S. cerevisiae

J Biol Chem, 1993 Apr 25, 268(12), 8665 - 8
Saccharomyces cerevisiae elongation factor 2 . Mutagenesis of the histidine precursor of diphthamide yields a functional protein that is resistant to diphtheria toxin; Phan LD et al.; Protein synthesis elongation factor 2 (EF-2) is the target of the ADP-ribosylating activity of diphtheria toxin which is responsible for cell killing . Diphthamide, an unique post-translationally modified histidine residue, is both required for and the site of this ADP-ribosylation . Although present in the EF-2 of all eukaryotes and archaebacteria, the function of diphthamide is unknown . Here we describe the site-specific mutagenesis of the histidine precursor of diphthamide, histidine 699, in yeast EF-2 . Plasmid-borne EFT was randomly mutagenized at the histidine 699 codon, and the technique of plasmid shuffling was utilized to select strains that were maintained by the mutant EFT . These mutants were screened for diphtheria toxin resistance . Sequence analysis of the EFT in 49 toxin-resistant isolates showed that histidine 699 had been replaced by 1 of 4 amino acids: asparagine, glutamine, leucine, or methionine . All 11 of the possible codons corresponding to these 4 amino acids were found . The growth rates of cells sustained by the mutant forms of EF-2 were slightly slower than those of isogenic wild-type cells . We conclude that despite its strict conservation and universal post-translational modification, the histidine precursor of diphthamide is not essential to the function of yeast EF-2 in protein synthesis.

Biochem Biophys Res Commun, 1993 Apr 15, 192(1), 143 - 50
Biochemical characterisation of the isolated Anc2 adenine nucleotide carrier from Saccharomyces cerevisiae mitochondria; Brandolin G et al.; The yeast mitochondrial adenine nucleotide carrier isoform encoded by the ANC2 gene has been specifically expressed in a yeast strain disrupted for the two other genes, ANC1 and ANC3 . Isolation of the carrier in a functional form was achieved by utilisation of a mixture of two detergents, dodecylmaltoside and Emulphogen . The intrinsic fluorescence of the Anc2 protein was specifically and rapidly enhanced upon addition of the transportable nucleotides ADP and ATP . Fluorescence enhancement was prevented or reversed by the addition of a stoichiometric amount of CATR . Addition of CATR alone elicited a dose-dependent decrease of fluorescence . The ANC2-specific yeast stain offers the means to study a single ADP/ATP carrier, with a well-defined amino acid sequence, suitable for analysis of substrate- or inhibitor-induced conformational changes.

J Biol Chem, 1993 Apr 15, 268(11), 8341 - 9
Phosphoglucomutase in Saccharomyces cerevisiae is a cytoplasmic glycoprotein and the acceptor for a Glc-phosphotransferase; Marchase RB et al.; UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins . The predominant acceptor in vertebrates and Paramecium tetraurelia is a cytoplasmic 62-kDa glycoprotein . To determine if the yeast Saccharomyces cerevisiae also possesses Glc-phosphotransferase activity, a crude cellular lysate was incubated with {beta-32P}UDP-Glc and analyzed . A phosphoglycoprotein having an apparent molecular mass of 62 kDa (pgp62) was found to be the predominant labeled macromolecule . Reconstitution experiments determined that both a soluble and membrane fraction were required for labeling, and suggested that the Glc-phosphotransferase is membrane-associated while pgp62 is cytoplasmic . The reaction is evolutionarily conserved to the extent that rat liver Glc-phosphotransferase was capable of recognizing the yeast acceptor and vice versa . The yeast 62-kDa acceptor was purified, and partial amino acid sequences showed a high level of identity with rabbit muscle phosphoglucomutase . Subsequently, both yeast and rabbit muscle phosphoglucomutase were found to be acceptors in the Glc-phosphotransferase reaction . The label was found on a tryptic peptide distinct from that containing the enzyme's active site serine . When phosphoglucomutase was overexpressed, an increase was seen in Glc-phosphotransferase acceptor activity and in specific metabolic labeling of the acceptor by glucose and mannose.

J Biol Chem, 1993 Apr 15, 268(11), 7728 - 32
Functional replacement of the Saccharomyces cerevisiae Trg1/Pdi1 protein by members of the mammalian protein disulfide isomerase family; Gunther R et al.; The TRG1/PDI1 gene of Saccharomyces cerevisiae is essential for growth and encodes a lumenal endoplasmic reticulum (ER) glycoprotein that is structurally related to thioredoxin and is involved in the secretory pathway . We have tested whether the yeast Trg1/Pdi1 protein can be replaced in vivo by three members of the mammalian thioredoxin-related protein family, protein disulfide isomerase (PDI), ERp72, and ERp61 . Multicopy plasmids containing galactose-inducible rodent PDI and ERp72 genes support germination and growth of haploid trg1 null mutants in galactose-containing media, whereas the ERp61 gene is inactive . Strains expressing PDI or ERp72 instead of Trg1 are thermosensitive . An overproduced mutant Trg1 protein lacking the HDEL retention signal supports growth, whereas a truncated version of the protein containing only one thioredoxin-like domain is inactive . The mammalian proteins were localized to both the soluble and microsomal membrane fraction of yeast cells . Our observations indicate that the two unglycosylated mammalian proteins PDI and ERp72 are capable of replacing at least some of the critical functions of Trg1, in spite of the fact that the three proteins diverge considerably in sequences surrounding the thioredoxin-related domains.

FEBS Lett, 1993 Apr 12, 320(3), 271 - 5
Two structural genes are encoding malate synthase isoenzymes in Saccharomyces cerevisiae; Fernandez E et al.; We report on the isolation of a gene encoding yeast malate synthase . A yeast genomic library was screened using a probe homologous to the yeast enzyme obtained by the polymerase chain reaction . The nucleotide sequence of the cloned gene was determined . Computer analysis showed that the isolated gene is identical to the one previously described as DAL7, which is involved in allantoin metabolism {Mol . Cel . Biol . 9 (1989) 3231-3243} . Enzymatic activities of multicopy transformants, Southern analysis and disruption mutants predict the existence of two genes encoding malate synthases that are differentially regulated at the transcriptional level.

Biochemistry, 1993 Apr 6, 32(13), 3261 - 70
Similarities and differences between yeast and vertebrate calmodulin: an examination of the calcium-binding and structural properties of calmodulin from the yeast Saccharomyces cerevisiae; Starovasnik MA et al.; The Ca(2+)-binding and structural properties of calmodulin (CaM) from the yeast Saccharomyces cerevisiae (yCaM) were analyzed by flow dialysis and NMR spectroscopy . Full-length yCaM and two truncated versions of yCaM were expressed in Escherichia coli and purified . yTR1 (residues 1-76) and yTR2 (residues 75-147) are similar to the vertebrate CaM fragments TR1 and TR2, which are generated by limited proteolysis with trypsin . As was found for the fragments of vertebrate CaM, the yCaM fragments retain native conformation and are useful for examining structure and metal-binding properties by NMR . Evidence for a short beta-sheet in each domain, as well as characteristic NOEs to aromatic residues, suggests that yCaM folds similarly to vertebrate CaM . Furthermore, although the previously considered "invariant" glycine at position 6 is replaced by a histidine in site II of yCaM, the far downfield chemical shift of His-61's amide proton suggests that this site adopts a conformation similar to that found in other EF-hand sites . Macroscopic Ca(2+)-binding constants were determined for yCaM by flow dialysis, revealing three high-affinity sites (dissociation constants were 5.2, 3.3, and 2.3 microM in the presence of 1 mM MgCl2 and 100 mM KCl) . Positive cooperativity was observed among all sites . Ca2+ binding was also monitored indirectly by one-dimensional NMR . Titrations of the fragment molecules reveal that two binding sites reside in the N-terminal domain (sites I and II) and one in the C-terminal domain (site III) . All three sites exhibit slow-exchange behavior in the intact protein, but site III exhibits fast-exchange behavior in the isolated C-terminal domain fragment (yTR2) . Thus, an interaction between the two domains of intact yCaM affects the behavior of site III . These results with yCaM differ from those of vertebrate CaM in terms of Ca(2+)-binding stoichiometry, affinity of sites I and II, relative affinity of sites in the N- and C-terminal domains, and the exchange behaviors observed.

J Biol Chem, 1993 Apr 5, 268(10), 7442 - 8
A distal heat shock element promotes the rapid response to heat shock of the HSP26 gene in the yeast Saccharomyces cerevisiae; Chen J et al.; Induction of heat shock genes is mediated by heat shock factor (HSF) . Our recent genomic footprinting experiments demonstrate that HSF binds constitutively to perfect and imperfect heat shock elements (HSEs) in the HSP26 gene in yeast . Site-directed mutagenesis of the single perfect HSE, previously reported to not be involved in regulating gene expression, significantly reduces the rate of response of the gene to heat shock . However, the same mutation only slightly reduced the rate of accumulation of HSP26 mRNA during heat shock . Genomic footprinting experiments indicate that this lag in response to heat shock is due to the failure of HSF to bind efficiently to the mutated HSE . The rate of response to heat shock of synthetic promoters containing one, two, three, or seven perfect HSEs was similar to that observed for the wild-type HSP26 gene . These results suggest that the rate of response to heat shock is correlated with HSF occupancy of HSEs, rather than the number of HSEs in a promoter . As with the wild-type and mutant HSP26 genes, the rate of accumulation of mRNA from synthetic promoters increased only moderately with an increase in the number of HSEs . These results suggest that as few as two HSE-HSF complexes are sufficient to saturate HSF's target in the basal transcription apparatus.

FEBS Lett, 1993 Apr 5, 320(2), 125 - 9
Precise mapping and molecular characterization of the MFT1 gene involved in import of a fusion protein into mitochondria in Saccharomyces cerevisiae; Ito M et al.; Garrett et al . {Mol . Gen . Genet . 225 (1991) 483-491} recently reported that an Atp2-lacZ fusion protein was transported into mitochondria in yeast, thus identifying the MFT1 (mitochondrial fusion targeting) gene as a genomic fragment which complements a mutation (mft1) that failed in targeting a fusion protein into mitochondria . They mapped this gene to the ORF, which we have independently identified as a gene homologous to the cyc07 gene, which is expressed specifically in the S phase during the plant cell cycle . We have mapped the MFT1 gene precisely and found that this gene should correspond to the neighboring ORF, rather than the ORF they identified.

J Biol Chem, 1993 Apr 5, 268(10), 7350 - 7
Isolation and characterization of an NTP-dependent 3'-exoribonuclease from mitochondria of Saccharomyces cerevisiae; Min J et al.; RNA turnover in eukaryotes is thought to require 3'-exonuclease activity but so far no RNase with that specificity has been isolated from a eukaryote . We report here on the purification and characterization of a 3'-exoribonuclease isolated from the mitochondria of Saccharomyces cerevisiae . In vitro the purified enzyme displayed an absolute requirement of NTPs for activity . Each of the eight standard ribo- and deoxyribonucleotides supported activity with Km values ranging from 20 to 90 microM . The enzyme also displayed RNA-stimulated NTPase activity . The NTP-dependent enzyme cofractionated with three polypeptides of molecular masses 75,000, 90,000, and 110,000 daltons, although the native enzyme appears to have a molecular mass of 160,000 daltons predicted from the Stokes radius . The possible functions of this enzyme in vivo in the regulated decay of mitochondrial RNAs are discussed.

Yeast, 1993 Apr, 9(4), 423 - 7
Sequence of the open reading frame of the FLO1 gene from Saccharomyces cerevisiae; Teunissen AW et al.; The cloned part of the flocculation gene FLO1 of Saccharomyces cerevisiae (Teunissen, A.W.R.H., van den Berg, J.A . and Steensma, H.Y . (1993) . Physical localization of the flocculation gene FLO1 on chromosome I of Saccharomyces cerevisiae, Yeast, in press) has been sequenced . The sequence contains a large open reading frame of 2685 bp . The amino acid sequence of the putative protein reveals a serine- and threonine-rich C-terminus (46%), the presence of repeated sequences and a possible secretion signal at the N-terminus . Although the sequence is not complete (we assume the missing fragment consists of repeat units), these data strongly suggest that the protein is located in the cell wall, and thus may be directly involved in the flocculation process.

Yeast, 1993 Apr, 9(4), 339 - 49
Control of Saccharomyces cerevisiae carboxypeptidase S (CPS1) gene expression under nutrient limitation; Bordallo J et al.; Expression of the vacuolar carboxypeptidase S (CPS1) gene in Saccharomyces cerevisiae is regulated by the availability of nutrients . Enzyme production is sensitive to nitrogen catabolite repression; i.e . the presence of ammonium ions maintains expression of the gene at a low level . Transfer of ammonium-glucose pre-grown cells to a medium deprived of nitrogen causes a drastic increase in CPS1 RNA level provided that a readily usable carbon source, such as glucose or fructose, is available to the cells . Derepression of the gene by nitrogen limitation is cycloheximide-insensitive . Neither glycerol, ethanol, acetate nor galactose support derepression of CPS1 expression under nitrogen starvation conditions . Non-metabolizable sugar analogs (2-deoxyglucose, 6-methyl-glucose or glucosamine) do not allow derepression of CPS1, showing that the process is energy-dependent . Production of carboxypeptidase yscS also increases several-fold when ammonium-pregrown cells are transferred to media containing glucose and a non-readily metabolizable nitrogen source such as proline, leucine, valine or leucyl-glycine . Analysis of CPS1 expression in RAS2+ (high cAMP) and ras2 mutant (low cAMP) strains and in cells grown at low temperature (23 degrees C) and in heat-shocked cells (38 degrees C) shows that steady-state levels of CPS1 mRNA are not controlled by a low cAMP level-signalling pathway.

Mol Microbiol, 1993 Apr, 8(1), 167 - 78
AUA1, a gene involved in ammonia regulation of amino acid transport in Saccharomyces cerevisiae; Sophianopoulou V et al.; In Saccharomyces cerevisiae the general amino acid (GAP1) permease catalyses active transport of apparently all amino acids across the plasma membrane . GAP1 activity is regulated by control of synthesis and control of activity in response to the nitrogen source supplied; ammonia and glutamine inactivate GAP1 function while proline and urea allow its maximum expression . We have isolated and characterized a gene, AUA1, involved in ammonia regulation of GAP1 activity . AUA1 is not essential for growth but overexpression of the AUA1 transcript in a high-copy vector or due to a regulatory mutation, aua1-1, present approximately 10 bp upstream from the start of AUA1 transcription, releases GAP1 activity from ammonia-inactivation without affecting GAP1 transcription . The aua1-1 mutation has no phenotype when ammonia is replaced by proline or glutamate as the nitrogen source or when it is present in a gap1 background . AUA1 expression is itself ammonia repressible in a wild-type strain but not in the aua1-1 mutant . The AUA1 gene sequence contains a unique short open reading frame of 94 codons corresponding to a polypeptide of 11,714 Da . This polypeptide is highly hydrophilic and extremely basic . The AUA1 product shows no significant similarity with any previously known protein sequence . Interestingly, a 10-amino acid segment of AUA1 is directly repeated in the most basic segment of the protein . Possible roles of AUA1 are discussed.

Mol Gen Genet, 1993 Apr, 238(3), 444 - 54
Promoter analysis of the PHO81 gene encoding a 134 kDa protein bearing ankyrin repeats in the phosphatase regulon of Saccharomyces cerevisiae; Ogawa N et al.; The PHO81 gene encoding one of the regulators of the phosphatase regulon in Saccharomyces cerevisiae was mapped 9.8 centimorgans distal from the ser2 locus on the right arm of chromosome VII . Determination of the nucleotide sequence of cloned PHO81 DNA revealed a 3537 bp open reading frame encoding a 134 kDa protein . This protein has six repeats of a 33-amino acid sequence homologous to the ankyrin repeat and an asparagine-rich region . Transcription of PHO81 is activated by Pho4 protein in cooperation with Pho2 (i.e., Bas2/Grf10) protein under the influence of the inorganic phosphate (Pi) concentration in the medium, through the PHO regulatory system . Major transcription initiation sites of PHO81, determined by primer extension analysis, are at nucleotide positions -66 and -65 relative to the ATG codon . Deletion analysis showed that a 95 bp region from nucleotide position -385 to -291 is essential for response to the Pi signals . Purified Pho4 protein protected a 19 bp region (positions -350 to -332) in the 95 bp fragment from DNase I digestion in vitro and the protected region includes the core sequence 5'-CACGTG-3', which is also observed in other genes of phosphate metabolism.

Mol Gen Genet, 1993 Apr, 238(3), 390 - 400
Expression of the Saccharomyces cerevisiae RAD50 gene during meiosis: steady-state transcript levels rise and fall while steady-state protein levels remain constant; Raymond WE et al.; In Saccharomyces cerevisiae, the RAD50 gene is required for repair of X-ray and MMS-induced DNA damage during vegetative growth, and for synaptonemal complex formation and genetic recombination during meiosis . We show below that the RAD50 gene encodes major and minor transcripts of 4.2 and 4.6 kb in length which differ primarily at their 5' ends . Steady-state levels of both RAD50 transcripts increase coordinately during meiosis, reaching maximal levels midway through meiotic prophase, about 3 or 4 h after transfer of cells to sporulation medium . The 5' ends of the major RAD50 transcript in both meiotic and vegetative cells map to the same cluster of sites approximately 20 bp upstream of the amino-terminal ATG of the RAD50 coding sequence . We conclude that the increased RAD50 transcript level observed during meiosis does not reflect utilization of a new promoter . In contrast, steady-state levels of Rad50 protein do not increase during meiosis . Thus, changes in RAD50 transcript levels are not necessarily accompanied by commensurate changes in Rad50 protein levels . Possible explanations are considered.

Eur J Biochem, 1993 Apr 1, 213(1), 547 - 53
Transcriptional control of AAC3 gene encoding mitochondrial ADP/ATP translocator in Saccharomyces cerevisiae by oxygen, heme and ROX1 factor; Sabova L et al.; The AAC3 gene of Saccharomyces cerevisiae encodes a mitochondrial ADP/ATP translocator which is subject to oxygen repression . Evidence is presented here, that the repression of AAC3 expression is dependent upon heme and the ROX1 factor . The promoter region of the AAC3 gene was isolated, sequenced, and deletion analysis was performed using lacZ as a reporter gene to determine the cis-acting regions responsible for the regulation of AAC3 expression . The results of the deletion analysis show that the negative control of the AAC3 gene by oxygen and ROX1 factor is mediated by an upstream repression sequence consisting of a T-rich segment adjacent to the consensus elements that are present in the 5' flanking regions of several other yeast genes . An additional upstream repressor site was located within the AAC3 promoter which, however, is not related either to oxygen or to ROX1 factor . The data presented here delineate the main cellular factors and DNA sequences involved in the regulatory mechanism by which an essential function for anaerobic cells growth, ADP/ATP translocation, is ensured . In addition, they show that the AAC3 gene belongs to the family of yeast genes including TIF51B, COX5b, HEM13 and CYC7 that are negatively regulated by oxygen and heme.

EMBO J, 1993 Apr, 12(4), 1459 - 66
The control in cis of the position and the amount of the ARG4 meiotic double-strand break of Saccharomyces cerevisiae; de Massy B et al.; During meiosis, a transient DNA double-strand break (DSB) occurs in the promoter region (positions -200/-185) of the Saccharomyces cerevisiae ARG4 gene and is a likely intermediate in the initiation of meiotic gene conversion events in this region . We report here a functional analysis of the ARG4 DSB based on the study of various deletions in this chromosomal region . We have identified several cis-acting elements located within the -465/+3 region of the ARG4 promoter that control the formation of this DSB . The -465/-317 region includes a transcription terminator and is necessary for a normal amount of ARG4 DSB, but not for its positioning . The -316/-140 region can be replaced by an unrelated DNA sequence where a meiotic DSB then occurs, suggesting that the site of DSB is not sequence-specific, but is positioned at a fixed distance from the adjacent -139/+3 region . Also, in all strains constructed, the amount of meiotic DSB is correlated with the frequency of gene conversion in ARG4, which provides a strong argument for the initiation of gene conversion by a DSB in this region of the yeast genome.

Genetics, 1993 Apr, 133(4), 851 - 63
Mutational analysis of pre-mRNA splicing in Saccharomyces cerevisiae using a sensitive new reporter gene, CUP1; Lesser CF et al.; We have developed a new reporter gene fusion to monitor mRNA splicing in yeast . An intron-containing fragment from the Saccharomyces cerevisiae ACT1 gene has been fused to CUP1, the yeast metallothionein homolog . CUP1 is a nonessential gene that allows cells to grow in the presence of copper in a dosage-dependent manner . By inserting previously characterized intron mutations into the fusion construct, we have established that the efficiency of splicing correlates with the level of copper resistance of these strains . A highly sensitive assay for 5' splice site usage was designed by engineering an ACT1-CUP1 construct with duplicated 5' splice sites; mutations were introduced into the upstream splice site in order to evaluate the roles of these highly conserved nucleotides in intron recognition . Almost all mutations in the intron portion of the 5' consensus sequence abolish recognition of the mutated site, while mutations in the exon portion of the consensus sequence have variable affects on cleavage at the mutated site . Interestingly, mutations at intron position 4 demonstrate that this nucleotide plays a role in 5' splice site recognition other than by base pairing with U1 snRNA . The use of CUP1 as a reporter gene may be generally applicable for monitoring cellular processes in yeast.

Genetics, 1993 Apr, 133(4), 837 - 49
A mutational analysis of killer toxin resistance in Saccharomyces cerevisiae identifies new genes involved in cell wall (1-->6)-beta-glucan synthesis; Brown JL et al.; Recessive mutations leading to killer resistance identify the KRE9, KRE10 and KRE11 genes . Mutations in both the KRE9 and KRE11 genes lead to reduced levels of (1-->6)-beta-glucan in the yeast cell wall . The KRE11 gene encodes a putative 63-kD cytoplasmic protein, and disruption of the KRE11 locus leads to a 50% reduced level of cell wall (1-->6)-glucan . Structural analysis of the (1-->6)-beta-glucan remaining in a kre11 mutant indicates a polymer smaller in size than wild type, but containing a similar proportion of (1-->6)- and (1-->3)-linkages . Genetic interactions among cells harboring mutations at the KRE11, KRE6 and KRE1 loci indicate lethality of kre11 kre6 double mutants and that kre11 is epistatic to kre1, with both gene products required to produce the mature glucan polymer at wild-type levels . Analysis of these KRE genes should extend knowledge of the beta-glucan biosynthetic pathway, and of cell wall synthesis in yeast.

