodes a nuclear protein in Xenopus. The ability of this cDNA to complement mitotic catastrophe mutations is independent of Wee1 kinase activity.

Amino Acid Sequence↗
PubMedDate not supplied

MRG1-1, a dominant allele that confers methomyl resistance in yeast expressing the cytoplasmic male sterility T-urf13 gene from maize.

We have previously described a eukaryotic heterologous expression system, with the urf13TW gene in yeast, which mimics the disease susceptibility associated with the Texas cytoplasmic male sterility in maize. This yeast model was used to isolate yeast nuclear mutants conferring methomyl resistance. The genetic strategy we have developed focused on screening for nuclear dominant yeast mutations which restore methomyl resistance. MRG1-1, a yeast nuclear dominant allele, was identified as a methomyl-resistance restorer. We have shown that methomyl resistance co-segregated with a pleiotropic phenotype in the heterozygous MRG1-1/MRG1 diploids, detectable even in the absence of the maize-derived mitochondrial protein and/or methomyl. We observed an increase in oxygen uptake, a significant decrease of the levels of cytochrome aa3, and a decrease in the growth yield. This phenotype is influenced by the carbon source and the results suggest a defect in the adaptation to the respiratory pathway in MRG1-1 yeast cells.

Alleles↗
PubMed1995

The complete mitochondrial DNA sequence of Hansenula wingei reveals new characteristics of yeast mitochondria.

The complete 27,694-bp mitochondrial (mt) DNA sequence of Hansenula wingei, which is a typical budding yeast and contains circular mitochondrial DNA, has been determined. The mt sequence contains genes encoding large and small ribosomal RNAs, 25 tRNAs, three subunits of cytochrome c oxidase (subunits 1, 2 and 3), three subunits of ATPase (subunits 6, 8 and 9), apocytochrome b, seven subunits of NADH dehydrogenase (subunits 1, 2, 3, 4, 4L, 5 and 6), and a ribosomal protein, VAR1. The VAR1 gene is considered to be a typical yeast type. This is consistent with data on DNA and the deduced amino-acid sequence homology comparisons of genes ubiquitous in yeast and fungi. However, we have identified seven genes encoding NADH dehydrogenase subunits, which are not found in other yeast mitochondrial genomes, thus placing the H. wingei mitochondrial genome in a unique position. In addition the H. wingei mitochondrial genome also encodes one tRNA pseudogene and one short unidentified ORF. The genome is compact with only two introns both of which contain an ORF. One intron lies in the large rRNA gene while the other is situated in the cytochrome c oxidase subunit-1 gene. The conserved nonanucleotide motif (A/T)TATAAG (T/A)(A/T), which is a transcription start signal in Saccharomyces cerevisiae mitochondria, has also been found in the H. wingei mitochondrial genome. The codon assignments for ATA and CTN in H. wingei mitochondria are different from those in S. cerevisiae mitochondria. These results indicate a unique and novel structure for the H. wingei mitochondrial genome in terms of characteristics which are typical for both yeast and for filamentous fungi. This is the first complete mt DNA sequence report in yeast.

Amino Acid Sequence↗
PubMed1990

Evolutionary conservation of transcriptional machinery between yeast and plants as shown by the efficient expression from the CaMV 35S promoter and 35S terminator.

Complementation of fission yeast mutants by plant genomic libraries could be a promising method for the isolation of novel plant genes. One important prerequisite is the functioning of plant promoters and terminators in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Therefore, we studied the expression of the bacterial beta-glucuronidase (GUS) reporter gene under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and 35S terminator. We show here that S. pombe initiates transcription at exactly the same start site as was reported for tobacco. The 35S CaMV terminator is appropriately recognized leading to a polyadenylated mRNA of the same size as obtained in plant cells transformed with the same construct. Furthermore, the GUS-mRNA is translated into fully functional GUS protein, as determined by an enzymatic assay. Interestingly, expression of the 35S promoter in the budding yeast S. cerevisiae was found to be only moderate and about hundredfold lower than in S. pombe. To investigate whether different transcript stabilities are responsible for this enormous expression difference in the two yeasts, the 35S promoter was substituted by the ADH (alcohol dehydrogenase) promoter from fission yeast. In contrast to the differential expression pattern of the 35S promoter, the ADH promoter resulted in equally high expression rates in both fission and budding yeast, comparable to the 35S promoter in S. pombe. Since the copy number of the 35S-GUS constructs differs only by a factor of two in the two yeasts, it appears that differential recognition of the 35S promoter is responsible for the different transcription rates.

Alcohol Dehydrogenase↗
PubMed1986

The yeast 2 micron plasmid: strategies for the survival of a selfish DNA.