Genetics, 1993 Apr, 133(4), 785 - 97
A conditional allele of the Saccharomyces cerevisiae HOP1 gene is suppressed by overexpression of two other meiosis-specific genes: RED1 and REC104; Hollingsworth NM et al.; The HOP1 gene of Saccharomyces cerevisiae is believed to encode a protein component of the synaptonemal complex, the structure formed when homologous chromosomes synapse during meiotic prophase . Five new mutant alleles (three conditional, two nonconditional) of HOP1 were identified by screening EMS-mutagenized cells for a failure to complement the spore viability defect of a hop1 null allele . Two high copy plasmids were found that partially suppress the temperature-sensitive spore inviability phenotype of one of these alleles, hop1-628 . The suppression is allele-specific; no effect of the plasmids is observed in hop1 null diploids . Mutation of either of the two suppressor genes results in recessive spore lethality, indicating that these genes play important roles during meiosis . The DNA sequence of one high copy suppressor gene matched that of RED1, a previously identified meiosis-specific gene . Our data strongly support the idea that RED1 protein is also a component of the synaptonemal complex and further suggest that the RED1 and HOP1 gene products may interact . The second suppressor maps to the right arm of chromosome VIII distal to CDC12 and is REC104, a meiosis-specific gene believed to act early in meiosis.

J Bacteriol, 1993 Apr, 175(7), 2102 - 6
Purification and characterization of the Saccharomyces cerevisiae BGL2 gene product, a cell wall endo-beta-1,3-glucanase; Mrsa V et al.; One of the major proteins of the Saccharomyces cerevisiae cell wall, a beta-glucanase (BGL2 gene product), has been isolated and purified to homogeneity under conditions for preserving enzyme activity . The study of enzyme properties of the protein revealed that it is an endo-beta-1,3-glucanase and not an exoglucanase as reported previously (F . Klebl and W . Tanner, J . Bacteriol . 171:6259-6264, 1989) . The examination of the glucanase structure showed that the lower apparent molecular mass of the protein (29 kDa) compared with what was calculated from the amino acid sequence of the enzyme (33.5 kDa) is due to anomalous migration in sodium dodecyl sulfate gels and not to posttranslational processing of the polypeptide chain . Of two potential N glycosylation sites at Asn-202 and Asn-284, only the latter site is glycosylated . The overproduction of the beta-glucanase from the high-copy-number plasmid brought about a significant decrease in the growth rate of transformed yeast cells.

J Bacteriol, 1993 Apr, 175(7), 1879 - 85
Physiological analysis of mutants indicates involvement of the Saccharomyces cerevisiae GPI-anchored protein gp115 in morphogenesis and cell separation; Popolo L et al.; This paper reports a phenotypic characterization of ggp1 mutants . The cloned GGP1 (GAS1) gene, which encodes a major GPI-anchored glycoprotein (gp115) of Saccharomyces cerevisiae of unknown function, was used to direct the inactivation of the chromosomal gene in haploid and diploid strains by gene replacement . The analysis of the null mutants reveals a reduction in the growth rate of 15 to 40% . Cells are round, with more than one bud, and extensively vacuolized . In the stationary phase, mutant cells are very large, arrest with a high percentage of budded cells (about 54 and 70% for haploid and diploid null mutants, respectively, in comparison with about 10 to 13% for control cells), and have reduced viability . The observed phenotype suggests defects in cell separation . Flow cytometric analysis of DNA reveals an increase in the fraction of cells in the G2+M+G1* compartment during exponential growth . Conjugation and sporulation are not affected . The exocellular location of gp115 led us to examine cell wall properties . Cell wall and septum ultrastructure of abnormally budded cells was analyzed by electron microscopy analysis, and no appreciable differences from wild-type cells were found . Microscopic analysis revealed an increase in chitin content and delocalization . In comparison with control cells, ggp1 null mutants are shown to be resistant to Zymolyase during the exponential growth phase . A fivefold overexpression of gp115 does not bring about any effects on cell growth parameters and cell wall properties.

Mol Cell Biol, 1993 Apr, 13(4), 2586 - 92
Genetic evidence for a role for MCM1 in the regulation of arginine metabolism in Saccharomyces cerevisiae; Messenguy F et al.; ARGRI, ARGRII, and ARGRIII regulatory proteins control the expression of arginine anabolic and catabolic genes in Saccharomyces cerevisiae . We have shown that MCM1 is part of the ARGR regulatory complex, by in vitro binding experiments, at the ARGR5,6 promoter . The participation of MCM1 in the regulation of arginine metabolism is confirmed by the behavior of an mcm1-gcn4 mutant, which is affected in the repression of arginine anabolic genes . In this mcm1 mutant, synthesis of the catabolic enzymes is rather constitutive, but this derepression requires the integrity of the ARGR system and of the target sequences of these proteins in the CAR1 promoter . Our in vitro binding experiments confirm the presence of MCM1 in the protein complex interacting with the promoters of the catabolic CAR1 and CAR2 genes . This is the first in vivo transcription role ascribed to MCM1 other than its role in the transcription of cell-type-specific genes.

Mol Cell Biol, 1993 Apr, 13(4), 2554 - 63
Cell surface anchorage and ligand-binding domains of the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin, a member of the immunoglobulin superfamily; Wojciechowicz D et al.; alpha-Agglutinin is a cell adhesion glycoprotein expressed on the cell wall of Saccharomyces cerevisiae alpha cells . Binding of alpha-agglutinin to its ligand a-agglutinin, expressed by a cells, mediates cell-cell contact during mating . Analysis of truncations of the 650-amino-acid alpha-agglutinin structural gene AG alpha 1 delineated functional domains of alpha-agglutinin . Removal of the C-terminal hydrophobic sequence allowed efficient secretion of the protein and loss of cell surface attachment . This cell surface anchorage domain was necessary for linkage to a glycosyl phosphatidylinositol anchor . A construct expressing the N-terminal 350 amino acid residues retained full a-agglutinin-binding activity, localizing the binding domain to the N-terminal portion of alpha-agglutinin . A 278-residue N-terminal peptide was inactive; therefore, the binding domain includes residues between 278 and 350 . The segment of alpha-agglutinin between amino acid residues 217 and 308 showed significant structural and sequence similarity to a consensus sequence for immunoglobulin superfamily variable-type domains . The similarity of the alpha-agglutinin-binding domain to mammalian cell adhesion proteins suggests that this structure is a highly conserved feature of adhesion proteins in diverse eukaryotes.

Mol Cell Biol, 1993 Apr, 13(4), 2441 - 55
Structural alterations of the nucleolus in mutants of Saccharomyces cerevisiae defective in RNA polymerase I; Oakes M et al.; We have previously constructed mutants of Saccharomyces cerevisiae in which the gene for the second-largest subunit of RNA polymerase I (Pol I) is deleted . In these mutants, rRNA is synthesized by RNA polymerase II from a hybrid gene consisting of the 35S rRNA coding region fused to the GAL7 promoter on a plasmid . These strains thus grow in galactose but not glucose media . By immunofluorescence microscopy using antibodies against the known nucleolar proteins SSB1 and fibrillarin, we found that the intact crescent-shaped nucleolar structure is absent in these mutants; instead, several granules (called mininucleolar bodies {MNBs}) that stained with these antibodies were seen in the nucleus . Conversion of the intact nucleolar structure to MNBs was also observed in Pol I temperature-sensitive mutants at nonpermissive temperatures . These MNBs may structurally resemble prenucleolar bodies observed in higher eukaryotic cells and may represent a constituent of the normal nucleolus . Furthermore, cells under certain conditions that inhibit rRNA synthesis did not cause conversion of the nucleolus to MNBs . Thus, the role of Pol I in the maintenance of the intact nucleolar structure might include a role as a structural element in addition to (or instead of) a functional role to produce rRNA transcripts . Our study also shows that the intact nucleolar structure is not absolutely required for rRNA processing, ribosome assembly, or cell growth and that MNBs are possibly functional in rRNA processing in the Pol I deletion mutants.

Mol Cell Biol, 1993 Apr, 13(4), 2324 - 31
Physical detection of heteroduplexes during meiotic recombination in the yeast Saccharomyces cerevisiae; Nag DK et al.; We describe a general physical method for detecting the heteroduplex DNA that is formed as an intermediate in meiotic recombination in the yeast Saccharomyces cerevisiae . We use this method to study the kinetic relationship between the formation of heteroduplex DNA and other meiotic events . We show that strains with the rad50, but not the rad52, mutation are defective in heteroduplex formation . We also demonstrate that, although cruciform structures can be formed in vivo as a consequence of heteroduplex formation between DNA strands that contain different palindromic insertions, small palindromic sequences in homoduplex DNA are rarely extruded into the cruciform conformation.

Mol Cell Biol, 1993 Apr, 13(4), 2172 - 81
Bipartite structure of an early meiotic upstream activation sequence from Saccharomyces cerevisiae; Bowdish KS et al.; Diploid a/alpha Saccharomyces cerevisiae cells cease mitotic growth and enter meiosis in response to starvation . Expression of meiotic genes depends on the IME1 gene product, which accumulates only in meiotic cells . We report here an analysis of the regulatory region of IME2, an IME1-dependent meiotic gene . Deletion and substitution studies identified a 48-bp IME1-dependent upstream activation sequence (UAS) . Activity of the UAS also requires the RIM11, RIM15, and RIM16 gene products, which are required for expression of the chromosomal IME2 promoter and for meiosis . Through a selection for suppressors that permit UAS activity in an ime1 deletion mutant, we identified recessive mutations in three genes, SIN3 (also called RPD1, UME4, and SDI1), RPD3, and UME6 (also called CAR80), that were previously known as negative regulators of other early meiotic genes . Mutational analysis of the IME2 UAS reveals two critical sequence elements: a G+C-rich sequence (called URS1), previously identified at many meiotic genes, and a newly described element, the T4C site, that we found at a subset of meiotic genes . In agreement with prior studies, URS1 mutations lead to elevated IME2 UAS activity in the absence of IME1 . However, the URS1 mutations prevent any further stimulation of UAS activity by IME1 . Repression through URS1 has been shown to require the UME6 gene product . We find that activation of the IME2 UAS by IME1 also requires the UME6 gene product . Thus, UME6 and the URS1 site both have dual negative and positive roles at the IME2 UAS . We propose that IME1 modifies UME6 to convert it from a negulator to a positive Regulor.

Mol Cell Biol, 1993 Apr, 13(4), 2126 - 33
U1 small nuclear ribonucleoprotein particle-protein interactions are revealed in Saccharomyces cerevisiae by in vivo competition assays; Stutz F et al.; Two highly conserved regions of the 586-nucleotide yeast (Saccharomyces cerevisiae) U1 small nuclear RNA (snRNA) can be mutated or deleted with little or no effect on growth rate: the universally conserved loop II (corresponding to the metazoan A loop) and the yeast core region (X . Liao, L . Kretzner, B . Seraphin, and M . Rosbash, Genes Dev . 4:1766-1774, 1990) . To examine the contribution of these regions to U1 small nuclear ribonucleoprotein particle (snRNP) activity, a competitor U1 gene, encoding a nonfunctional U1 snRNA molecule, was introduced into a number of strains carrying a U1 snRNA gene with loop II or yeast core mutations . The presence of the nonfunctional U1 gene lowered the growth rate of these mutant strains but not wild-type strains, consistent with the notion that mutant U1 RNAs are less active than wild-type U1 snRNAs . A detailed analysis of the U1 snRNA levels and half-lives in a number of merodiploid strains suggests that these mutant U1 snRNAs interact with U1 snRNP proteins less well than do their wild-type counterparts . Competition for protein factors during snRNP assembly could account for a number of previous observations in both yeast and mammalian cells.

Mol Cell Biol, 1993 Apr, 13(4), 2069 - 80
Pheromone-induced signal transduction in Saccharomyces cerevisiae requires the sequential function of three protein kinases; Zhou Z et al.; Protein phosphorylation plays an important role in pheromone-induced differentiation processes of haploid yeast cells . Among the components necessary for signal transduction are the STE7 and STE11 kinases and either one of the redundant FUS3 and KSS1 kinases . FUS3 and presumably KSS1 are phosphorylated and activated during pheromone induction by a STE7-dependent mechanism . Pheromone also induces the accumulation of STE7 in a hyperphosphorylated form . This modification of STE7 requires the STE11 kinase, which is proposed to act before STE7 during signal transmission . Surprisingly, STE7 hyperphosphorylation also requires a functional FUS3 (or KSS1) kinase . Using in vitro assays for FUS3 phosphorylation, we show that pheromone activates STE7 even in the absence of FUS3 and KSS1 . Therefore, STE7 activation must precede modification of FUS3 (and KSS1) . These findings suggest that STE7 hyperphosphorylation is a consequence of its activation but not the determining event.

Mol Cell Biol, 1993 Apr, 13(4), 2050 - 60
Function of the ste signal transduction pathway for mating pheromones sustains MAT alpha 1 transcription in Saccharomyces cerevisiae; Mukai Y et al.; Sterile mutants of Saccharomyces cerevisiae were isolated from alpha * cells having the a/alpha aar1-6 genotype (exhibiting alpha mating ability and weak a mating ability as a result of a defect in a1-alpha 2 repression) . Among these sterile mutants, we found two ste5 mutants together with putative ste7, ste11, and ste12 mutants of the signal transduction pathway of mating pheromones . The amino acid sequence of the Ste5p protein predicted from the nucleotide sequence of a cloned STE5 DNA has a domain rich in acidic amino acids close to its C terminus, a cysteine-rich sequence, resembling part of a zinc finger structure, in its N-terminal half, and a possible target site of cyclic AMP-dependent protein kinase at its C terminus . Northern (RNA) blot analysis revealed that STE5 transcription is under a1-alpha 2-Aar1p repression . The MAT alpha 1 cistron has a single copy of the pheromone response element in its 5' upstream region, and its basal level of transcription was reduced in these ste mutant cells . However, expression of the MAT alpha 1 cistron was not enhanced appreciably by pheromone signals . One of the ste5 mutant alleles conferred a sterile phenotype to a/alpha aar1-6 cells but a mating ability to MATa cells.

Mol Cell Biol, 1993 Apr, 13(4), 2041 - 9
The COT2 gene is required for glucose-dependent divalent cation transport in Saccharomyces cerevisiae; Conklin DS et al.; Eleven cobalt-tolerant mutants were found to belong to a single complementation group, cot2 . In addition to cobalt, the cot2 mutants were found to tolerate increased levels of the divalent cations Zn2+, Mn2+, and Ni2+ as well . All of the cot2 mutants exhibited a wiener-shaped cellular morphology that was exacerbated by the carbon and nitrogen source but was unaffected by metals . The rate of glucose-dependent transport of cobalt into cells was reduced in strains that carry mutations in the COT2 gene . COT2 is not essential for growth . Strains that carry a COT2 allele conferring complete loss of function are viable and exhibit phenotypes similar to those of spontaneous cot2 mutations . The sequence of the COT2 gene shows that it is identical to GRR1, which encodes a protein required for glucose repression . The glucose dependence of the transport defect implies that cot2 mutations affect the link between glucose metabolism and divalent cation active transport.

Yeast, 1993 Apr, 9(4), 399 - 409
Targeting of a heterologous protein to the cell wall of Saccharomyces cerevisiae; Schreuder MP et al.; The sexual adhesion protein of Saccharomyces cerevisiae MAT alpha cells, alpha-agglutinin, could not be extracted from the cell wall with hot sodium dodecyl sulfate (SDS), but became soluble after digestion of the cell wall with laminarinase . This indicates that it is intimately associated with cell wall glucan . A fusion protein was constructed consisting of the signal sequence of yeast invertase, guar alpha-galactosidase, and the C-terminal half of the alpha-agglutinin . Most of the fusion protein was incorporated in the cell wall . A small amount could be extracted with SDS, but most of it could only be extracted with laminarinase . On the other hand, cells containing a construct consisting of the signal sequence of invertase and alpha-galactosidase released most of the alpha-galactosidase into the medium and all cell wall-associated alpha-galactosidase was released by SDS . Labelling with antibodies showed that the alpha-galactosidase part of the fusion protein was exposed on the surface of the cell wall . The results demonstrate that the C-terminal half of the alpha-agglutinin contains the information needed to incorporate a protein into the cell wall.

Mol Gen Genet, 1993 Apr, 238(1-2), 6 - 16
Characterization of the MKS1 gene, a new negative regulator of the Ras-cyclic AMP pathway in Saccharomyces cerevisiae; Matsuura A et al.; In order to isolate genes that function downstream of the Ras-cAMP pathway in Saccharomyces cerevisiae, a YEp13-based genomic library was screened for clones that inhibit growth of cells with diminished A-kinase activity . One such gene, MKS1, was found to encode a hydrophilic 52 kDa protein that shares weak homology with the yeast SPT2/SIN1 gene product . Three lines of evidence suggest that the MKS1 gene product is a negative regulator downstream of the Ras-cAMP pathway: (i) overexpression of MKS1 inhibits growth of cyr1 disruptant cells on YPD medium containing a low concentration of cAMP; (ii) overexpression of MKS1 does not affect TPK1 expression; and (iii) the temperature-sensitive cyr1-230 mutation is partially suppressed by mks1 disruption . The mks1 mutant shows similar phenotypes to gal11/spt13, i.e., it cannot grow on YPGal containing ethidium bromide at 25 degrees C, or on YPGly or SGal at 37 degrees C . The mks1 gal11 double mutant shows more marked phenotypic changes than the single mutants . These results suggest that MKS1 is involved in transcriptional regulation of several genes by cAMP.

Eur J Biochem, 1993 Apr 1, 213(1), 641 - 8
Synthesis of human parathyroid-hormone-related protein(1-141) in Saccharomyces cerevisiae . A correct amino-terminal processing vital for the hormone's biological activity is obtained by an ubiquitin fusion protein approach; Rian E et al.; Gene fusions have been widely used in heterologous expression systems as a technique to stabilize the recombinant product against proteolysis, increase the translational initiation efficiency or to serve as an affinity handle for the purification of the protein . A further advantage is the potential to generate an authentic amino terminus of the foreign protein when this is vital for its biological activity, such as for the ability of human parathyroid-hormone-related protein (hPTHrP) to mediate activation of adenylate cyclase . We report here the construction and utility of a ubiquitin fusion protein system for production of the otherwise short-lived hPTHrP(1-141) as a carboxyl extension to ubiquitin in yeast . A hybrid gene containing the hPTHrP(1-141) cDNA coding region fused in-frame to the 3' end of the yeast ubiquitin cDNA was constructed and expressed under the control of the regulatable yeast metallothionein promoter . The recombinant protein was purified to homogeneity and finally characterized by N-terminal amino acid sequencing and amino acid composition analysis, demonstrating that the fusion protein was cleaved correctly and quantitatively in vivo by an ubiquitin-specific yeast endoprotease to generate authentic hPTHrP(1-141) . hPTHrP(1-141) stimulated adenylate cyclase in rat osteosarcoma cell membranes to the same extent as equimolar amounts of recombinant human parathyroid hormone(1-84) and {Tyr34}hPTHrP(1-34)amide . Thus, this expression cloning strategy permits the production of authentic, biologically active recombinant hPTHrP(1-141), and the procedure can easily be adapted to make PTHrP analogues for further studies of its domain-specific activities and biological roles.

Eur J Biochem, 1993 Apr 1, 213(1), 137 - 45
Rapid screening of cytochromes of respiratory mutants of Saccharomyces cerevisiae . Application to the selection of strains containing novel forms of cytochrome-c oxidase; Brown S et al.; A technique has been developed for the direct analysis by visible spectrophotometry of yeast spots growing on agar plates . This allows rapid semi-quantitative estimations of cytochromes c, b and oxidase and permits the identification of strains with impaired respiratory electron flow . Results of screening of 105 mutants are presented . There appears to be a correlation between the exonic location of the mutation in COX1 of oxidase and the level of optically detectable enzyme . Mutations in cytochrome b of the bc1 complex also affect the level of expression of cytochrome oxidase and can cause either an increased or decreased level of expression of oxidase relative to the wild-type strain . Twelve strains selected by the rapid level-1 screening were grown as lawns on sections of an agar plate and resuspended for a second level of screening . Quantitative estimates have been made of the concentrations of cytochromes, the turnover number of cytochrome oxidase and the kinetics of recombination of carbon monoxide with oxidase after flash photolysis . This confirmed the validity of the rapid screening procedure, and we have identified several strains which contain high levels of a mutant form of cytochrome oxidase with properties worthy of further investigation.

Eur J Biochem, 1993 Apr 1, 213(1), 129 - 35
Genetic screening in Saccharomyces cerevisiae for large numbers of mitochondrial point mutations which affect structure and function of catalytic subunits of cytochrome-c oxidase; Meunier B et al.; A new search for mitochondrial respiratory deficient mutants (Mit-) has been undertaken in order to accumulate a large number of point mutations in the coding portions of cytochrome-c-oxidase catalytic subunits and cytochrome b . Therefore, a mitochondrial DNA which retains the exons and lacks all the introns of the cytochrome oxidase subunit I and of the cytochrome-b split genes has been introduced into a strain carrying a nuclear recessive mutation affecting the adenine-nucleotide translocator, the op1 mutation, which is known to prevent the accumulation of large deletion petite mutants and this was used as the parental strain . After a moderate MnCl2 mutagenesis in order to limit multiple mutations, 105 Mit- mutants were isolated from 15,000 mutagenised clones in Saccharomyces cerevisiae . Mutations were mapped to the three catalytic subunits encoding genes (COX1, COX2 and COX3) of the cytochrome-c oxidase (70 mutations) and to the cytochrome-b gene (15 mutations) . More than 50% of the mutants tested still exhibited mitochondrial translation products (subunits I, II and III), suggesting that they carry a missense mutation, rather than a nonsense mutation which would normally have led to a truncated protein . Mutations in the COX1 gene were allocated to four different subregions corresponding to exons 4 and 8 or to groups of exons, exons 1, 2, 3 or exons 5, 6, 7 . Seven missense monosubstitution mutations and two frameshift mutations were also identified . The amino acid changes of the missense mutations were located in the vicinity of the CuB-heme alpha 3 binuclear centre ligands.

EMBO J, 1993 Apr, 12(4), 1467 - 73
Pathway correcting DNA replication errors in Saccharomyces cerevisiae; Morrison A et al.; Mutation of predicted 3'-->5' exonuclease active site residues of Saccharomyces cerevisiae POL3 DNA polymerase (delta) or deletion of the PMS1 mismatch repair gene lead to relative (to wild type) spontaneous mutation rates of approximately 130 and 41, respectively, measured at a URA3 reporter gene inserted near to a defined replication origin . The POL3 exonuclease-deficient mutant pol3-01 generated most classes of single base mutation in URA3, indicating a broad specificity that generally corresponds to that of the PMS1 system . pol3-01 pms1 haploid cells ceased growth after a few divisions with no unique terminal cell morphology . A pol3-01/pol3-01 pms1/pms1 diploid was viable and displayed an estimated URA3 relative mutation rate of 2 x 10(4), which we calculate to be catastrophically high in a haploid . The relationship between the relative mutation rates of pol3-01 and pms1 was multiplicative, indicating action in series . The PMS1 transcript showed the same cell cycle periodicity as those of a set of DNA replication genes that includes POL3, suggesting PMS1 is co-regulated with these genes . We propose that the POL3 3'-->5' exonuclease and the PMS1 mismatch repair system act on a common pathway analogous to the dnaQ-->mutHLS pathway of DNA replication error correction in Escherichia coli.

Mol Cell Biol, 1993 Apr, 13(4), 2091 - 103
Interspersion of an unusual GCN4 activation site with a complex transcriptional repression site in Ty2 elements of Saccharomyces cerevisiae; Turkel S et al.; Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2 . The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII) . In addition, the negative region includes at least two downstream activation sites (DASs) . This paper concerns the characterization of DASI . A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation . We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell . We have termed the two sites GCN4 binding sites (GBS1 and GBS2) . GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site . GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity . Nucleotides 13 to 36 of DASI overlap DRSII . Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements . We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4) . One of these is repeated in the 12 bp immediately adjacent to DASI . Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators . The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins.

Mol Cell Biol, 1993 Apr, 13(4), 2081 - 90
Three downstream sites repress transcription of a Ty2 retrotransposon in Saccharomyces cerevisiae; Farabaugh PJ et al.; Transcription of Ty1 and Ty2 retrotransposons of the yeast Saccharomyces cerevisiae is modulated by multiple downstream regulatory sites . Both transposon families include a positively acting site within the transcribed region which resembles a higher eukaryotic enhancer . We have demonstrated the existence of a repression site distal to the enhancer of the Ty2-917 element . Here we describe experiments investigating the internal structure of this site . We show that this 200-bp region includes three distinct repression sites which we term DRSI (downstream repression site I), DRSII, and DRSIII . Individually each site causes almost twofold repression, and together the sites repress eightfold . Unexpectedly, when the entire region encompassing the DRS sites is moved outside the transcription unit, it acts as a qualitatively positively acting element . In this context the DRS sites still repress transcription, since eliminating them increases transcription further . That the region can activate transcription implies that it includes activation sites in addition to the three repression sites . The change from qualitatively negatively acting to positively acting must reflect a change in the relative effects of the multiple positive and negative sites; when moved outside the transcription unit, the activators predominate . Importantly, DRSII and DRSIII repress transcription autonomously when inserted upstream of a heterologous promoter activated by the transcriptional activator GCN4, showing that they are indeed transcriptional repression sites.

Yeast, 1993 Apr, 9(4), 419 - 21
Genetic and physical localization of the acetyl-coenzyme A synthetase gene ACS1 on chromosome I of Saccharomyces cerevisiae; Steensma HY et al.; The ACS1 gene, encoding acetyl-coenzyme A synthetase, was mapped genetically at the left arm of chromosome I between pURA3 and PYK1 at 19 and 28 cM respectively . Comparison with the physical map defined a recombinational 'hot-spot' in this region in addition to the one between CDC24 and PYK1.

Gene, 1993 Mar 30, 125(2), 115 - 23
Effect of a pmr 1 disruption and different signal sequences on the intracellular processing and secretion of Cyamopsis tetragonoloba alpha-galactosidase by Saccharomyces cerevisiae; Harmsen MM et al.; We fused the yeast-derived sequences encoding the invertase, acid phosphatase and alpha-factor pre- and prepro-signal peptides (SP) to the Cyamopsis tetragonoloba (guar plant) alpha-galactosidase(alpha Gal)-encoding gene and expressed these gene fusions in yeast . Whereas the amount of fusion protein produced by each of the constructs did not vary significantly, the secretion efficiency of the fusion protein that carried the SP of the prepro-alpha-factor (MF alpha 1) was consistently found to be about 10% higher than that of the other fusions (99% vs . 90%) . Furthermore, when the secretion of alpha Gal was directed by the invertase (SUC2) SP, the intracellular enzyme localized to the endoplasmic reticulum (ER), whereas use of the MF alpha 1 SP caused the intracellular enzyme to be outer-chain-glycosylated and processed by the KEX2 endoproteinase, implying that it had passed the ER . These results suggest that the pro-peptide of MF alpha 1 stimulates the efflux of the heterologous protein from the ER . Null mutants of PMR1 (encoding a Ca(2+)-dependent ATPase) are known to give higher secretion efficiencies for a number of different heterologous proteins . Therefore, we also studied the secretion of alpha Gal in a pmr 1 disruption mutant . Structural analysis of the enzyme secreted by the mutant cells showed that it was completely processed by KEX2 and outer-chain-glycosylated, although the length of the outer-chain carbohydrate moiety was reduced when compared with the enzyme secreted by wild-type cells . These results contradict the hypothesis advanced by Rudolph et al . {Cell 58 (1989) 133-145} that disruption of PMR1 causes the secretory pathway to bypass the Golgi apparatus.

Gene, 1993 Mar 30, 125(2), 185 - 9
Characterization of a lysine-to-glutamic acid mutation in a conservative sequence of farnesyl diphosphate synthase from Saccharomyces cerevisiae; Blanchard L et al.; The mutant gene erg20-2 was isolated from a yeast strain defective in farnesyl diphosphate synthase (FPPS) . This strain had the unusual property of excreting prenyl alcohols such as geraniol . The nucleotide (nt) sequence, compared with that of the wild-type gene, showed a single nt change, resulting in a Lys197-->Glu substitution in FPPS which is directly involved in terpenic alcohol formation . In addition, disruption of ERG20 revealed that in yeast no other prenyl transferase is able to synthesize the FPP molecules required for essential non-sterol metabolites.

J Biol Chem, 1993 Mar 25, 268(9), 6453 - 62
Inhibition of translational initiation in the yeast Saccharomyces cerevisiae as a function of the stability and position of hairpin structures in the mRNA leader; Vega Laso MR et al.; A new modular in vivo/in vitro expression system was constructed which facilitates studies of the control and regulation of gene expression in the yeast Saccharomyces cerevisiae . We studied the influence of stem-loop structures inserted into the non-translated leader region upon the steady-state levels and translation of mRNAs bearing the cat gene from the bacterial transposon Tn9 . mRNA abundance changed relatively little in response to alterations in the leader sequence and structure, whereas stem-loop structures clearly inhibited translation to a degree that was dependent upon the predicted stability as well as the position of the inserted secondary structure . A stem-loop structure with a predicted stability greater than -28 kcal mol-1 and with a stem comprising at least 15 (mainly G/C) base pairs inhibited translation in vivo by at least 98% . A stem-loop structure with a predicted stability of approximately -14 kcal mol-1, whose stem comprised at least six G/C base pairs, inhibited translation in vivo by at least 66% . The hairpins were more inhibitory when placed close to the start codon than when positioned near the 5' end of the leader . An mRNA showing extensive complementarity between the leader and trailer regions was not only poorly translated but also had a steady-state level at least three times higher than the average for all the cat constructs examined . Translation of the various mRNAs in a yeast cell-free system followed qualitatively the same pattern as the results obtained in vivo . The stem-loop structures were far less inhibitory in a reticulocyte lysate system . Overall, the data are likely to reflect the full spectrum of translational activities of yeast mRNAs in vivo determined by secondary structure and emphasize the importance of translation as a control step in gene expression.

J Biol Chem, 1993 Mar 25, 268(9), 6248 - 55
Regulation of the 45- and 55-kDa forms of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae by nucleotides; Buxeda RJ et al.; The synthesis of phosphatidylinositol (PI) 4-phosphate and PI 4,5-bisphosphate in the yeast Saccharomyces cerevisiae is stimulated by glucose . PI 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) catalyzes the committed step in the synthesis of these phosphoinositides . Previous studies have suggested that the glucose effect on phosphoinositide synthesis is mediated by cellular levels of ATP and ADP and by the RAS/cAMP pathway . Using purified preparations of the membrane-associated 45- and 55-kDa forms of PI 4-kinase, we examined the regulation of these activities by nucleotides and cAMP-dependent protein kinase . MgADP was a potent inhibitor of both forms of the enzyme . Detailed kinetic analyses of the 45- and 55-kDa enzymes using Triton X-100/PI-mixed micelles showed that MgADP was a competitive inhibitor (Ki = 0.14 and 0.25 mM, respectively) with respect to MgATP and a noncompetitive inhibitor (Ki = 1.3 and 0.9 mM, respectively) with respect to PI . The Ki values for MgADP were about 2-fold lower than the Km values the enzymes have for their substrate MgATP and about 2-fold lower than the cellular concentration of ADP . The 45- and 55-kDa forms of PI 4-kinase activity were regulated differentially by CTP, an important nucleotide involved in phospholipid biosynthesis . Whereas the 55-kDa PI 4-kinase was inhibited by CTP, the 45-kDa enzyme was unaffected by CTP . CTP was a mixed type of inhibitor (Ki = 1.5 mM) with respect to MgATP and a noncompetitive inhibitor (Ki = 4 mM) with respect to PI . The Ki value for CTP was 4-fold higher than the Km value for MgATP and 7-fold higher than the cellular concentration of CTP . The 45- and 55-kDa PI 4-kinases were neither phosphorylated nor regulated by cAMP-dependent protein kinase . These results did not support the previous conclusion that PI 4-phosphate synthesis was mediated by the RAS/cAMP pathway . Our kinetic studies supported the conclusion that the glucose effect on the synthesis of PI 4-phosphate was mediated by cellular levels of ATP and ADP through the regulation of membrane-associated PI 4-kinase activity.

Biochim Biophys Acta, 1993 Mar 21, 1156(3), 263 - 6
Role of the trehalose carrier in dehydration resistance of Saccharomyces cerevisiae; Eleutherio EC et al.; Yeast cells are well known for their ability to survive complete dehydration, a phenomenon that is directly linked to the presence of the sugar trehalose in these cells . This sugar apparently endows the cells with the capacity to survive dehydration . Previous studies on in vitro models showed that trehalose must be present on both sides of the bilayer to stabilize dry membranes . The present report demonstrates that a specific trehalose carrier seems to enable the sugar to protect the yeast cell membrane by translocating trehalose from the cytosol to the extracellular environment . Saccharomyces cerevisiae mutant strains which lack the trehalose carrier did not survive after dehydration although they accumulated endogenous trehalose . Furthermore, when carrier mutants were dehydrated in the presence of exogenous trehalose the cells became more resistant showing increased survival.

FEMS Microbiol Lett, 1993 Mar 15, 108(1), 23 - 6
Acetoin production in Saccharomyces cerevisiae wine yeasts; Romano P et al.; One hundred strains of Saccharomyces cerevisiae were examined for the capacity to produce acetoin in synthetic medium and in grape must . The low production of acetoin was found to be the more common pattern in this species . Most strains exhibited a similar distribution in both media, production ranging from non-detectable amounts to 12 mg l-1 . Only four strains produced high quantities of acetoin, up to 29.5 mg l-1 in synthetic medium and up to 194.6 mg l-1 in grape must . This biometric study showed the existence of two phenotypes, "low and high acetoin production", that could be selected for conferring a desirable flavour of the final product.

Biochem Biophys Res Commun, 1993 Mar 15, 191(2), 750 - 8
Characterization of DNA binding properties of Yp20: an abundant nuclear protein isolated from Saccharomyces cerevisiae; Bartuv J et al.; The aim of this study was to elucidate the function of Yp20 (yeast 20 kDa protein) which is an abundant basic DNA-binding protein copurified with yeast chromatin . The work presented here shows that Yp20 is a sequence specific DNA binding protein . DNA binding activity was extremely thermostable . The affinity of binding to TRP1 was higher than the affinity of binding to the B domain of ARS1 . The dissociation half time of Yp20-DNA complexes was less than 1 min . Yp20 showed no homology to a similar abundant 21 kDa ARS binding factor II (ABFII), previously described . Competitive gel retardation assays revealed two different regions that were protected by Yp20 . One was overlapping the ABF1 binding site on ARS1 and another protected region was found upstream to the translational start codon of the TRP1 gene . It thus appears that Yp20 may have a role in DNA replication and/or transcription.

J Biol Chem, 1993 Mar 15, 268(8), 5754 - 62
Isolation and immunochemical characterization of eukaryotic translation initiation factor 5 from Saccharomyces cerevisiae; Chakravarti D et al.; Eukaryotic translation initiation factor 5 (eIF-5), which catalyzes the hydrolysis of GTP bound to the 40 S ribosomal initiation complex has been purified from yeast cell lysates . The purified factor eluted from gel filtration columns as a protein of apparent M(r) = 45,000-50,000 . However, when the purified preparation was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two distinct polypeptides of apparent M(r) = 54,000 and 56,000 were observed . Each of the two polypeptides individually was found to contain eIF-5 activity, and they were immunologically related to each other . In less pure preparations of yeast eIF-5, however, a significant proportion of eIF-5 activity eluted from gel filtration columns as a protein of M(r) > 140,000 . Immunochemical methods were therefore employed to determine the molecular structure of eIF-5 in crude yeast cell lysates . Antisera against purified yeast eIF-5 were prepared in rabbits and shown to be highly potent in inhibiting eIF-5-mediated 80 S initiation complex formation . When crude eIF-5 preparations, as well as yeast cells that were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate, were analyzed by Western blots probed with affinity-purified anti-eIF-5 antibodies, a major immunoreactive polypeptide (apparent M(r) = 54,000) and a minor band (apparent M(r) = 56,000) were observed . No precursor forms of molecular weight higher than 56,000 were detected in any preparations . These results suggest that yeast eIF-5 is a monomeric protein of apparent M(r) = 50,000-56,000.

J Biol Chem, 1993 Mar 15, 268(8), 5728 - 34
Functional characterization of flavin-containing monooxygenase 1B1 expressed in Saccharomyces cerevisiae and Escherichia coli and analysis of proposed FAD- and membrane-binding domains; Lawton MP et al.; A cDNA encoding the flavin-containing monooxygenase of rabbit lung (FMO 1B1) was expressed in yeast and Escherichia coli and the recombinant enzymes characterized . A high copy, isopropyl-1-thio-beta-D-galactopyranoside (IPTG)-inducible E . coli expression vector, pKKHC, was used for expression in E . coli strain JM109, and a galactose-inducible vector, YEp53, was used for expression in yeast strain 334 . Following transcriptional induction with IPTG or galactose, subcellular fractions were prepared and analyzed immunochemically and catalytically . Antibodies to rabbit FMO 1B1 were used to detect the recombinant proteins in the 100,000 x g pellet prepared from the 10,000 x g supernatant fraction of yeast homogenates and the 2,000 x g supernatant fraction of E . coli homogenates . No FMO 1B1 was detected in cytosol . Mobilities of the recombinant proteins in SDS-polyacrylamide gel electrophoresis appeared identical to that of the native microsomal enzyme . Catalytic similarity to the native FMO 1B1 was demonstrated by the ability of the expressed enzymes to metabolize methimazole, thiourea, dimethylaniline, and cysteamine, but not chlorpromazine or imipramine . In addition, the recombinant enzymes exhibited a number of the unique physical properties associated with FMO 1B1, including stability to elevated temperature and activation by sodium cholate and magnesium chloride . Based on the specific content of FAD, the level of expression was estimated to be approximately 2% of the total protein in the E . coli 100,000 x g particulate fraction and 1% in the fraction from yeast . To demonstrate the utility of the E . coli expression system for studying structure/function relationships of the flavin-containing monooxygenase, two mutant FMOs were expressed and characterized . One mutant, formed by deletion of a putative membrane-anchoring peptide (the 26 carboxyl-terminal amino acids) was tested for membrane association . No difference in the subcellular distribution was found between the truncated and unmodified proteins, suggesting that the 26-residue COOH-terminal peptide is not important in membrane association . Catalytic analysis of the truncated FMO 1B1 established its functional similarity to the full-length protein, indicating that the COOH terminus does not contribute to any of the unique properties of the lung enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1993 Mar 5, 268(7), 4832 - 8
Functional expression of human cathepsin S in Saccharomyces cerevisiae . Purification and characterization of the recombinant enzyme; Bromme D et al.; A cDNA encoding the human lysosomal cysteine proteinase cathepsin S precursor has been expressed in yeast using the pVT100-U expression vector containing the alpha-factor promoter . The procathepsin S gene was expressed either as a fusion protein with the pre-region or with the prepro-region of the yeast alpha-factor precursor gene . Following in vitro processing both constructs gave an identical active mature enzyme with a molecular weight of 24,000 . After prolonged cultivation of the cells the recombinant protein is also found as an active proteinase in the culture supernatant . The precursor can be activated in vitro at pH 4.5 and 40 degrees C under reducing conditions . The in vitro activated enzyme has a 6-amino acid NH2-terminal extension when compared with the native bovine enzyme . The purified enzyme displays a bell-shaped pH activity profile with a pH optimum of 6.5 and pK values of 4.5 and 7.8 . The isoelectric point of the recombinant human cathepsin S is between 8.3 and 8.6 and about 1.5 pH units higher than for the bovine enzyme . The kinetic data for several synthetic substrates and inhibitors reveal a preference for smaller amino acid residues in the binding subsites S2 and S3 of cathepsin S . Like the bovine enzyme, the recombinant human cathepsin S is characterized by a broader range of pH stability (pH 5-7.5) than cathepsins B and L.

J Membr Biol, 1993 Mar, 132(3), 183 - 99
Gating and conductance in an outward-rectifying K+ channel from the plasma membrane of Saccharomyces cerevisiae; Bertl A et al.; The plasma membrane of the yeast Saccharomyces cerevisiae has been investigated by patch-clamp techniques, focusing upon the most conspicuous ion channel in that membrane, a K(+)-selective channel . In simple observations on inside-out patches, the channel is predominantly closed at negative membrane voltages, but opens upon polarization towards positive voltages, typically displaying long flickery openings of several hundred milliseconds, separated by long gaps (G) . Elevating cytoplasmic calcium shortens the gaps but also introduces brief blocks (B, closures of 2-3 msec duration) . On the assumption that the flickery open intervals constitute bursts of very brief openings and closings, below the time resolution of the recording system, analysis via the beta distribution revealed typical closed durations (interrupts, I) near 0.3 msec, and similar open durations . Overall behavior of the channel is most simply described by a kinetic model with a single open state (O), and three parallel closed states with significantly different lifetimes: long (G), short (B) and very short (I) . Detailed kinetic analysis of the three open/closed transitions, particularly with varied membrane voltage and cytoplasmic calcium concentration, yielded the following stability constants for channel closure: K1 = 3.3 x e-zu in which u = eVm/kT is the reduced membrane voltage, and z is the charge number; KG = 1.9 x 10(-4) ({Ca2+}.ezu)-1; and KB = 2.7 x 10(3)({Ca2+}.ezu)2 . Because of the antagonistic effects of both membrane voltage (Vm) and cytoplasmic calcium concentration ({Ca2+}cyt) on channel opening from the B state, compared with openings from the G state, plots of net open probability (Po) vs . either Vm or {Ca2+} are bell-shaped, approaching unity at low calcium (microM) and high voltage (+150 mV), and approaching 0.25 at high calcium (10 mM) and zero voltage . Current-voltage curves of the open channel are sigmoid vs . membrane voltage, saturating at large positive or large negative voltages; but time-averaged currents, along the rising limb of Po (in the range 0 to +150 mV, for 10 microM {Ca2+}) make this channel a strong outward rectifier . The overall properties of the channel suggest that it functions in balancing charge movements during secondary active transport in Saccharomyces.

Yeast, 1993 Mar, 9(3), 221 - 34
Reduced efficiency in the glycosylation of the first sequon of Saccharomyces cerevisiae exoglucanase leads to the synthesis and secretion of a new glycoform of the molecule; Basco RD et al.; In addition to exoglucanases (EXGs) I and II, old cultures of Saccharomyces cerevisiae secreted into the culture medium a new immunologically-related material that exhibited exoglucanase activity . The new exoglucanase (EXGII1/2) was purified from stationary-phase cultures . It turned out to be a glycoprotein whose protein portion was identical to that of the other two isoenzymes in terms of ionic properties, size, amino acid composition and NH2-terminal sequence (25 residues) . Disruption of the structural gene encoding EXGs I and II resulted in a strain unable to secrete all three isoenzymes . EXGII1/2 was indistinguishable in terms of molecular weight from the single intermediate detected during the deglycosylation (mediated by endo H) of EXGII by sodium dodecyl sulphate-polyacrylamide gel electrophoresis . Thus, the new isoenzyme contains only one of the two slightly elongated mannan inner cores present in enzyme II . Two intermediates were, however, detected when the deglycosylation of EXGII was monitored by ion-exchange chromatography (high-pressure liquid chromatography) . Site-directed mutagenesis indicated that the major intermediate, which eluted at about the same position as enzyme II1/2, corresponded to protein molecules carrying the oligosaccharide attached to the Asn of the second sequon, whereas the minor one carried the oligosaccharide in the first potential glycosylation site . Several lines of evidence indicate that EXGII1/2 is a biosynthetic product resulting from an imbalance between the rate of protein synthesis and the glycosylation capabilities of the glycosylation machinery.