The designation of the yeast 2 mu circle as a "selfish" DNA molecule has been confirmed by demonstrating that the plasmid is lost with exponential kinetics from haploid yeast populations grown in continuous culture. We show that plasmid-free yeast cells have a growth rate advantage of some 1.5%-3% over their plasmid-containing counterparts. This finding makes the ubiquity of this selfish DNA in yeast strains puzzling. Two other factors probably account for its survival. First, the rate of plasmid loss was reduced by allowing haploid populations to enter stationary phase periodically. Second, it was not possible to isolate a plasmid-free segregant from a diploid yeast strain. Competition experiments demonstrated that stability in a diploid is conferred at the level of segregation and that plasmid-free diploid cells are at a selective advantage compared with their plasmid-containing counterparts. Yeast cells in nature are usually homothallic and must frequently pass through both diploid and stationary phases. The 2 mu plasmid appears to have evolved a survival strategy which exploits these two features of its host's life cycle.

DNA, Fungal↗
PubMed1984

Cloning and restriction analysis of the hexokinase PII gene of the yeast Saccharomyces cerevisiae.

Carbon catabolite repression in yeast depends on catalytic active hexokinase isoenzyme PII ( Entian 1980a ). A yeast strain lacking hexokinase isoenzymes PI and PII was transformed, using a recombinant pool with inserts of yeast nuclear DNA up to 10 kbp in length. One hundred transformants for hexokinase were obtained. All selected plasmids coded for hexokinase isoenzyme PII, none for hexokinase isoenzyme PI, and carbon catabolite repression was restored in the transformants. Thirty-five independently isolated stable plasmids were investigated further. Analysis with the restriction enzyme EcoRI showed that these plasmids fell into two classes with different restriction behaviour. One representative of each class was amplified in Escherichia coli and transferred back into the yeast hexokinase-deficient strain with concomitant complementation of the nuclear mutation. The two types of insert were analysed in detail with 16 restriction enzymes, having 0-3 cleavage sites on transformant vector YRp7 . The plasmids differed from each other by the orientation of the yeast insert in the vector. After yeast transformation with fragments of one plasmid the hexokinase PII gene was localised within a region of 1.65 kbp.

Cloning, Molecular↗
PubMed1979

Evidence that the ribosomal DNA genes of yeast are not on chromosome I.

Several workers have reported that most of the ribosomal DNA genes (rDNA) of the yeast Saccharomyces cerevisiae are located on chromosome I. More recently, data indicating that the yeast rDNA genes are located on chromosome XII has been presented. In this report, we present additional evidence indicating that most of the yeast rDNA genes are not on chromosome I. Starting from a diploid yeast strain, we isolated ten strains which were monosomic (2n-1) for chromosome I. We found that each of these ten strains contained two copies of the rDNA-containing chromosome. In addition, we show that the earlier evidence indicating that the yeast rDNA genes were on chromosome I cannot be explained by a difference in the yeast strains which were used in the different experiments.

Aneuploidy↗
PubMed1985

Formation and stability of linear plasmids in a recombination deficient strain of yeast.

Natural termini from macronuclear DNA of the ciliated protozoans Tetrahymena thermophila and Oxytricha fallax can support telomere formation yeast. However, plasmids carrying these ciliate termini are modified by the addition of DNA which hybridizes to the synthetic oligonucleotide poly [d(C-A]), a sequence which also hybridizes to terminal restriction fragments from yeast chromosomes but not to Tetrahymena or Oxytricha macronuclear DNAs. Thus, in yeast, the creation of new telomeres on ciliate termini involves the acquisition of yeast-specific terminal sequences presumably by either recombination or non-templated DNA synthesis. The RAD52 gene is required for the majority of yeast mitotic and meiotic recombination events. Moreover, the absence of an active RAD52 gene product results in high rates of chromosome loss. Here we demonstrate that terminal restriction fragments from Tetrahymena macronuclear ribosomal DNA (rDNA) support the formation of modified telomeres in a yeast strain carrying a defect in the RAD52 gene. Moreover, linear plasmids bearing these modified ciliate termini are stably propagated in rad52- cells.

Animals↗
PubMed1982

Cysteine-independent and cysteine-requiring yeast-strains of Histoplasma capsulatum.

Recently we described a strain of Histoplasma capsulatum, designated H-35, which is able to grow as yeast on a minimal medium consisting of inorganic salts, glucose and a trace of biotin. Using this strain as a prototrophic wild type we sought auxotrophic mutants. Mutagenized yeast-cells were starved for inorganic sulfate in sulfur-free minimal medium. Sulfate was then added, and growing prototrophic cells were killed by addition of amphotericin B. After 24 hours non-growing auxotrophs were 'rescued' by removal of amphotericin and addition of yeast extract. This 'mutant enrichment' cycle was repeated two additional times, after which the cells were plated on blood agar and 800 yeast-colonies were picked. Seventeen of these yeast-strains required cysteine for growth, as compared with strain H-35, which grew as yeast on minimal medium.

Amphotericin B↗