Mol Gen Genet, 1993 Mar, 237(3), 463 - 6
Characterization of the cyr1-2 UGA mutation in Saccharomyces cerevisiae; Morishita T et al.; cyr1-2 is a temperature-sensitive mutation of the yeast adenylate cyclase structural gene, CYR1 . The cyr1-2 mutation has been suggested to be a UGA mutation since a UGA suppressor SUP201 has been isolated as a suppressor of the cyr1-2 mutation . Construction of chimeric genes restricted the region containing the cyr1-2 mutation, and the cyr1-2 UGA mutation was identified at codon 1282, which lies upstream of the region coding for the catalytic domain of adenylate cyclase . Alterations in the region upstream of the cyr1-2 mutation site result in null mutations . The complete open reading frame of the cyr1-2 gene expressed under the control of the GAL1 promoter complemented cyr1-d1 in a galactose-dependent manner . These results suggest that at the permissive temperature weak readthrough occurs at the cyr1-2 mutation site to produce low levels of active adenylate cyclase . An endogenous suppressor in yeast cells is assumed to be responsible for this readthrough.

Mol Gen Genet, 1993 Mar, 237(3), 449 - 59
Molecular and genetic characterization of SPT4, a gene important for transcription initiation in Saccharomyces cerevisiae; Malone EA et al.; Mutations in the SPT4 gene of Saccharomyces cerevisiae were isolated as suppressors of delta insertion mutations that interfere with adjacent gene transcription . Recent genetic evidence indicates that the SPT4 protein functions with two other proteins, SPT5 and SPT6, in some aspect of transcription initiation . In this work we have characterized the SPT4 gene and we demonstrate that spt4 mutations, like spt5 and spt6 mutations, cause changes in transcription . Using the cloned SPT4 gene, spt4 null mutations were constructed; in contrast to spt5 and spt6 null mutants, which are inviable, spt4 null mutants are viable and have an Spt- phenotype . The DNA sequence of the SPT4 gene predicts a protein product of 102 amino acids that contains four cysteine residues positioned similarly to those of zinc binding proteins . Mutational analysis suggests that at least some of these cysteines are essential for SPT4 function . Genetic mapping showed that SPT4 is a previously unidentified gene that maps to chromosome VII, between ADE6 and CLY8.

Mol Gen Genet, 1993 Mar, 237(3), 375 - 84
Post-transcriptional regulation of IME1 determines initiation of meiosis in Saccharomyces cerevisiae; Sherman A et al.; The IME1 gene of Saccharomyces cerevisiae is required for initiation of meiosis . Transcription of IME1 is detected under conditions which are known to induce initiation of meiosis, namely starvation for nitrogen and glucose, and the presence of MATa1 and MAT alpha 2 gene products . In this paper we show that IME1 is also subject to translational regulation . Translation of IME1 mRNA is achieved either upon nitrogen starvation, or upon G1 arrest . In the presence of nutrients, constitutively elevated transcription of IME1 is also sufficient for the translation of IME1 RNA . Four different conditions were found to cause expression of Ime1 protein in vegetative cultures: elevated transcription levels due to the presence of IME1 on a multicopy plasmid; elevated transcription provided by a Gal-IME1 construct; G1 arrest due to alpha-factor treatment; G1 arrest following mild heat-shock treatment of cdc28 diploids . Using these conditions, we obtained evidence that starvation is required not only for transcription and efficient translation of IME1, but also for either the activation of Ime1 protein or for the induction/activation of another factor that, either alone or in combination with Ime1, induces meiosis.

J Gen Microbiol, 1993 Mar, 139 ( Pt 3), 501 - 7
Saccharomyces cerevisiae has an inducible response to menadione which differs from that to hydrogen peroxide; Flattery-O'Brien J et al.; Exponential phase cells of Saccharomyces cerevisiae treated with the superoxide free-radical generating agent menadione (MD; 0.2 mM) for 60 min adapted to become resistant to the lethal effects of a higher concentration of MD (4 mM) . Inhibition of protein synthesis by treatment with cycloheximide totally prevented the adaptation to MD, indicating that this is an inducible response completely dependent on protein synthesis; this differs from the situation with peroxide in which only some of the adaptive response is cycloheximide-sensitive . Cells subjected to heat shock (23 to 37 degrees C) or treatment with hydrogen peroxide (H2O2; 0.2 mM, 60 min) became more resistant to 4 mM-MD; however, MD pretreatment did not induce any thermotolerance or resistance to peroxide . These differences between the response to MD and H2O2 were reflected in the results of L-{35S}methionine labelling studies . Using one-dimensional electrophoresis, only one polypeptide (60 kDa) was seen to be induced by 0.2 mM-MD and this was also induced by heat shock but not peroxide stress . With heat shock or peroxide treatment the induction of at least 10 polypeptides was detected using this approach . Using an isogenic petite strain, it was found that functional mitochondria were needed for conferring full resistance to MD, but that induction of the adaptive response was not dependent on mitochondrial function.

Mol Microbiol, 1993 Mar, 7(5), 683 - 92
Deletion analysis of the SUP35 gene of the yeast Saccharomyces cerevisiae reveals two non-overlapping functional regions in the encoded protein; Ter-Avanesyan MD et al.; SUP35 is an omnipotent suppressor gene of Saccharomyces cerevisiae coding for a protein consisting of a C-terminal part similar to the elongation factor EF-1 alpha and a unique N-terminal sequence of 253 amino acids . Twelve truncated versions of the SUP35 gene were generated by the deletion of fragments internal to the coding sequence . Functional studies of these deletion mutants showed that: (i) only the EF-1 alpha-like C-terminal part of the Sup35 protein is essential for the cell viability; (ii) overexpression of either the N-terminal part of the Sup35 protein or the full-length Sup35 protein decreases translational fidelity, resulting in omnipotent suppression and reduced growth of {psi+} strains; (iii) expression of the C-terminal part of the Sup35 protein generates an antisuppressor phenotype; and (iv) both the N- or C-terminal segments of the Sup35 protein can bind to 80S ribosomes . Thus, the data obtained define two domains within the Sup35 protein which are responsible for different functions.

EMBO J, 1993 Mar, 12(3), 1209 - 18
Evidence for an interaction between the CYP1(HAP1) activator and a cellular factor during heme-dependent transcriptional regulation in the yeast Saccharomyces cerevisiae; Fytlovich S et al.; Previously, it was shown that the CYP1(HAP1) gene product mediates the transcription of several oxygen-regulated genes through a metabolic co-effector, heme, in the yeast Saccharomyces cerevisiae . This study investigates the overproduction of the CYP1 protein when the CYP1(HAP1) gene is placed under the control of the GAL10-CYC1 hybrid promoter (either at the locus of the CYP1(HAP1) gene or cloned in a high-copy-number plasmid) . In these conditions, the CYP1 protein is detected by Western blot analysis and has a molecular mass in agreement with the open reading frame sequence . Band-shift experiments show that the CYP1(HAP1) protein is able to interact specifically with its target sequences in vitro without addition of hemin, and forms a large complex with one or several unidentified factors denoted as X . Addition of hemin allows the formation of a new complex which has a lower molecular mass . The internal deletion of the seven repeated amino acid sequences containing the KCPVDH motif in the CYP1(HAP1) protein modifies the heme responsiveness phenomenon observed in vitro in the band-shift experiments and in vivo in the transcription of the CYB2, CYC1, CYP3(CYC7) and ERG11 genes . On the basis of these data, we propose a new model for heme-induced activation of the CYP1 protein.

Genetics, 1993 Mar, 133(3), 489 - 98
a/alpha-control of DNA repair in the yeast Saccharomyces cerevisiae: genetic and physiological aspects; Heude M et al.; It has long been known that diploid strains of yeast are more resistant to gamma-rays than haploid cells, and that this is in part due to heterozygosity at the mating type (MAT) locus . It is shown here that the genetic control exerted by the MAT genes on DNA repair involves the a1 and alpha 2 genes, in a RME1-independent way . In rad18 diploids, affected in the error-prone repair, the a/alpha effects are of a very large amplitude, after both UV and gamma-rays, and also depends on a1 and alpha 2 . The coexpression of a and alpha in rad18 haploids suppresses the sensitivity of a subpopulation corresponding to the G2 phase cells . Related to this, the coexpression of a and alpha in RAD+ haploids depresses UV-induced mutagenesis in G2 cells . For srs2 null diploids, also affected in the error-prone repair pathway, we show that their G1 UV sensitivity, likely due to lethal recombination events, is partly suppressed by MAT homozygosity . Taken together, these results led to the proposal that a1-alpha 2 promotes a channeling of some DNA structures from the mutagenic into the recombinational repair process.

Genetics, 1993 Mar, 133(3), 469 - 87
Replication-dependent sister chromatid recombination in rad1 mutants of Saccharomyces cerevisiae; Kadyk LC et al.; Homolog recombination and unequal sister chromatid recombination were monitored in rad1-1/rad1-1 diploid yeast cells deficient for excision repair, and in control cells, RAD1/rad1-1, after exposure to UV irradiation . In a rad1-1/rad1-1 diploid, UV irradiation stimulated much more sister chromatid recombination relative to homolog recombination when cells were irradiated in the G1 or the G2 phases of the cell cycle than was observed in RAD1/rad1-1 cells . Since sister chromatids are not present during G1, this result suggested that unexcised lesions can stimulate sister chromatid recombination events during or subsequent to DNA replication . The results of mating rescue experiments suggest that unexcised UV dimers do not stimulate sister chromatid recombination during the G2 phase, but only when they are present during DNA replication . We propose that there are two types of sister chromatid recombination in yeast . In the first type, unexcised UV dimers and other bulky lesions induce sister chromatid recombination during DNA replication as a mechanism to bypass lesions obstructing the passage of DNA polymerase, and this type is analogous to the type of sister chromatid exchange commonly observed cytologically in mammalian cells . In the second type, strand scissions created by X-irradiation or the excision of damaged bases create recombinogenic sites that result in sister chromatid recombination directly in G2 . Further support for the existence of two types of sister chromatid recombination is the fact that events induced in rad1-1/rad1-1 were due almost entirely to gene conversion, whereas those in RAD1/rad1-1 cells were due to a mixture of gene conversion and reciprocal recombination.

Eur J Biochem, 1993 Mar 1, 212(2), 521 - 8
Cloning and expression of Saccharomyces cerevisiae copper-metallothionein gene in Escherichia coli and characterization of the recombinant protein; Sayers Z et al.; The gene sequences for intact and truncated forms of copper-binding metallothionein from Saccharomyces cerevisiae were cloned and overexpressed in Escherichia coli BL21(DE3)pLysE cells . In contrast to several other genes, the intact and truncated metallothionein genes are amplified in the polymerase chain reaction when Mg2+ is replaced by Co2+ . The recombinant truncated protein binds copper in vivo and in vitro . A ratio of 8 Cu/12 cysteines was determined from atomic absorption, X-ray fluorescence and amino acid analysis . Extended X-ray absorption spectroscopy indicates that all Cu is in Cu(I) form and coordinated to three S atoms.

Eur J Biochem, 1993 Mar 1, 212(2), 315 - 23
Disruption of TPS2, the gene encoding the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae, causes accumulation of trehalose-6-phosphate and loss of trehalose-6-phosphate phosphatase activity; De Virgilio C et al.; Preparations of the trehalose-6-phosphate synthase/phosphatase complex from Saccharomyces cerevisiae contain three polypeptides with molecular masses 56, 100 and 130 kDa, respectively . Recently, we have cloned the gene for the 56-kDa subunit of this complex (TPS1) and found it to be identical with CIF1, a gene essential for growth on glucose and for the activity of trehalose-6-phosphate synthase . Peptide sequencing of the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex (TPS2) revealed one sequence to be 100% identical with the deduced amino acid sequence of the upstream region of PPH3 on the right arm of chromosome IV . This sequence was used to clone an upstream region of PPH3 containing an open reading frame of 2685 nucleotides, predicted to encode a polypeptide of 102.8 kDa . The N-terminal sequence, as well as three internal amino acid sequences, obtained from peptide sequencing of the 100-kDa subunit, were identical with specific regions of the deduced amino acid sequence . Thus, the sequence cloned represents TPS2, the gene encoding the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex . Interestingly, a stretch of about 500 amino acids from the first part of TPS2 was 33% identical with the entire TPS1 sequence . Disruption of TPS2 had no effect on trehalose-6-phosphate synthase activity but caused complete loss of trehalose-6-phosphate phosphatase activity, measured in vitro, and accumulation of excessive amounts of trehalose-6-phosphate instead of trehalose upon heat shock or entrance into stationary phase in vivo . These results suggest that TPS2 codes for the structural gene of the trehalose-6-phosphate phosphatase . Heat shock induced an increase in trehalose-6-phosphate phosphatase activity and this was preceded by an accumulation in TPS2 mRNA, suggesting that the trehalose-6-phosphate phosphatase is subjected to transcriptional control under heat-shock conditions.

Mol Cell Biol, 1993 Mar, 13(3), 1805 - 14
Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein; Wang H et al.; The yeast SIN3 gene (also known as SDI1, UME4, RPD1, and GAM2) has been identified as a transcriptional regulator . Previous work has led to the suggestion that SIN3 regulates transcription via interactions with DNA-binding proteins . Although the SIN3 protein is located in the nucleus, it does not bind directly to DNA in vitro . We have expressed a LexA-SIN3 fusion protein in Saccharomyces cerevisiae and show that this fusion protein represses transcription from heterologous promoters that contain lexA operators . The predicted amino acid sequence of the SIN3 protein contains four copies of a paired amphipathic helix (PAH) motif, similar to motifs found in HLH (helix-loop-helix) and TPR (tetratricopeptide repeat) proteins, and these motifs are proposed to be involved in protein-protein interactions . We have conducted a deletion analysis of the SIN3 gene and show that the PAH motifs are required for SIN3 activity . Additionally, the C-terminal region of the SIN3 protein is sufficient for repression activity in a LexA-SIN3 fusion, and deletion of a PAH motif in this region inactivates this repression activity . A model is presented in which SIN3 recognizes specific DNA-binding proteins in vivo in order to repress transcription.

Mol Cell Biol, 1993 Mar, 13(3), 1489 - 96
A DNA polymerase mutation that suppresses the segregation bias of an ARS plasmid in Saccharomyces cerevisiae; Houtteman SW et al.; Yeast autonomously replicating sequence (ARS) plasmids exhibit an unusual segregation pattern during mitosis . While the nucleus divides equally into mother and daughter cells, all copies of the ARS plasmid will often remain in the mother cell . A screen was designed to isolate mutations that suppress this segregation bias . A plasmid with a weak ARS (wARS) that displayed an extremely high segregation bias was constructed . When cells were grown under selection for the wARS plasmid, the resulting colonies grew slowly and had abnormal morphology . A spontaneous recessive mutation that restored normal colony morphology was identified . This mutation suppressed plasmid segregation bias, as indicated by the increased stability of the wARS plasmid in the mutant cells even though the plasmid was present at a lower copy number . An ARS1 plasmid was also more stable in mutant cells than in wild-type cells . The wild-type allele for this mutant gene was cloned and identified as POL delta (CDC2) . This gene encodes DNA polymerase delta, which is essential for DNA replication . These results indicate that DNA polymerase delta plays some role in causing the segregation bias of ARS plasmids.

Curr Genet, 1993 Mar, 23(3), 187 - 91
Saccharomyces cerevisiae phosphoglucose isomerase and fructose bisphosphate aldolase can be replaced functionally by the corresponding enzymes of Escherichia coli and Drosophila melanogaster; Boles E et al.; Two glycolytic enzymes, phosphoglucose isomerase and fructose-1,6-bisphosphate aldolase, of Saccharomyces cerevisiae could be replaced by their heterologous counterparts from Escherichia coli and Drosophila melanogaster . Both heterologous enzymes, which show respectively little and no sequence homology to the corresponding yeast enzymes, fully restored wild-type properties when their genes were expressed in yeast deletion mutants . This result does not support notions of an obligatory formation of glycolytic multi-enzyme aggregates in yeast; nor does it support possible regulatory functions of yeast phosphoglucose isomerase.

Mol Microbiol, 1993 Mar, 7(6), 831 - 5
The Rad3 protein from Saccharomyces cerevisiae: a DNA and DNA:RNA helicase with putative RNA helicase activity; Deschavanne PJ et al.; The Rad3 protein from Saccharomyces cerevisiae is a DNA helicase which participates in the repair of ultraviolet-irradiated DNA and is inhibited in the presence of DNA containing thymine dimers . This protein is also involved in mitotic recombination and spontaneous mutagenesis and is essential for cell viability in the absence of DNA damage . Furthermore, the Rad3 protein also exhibits a DNA:RNA helicase activity in which there is a significant preference for a partial DNA:RNA hybrid rather than a partial duplex DNA substrate, which suggests that this protein might be involved in DNA repair within transcriptionally active genes . Finally, the Rad3 protein contains the DEAH motif and shares high amino acid sequence similarity with the DEAD family of RNA helicase proteins, suggesting that it might also possess an RNA helicase activity.

Proc Natl Acad Sci U S A, 1993 Mar 1, 90(5), 1804 - 8
The putative phosphoinositide-specific phospholipase C gene, PLC1, of the yeast Saccharomyces cerevisiae is important for cell growth; Yoko-o T et al.; Using the polymerase chain reaction technique, we have isolated a gene that encodes a putative phosphoinositide-specific phospholipase C (PLC) in the yeast Saccharomyces cerevisiae . The nucleotide sequence indicates that the gene encodes a polypeptide of 869 amino acid residues with a calculated molecular mass of 101 kDa . This polypeptide has both the X and Y regions conserved among mammalian PLC-beta, -gamma, and -delta, and the structure is most similar to that of mammalian PLC-delta . This putative yeast PLC gene has been designated PLC1 . Disruption of PLC1 results in slow growth or lethality for cells, depending on their genetic background and the medium, indicating that PLC1 is important for cell growth . Expression of rat PLC-delta 1 cDNA suppressed the growth defect of plc1 disruptants, strongly suggesting that PLC1 encodes PLC.

Curr Genet, 1993 Mar, 23(3), 201 - 4
Messenger RNA 3'-end formation of a DNA fragment from the human c-myc 3'-end region in Saccharomyces cerevisiae; Irniger S et al.; We have tested the functioning of the human c-myc polyadenylation signal in Saccharomyces cerevisiae . A DNA fragment containing the two AATAAA polyadenylation signals of the c-myc gene was inserted into a plasmid designed for the in-vivo testing of polyadenylation signals in yeast . The c-myc fragment had a partial capacity for directing mRNA 3'-end formation in yeast . The 3'-endpoints were 50-100 bp distant from the mRNA 3'-ends mapped in humans . This human DNA fragment is therefore unspecifically functional in yeast, indicating that other sequence elements than the human polyadenylation signal, AATAAA, are necessary for 3'-end formation.

Appl Microbiol Biotechnol, 1993 Mar, 38(6), 795 - 8
Effect of n-alcohols on the electrotransformation and permeability of Saccharomyces cerevisiae; Ganeva VJ et al.; The correlation between electrotransformation and electropermeability was studied in yeast cells following the modification of their membranes by treatment with n-alcohols . It was found that the number of transformed cells decreased with increase of chain length of the alcohols used as follows: methanol < ethanol < propanol < butanol . The electropermeability was unaffected by the prepulse n-alcohol treatment . The lack of a unidirectional link between permeability and transformation leads to the assumption that the mechanism of DNA introduction into the cell could not be interpreted solely as a result of the existence of pores.

Genetics, 1993 Mar, 133(3), 509 - 16
PET111 acts in the 5'-leader of the Saccharomyces cerevisiae mitochondrial COX2 mRNA to promote its translation; Mulero JJ et al.; PET111 is a yeast nuclear gene specifically required for the expression of the mitochondrial gene COX2, encoding cytochrome c oxidase subunit II (coxII) . Previous studies have shown that PET111 activates translation of the COX2 mRNA . To map the site of PET111 action we have constructed, in vitro, genes coding for chimeric mRNAs, introduced them into mitochondria by transformation and studied their expression . Translation of a chimeric mRNA with the 612-base 5'-untranslated leader of the COX3 mRNA fused precisely to the structural gene for the coxII-precursor protein is independent of PET111, but does require a COX3 mRNA-specific translational activator known to work on the COX3 5'-leader . This result demonstrates that PET111 is not required for any post-translational step . Translation of a chimeric mRNA with the 54-base 5'-leader of the COX2 mRNA fused precisely to the structural gene for cytochrome c oxidase subunit III was dependent on PET111 activity . These results demonstrate that PET111 acts specifically at a site in the short COX2 5'-leader to activate translation of downstream coding sequences.

J Cell Biol, 1993 Mar, 120(5), 1203 - 15
The a-factor transporter (STE6 gene product) and cell polarity in the yeast Saccharomyces cerevisiae; Kuchler K et al.; STE6 gene product is required for secretion of the lipopeptide mating pheromone a-factor by Saccharomyces cerevisiae MATa cells . Radiolabeling and immunoprecipitation, either with specific polyclonal antibodies raised against a TrpE-Ste6 fusion protein or with mAbs that recognize c-myc epitopes in fully functional epitope-tagged Ste6 derivatives, demonstrated that Ste6 is a 145-kD phosphoprotein . Subcellular fractionation, various extraction procedures, and immunoblotting showed that Ste6 is an intrinsic plasma membrane-associated protein . The apparent molecular weight of Ste6 was unaffected by tunicamycin treatment, and the radiolabeled protein did not bind to concanavalin A, indicating that Ste6 is not glycosylated and that glycosylation is not required either for its membrane delivery or its function . The amino acid sequence of Ste6 predicts two ATP-binding folds; correspondingly, Ste6 was photoaffinity-labeled specifically with 8-azido-{alpha-32P}ATP . Indirect immunofluorescence revealed that in exponentially growing MATa cells, the majority of Ste6 showed a patchy distribution within the plasma membrane, but a significant fraction was found concentrated in a number of vesicle-like bodies subtending the plasma membrane . In contrast, in MATa cells exposed to the mating pheromone alpha-factor, which markedly induced Ste6 production, the majority of Ste6 was incorporated into the plasma membrane within the growing tip of the elongating cells . The highly localized insertion of this transporter may establish pronounced anisotropy in a-factor secretion from the MATa cell, and thereby may contribute to the establishment of the cell polarity which restricts partner selection and cell fusion during mating to one MAT alpha cell.

Eur J Biochem, 1993 Feb 15, 212(1), 113 - 20
Purification and characterization of recombinant human beta 1-4 galactosyltransferase expressed in Saccharomyces cerevisiae; Krezdorn CH et al.; A protease-defective strain of Saccharomyces cerevisiae (BT 150) was used to express full-length cDNA of HeLa cell beta-D-N-acetylglucosaminide-beta-1,4-galactosyltransferase (gal-T) . To ascertain import of the recombinant gal-T into the secretory pathway of yeast cells, metabolically labeled enzyme was immunoprecipitated from extracts of yeast transformants, analysed by SDS/PAGE/fluorography and tested for sensitivity to treatment with endoglycosidase-H . Untreated recombinant gal-T had an apparent molecular mass of 48 kDa, which was reduced to 47 kDa after treatment, indicating that the recombinant enzyme was N-glycosylated and, therefore, competent for translocation across the membranes of the endoplasmic reticulum . Using specific gal-T assays employing N-acetylglucosamine or glucose in combination with alpha-lactalbumin as exogenous acceptor substrates, recombinant gal-T enzyme activity could readily be detected in crude homogenates . Analysis of the disaccharide products by 1H-NMR spectroscopy demonstrated that only beta-1-4 linkages were formed by the recombinant gal-T . The recombinant gal-T was detergent solubilized and subsequently purified by affinity chromatography on N-acetylglucosamine-derivatized Sepharose followed by alpha-lactalbumin-Sepharose . The purified enzyme preparation had a specific activity comparable to that of the soluble gal-T isolated from human milk . Furthermore, kinetic parameters determined for both acceptor and donor substrates of both enzymes differed only slightly . This work shows that yeast provides an appropriate host system for the heterologous expression of mammalian glycosyltransferases.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1252 - 6
Characterization of PDH beta 1, the structural gene for the pyruvate dehydrogenase beta subunit from Saccharomyces cerevisiae; Miran SG et al.; The gene encoding the pyruvate dehydrogenase (PDH) beta subunit (E1 beta) of the PDH complex from Saccharomyces cerevisiae has been cloned, sequenced, disrupted, and expressed . Two overlapping DNA fragments were generated from a yeast genomic DNA library by the polymerase chain reaction with synthetic oligonucleotide primers based on amino acid sequences of the yeast and human E1 beta subunits . The DNA fragments were subcloned and sequenced . The composite sequence has an open reading frame of 1098 nucleotides encoding a putative presequence of 33 amino acid residues and a mature protein of 333 residues with a calculated M(r) = 36,486 . Yeast and human E1 beta exhibit 62% sequence identity . The size of the mRNA is approximately 1.5 kilobases . Hybridization analysis showed that the E1 beta gene (PDH beta 1) is localized to chromosome II . Disruption of PDH beta 1 is not lethal under vegetative growth conditions . The null mutant transformed with PDH beta 1 on a unit-copy plasmid produced mature E1 beta and a functional PDH complex.

J Biol Chem, 1993 Feb 15, 268(5), 3594 - 603
Repression and redirection of Saccharomyces cerevisiae tRNA synthesis from upstream of the transcriptional start site; Leveillard T et al.; Derivatives of the Saccharomyces cerevisiae SUP4 tRNATyr gene with binding sites for the transcription regulatory protein GCN4 located upstream of the transcriptional start site have been constructed . The effect of GCN4 on transcription of these genes by purified RNA polymerase III and transcription factors (TF) IIIB and IIIC has been analyzed . GCN4 effectively blocks initiation of transcription only when prebound to sites that overlap with the binding site of TFIIIB . Residual GCN4-repressed transcription is significantly redirected to nearby downstream sites, the selection of which depends on the location of bound GCN4 . That prebound repressing GCN4 redirects, instead of merely blocking, the TFIIIC-dependent interaction of TFIIIB with DNA has been directly demonstrated by footprinting . The effect of GCN4 on transcription persists after it has been stripped off its DNA-binding site: once it has been redirected, DNA-bound TFIIIB remains in place, a consequence of the fact that it binds extraordinarily tightly to DNA without recognizing specific DNA sequence.

J Biol Chem, 1993 Feb 15, 268(5), 3456 - 62
Stimulation of calcium uptake in Saccharomyces cerevisiae by bovine protein kinase C alpha; Riedel H et al.; Ca2+ plays essential roles as a second messenger often in synergism with the calcium- and phospholipid-dependent phorbol ester receptor, protein kinase C (PKC), which stimulates Ca2+ influx in various cell types in a potential positive feedback mechanism . To address the compatibility of these mechanisms between lower eukaryotes and mammals, we have stably expressed bovine PKC alpha in the yeast Saccharomyces cerevisiae . We find that phorbol ester binding sites are created which stimulate a specific calcium- and phospholipid-dependent catalytic activity in vitro . Phorbol ester activation in vivo stimulates PKC down-regulation, uptake of extracellular Ca2+, Ca2+ dependence of cell viability, and changes in cell morphology . This may represent activation of a putative PKC-mediated signaling pathway utilized by functional yeast homologs of mammalian PKC isoforms . These are suggested by some protein data; however, their genes have not yet been characterized (Simon, A . J., Milner, Y., Saville, S . P., Dvir, A., Mochly-Rosen, D., and Orr, E . (1991) Proc . R . Soc . Lond . B 243, 165-171) . Our findings indicate that bovine PKC alpha is functional in yeast and stimulates calcium uptake in a manner similar to some of its responses in mammalian cells, which suggests compatible aspects of higher and lower eukaryotic signaling pathways and the feasibility of dissecting parts of the action of common signaling mediators in a simple genetic model.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1290 - 4
Futile cycles in Saccharomyces cerevisiae strains expressing the gluconeogenic enzymes during growth on glucose; Navas MA et al.; The systems which control the levels of the gluconeogenic enzymes in Saccharomyces cerevisiae have been bypassed to ascertain their physiological significance . The coding regions of the genes FBP1 and PCK1, which encode fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, have been put under the control of the promoter of ADC1 (alcohol dehydrogenase I), a gene not repressed by glucose, and introduced into yeast in multicopy plasmids . The transformed yeast cells show high levels of the gluconeogenic enzymes during growth on glucose . Generation time and growth yield of yeast expressing either fructose-1,6-bisphosphatase or phosphoenolpyruvate carboxykinase are not significantly different from those of the wild-type strain . For a strain expressing both enzymes the increase in generation time is about 20% and the decrease in growth yield around 30% . The concentration of ATP is about 1.5 mM in the growing cells of the different strains . The extent of in vivo cycling was measured by 13C NMR in cell-free extracts from yeast growing on {6-13C}glucose . Cycling between fructose-6-phosphate and fructose-1,6-bisphosphate is < 2%, most likely due to the very strong inhibition of fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate . Cycling between phosphoenolpyruvate and pyruvate is low, but a precise figure could not be obtained due to poor equilibration of label between carbons 2 and 3 of oxaloacetate.

Gene, 1993 Feb 14, 124(1), 141 - 2
The sequence of Saccharomyces cerevisiae cloning vector pCS19 allowing direct selection for DNA inserts; Sengstag C; The complete nucleotide sequence of plasmid pCS19 has been determined . This centromeric Saccharomyces cerevisiae cloning vector was designed for the construction of gene libraries, since it allows direct selection for DNA inserts.

Biochemistry, 1993 Feb 9, 32(5), 1294 - 301
Phosphomannose isomerase from Saccharomyces cerevisiae contains two inhibitory metal ion binding sites; Wells TN et al.; Phosphomannose isomerase (PMI) from Saccharomyces cerevisiae is a zinc-dependent metalloenzyme . Besides its role in catalysis, zinc is also a potent inhibitor of the enzyme . The inhibition is competitive with the substrate mannose 6-phosphate, with Kis = 6.4 microM in 50 mM Tris-HCl buffer, pH 8.0, at 37 degrees C . This inhibition constant is 4 orders of magnitude smaller than for group II divalent cations, indicating that the binding is not primarily electrostatic . Micromolar inhibition is also observed with ions of the other metals of the electronic configuration d10 . Under identical conditions, cadmium is a predominantly competitive inhibitor with Kis = 19.5 microM . Inhibition by mercury is predominantly competitive with Kis = 6.0 microM but shows a hyperbolic Dixon plot . Theorell-Yonetani double-inhibition analysis shows that zinc and cadmium ions are mutually exclusive inhibitors against mannose 6-phosphate . However, analysis of zinc and mercury double inhibition shows that they can simultaneously bind in the mannose 6-phosphate binding pocket, with only a small mutual repulsion . Inhibition of the enzyme by cadmium and zinc ions is strongly pH dependent with pKa = 9.2 for cadmium and one pKa at 6.6 and two at 8.9 for zinc . The inhibitory species are the monohydroxide forms, Zn(OH)+ and Cd(OH)+ . However, inhibition by mercury is relatively pH-independent, consistent with the neutral Hg(OH)2 being the inhibitory species . In all three cases, the metal ion binding causes a conformational change in the enzyme as judged by tryptophan fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1993 Feb 5, 268(4), 2451 - 7
The activity-controlling phosphorylation site is not the same in the four acidic ribosomal proteins from Saccharomyces cerevisiae; Naranda T et al.; By using site-directed mutagenesis and chemical analysis of phosphopeptides, a unique phosphorylation site has been shown at serine 73 in the amino acid sequence of the Saccharomyces cerevisiae acidic ribosomal protein YP1 beta (L44') . The mutation in this position prevents in vitro phosphorylation by protein kinases that modify the wild-type polypeptide . The unphosphorylatable mutated protein is unable to bind to the ribosomes and to rescue the growth deficiency of yeast strains in which the corresponding original gene is inactivated by gene disruption . Sequencing of tryptic phosphopeptides has shown that acidic proteins YP1 alpha and YP2 alpha (L44) are also phosphorylated at positions near the carboxyl end . These results contrast with the data indicating that in the highly homologous protein YP2 beta, phosphorylation takes place at serine 19, close to the amino terminus . The results show that phosphorylation is definitely required for the biological activity of these ribosomal proteins . However, the differences in the phosphorylation sites suggest that the effect of this modification is not the same in all of them, confirming the heterologous role of these peculiar ribosomal components . In fact, the different context of the modification sites in the four polypeptides suggests the existence of more than one protein kinase specific for this set of proteins.

Biochem Mol Biol Int, 1993 Feb, 29(2), 375 - 85
Effect of anisotropic inhibitors of ATP synthesis in mitochondria of Saccharomyces cerevisiae . Comparison between a wild-type and a mutant strain altered in the stoichiometry of the MT DNA encoded proteolipids of ATP synthase; Manon S et al.; The effect of a group of non-usual inhibitors of ATP synthesis was investigated in yeast mitochondria . Tetraphenylphosphonium, tetraphenylarsonium and ethidium decreased the rate of ATP synthesis but did not decrease the ATP/O ratio . They did not inhibit the ATPase activity and the translocase, and did not decrease the proton-motive force . They inhibited phosphate transport under conditions in which it was strictly energy-dependent, but did not under conditions in which it was energy-independent . These inhibitors had a poor effect on ATP synthesis in mitochondria isolated from a nuclear mutant which contained a reduced amount of the subunits 8 and 6 of the Fo part of the ATP synthase as compared to wild-type and which were unable to support any energy-dependent phosphate-transport . Thus, it appeared that the phosphate-dependence of ATP synthesis was altered in the same way by both tetraphenylphosphonium and the mutation.

Genetika, 1993 Feb, 29(2), 246 - 56
{Rec41--a new gene, participating in the control of recombination in Saccharomyces cerevisiae yeast}; Chepurnaia OV et al.; We isolated a collection of rec- mutants . The mutant rec41 from this collection was studied in detail . The mutant demonstrated a reduced level of interplasmid recombination, did not grow at the elevated temperature (36 degrees C) and was sensitive to gamma-rays but not to ultraviolet irradiation . The rec41-1 mutation behaved as a semidominant nuclear mutation . Our study on inter- and intraplasmid recombination suggested that the rec41-1 mutation decreased the level of crossing over events significantly and the frequency of gene conversion events at a lower degree . We postulate that rec41 mutation reduces the efficiency of double-stranded break repair and this results in high sensitivity of mutant cells to methylenmethan-sulfonate and ionizing radiation . Using gamma-sensitivity of the mutant cells we isolated a rec41-1 complementing plasmid from a yeast genomic DNA library . The mapping of plasmid integration site allowed us to localize the REC41 gene on the left arm of the VII chromosome nearby LYS4 and CDC43 genes.

Genetika, 1993 Feb, 29(2), 197 - 211
{Genetic control of mitotic recombination in Saccharomyces cerevisiae yeast}; Korolev VG; A review of research on genetic control of mitotic recombination in the yeast Saccharomyces cerevisiae is presented . The genes controlling different stages of mitotic recombination have been considered . Possible relationship of the recombination and repair functions of these genes is under discussion.

Yeast, 1993 Feb, 9(2), 165 - 73
The role of ALA-S and ALA-D in regulating porphyrin biosynthesis in a normal and a HEM R+ mutant strain of Saccharomyces cerevisiae; Correa Garcia S et al.; Catabolite repression and derepression on delta-aminolevulinate synthase (ALA-S) and delta-aminolevulinate dehydratase (ALA-D) in a normal yeast strain, D27, and its derived D27/C6 (HEM R+) were investigated . ALA-S and ALA-D activities and intracellular ALA (I-ALA) at different physiological states of the cells were measured . In YPD medium, under conditions of repression and when glucose was exhausted, both strains behaved identically as if the mutation was not expressed . In YPEt medium, however, both ALA-S and ALA-D activities were higher than in YPD, but the I-ALA content and the enzymic activity profiles shown by the two strains were quite different . It appears, therefore, that the mutation causes a deregulation of ALA-S, so that its activity is kept at a high level throughout the cell cycle . This would explain the increased levels of cytochromes present in the mutant . This mutation may affect some regulatory aspect of ALA formation and renders an ALA-S of high activity; moreover, this enzyme species seems to be more stable than in the normal strain.

Yeast, 1993 Feb, 9(2), 151 - 63
Cloning and genetic characterization of a calcium- and phospholipid-binding protein from Saccharomyces cerevisiae that is homologous to translation elongation factor-1 gamma; Kambouris NG et al.; We have isolated a gene (CAM1) from the yeast Saccharomyces cerevisiae that encodes a protein homologous to the translational cofactor elongation factor-1 gamma (EF-1 gamma) first identified in the brine shrimp Artemia salina . The predicted Cam1 amino acid sequence consists of 415 residues that share 32% identity with the Artemia protein, increasing to 72% when conservative substitutions are included . The calculated M(r) of Cam1p (47,092 Da) is in close agreement with that of EF-1 gamma (M(r) = 49,200 Da), and hydropathy plots of each protein exhibit strikingly similar profiles . Disruption of the CAM1 locus yields four viable meiotic progeny, indicating that under normal growth conditions the Cam1 protein is non-essential . Attempts to elicit a translational phenotype have been unsuccessful . Since EF-1 gamma participates in the regulation of a GTP-binding protein (EF-1 alpha), double mutants with cam1 disruptions and various mutant alleles of known GTP-binding proteins were constructed and examined . No evidence was found for an interaction of CAM1 with TEF1, TEF2, SEC4, YPT1, RAS1, RAS2, CDC6, ARF1, ARF2 or CIN4 . The possibility that Cam1p may play a redundant role in the regulation of protein synthesis or another GTP-dependent process is discussed.

Yeast, 1993 Feb, 9(2), 141 - 50
Cloning and genetic analysis of the gene encoding a new protein kinase in Saccharomyces cerevisiae; Kambouris NG et al.; We have isolated a single gene from the yeast Saccharomyces cerevisiae encoding a potential 800 amino acid polypeptide of calculated M(r) 90,098 Da . This protein consists of an N-terminal region that shares significant homology with the catalytic domains of several serine- and threonine-specific protein kinases, as well as a large, unique, C-terminal domain of unknown function . Haploid disruption mutants are viable and do not exhibit any readily observable growth defects under varying conditions of temperature, nutrients or osmotic strength . Due to the apparent structural similarity between this kinase and the protein products of the KIN1 and KIN2 genes, we have chosen to name this new gene KIN3.

Yeast, 1993 Feb, 9(2), 111 - 9
Galactose inhibition of the constitutive transport of hexoses in Saccharomyces cerevisiae; Nevado J et al.; The relationship between the pathways of glucose and galactose utilization in Saccharomyces cerevisiae has been studied . Galactose (which is transported and phosphorylated by inducible systems) is a strong inhibitor of the utilization of glucose, fructose and mannose (which have the same constitutive transport and phosphorylation systems) . Conversely, all these three hexoses inhibit the utilization of galactose, though with poor efficiency . These cross-inhibitions only occur in yeast adapted to galactose or in galactose-constitutive mutants . The efficiency of galactose as inhibitor is even greater than the efficiencies of the other three hexoses to inhibit the utilization of each other . Phosphorylation is not involved in the inhibition and the transport of sugars is the affected step . The cross-inhibitions between galactose and either glucose, fructose or mannose do not implicate utilization of one hexose at the expense of the other, as it occurs in the mutual interactions between the latter three sugars . It seems that, by growing the yeast in galactose, a protein component is synthesized, or alternatively modified, that once bound to either galactose or any one of the other three hexoses (glucose, fructose or mannose), cross-interacts respectively with the constitutive or the inducible transport systems, impairing their function.

Genome, 1993 Feb, 36(1), 32 - 42
Sequencing of chromosome I from Saccharomyces cerevisiae: analysis of a 32 kb region between the LTE1 and SPO7 genes; Ouellette BF et al.; The DNA sequencing and preliminary functional analysis of a 32 kb section of yeast chromosome I has been completed . This region lies on the left arm of the chromosome between the LTE1 and SPO7 genes and contains 14 open reading frames (ORFs) positioned closely together, with an average spacing of approximately 350 nucleotides between coding regions . Three of these ORFs correspond to previously identified genes, a further three show significant homology with other proteins, while the remaining eight ORFs share no significant homology to genes in the databases.

Chromosoma, 1993 Feb, 102(3), 207 - 17
Saccharomyces cerevisiae linear chromosome stability (lcs) mutants increase the loss rate of artificial and natural linear chromosomes; Runge KW et al.; We isolated mutants of Saccharomyces cerevisiae that lose a 100 kb linear yeast artificial chromosome (YAC) at elevated rates . Mutations in two of these LCS (linear chromosome stability) genes had little or no effect on the loss rate of a circular YAC that had the same centromere and origin of replication as present on the linear YAC . Moreover, mutations in these LCS genes also increased the loss rate of an authentic linear yeast chromosome, chromosome III, but had only small effects on the loss rate of a circular derivative of chromosome III . As these mutants preferentially destabilize linear chromosomes, they may affect chromosome stability through interactions at telomeres . Telomeres are thought to be essential for the protection and complete replication of chromosome ends . The cytological properties of telomeres suggest that these structures may play additional roles in chromosome function . The lengths of the terminal C1-3A repeats at the ends of yeast chromosomes were unaltered in the linear preferential lcs mutants, suggesting that these mutants do not affect the replication or protection of telomeric DNA . Thus, the linear-preferential lcs mutants may identify a role for telomeres in chromosome stability that is distinct from their function in the replication and protection of chromosomal termini.

Mol Gen Genet, 1993 Feb, 237(1-2), 17 - 25
Cloning and expression of the UGA4 gene coding for the inducible GABA-specific transport protein of Saccharomyces cerevisiae; Andre B et al.; Transport of 4-aminobutyric acid (GABA) in Saccharomyces cerevisiae is mediated by three transport systems: the general amino acid permease (GAP1 gene), the proline permease (PUT4 gene), and a specific GABA permease (UGA4 gene) which is induced in the presence of GABA . The UGA4 gene encoding the inducible GABA-specific transporter was cloned and sequenced and its expression analyzed . The predicted amino acid sequence shows that UGA4 encodes a 62 kDa protein having 9-12 putative membrane-spanning regions . The predicted UGA4 protein shares significant sequence similarity with the yeast choline transporter (CTR gene), exhibiting but limited similarity to the previously reported GABA transporters, i.e . the yeast GAP1 and PUT4 permeases and the rat brain GAT-1 transporter . Induction of UGA4 in the presence of GABA is exerted at the level of UGA4 mRNA accumulation, most probably at the level of transcription itself . This induction is conferred by the 5' flanking region and requires the integrity of two positive regulatory proteins, the inducer-specific factor UGA3 and the pleiotropic factor UGA35/DURL/DAL81 . In the absence of the pleiotropic UGA43/DAL80 repressor, UGA4 is constitutively expressed at high level.

Photochem Photobiol, 1993 Feb, 57(2), 272 - 8
Attempted biostimulation of division in Saccharomyces cerevisiae using red coherent light; Quickenden TI et al.; Replicate cultures of the yeast Saccharomyces cerevisiae were irradiated with 632.8 nm coherent light from He-Ne lasers at irradiances of 6.5 x 10(15) and 1.0 x 10(16) photons s-1 cm-2 . Irradiation periods ranged from 0 to 652 min, and cultures were grown until well into the exponential phase . Unirradiated control cultures were grown alongside the irradiated cultures under otherwise identical conditions . The extents of growth in the control and irradiated cultures were compared spectrophotometrically at the end of each experiment . Contrary to the expectations of Karu et al . (e.g . Karu, 1988, Lasers Life Sci . 2, 53-74) no growth enhancement was found in the irradiated cultures, but a mild inhibitory effect was observed.

Genes Dev, 1993 Feb, 7(2), 241 - 9
Homology with Saccharomyces cerevisiae RNA14 suggests that phenotypic suppression in Drosophila melanogaster by suppressor of forked occurs at the level of RNA stability; Mitchelson A et al.; The suppressor of forked {su(f)} locus of Drosophila melanogaster encodes at least one cell-autonomous vital function . Mutations at su(f) can affect the expression of unlinked genes where retroviral-like transposable elements are inserted . Changes in phenotype are correlated with changes in mRNA profiles, indicating that su(f) affects the production and/or stability of mRNAs . We have cloned the su(f) gene by P-element transposon tagging . Alterations in the DNA map of eight lethal alleles were detected in a 4.3-kb region . P-element-mediated transformation using a fragment including this interval rescued all aspects of the su(f) mutant phenotype . The gene is transcribed to produce a major 2.6-kb RNA and minor RNAs of 1.3 and 2.9 kb, which are present throughout development, being most abundant in embryos, pupae, and adult females . The major predicted gene product is an 84- kD protein that is homologous to RNA14 of Saccharomyces cerevisiae, a vital gene where mutation affects mRNA stability . This suggests that phenotypic modification by su(f) occurs at the level of RNA stability.

Genetics, 1993 Feb, 133(2), 171 - 82
Telomere-mediated plasmid segregation in Saccharomyces cerevisiae involves gene products required for transcriptional repression at silencers and telomeres; Longtine MS et al.; Plasmids that contain Saccharomyces cerevisiae TG1-3 telomere repeat sequences (TRS plasmids) segregate efficiently during mitosis . Mutations in histone H4 reduce the efficiency of TRS-mediated plasmid segregation, suggesting that chromatin structure is involved in this process . Sir2, Sir3 and Sir4 are required for the transcriptional repression of genes located at the silent mating type loci (HML and HMR) and at telomeres (telomere position effect) and are also involved in the segregation of TRS plasmids, indicating that TRS-mediated plasmid segregation involves factors that act at chromosomal telomeres . TRS plasmid segregation differes from the segregation of plasmids carrying the HMR E silencing region: HMR E plasmid segregation function is completely dependent upon Sir2, Sir3 and Sir4, involves Sir1 and is not influenced by mutations in RAP 1 that eliminate TRS plasmid segregation . Mutations in SIR1, SIN1, TOP1, TEL1 and TEL2 do not influence TRS plasmid segregation . Unlike transcriptional repression at telomeres, TRS plasmids retain partial segregation function in sir2, sir3, sir4, nat1 and ard1 mutant strains . Thus it is likely that TRS plasmid segregation involves additional factors that are not involved in telomere position effect.

Genetics, 1993 Feb, 133(2), 149 - 58
Meiotic nondisjunction and recombination of chromosome III and homologous fragments in Saccharomyces cerevisiae; Goldway M et al.; A yeast strain, in which nondisjunction of chromosome III at the first meiotic division could be assayed, was constructed . Using chromosome fragmentation plasmids, chromosomal fragments (CFs) were derived in isogenic strains from six sites along chromosome III and one site on chromosome VII . Whereas the presence of the CFs derived from chromosome III increased considerably the meiosis I nondisjunction of that chromosome, the CF derived from chromosome VII had no effect on chromosome III segregation . The effects of the chromosome III-derived fragments were not linearly related to fragment length . Two regions, one of 12 kb in size located at the left end of the chromosome, and the other of 5 kb, located at the center of the right arm, were found to have profound effects on chromosome III nondisjunction . Most disomics arising from meioses in strains containing chromosome III CFs did not contain the CF; thus it appears that the two chromosome III homologs had segregated away from the CF . Among the disomics, recombination between the homologous chromosomes III was lower than expected from the genetic distance, while recombination between one of the chromosomes III and the fragment was frequent . We suggest that there are sites along the chromosome that are more involved than others in the pairing of homologous chromosomes and that the pairing between fragment and homologs involves recombination among these latter elements.

Arch Biochem Biophys, 1993 Feb 1, 300(2), 724 - 33
The structural requirements of sterols for membrane function in Saccharomyces cerevisiae; Nes WD et al.; Cultures of Saccharomyces cerevisiae strain GL7 auxotrophic for sterol were incubated with a series of sterols and sterol-like molecules (tetracyclic and pentacyclic triterpenoids) in order to determine the structural requirements of sterols for bulk membrane function . For growth support, the 3 beta-OH group could not be replaced by H, OMe, OBu, NH2, NHOH, OAc, keto, or 3 alpha-OH . A methyl group at C-14 was neither deleterious nor essential for activity . Removal of the C-4 geminal methyl group was obligatory for activity . Thus, no sterol-like triterpenoid supported growth (e.g., tetrahymanol, lanosterol, and cycloartenol) . Growth support required a sterol with the longest methylene segment extending from C-20 not to exceed six contiguous C-atoms and the stereochemistry must be C-20 R . No significance could be attributed to branching at C-20 (i.e., to C-21), C-24 (when alkylated), or C-25 (regarding the isopropyl group) . Double bonds in the nucleus were not essential for activity since cholestanol supported growth . In several incubations, the addition of trace levels of dietary ergosterol (0.5 microgram/ml) to the medium was necessary to promote growth and transformation of the bulk sterol to a membrane competent sterol(s).

J Bacteriol, 1993 Feb, 175(4), 941 - 51
Participation of RAP1 protein in expression of the Saccharomyces cerevisiae arginase (CAR1) gene; Kovari LZ et al.; Regulated expression of the inducible arginase (CAR1) gene of Saccharomyces cerevisiae has been shown to require three upstream activation sequences (UASs) and an upstream repression sequence, URS1 . Two of the UAS elements, UASC1 and UASC2, operate in an inducer-independent manner, while the third, UASI, is inducer dependent . UASC1 and UASC2 were previously shown to contain ABF-1 binding sites that were required for normal transcription . In this work, we demonstrate that UASC1 and UASC2 also contain two and three sites, respectively, that are able to bind RAP1 protein . RAP1 binding to these sites, however, is significantly weaker than that to sites in TEF2 and HMRE . The effects of mutating the sites individually or in combination suggest that at least three of them, two in UASC1 and one in UASC2, probably participate in CAR1 expression.

J Cell Biol, 1993 Feb, 120(3), 665 - 73
Two independent peroxisomal targeting signals in catalase A of Saccharomyces cerevisiae; Kragler F et al.; In contrast to many other peroxisomal proteins catalase A contains at least two peroxisomal targeting signals each sufficient to direct reporter proteins to peroxisomes . One of them resides at the extreme carboxy terminus constituting a new variant of this signal, -SSNSKF, not active in monkey kidney cells (Gould, S . J., G . A . Keller, N . Hosken, J . Wilkinson, and S . Subramani 1989 . J . Cell Biol . 108:1657-1664) . However, this signal is completely dispensable for import of catalase A itself . In its amino-terminal third this protein contains another peroxisomal targeting signal sufficient to direct reporter proteins into microbodies . This internal signal depends on the context . The nature of this targeting signal might be a short defined sequence or a structural feature recognized by import factors . In addition, we have demonstrated that the carboxy-terminal seven amino acids of citrate synthase of Saccharomyces cerevisiae encoded by CIT2 and containing the canonical -SKL represents a targeting signal sufficient to direct reporter proteins to peroxisomes.

Mol Cell Biol, 1993 Feb, 13(2), 1212 - 21
p62cdc23 of Saccharomyces cerevisiae: a nuclear tetratricopeptide repeat protein with two mutable domains; Sikorski RS et al.; CDC23 is required in Saccharomyces cerevisiae for cell cycle progression through the G2/M transition . The CDC23 gene product contains tandem, imperfect repeats, termed tetratricopeptide repeats, (TPR) units common to a protein family that includes several other nuclear division CDC genes . In this report we have used mutagenesis to probe the functional significance of the TPR units within CDC23 . Analysis of truncated derivatives indicates that the TPR block of CDC23 is necessary for the function or stability of the polypeptide . In-frame deletion of a single TPR unit within the repeat block proved sufficient to inactivate CDC23 in vivo, though this allele could rescue the temperature-sensitive defect of a cdc23 point mutant by intragenic complementation . By both in vitro and in vivo mutagenesis techniques, 17 thermolabile cdc23 alleles were produced and examined . Fourteen alleles contained single amino acid changes that were found to cluster within two distinct mutable domains, one of which encompasses the most canonical TPR unit found in CDC23 . In addition, we have characterized CDC23 as a 62-kDa protein (p62cdc23) that is localized to the yeast nucleus . Our mutagenesis results suggest that TPR blocks form an essential domain within members of the TPR family.

Mol Cell Biol, 1993 Feb, 13(2), 1069 - 77
Analysis of the SWI4/SWI6 protein complex, which directs G1/S-specific transcription in Saccharomyces cerevisiae; Sidorova J et al.; SWI4 and SWI6 play a crucial role in START-specific transcription in Saccharomyces cerevisiae . SWI4 and SWI6 form a specific complex on the SCB (SWI4/6-dependent cell cycle box) sequences which have been found in the promoters of HO and G1 cyclin genes . Overproduction of SWI4 eliminates the SWI6 dependency of HO transcription in vivo and results in a new SWI6-independent, SCB-specific complex in vitro, which is heterogeneous and reacts with SWI4 antibodies . The C terminus of SWI4 is not required for SWI6-independent binding of SWI4 to SCB sequences, but it is necessary and sufficient for association with SWI6 . Both SWI4 and SWI6 contain two copies of a 33-amino-acid TPLH repeat, which has been implicated in protein-protein interactions in other proteins . These repeats are not required for the SWI4-SWI6 association . Alanine substitutions in both TPLH repeats of SWI6 reduce its activity but do not affect the stability of the protein or its association with SWI4 . However, these mutations reduce the ability of the SWI4/6 complex to bind DNA . Deletion of the lucine zipper motif in SWI6 also allows SWI4/6 complex formation, but it eliminates the DNA-binding ability of the SWI4/6 complex . This indicates that the integrity of two different regions of SWI6 is required for DNA binding by the SWI4/6 complex . From these data, we propose that the sequence-specific DNA-binding domain resides in SWI4 but that SWI6 controls the accessibility of this domain in the SWI4/6 complex.

Mol Cell Biol, 1993 Feb, 13(2), 1051 - 8
DNA repair synthesis during base excision repair in vitro is catalyzed by DNA polymerase epsilon and is influenced by DNA polymerases alpha and delta in Saccharomyces cerevisiae; Wang Z et al.; Base excision repair is an important mechanism for correcting DNA damage produced by many physical and chemical agents . We have examined the effects of the REV3 gene and the DNA polymerase genes POL1, POL2, and POL3 of Saccharomyces cerevisiae on DNA repair synthesis is nuclear extracts . Deletional inactivation of REV3 did not affect repair synthesis in the base excision repair pathway . Repair synthesis in nuclear extracts of pol1, pol2, and pol3 temperature-sensitive mutants was normal at permissive temperatures . However, repair synthesis in pol2 nuclear extracts was defective at the restrictive temperature of 37 degrees C and could be complemented by the addition of purified yeast DNA polymerase epsilon . Repair synthesis in pol1 nuclear extracts was proficient at the restrictive temperature unless DNA polymerase alpha was inactivated prior to the initiation of DNA repair . Thermal inactivation of DNA polymerase delta in pol3 nuclear extracts enhanced DNA repair synthesis approximately 2-fold, an effect which could be specifically reversed by the addition of purified yeast DNA polymerase delta to the extract . These results demonstrate that DNA repair synthesis in the yeast base excision repair pathway is catalyzed by DNA polymerase epsilon but is apparently modulated by the presence of DNA polymerases alpha and delta.

Yeast, 1993 Feb, 9(2), 127 - 39
Purification and characterization of highly active and stable polyphosphatase from Saccharomyces cerevisiae cell envelope; Andreeva NA et al.; Saccharomyces cerevisiae cell envelope polyphosphatase was isolated in highly active and stable form by extraction from cells with zwittergent TM-314 followed by chromatography of the extract on phosphocellulose and QAE-Sephadex in the presence of 5 mM-MgCl2, 0.5 mM-EDTA and 0.1% Triton X-100 . The enzyme possessed a specific activity of 220 U/mg and after 30 days retained 87% of its activity at -20 degrees C . Polyphosphatase molecular mass was determined to be about 40 kDa by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate . The enzyme hydrolysed polyphosphates with various chain lengths (n = 3-208), had low activity for GTP and did not split pyrophosphate, ATP and p-nitrophenylphosphate . On polyphosphates with chain lengths n = 3, 9 and 208, Km values were 1.7 x 10(-4), 1.5 x 10(-5) and 8.8 x 10(-7) M respectively . Polyphosphatase was most active and stable at pH 6.0-8.0 . The enzyme showed maximal activity at 50 degrees C . The time of half inactivation of polyphosphatase at 40, 45 and 50 degrees C was 45, 10 and 3 min, respectively . In the absence of divalent cations and also with Ca2+ or Cu2+, the enzyme showed practically no activity . The ability of divalent cations to activate polyphosphatase was reduced in the following order: Co2+ > Mg2+ > Mn2+ > Fe2+ > Zn2+ . Polyphosphatase was completely inhibited by 1 mM-ammonium molybdate and 50 microM-Zn2+ or Cu2+ (in the presence of Mg2+).

Mol Biol Cell, 1993 Feb, 4(2), 195 - 208
Regulation of Saccharomyces cerevisiae CDC7 function during the cell cycle; Yoon HJ et al.; The yeast Cdc7 function is required for the G1/S transition and is dependent on passage through START, a point controlled by the Cdc28/cdc2/p34 protein kinase . CDC7 encodes a protein kinase activity, and we now show that this kinase activity varies in the cell cycle but that protein levels appear to remain constant . We present several lines of evidence that periodic activation of CDC7 kinase is at least in part through phosphorylation . First, the kinase activity of the Cdc7 protein is destroyed by dephosphorylation of the protein in vitro with phosphatase . Second, Cdc7 protein is hypophosphorylated and inactive as a kinase in extracts of cells arrested at START but becomes active and maximally phosphorylated subsequent to passage through START . The phosphorylation pattern of Cdc7 protein is complex . Phosphopeptide mapping reveals four phosphopeptides in Cdc7 prepared from asynchronous yeast cells . Both autophosphorylation and phosphorylation in trans appear to contribute to this pattern . Autophosphorylation is shown to occur by using a thermolabile Cdc7 protein . A protein in yeast extracts can phosphorylate and activate Cdc7 protein made in Escherichia coli, and phosphorylation is thermolabile in cdc28 mutant extracts . Cdc7 protein carrying a serine to alanine change in the consensus recognition site for Cdc28 kinase shows an altered phosphopeptide map, suggesting that this site is important in determining the overall Cdc7 phosphorylation pattern.

Mol Cell Biol, 1993 Feb, 13(2), 1034 - 41
Heat shock-mediated cell cycle blockage and G1 cyclin expression in the yeast Saccharomyces cerevisiae; Rowley A et al.; For cells of the yeast Saccharomyces cerevisiae, heat shock causes a transient inhibition of the cell cycle-regulatory step START . We have determined that this heat-induced START inhibition is accompanied by decreased CLN1 and CLN2 transcript abundance and by possible posttranscriptional changes to CLN3 (WHI1/DAF1) cyclin activity . Persistent CLN2 expression from a heterologous promoter or the CLN2-1 or CLN3-1 alleles that are thought to encode cyclin proteins with increased stability eliminated heat-induced START inhibition but did not affect other aspects of the heat shock response . Heat-induced START inhibition was shown to be independent of functions that regulate cyclin activity under other conditions and of transcriptional regulation of SWI4, an activator of cyclin transcription . Cells lacking Bcy1 function and thus without cyclic AMP control of A kinase activity were inhibited for START by heat shock as long as A kinase activity was attenuated by mutation . We suggest that heat shock mediates START blockage through effects on the G1 cyclins.

Mol Cell Biol, 1993 Feb, 13(2), 1023 - 33
A mobile group I intron from Physarum polycephalum can insert itself and induce point mutations in the nuclear ribosomal DNA of saccharomyces cerevisiae; Muscarella DE et al.; Pp LSU3 is a mobile group I intron in the extrachromosomal nuclear ribosomal DNA (rDNA) of Physarum polycephalum . As found for other mobile introns, Pp LSU3 encodes a site-specific endonuclease, I-Ppo, which mediates "homing" to unoccupied target sites in Physarum rDNA . The recognition sequence for this enzyme is conserved in all eucaryotic nuclear rDNAs . We have introduced this intron into a heterologous species, Saccharomyces cerevisiae, in which nuclear group I introns have not been detected . The expression of Pp LSU3, under control of the inducible GAL10 promoter, was found to be lethal as a consequence of double-strand breaks in the rDNA . However, surviving colonies that are resistant to the lethal effects of I-Ppo because of alterations in the rDNA at the cleavage site were recovered readily . These survivors are of two classes . The first comprises cells that acquired one of three types of point mutations . The second comprises cells in which Pp LSU3 became inserted into the rDNA . In both cases, each resistant survivor appears to carry the same alterations in all approximately 150 rDNA repeats . When it is embedded in yeast rDNA, Pp LSU3 leads to the synthesis of I-Ppo and appears to be mobile in appropriate genetic crosses . The existence of yeast cells carrying a mobile intron should allow dissection of the steps that allow expression of the highly unusual I-Ppo gene.

Appl Microbiol Biotechnol, 1993 Feb, 38(5), 624 - 30
Biologically active human and mouse nerve growth factors secreted by the yeast Saccharomyces cerevisiae; Nishizawa M et al.; Nerve growth factor (NGF) is a trophic agent that is essential for the development and survival of sympathetic and sensory nerves . A chemically-synthesized DNA fragment encoding human NGF (hNGF) and a cDNA encoding mouse NGF (mNGF) were engineered for expression in the yeast, Saccharomyces cerevisiae . Expression and secretion of hNGF and mNGF was attempted under the direction of the yeast PGK promoter and with various leader sequences . Among the leader sequences tested, that of the yeast alpha-factor successfully directed secretion of both hNGF and mNGF that were correctly processed . The content of the recombinant NGF (reNGF) in the culture supernatant was estimated to be 1 microgram/ml . The yeast-produced reNGF was able to bind to NGF receptors in rat pheochromocytoma (PC12) cells as efficiently as the standard mNGF, and partially purified reNGF could induce neurite outgrowth of PC12 cells . Thus, we have demonstrated that biologically active human and mouse reNGF can be produced in yeast cells.

J Biotechnol, 1993 Feb, 27(3), 295 - 305
Phenotypic effects in Saccharomyces cerevisiae after regulated expression of beta-(1,3)-glucanase from Nicotiana plumbaginifolia; Demolder J et al.; A recombinant yeast strain was constructed of which the cell wall porosity could be reversibly and conditionally modulated . This strain expresses the Nicotiana plumbaginifolia beta-(1,3)-glucanase under control of the Saccharomyces cerevisiae GAL1 promoter and the mating factor alpha 1 prepro-sequence . The following phenotypic effects were observed after expression of this enzyme: (a) expressed beta-(1,3)-glucanase is toxic to the producing yeast cells, which is reflected by a strong growth inhibition; as beta-(1,3)-glucanase could be detected only inside the cells, it seems to interfere with cell wall growth from within the cell; (b) after induction of glucanase, the recombinant strain lost up to 20% of some periplasmic enzymes, as evidenced by the release of normally periplasmic-associated invertase; (c) preliminary growth in a synthetic medium containing galactose significantly increased the transformation efficiency of the recombinant yeast strain.

Gene, 1993 Jan 30, 123(2), 283 - 5
Characterization of cDNA clones encoding the human homologue of Saccharomyces cerevisiae ribosomal protein L30; Johnson KR; We have isolated cDNA clones encoding the human homologue (hL30) of yeast ribosomal protein (r-protein) L30 . The hL30 nucleotide (nt) sequence shows high homology to the yeast sequences and also to a partial Xenopus laevis sequence previously identified as an immunoglobulin heavy chain . The 5' end of hL30 is pyrimidine-rich, as is the case for most other mammalian r-protein mRNAs . The open reading frame consists of 157 codons with a C-terminal region that is different from corresponding regions of the yeast proteins . In several human tissue culture cells, the mRNA encoding hL30 is approx . 700 nt in length.

FEBS Lett, 1993 Jan 25, 316(2), 165 - 9
Cloning and characterization of the elongation factor EF-1 beta homologue of Saccharomyces cerevisiae . EF-1 beta is essential for growth; Hiraga K et al.; A Saccharomyces cerevisiae cDNA homologue of the elongation factor EF-1 beta was found among the clones obtained by immunoscreening of a yeast cDNA expression library with an antibody against calmodulin affinity-purified proteins . The cDNA encoded a protein of 206 amino acids which was highly homologous (about 70% homology) with Artemia salina and human EF-1 beta . A protein with an apparent molecular mass of 33,000, significantly larger than that expected from the gene, was identified by Western blotting . Gene disruption experiments indicated that EF-1 beta is essential for growth.

Proc Natl Acad Sci U S A, 1993 Jan 15, 90(2), 452 - 6
A dominant truncation allele identifies a gene, STE20, that encodes a putative protein kinase necessary for mating in Saccharomyces cerevisiae; Ramer SW et al.; This work reports the identification, characterization, and nucleotide sequence of STE20, a newly discovered gene involved in the Saccharomyces cerevisiae mating response pathway, to date one of the best understood signal transduction pathways . STE20 encodes a putative serine/threonine-specific protein kinase with a predicted molecular mass of 102 kDa . Its expression pattern is similar to that of several other protein kinases in the mating response pathway . Deletion of the kinase domain of STE20 causes sterility in both haploid mating types . This sterility can be partially suppressed by high-level production of STE12 but is not suppressible by high levels of STE4 or a dominant STE11 truncation allele . A truncation allele of STE20 was isolated that can activate the mating response pathway in the absence of exogenous mating pheromone . This allele causes dominant growth arrest that cannot be suppressed by deletions of STE4, STE5, STE7, STE11, or STE12 . The allele is able to suppress the mating defect of a strain in which the STE20 kinase domain has been deleted, but not the mating defects of strains carrying mutations in STE4, STE5, STE7, STE11, or STE12.

J Biol Chem, 1993 Jan 15, 268(2), 961 - 7
VMA12 is essential for assembly of the vacuolar H(+)-ATPase subunits onto the vacuolar membrane in Saccharomyces cerevisiae; Hirata R et al.; vma12 mutants of the yeast Saccharomyces cerevisiae, which were originally identified as calcium-sensitive (cls) mutants that were also respiratory deficient (Pet-), have a defect in vacuolar membrane H(+)-ATPase activity (Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku, Y . (1991) J . Biol . Chem . 266, 13971-13977) . The VMA12 gene was cloned by complementation of the growth defects of vma12 mutants . The nucleotide sequence of the gene predicts a polypeptide of 215 amino acids (25.2 kDa) with two putative membrane-spanning domains . A null vma12 mutant, constructed by chromosomal deletion of the gene, is viable but has completely lost the vacuolar membrane H(+)-ATPase activity and exhibits the same growth defects as observed for the original vma12 mutants . Synthesis and targeting of the subunits of the H(+)-ATPase in the delta vma12 mutant cells were examined by Western blotting analyses of whole cell and vacuolar membrane protein extracts . None of the peripheral membrane subunits that we analyzed (the 69-, 60-, 42-, and 27-kDa subunits) was detected in the vacuolar membrane fractions, although the cellular levels of these polypeptides appeared to be normal . The 100- and 17-kDa integral membrane subunits of the enzyme were absent or present at a substantially reduced level in mutant vacuolar membrane fractions . Anti-Vma12p antibodies recognized a vacuolar protein with the expected molecular mass of 25 kDa . However, the Vma12 protein was not detected in the vacuolar membrane ATPase complex that had been solubilized with a zwitterionic detergent, ZW3-14, and purified by glycerol gradient centrifugation (Kane, P . M., Yamashiro, C . T., and Stevens, T . H . (1989) J . Biol . Chem . 264, 19236-19244) . These results indicate that the VMA12 gene product is not a component of the active vacuolar ATPase complex and instead suggest that this protein is required during the process of assembly and/or targeting of the enzyme complex to the vacuolar membrane.

J Biol Chem, 1993 Jan 15, 268(2), 1252 - 9
Purification and characterization of the SRS2 DNA helicase of the yeast Saccharomyces cerevisiae; Rong L et al.; The SRS2 gene of Saccharomyces cerevisiae was identified through mutational analysis as a suppressor of radiation-sensitive mutations in the error-prone repair pathway and by a hyper-recombination phenotype . Comparison of the derived amino acid sequence revealed the gene to have high homology to the bacterial DNA helicases UvrD and Rep (Aboussekhra, A., Chanet, R., Zgaga, Z., Cassier-Chauvat, C., Heude, M., and Fabre, F . (1989) Nucleic Acids Res . 17, 7211-7219) . We have purified the SRS2 protein from Escherichia coli extracts by tagging the SRS2 gene with 6 carboxyl-terminal histidine residues and overexpressing the tagged protein in a pET-3c vector . Extracts were passed over a metal-chelating affinity chromatography column followed by gel filtration to obtain an enriched protein fraction . Sephacryl gel filtration of pooled fractions containing the SRS2 protein yielded purified SRS2 protein by Coomassie Blue stain of SDS-polyacrylamide gel electrophoresis gels . The purified SRS2 protein was found to have in vitro DNA-dependent ATPase and DNA helicase activities . The polarity of the helicase activity was determined to be 3' to 5', the same polarity as that found for the UvrD and Rep proteins . The carboxyl-terminal region of the protein is shown to contain a sequence for nuclear localization . Expression of the SRS2 in yeast was examined and found to be extremely low.

J Biol Chem, 1993 Jan 15, 268(2), 982 - 90
Purification and characterization of the Saccharomyces cerevisiae DNA polymerase delta overproduced in Escherichia coli; Brown WC et al.; In order to further define the enzymatic properties of yeast DNA polymerase delta, the Saccharomyces cerevisiae POL3 gene, whose expression is highly toxic to bacteria in most cloning vectors, was cloned into a new T7 expression vector (W . C . Brown and J . L . Campbell, submitted for publication) which allowed efficient overexpression in bacteria . Fifteen mg of polymerase were obtained from 3 g of cells . Since the protein is produced in insoluble form, to obtain active polymerase, inclusion bodies were solubilized with urea . DNA polymerase delta (124 kDa) was purified in the presence of urea and then renatured by dialysis against buffers containing decreasing concentrations of urea . Optimal protein concentration for refolding was 5 micrograms/ml . By several criteria the enzyme obtained is comparable with that from yeast: specific activity, electrophoretic mobility, template preference, sensitivity to inhibitors, and processivity . The electrophoretic mobility suggests that, unlike DNA polymerase alpha, polymerase delta is not posttranslationally modified in yeast . Polyclonal antibody was raised against the full-length DNA polymerase delta from bacteria and shown to cross-react with the protein purified from yeast on protein blots . The renatured protein also exhibits an exonucleolytic activity . Further examination of this nuclease determined it to be a 3' to 5' exonuclease with the characteristics of a proofreading activity . The presence of this nuclease in the highly purified bacterial polymerase provides biochemical confirmation of earlier genetic evidence (Simon, M., Giot, L., and Faye, G . (1991) EMBO J . 10, 2165-2170) that suggested that DNA polymerase delta's core catalytic subunit contains an intrinsic 3' to 5' exonuclease.

Science, 1993 Jan 8, 259(5092), 216 - 9
An inhibitor of p34CDC28 protein kinase activity from Saccharomyces cerevisiae; Mendenhall MD; The p34CDC28 protein from Saccharomyces cerevisiae is a homolog of the p34cdc2 protein kinase, a fundamental regulator of cell division in all eukaryotic cells . Once activated it initiates the visible events of mitosis (chromosome condensation, nuclear envelope breakdown, and spindle formation) . The p34CDC28 protein also has a critical role in the initiation of DNA synthesis . The protein kinase activity is regulated by cycles of phosphorylation and dephosphorylation and by periodic association with cyclins . An endogenous 40-kilodalton protein (p40) originally identified as a substrate of the p34CDC28 protein kinase was purified . The p40 protein bound tightly to p34CDC28 and inhibited the activity of the kinase . The p40 protein may provide another mechanism to regulate p34CDC28 protein kinase activity.

J Biol Chem, 1993 Jan 5, 268(1), 217 - 20
Identity of Saccharomyces cerevisiae tRNA(Trp) is not changed by an anticodon mutation that creates an amber suppressor; Yesland KD et al.; A C35-->T mutation in an Escherichia coli tRNA(Trp) gene creates an amber suppressor which efficiently inserts glutamine in response to UAG codons in vivo (Soll, L., and Berg, P . (1969) Nature 223, 1340-1342) . We have introduced the same change in a yeast tRNA(Trp) gene and demonstrated that the tRNA acts as an efficient amber suppressor in vivo . Amino acid sequence analyses were performed on chitinase produced by cells carrying the corresponding gene with a UAG codon at position 8 of the mature protein plus the mutant tRNA(Trp) gene . In contrast to comparable experiments with E . coli, tryptophan is inserted at a frequency > or = 80% by the yeast suppressor tRNA(Trp) . Furthermore, in vitro charging experiments with the mutant tRNA(Trp) reveal no detectable increase in glutamine acceptor activity results from the C35-->T transition . The identity elements in E . coli tRNA(Gln) are well characterized (Jahn, M., Rogers, J., and Soll, D . (1991) Nature 352, 258-260) . Sequence comparisons of the tRNA(Trp) and tRNA(Gln) molecules from E . coli reveal that the amber suppressor tRNA(Trp) has four of five identity elements required for glutaminyl-tRNA synthetase recognition . A similar comparison in the yeast system shows only two of the five potential identity elements are present . We conclude that, in spite of substantial structural similarities between yeast and E . coli aminoacyl-tRNA synthetases, fundamental differences can exist with regard to tRNA recognition.

J Biol Chem, 1993 Jan 5, 268(1), 153 - 60
Cloning and characterization of the Saccharomyces cerevisiae gene encoding NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase; West MG et al.; Saccharomyces cerevisiae possess a monofunctional, cytoplasmic NAD-dependent 5,10-methylenetetrahydrofolate (THF) dehydrogenase that converts 5,10-methylene-THF to 5,10-methenyl-THF (Barlowe, C . K., and Appling, D.R . (1990) Biochemistry 29, 7089-7094) . We have now isolated the gene encoding this enzyme from a yeast genomic library using oligonucleotide probes based on internal peptide sequences from the purified protein . Nucleotide sequence analysis reveals a 320-amino acid open reading frame that contains both of the internal peptide sequences . The predicted molecular weight (36,236) is consistent with the estimated size (33,000-38,000) of the purified protein . Disruption of the chromosomal copy of the gene resulted in loss of NAD-dependent 5,10-methylene-THF dehydrogenase activity and led to a purine requirement in certain genetic backgrounds, confirming a role for this enzyme in the oxidation of cytoplasmic one-carbon units . A single gene was mapped to chromosome XI by hybridization to a yeast chromosomal blot . We propose MTD1 as the name for this gene . Northern analysis of total yeast RNA revealed a single transcript of approximately 1,100 nucleotides . Multiple transcription initiation sites were identified between 58 and 83 base pairs upstream of the start of translation . The amino acid sequences derived from the nucleic acid sequences of seven other methylene-THF dehydrogenases cloned to date have been found to be highly homologous . Although the predicted amino acid sequence of the yeast NAD-dependent enzyme shows slight homology to the other sequences, it appears to be only distantly related to the other 5,10-methylene-THF dehydrogenases.

FEBS Lett, 1993 Jan 4, 315(2), 197 - 200
Regulation of Cu,Zn- and Mn-superoxide dismutase transcription in Saccharomyces cerevisiae; Galiazzo F et al.; The regulation of Cu,Zn- and Mn-superoxide dismutases (SOD) was investigated by Northern blotting and gene fusions of SOD1 and SOD2 promoters with the beta-galactosidase reporter gene . Cu,ZnSOD expression was increased 3-fold under glucose derepressing conditions, and decreased 4- to 6-fold by oxygen or heme deficiency . MnSOD expression was increased 5-fold by glucose derepression, and decreased 8- to 10-fold by anaerobiosis and 4- to 5-fold by heme deficiency . Induction by paraquat was modest, about 50% for SOD1 and 100% for SOD2; it was apparently independent of the respiratory chain function.

Biochimie, 1993, 75(3-4), 209 - 24
Two complementary approaches to study peroxisome biogenesis in Saccharomyces cerevisiae: forward and reversed genetics; Kunau WH et al.; In order to investigate the mechanisms of peroxisome biogenesis and to identify components of the peroxisomal import machinery we studied these processes in the yeast Saccharomyces cerevisiae . The forward genetic approach has led to pas-mutants (peroxisomal assembly) which fall into 12 complementation groups and allowed to identify 10 of the corresponding wild-type PAS genes (PAS 1-7, 9, 11 and 12) . Recent sequence analysis data of some of these genes are beginning to provide first hints as to the possible function of their gene products . The PAS genes and their corresponding mutants are presently used to address some important questions of peroxisomal biogenesis . Reversed genetics has been started as a complementary approach to characterize especially the function of peroxisomal membrane proteins . For this purpose we describe a technique to isolate highly purified peroxisomes . This led to the identification of 21 polypeptides as constituents of this organelle . Some of them are presently sequenced.

Folia Microbiol (Praha), 1993, 38(1), 59 - 67
Ultracytochemical localization of dihydroorotate dehydrogenase in mitochondria and vacuoles of Saccharomyces cerevisiae; Vorisek J et al.; The coenzyme-independent dihydroorotate dehydrogenase (EC 1.3.3.1) linking the pyrimidine biosynthetic pathway to the respiratory chain, was ultracytochemically localized by the tetrazolium method in derepressed exponential-phase cultures of Saccharomyces cerevisiae . Biochemical analysis showed a considerable variation of this enzyme activity in inverse proportion to the aeration of the yeast cultures . The assay also showed that after prefixation of yeast cells with 1% glutaraldehyde at 0 degrees C for 20 min, approximately one-half of the enzyme activity was preserved . The cytochemical reaction mixture contained dihydroorotate (2 mmol/L), thiocarbamyl nitroblue tetrazolium (0.44 mmol/L), phenazine methosulfate (0.16 mmol/L) and KCN (1.7 mmol/L) in Tris-HCl buffer (100 mmol/L) of pH 8.0 . The osmicated formazan deposits features envelopes of mitochondria and of nuclei and were prominent in the mitochondrial inclusions and in the vacuolar membranes . The latter sites of dihydroorotate dehydrogenase activity represent biosynthetic activity in yeast vacuoles, still generally assumed to function as yeast lysosomes and storage organelles . In the light of the generally observed invasions of juvenile yeast vacuoles into mitochondria, the enzymic sites observed in mitochondrial inclusion were considered as evidence of the interactions of yeast vacuoles and mitochondria . Transfer of vacuolar membranes with dihydroorotate dehydrogenase activity into mitochondrial matrix is suggested.

C R Acad Sci III, 1993, 316(1), 13 - 9
{Purification of leucine-endopeptidase from Saccharomyces cerevisiae inner mitochondrial membrane}; Pirman A et al.; Several proteolytic activities have been extracted from isolated yeast mitochondrial inner membranes, in the presence of Tween 20 . Absence of matrix and cytoplasmic contamination has been checked . A leucine-endopeptidase has been purified to homogeneity . It has an apparent molecular mass of 66 kDa and a pHi near 8.6 . It is a serine-protease, which hydrolyses synthetic polypeptides whose leucine carboxylic group is engaged, as well as various macromolecular proteins; its optimal pH is 7.2.

Microbios, 1993, 73(296), 199 - 203
Glucose uptake in the yeast Saccharomyces cerevisiae; Ota A; The glucose uptake of Saccharomyces cerevisiae cells in sporulation medium for 3 h was higher than that of cells in 5.5 mM glucose medium . This difference might be due to the change in cell structure which is necessary for the sporulation process . In both types of cells, the glucose uptake activity became higher as the glucose concentration of the medium decreased.

Mol Microbiol, 1993 Jan, 7(2), 215 - 28
The general amino acid control regulates MET4, which encodes a methionine-pathway-specific transcriptional activator of Saccharomyces cerevisiae; Mountain HA et al.; A met4 mutant of Saccharomyces cerevisiae was unable to transcribe a number of genes encoding enzymes of the methionine biosynthetic pathway . The sequence of the cloned MET4 gene allowed the previously sequenced flanking LEU4 and POL1 genes to be linked to MET4 into a 10,327 bp contiguous region of chromosome XIV . From the sequence and mapping of the transcriptional start points, MET4 is predicted to encode a protein of 634 amino acids (as opposed to 666 amino acids published by others) with a leucine zipper domain at the C-terminus, preceded by both acidic and basic regions . Thus, MET4 belongs to the family of basic leucine zipper trans-activator proteins . Disruption of MET4 resulted in methionine auxotrophy with no other phenotype . Transcriptional studies showed that MET4 was regulated by the general amino acid control and hence by another bZIP protein encoded by GCN4 . GCN4 binding sequences are present between the divergently transcribed MET4 and LEU4 genes . Over-expression of MET4 resulted in leaky expression from the otherwise tightly regulated MET3 promoter under its control . The presence of consensus sequences for other potential regulatory elements in the MET4 promoter suggests a complex regulation of this gene.

Environ Mol Mutagen, 1993, 21(2), 180 - 92
Analysis of the six additional chemicals for in vitro assays of the European Economic Communities' EEC aneuploidy programme using Saccharomyces cerevisiae D61.M and the in vitro porcine brain tubulin assembly assay; Albertini S et al.; We tested six additional chemicals (acetaldehyde, benomyl, diethylstilboestrol, diethylstilboestrol dipropionate, griseofulvin, and mercaptoethanol) for in vitro systems of the coordinated programme to study aneuploidy induction sponsored by the Commission of the European Communities in two in vitro test systems . Using Saccharomyces cerevisiae D61.M (mitotic chromosomal malsegregation assay), benomyl showed a dose-dependent increase in the frequency of chromosomal malsegregation with a lowest effective dose tested (LEDT) of 30 micrograms/ml (0.1 mM) . Diethylstilboestrol (DES) showed solvent-dependent effects . DES dissolved in ethanol induced an increase in chromosomal malsegregation as well as in the frequency of total resistant colonies (mutations and recombinations) with a LEDT around 13 micrograms/ml (0.048 mM) . Using dimethylsulfoxide as the solvent, no increases were observed with DES up to 333 micrograms/ml (1.24 mM) . Acetaldehyde induced an increase in chromosomal malsegregation with the cold treatment protocol (LEDT: 1.25 microliters/ml (21 mM) and 0.75 microliters/ml (13 mM), respectively) but no increase with the overnight protocol (highest dose tested (HDT): 1.75 microliters/ml; 30 mM) . Concerning the frequency of total cycloheximide-resistant colonies (mutations and recombinations) increases were obtained with both protocols . The other three compounds were negative when tested up to toxic doses (survival below 10%), up to the maximum solubility in the solvent used or up to heavy precipitation in the incubation mix . The HDT were 333 micrograms/ml (0.88 mM) for diethylstilboestrol dipropionate, 1,600 micrograms/ml (4.5 mM) for griseofulvin and 0.5 microliters/ml (7 mM) for mercaptoethanol . Concerning effects on porcine brain tubulin assembly in vitro, diethylstilboestrol and griseofulvin inhibited the assembly process . The IC30% (30% inhibition concentration) values were 12.5 microM and 100 microM for DES and griseofulvin, respectively . Mercaptoethanol showed no effects up to 50 mM.

Yeast, 1993 Jan, 9(1), 59 - 69
Human catalase is imported and assembled in peroxisomes of Saccharomyces cerevisiae; de Hoop MJ et al.; To study the conservation of peroxisomal targeting signals, we have determined the intracellular localization of human peroxisomal catalase when expressed in yeast . Using immunofluorescence, differential centrifugation and immunoelectron microscopy, we show that the protein is targeted to the peroxisomes of the heterologous cell and assembled in its active tetrameric form . These data show the conservation of the catalase targeting signal and import specificity between human and yeast peroxisomes.

Yeast, 1993 Jan, 9(1), 1 - 10
Physical localization of the flocculation gene FLO1 on chromosome I of Saccharomyces cerevisiae; Teunissen AW et al.; The genetics of flocculation in the yeast Saccharomyces cerevisiae are poorly understood despite the importance of this property for strains used in industry . To be able to study the regulation of flocculation in yeast, one of the genes involved, FLO1, has been partially cloned . The identity of the gene was confirmed by the non-flocculent phenotype of cells in which the C-terminal part of the gene had been replaced by the URA3 gene . Southern blots and genetic crosses showed that the URA3 gene had integrated at the expected position on chromosome I . A region of approximately 2 kb in the middle of the FLO1 gene was consistently deleted during propagation in Escherichia coli and could not be isolated . Plasmids containing the incomplete gene, however, were still able to cause weak flocculation in a non-flocculent strain . The 3' end of the FLO1 gene was localized at approximately 24 kb from the right end of chromosome I, 20 kb centromere-proximal to PHO11 . Most of the newly isolated chromosome I sequences also hybridized to chromosome VIII DNA, thus extending the homology between the right end of chromosome I and chromosome VIII to approximately 28 kb.

J Membr Biol, 1993 Jan, 131(2), 115 - 27
Anomalously slow mobility of fluorescent lipid probes in the plasma membrane of the yeast Saccharomyces cerevisiae; Greenberg ML et al.; We measured the lateral mobility of two fluorescent lipid probes dioctadecylindocarbocyanine (diI) and tetramethyl rhodamine phosphatidylethanolamine (R-PE) in the plasma membranes of Saccharomyces cerevisiae ino1 and opi3 spheroplasts . These are well-characterized strains with mutations in the inositol and phosphatidylcholine biosynthetic pathways . Membrane phospholipid composition was altered by growing these mutants in the presence or absence of inositol and choline . Lateral mobility was measured by fluorescence recovery after photobleaching (FRAP) . Microscopic fluorescence polarization employing CCD digital imaging produced an ordered orientation distribution of the lipid probe diI, confirming that at least one of the probes was largely incorporated into the bilayer membrane . Our results demonstrated anomalously slow mobility of both lipid probes for both mutants, regardless of whether the lipid composition was near normal or dramatically altered in relative composition of phosphatidylinositol and phosphatidylcholine . Trypsinization of the spheroplasts to remove surface proteins resulted in markedly increased lateral mobility . However, even in trypsinized spheroplasts, mobility was still somewhat lower than the mobility observed in the membrane of mammalian cells, such as rat smooth muscle culture cells tested here for comparison.

Mol Gen Genet, 1993 Jan, 236(2-3), 443 - 7
A pair of putative protein kinase genes (YPK1 and YPK2) is required for cell growth in Saccharomyces cerevisiae; Chen P et al.; Probes derived from cDNAs encoding isozymes of rat protein kinase C (PKC) were used to screen the genome of the budding yeast Saccharomyces cerevisiae . We reported previously the isolation of the yeast PKC1 gene, a homolog of the alpha, beta, and gamma subspecies of mammalian PKC . Here we report the isolation and genetic characterization of a pair of previously described genes (YPK1 and YPK2) which are predicted to encode protein kinases that share 90% amino acid identity with each other and 44-46% identity with various isozymes of PKC throughout their putative catalytic domains . Deletion of YPK2 resulted in no apparent phenotypic defect, but loss of YPK1 resulted in slow growth . Cells deleted for both YPK1 and YPK2 were defective in vegetative growth, indicating that the protein kinases predicted to be encoded by these genes are functionally overlapping and play an essential role in the proliferation of yeast cells . The YPK1 gene was mapped to the left arm of chromosome XI and YPK2 was mapped to the right arm of chromosome XIII.

Mol Gen Genet, 1993 Jan, 236(2-3), 363 - 8
Differential expression of two genes encoding isoforms of the ATPase involved in sodium efflux in Saccharomyces cerevisiae; Garciadeblas B et al.; The ENA2 gene encoding a P-type ATPase involved in Na+ and Li+ effluxes in Saccharomyces cerevisiae has been isolated . The putative protein encoded by ENA2 differs only in thirteen amino acids from the protein encoded by ENA1/PMR2 . However, ENA2 has a very low level of expression and for this reason did not confer significant Li+ tolerance on a Li+ sensitive strain . ENA1 and ENA2 are the first two units of a tandem array of four highly homologous genes with probably homologous functions.

Mol Gen Genet, 1993 Jan, 236(2-3), 283 - 8
A gene, SMP2, involved in plasmid maintenance and respiration in Saccharomyces cerevisiae encodes a highly charged protein; Irie K et al.; The smp2 mutant of Saccharomyces cerevisiae shows increased stability of the heterologous plasmid pSR1 and YRp plasmids . A DNA fragment bearing the SMP2 gene was cloned by its ability to complement the slow growth of the smp2 smp3 double mutant (smp3 is another mutation conferring increased stability of plasmid pSR1) . The nucleotide sequence of SMP2 indicated that it encodes a highly charged 95 kDa protein . Disruption of the genomic SMP2 gene resulted in a respiration-deficient phenotype, although the cells retained mitochondrial DNA, and showed increased stability of pSR1 like the original smp2 mutant . The fact that the smp2 mutant is not always respiration deficient and shows increased pSR1 stability even in a rho0 strain lacking mitochondrial DNA suggested that the function of the Smp2 protein in plasmid maintenance is independent of respiration . The SMP2 locus was mapped at a site 71 cM from lys7 and 21 cM from ilv2/SMR1 on the right arm of chromosome XIII.

Mol Gen Genet, 1993 Jan, 236(2-3), 214 - 8
Gene SNQ2 of Saccharomyces cerevisiae, which confers resistance to 4-nitroquinoline-N-oxide and other chemicals, encodes a 169 kDa protein homologous to ATP-dependent permeases; Servos J et al.; The yeast gene SNQ2 confers hyper-resistance to the mutagens 4-nitroquinoline-N-oxide (4-NQO) and Triaziquone, as well as to the chemicals sulphomethuron methyl and phenanthroline when present in multiple copies in transformants of Saccharomyces cerevisiae . Subcloning and sequencing of a 5.5 kb yeast DNA fragment revealed that SNQ2 has an open reading frame of 4.5 kb . The putative encoded polypeptide of 1501 amino acids has a predicted molecular weight of 169 kDa and has several hydrophobic regions . Northern analysis showed a transcript of 5.5 kb . Haploid cells with a disrupted SNQ2 reading frame are viable . The SNQ2-encoded protein has domains believed to be involved in ATP binding and is likely to be membrane associated . It most probably serves as an ATP-dependent permease.

Chromosoma, 1993 Jan, 102(2), 129 - 36
The replication behavior of Saccharomyces cerevisiae DNA in human cells; Tran CT et al.; We studied the replication of random genomic DNA fragments from Saccharomyces cerevisiae in a long-term assay in human cells . Plasmids carrying large yeast DNA fragments were able to replicate autonomously in human cells . Efficiency of replication of yeast DNA fragments was comparable to that of similarly sized human DNA fragments and better than that of bacterial DNA . This result suggests that yeast genomic DNA contains sequence information needed for replication in human cells . To examine whether DNA replication in human cells would initiate specifically at a yeast origin of replication, we monitored initiation on a plasmid containing the yeast 2-micron autonomously replicating sequence (ARS) in yeast and human cells . We found that while replication initiates at the 2-micron ARS in yeast, it does not preferentially initiate at the ARS in human cells . This result suggests that the sequences that direct site specific replication initiation in yeast do not function in the same way in human cells, which initiate replication at a broader range of sequences.

Curr Genet, 1993 Jan, 23(1), 66 - 91
A comprehensive compilation of 1001 nucleotide sequences coding for proteins from the yeast Saccharomyces cerevisiae (= ListA2)
Mosse MO, Linder P, Lazowska J, Slonimski PP.
The amount of nucleotide sequence data is increasing exponentially . We therefore continued our effort to make a comprehensive database for the yeast Saccharomyces cerevisiae . In this database (ListA2) we have compiled 1001 protein coding sequences from this organism . Each sequence has been attributed a single genetic name and in the case of allelic duplicated sequences, synonyms are given, if necessary . For the nomenclature we have introduced a standard principle for naming gene sequences based on priority rules . We have also applied a simple method to distinguish duplicated sequences of one and the same gene from non-allelic sequences of duplicated genes . By using these principles we have sorted out a lot of confusion in the literature and databanks . Along with the genetic name, the mnemonic from the EMBL databank, the codon bias, reference of the publication of the sequence and the EMBL accession numbers are included for each entry . The database is available on request.

Genetics, 1993 Jan, 133(1), 51 - 66
Identification of new genes required for meiotic recombination in Saccharomyces cerevisiae; Ajimura M et al.; Mutants defective in meiotic recombination were isolated from a disomic haploid strain of Saccharomyces cerevisiae by examining recombination within the leu2 and his4 heteroalleles located on chromosome III . The mutants were classified into two new complementation groups (MRE2 and MRE11) and eight previously identified groups, which include SPO11, HOP1, REC114, MRE4/MEK1 and genes in the RAD52 epistasis group . All of the mutants, in which the mutations in the new complementation groups are homozygous and diploid, can undergo premeiotic DNA synthesis and produce spores . The spores are, however, not viable . The mre2 and mre11 mutants produce viable spores in a spo13 background, in which meiosis I is bypassed, suggesting that these mutants are blocked at an early step in meiotic recombination . The mre2 mutant does not exhibit any unusual phenotype during mitosis and it is, thus, considered to have a mutation in a meiosis-specific gene . By contrast, the mre11 mutant is sensitive to damage to DNA by methyl methanesulfonate and exhibits a hyperrecombination phenotype in mitosis . Among six alleles of HOP1 that were isolated, an unusual pattern of intragenic complementation was observed.

Genetics, 1993 Jan, 133(1), 39 - 49
A Saccharomyces cerevisiae RAD52 allele expressing a C-terminal truncation protein: activities and intragenic complementation of missense mutations; Boundy-Mills KL et al.; A nonsense allele of the yeast RAD52 gene, rad52-327, which expresses the N-terminal 65% of the protein was compared to two missense alleles, rad52-1 and rad52-2, and to a deletion allele . While the rad52-1 and the deletion mutants have severe defects in DNA repair, recombination and sporulation, the rad52-327 and rad52-2 mutants retain either partial or complete capabilities in repair and recombination . These two mutants behave similarly in most tests of repair and recombination during mitotic growth . One difference between these two alleles is that a homozygous rad52-2 diploid fails to sporulate, whereas the homozygous rad52-327 diploid sporulates weakly . The low level of sporulation by the rad52-327 diploid is accompanied by a low percentage of spore viability . Among these viable spores the frequency of crossing over for markers along chromosome VII is the same as that found in wild-type spores . rad52-327 complements rad52-2 for repair and sporulation . Weaker intragenic complementation occurs between rad52-327 and rad52-1.

Mol Cell Biol, 1993 Jan, 13(1), 649 - 58
The REB1 site is an essential component of a terminator for RNA polymerase I in Saccharomyces cerevisiae; Lang WH et al.; We have identified a terminator for transcription by RNA polymerase I in the genes coding for rRNA of the yeast Saccharomyces cerevisiae . The terminator is located 108 bp downstream of the 3' end of the mature 25S rRNA and shares several characteristics with previously studied polymerase I terminators in the vertebrates . For example, the yeast terminator is orientation dependent, is inhibited by its own sequence, and forms RNA 3' ends 17 +/- 2 bp upstream of an essential protein binding site . The recognition sequence for binding of the previously cloned REB1 protein (Q . Ju, B . E . Morrow, and J . R . Warner, Mol . Cell . Biol . 10:5226-5234, 1990) is an essential component of the terminator . In addition, the efficiency of termination depends upon sequence context extending at least 12 bp upstream of the REB1 site.

Mol Cell Biol, 1993 Jan, 13(1), 638 - 48
Roles of multiple glucose transporters in Saccharomyces cerevisiae; Ko CH et al.; In Saccharomyces cerevisiae, TRK1 and TRK2 are required for high- and low-affinity K+ transport . Among suppressors of the K+ transport defect in trk1 delta trk2 delta cells, we have identified members of the sugar transporter gene superfamily . One suppressor encodes the previously identified glucose transporter HXT1, and another encodes a new member of this family, HXT3 . The inferred amino acid sequence of HXT3 is 87% identical to that of HXT1, 64% identical to that of HXT2, and 32% identical to that of SNF3 . Like HXT1 and HXT2, overexpression of HXT3 in snf3 delta cells confers growth on low-glucose or raffinose media . The function of another new member of the HXT superfamily, HXT4 (previously identified by its ability to suppress the snf3 delta phenotype; L . Bisson, personal communication), was revealed in experiments that deleted all possible combinations of the five members of the glucose transporter gene family . Neither SNF3, HXT1, HXT2, HXT3, nor HXT4 is essential for viability . snf3 delta hxt1 delta hxt2 delta hxt3 delta hxt4 delta cells are unable to grow on media containing high concentrations of glucose (5%) but can grow on low-glucose (0.5%) media, revealing the presence of a sixth transporter that is itself glucose repressible . This transporter may be negatively regulated by SNF3 since expression of SNF3 abolishes growth of hxt1 delta hxt2 delta hxt3 delta hxt4 delta cells on low-glucose medium . HXT1, HXT2, HXT3, and HXT4 can function independently: expression of any one of these genes is sufficient to confer growth on medium containing at least 1% glucose . A synergistic relationship between SNF3 and each of the HXT genes is suggested by the observation that SNF2 hxt1 delta hxt2 delta hxt3 delta hxt4 delta cells and snf3 delta HXT1 HXT2 HXT3 HXT4 cells are unable to grow on raffinose (low fructose) yet SNF3 in combination with any single HXT gene is sufficient for growth on raffinose . HXT1 and HXT3 are differentially regulated . HXT1::lacZ is maximally expressed during exponential growth whereas HXT3::lacZ is maximally expressed after entry into stationary phase.

Mol Cell Biol, 1993 Jan, 13(1), 496 - 505
DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae; Budd ME et al.; Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomyces cerevisiae . We have investigated whether DNA polymerases epsilon and delta are required for DNA replication . Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique . Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature . The only DNA synthesis observed is a reduced quantity of short DNA fragments . The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions . The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation . By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature . We have reinvestigated the nature of DNA synthesis in pol3 mutants . We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block . These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.

Mol Cell Biol, 1993 Jan, 13(1), 462 - 72
Estradiol-inducible squelching and cell growth arrest by a chimeric VP16-estrogen receptor expressed in Saccharomyces cerevisiae: suppression by an allele of PDR1; Gilbert DM et al.; We have constructed and characterized a flexible system for analyzing the phenomenon of squelching and estrogen receptor function in the yeast Saccharomyces cerevisiae . The A/B region of the human estrogen receptor was replaced with the transcriptional activating domain of VP16 and expressed in yeast cells from high-copy-number plasmids . Addition of hormone resulted in an immediate inhibition of expression (squelching) of a chromosomally integrated GAL1:lacZ reporter gene and the eventual arrest of cell growth (toxicity) . In order to determine whether a relationship exists between toxicity and squelching, mutations were made in this chimeric receptor (VEO) and their effects on transcriptional activation, squelching, and toxicity were compared . A direct correlation was found between mutations in VEO that reduced VP16 transactivation ability in yeast cells and those that reduced both squelching and toxicity . Surprisingly, mutations in the DNA binding domain (DBD) of VEO dramatically reduced squelching and completely relieved toxicity, suggesting a role for the DBD in squelching and strengthening the correlation between squelching and toxicity . To demonstrate the utility of this system for carrying out genetic selection, a plasmid-based yeast genomic bank was screened for genes that can relieve the toxicity of VEO by means of an elevated copy number, resulting in the repeated cloning of an allele of the PDR1 (pleiotropic drug resistance) gene . We present evidence that mutations in PDR1 can modulate the intracellular availability of estradiol by the same mechanism that leads to multiple drug resistance in yeast cells . Taken together, our results provide evidence that cell growth arrest occurs when squelching exceeds a certain threshold and that strong squelching requires both a DBD and a transcriptional activating domain . Furthermore, we show that growth arrest can provide a useful phenotype for carrying out the genetic analysis of both squelching and estrogen receptor function in yeast cells.

Mol Cell Biol, 1993 Jan, 13(1), 373 - 82
Timing of molecular events in meiosis in Saccharomyces cerevisiae: stable heteroduplex DNA is formed late in meiotic prophase; Goyon C et al.; To better understand the means by which chromosomes pair and recombine during meiosis, we have determined the time of appearance of heteroduplex DNA relative to the times of appearance of double-strand DNA breaks and of mature recombined molecules . Site-specific double-strand breaks appeared early in meiosis and were formed and repaired with a timing consistent with a role for breaks as initiators of recombination . Heteroduplex-containing molecules appeared about 1 h after double-strand breaks and were followed shortly by crossover products and the first meiotic nuclear division . We conclude that parental chromosomes are stably joined in heteroduplex-containing structures late in meiotic prophase and that these structures are rapidly resolved to yield mature crossover products . If the chromosome pairing and synapsis observed earlier in meiotic prophase is mediated by formation of biparental DNA structures, these structures most likely either contain regions of non-Watson-Crick base pairs or contain regions of heteroduplex DNA that either are very short or dissociate during DNA purification . Two loci were examined in this study: the normal ARG4 locus, and an artificial locus consisting of an arg4-containing plasmid inserted at MAT . Remarkably, sequences in the ARG4 promoter that suffered double-strand cleavage at the normal ARG4 locus were not cut at significant levels when present at MAT::arg4 . These results indicate that the formation of double-strand breaks during meiosis does not simply involve the specific recognition and cleavage of a short nucleotide sequence.

Mol Cell Biol, 1993 Jan, 13(1), 114 - 22
Gene RRN4 in Saccharomyces cerevisiae encodes the A12.2 subunit of RNA polymerase I and is essential only at high temperatures; Nogi Y et al.; We have previously isolated mutants of Saccharomyces cerevisiae that are primarily defective in transcription of 35S rRNA genes by RNA polymerase I and have identified genes (RRN1 to RRN9) involved in this process . We have now cloned the RRN4 gene by complementation of the temperature-sensitive phenotype of the rrn4-1 mutant and have determined its complete nucleotide sequence . The following results demonstrate that the RRN4 gene encodes the A12.2 subunit of RNA polymerase I . First, RRN4 protein expressed in Escherichia coli reacted with a specific antiserum against A12.2 . Second, amino acid sequences of three tryptic peptides obtained from A12.2 were determined, and these sequences are found in the deduced amino acid sequence of the RRN4 protein . The amino acid sequence of the RRN4 protein (A12.2) is similar to that of the RPB9 (B12.6) subunit of yeast RNA polymerase II; the similarity includes the presence of two putative zinc-binding domains . Thus, A12.2 is a homolog of B12.6 . We propose to rename the RRN4 gene RPA12 . Deletion of RPA12 produces cells that are heat but not cold sensitive for growth . We have found that in such null mutants growing at permissive temperatures, the cellular concentration of A190, the largest subunit of RNA polymerase I, is lower than in the wild type . In addition, the temperature-sensitive phenotype of the rpa12 null mutants can be partially suppressed by RPA190 (the gene for A190) on multicopy plasmids . These results suggest that A12.2 plays a role in the assembly of A190 into a stable polymerase I structure.

Mol Cell Biol, 1993 Jan, 13(1), 1 - 8
Interactions among the subunits of the G protein involved in Saccharomyces cerevisiae mating; Clark KL et al.; The SCG1 (GPA1), STE4, and STE18 genes of Saccharomyces cerevisiae encode mating-pathway components whose amino acid sequences are similar to those of the alpha, beta, and gamma subunits, respectively, of mammalian G proteins . Genetic evidence suggests that the STE4 and STE18 gene products interact . The mating defects of a set of ste4 mutants were partially suppressed by the overexpression of STE18, and, moreover, a combination of partially defective ste4 and ste18 alleles created a totally sterile phenotype, whereas such synthetic sterility was not observed when the ste18 allele was combined with a weakly sterile ste11 allele . Others have provided genetic evidence consistent with an interaction between the SCG1 (GPA1) and STE4 gene products . We have examined the physical interactions of these subunits by using an in vivo protein association assay . The STE4 and STE18 gene products associated with each other, and this association was disrupted by a mutation in the STE4 gene product whose phenotype was partially suppressed by overexpression of STE18 . The STE4 and SCG1 (GPA1) gene products also interacted in the assay, whereas we detected no association of the SCG1 (GPA1) and STE18 gene products.

J Bacteriol, 1993 Jan, 175(1), 64 - 73
Regulatory circuit for responses of nitrogen catabolic gene expression to the GLN3 and DAL80 proteins and nitrogen catabolite repression in Saccharomyces cerevisiae; Daugherty JR et al.; We demonstrate that expression of the UGA1, CAN1, GAP1, PUT1, PUT2, PUT4, and DAL4 genes is sensitive to nitrogen catabolite repression . The expression of all these genes, with the exception of UGA1 and PUT2, also required a functional GLN3 protein . In addition, GLN3 protein was required for expression of the DAL1, DAL2, DAL7, GDH1, and GDH2 genes . The UGA1, CAN1, GAP1, and DAL4 genes markedly increased their expression when the DAL80 locus, encoding a negative regulatory element, was disrupted . Expression of the GDH1, PUT1, PUT2, and PUT4 genes also responded to DAL80 disruption, but much more modestly . Expression of GLN1 and GDH2 exhibited parallel responses to the provision of asparagine and glutamine as nitrogen sources but did not follow the regulatory responses noted above for the nitrogen catabolic genes such as DAL5 . Steady-state mRNA levels of both genes did not significantly decrease when glutamine was provided as nitrogen source but were lowered by the provision of asparagine . They also did not respond to disruption of DAL80.

Arch Microbiol, 1993, 159(3), 220 - 4
Metabolic effects of benzoate and sorbate in the yeast Saccharomyces cerevisiae at neutral pH; Burlini N et al.; Preincubation of yeast cells in the presence of benzoate or sorbate at an extracellular pH value of 6.8 elicited a set of metabolic effects on sugar metabolism, which became apparent after the subsequent glucose addition . They can be summarized as follows: a) reduced glucose consumption; b) inhibition of glucose- and fructose-phosphorylating activities; c) suppression of glucose-triggered peak of hexoses monophosphates; d) substantial reduction of glucose-triggered peak of fructose 2,6-bisphosphate; e) block of catabolite inactivation of fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxy-kinase, but not of cytoplasmic malate dehydrogenase . On the whole this pattern resulted in prevention of glucose-induced switch of metabolism from a gluconeogenetic to a glycolytic state . Our data also show that, unlike former assumptions, intracellular acidification is not likely to mediate the bulk of metabolic effects of benzoate and sorbate, since under our working conditions intracellular pH kept close to neutrality.

Curr Genet, 1993 Jan, 23(1), 92 - 4
The STA2 and MEL1 genes of Saccharomyces cerevisiae are idiomorphic; Lyness CA et al.; The STA2 (glucoamylase) gene of Saccharomyces cerevisiae has been mapped close to the end of the left arm of chromosome II . Meiotic analysis of a cross between a haploid strain containing STA2, and another strain carrying the melibiase gene MEL1 (which is known to be at the end of the left arm of chromosome II) produced parental ditype tetrads only . Since there is no significant DNA sequence similarity between the STA2 and MEL1 genes, or their respective flanking regions, we conclude that these two genes are carried by separate non-hybridizing sequences of chromosomal DNA, either of which can reside at the end of the left arm of chromosome II . By analogy with the mating-type locus of Neurospora crassa, we suggest that the STA2 and MEL1 genes are idiomorphs with respect to one another.

Mol Cell Biol, 1993 Jan, 13(1), 63 - 71
SPT13 (GAL11) of Saccharomyces cerevisiae negatively regulates activity of the MCM1 transcription factor in Ty1 elements; Yu G et al.; The Ty transposable elements of Saccharomyces cerevisiae consist of a single large transcription unit whose expression is controlled by a combination of upstream and downstream regulatory sequences . Errede (B . Errede, Mol . Cell . Biol . 13:57-62, 1993) has shown that among the downstream control sequences is a binding site for the transcription factor, MCM1 . A small restriction fragment containing the Ty1 MCM1-binding site exhibits very weak activation of heterologous gene expression . The absence of SPT13 (GAL11) causes a dramatic increase in activity directed by these sequences . This effect is mediated through the MCM1-binding site itself . MCM1 mRNA and protein levels, as well as its affinity for its binding site, are unchanged in the absence of SPT13 . Our results suggest that SPT13 has a role in the negative control of MCM1 activity that is likely to be posttranslational . A role for SPT13 in the negative regulation of the activity of the Ty1 MCM1-binding site is consistent with our previous proposal that spt13-mediated suppression of Ty insertion mutations could be attributed to the loss of negative regulation of genes adjacent to Ty elements.

Mol Cell Biol, 1993 Jan, 13(1), 521 - 32
Suppressors of clathrin deficiency: overexpression of ubiquitin rescues lethal strains of clathrin-deficient Saccharomyces cerevisiae; Nelson KK et al.; Clathrin-mediated vesicular transport is important for normal growth of the yeast Saccharomyces cerevisiae . Previously, we identified a genetic locus (SCD1) that influences the ability of clathrin heavy-chain-deficient (Chc-) yeast cells to survive . With the scd1-v allele, Chc- yeast cells are viable but grow poorly; with the scd1-i allele, Chc- cells are inviable . To identify the SCD1 locus and other genes that can rescue chc1 delta scd1-i cells to viability, a multicopy suppressor selection strategy was developed . A strain of scd1-i genotype carrying the clathrin heavy-chain gene under GAL1 control (GAL1:CHC1) was transformed with a YEp24 yeast genomic library, and colonies that could grow on glucose were selected . Plasmids from six distinct genetic loci, none of which encoded CHC1, were recovered . One of the suppressor loci was shown to be UBI4, the polyubiquitin gene . UBI4 rescues only in high copy number and is not allelic to SCD1 . The conjugation of ubiquitin to intracellular proteins can mediate their selective degradation . Since UBI4 is required for survival of yeast cells under stress and is induced during starvation, ubiquitin expression in GAL1:CHC1 cells was examined . After a shift to growth on glucose to repress synthesis of clathrin heavy chains, UBI4 mRNA levels were elevated > 10-fold, whereas the quantity of free ubiquitin declined severalfold relative to that of Chc+ cells . In addition, novel higher-molecular-weight ubiquitin conjugates appeared in clathrin-deficient cells . We suggest that higher levels of ubiquitin are required for turnover of mislocalized or improperly processed proteins that accumulate in the absence of clathrin and that ubiquitin may play a general role in turnover of proteins in the secretory or endocytic pathway.

Mol Cell Biol, 1993 Jan, 13(1), 39 - 43
The Saccharomyces cerevisiae SDC25 C-domain gene product overcomes the dominant inhibitory activity of Ha-Ras Asn-17; Schweighoffer F et al.; The carboxy-terminal part of the Saccharomyces cerevisiae SDC25 gene product (SDC25 C domain) can elicit activation of mammalian Ras proteins . Specifically, SDC25 C domain functions as an exchange factor for cellular Ras proteins in CHO cells . In this study, we used the dominant inhibitory Ha-Ras Asn-17 mutant and SDC25 C domain to further investigate the interaction between cellular Ras proteins and their putative endogenous guanine nucleotide-releasing factors . Transcription from the polyomavirus thymidine kinase gene (Py tk) promoter is strongly inhibited by the expression of Ha-Ras Asn-17 in NIH 3T3 cells . Coexpression of SDC25 C domain overcomes the negative effect of the Ras mutant on the Py tk promoter . On the other hand, transactivation of the Ras-responsive element of the Py tk promoter induced by SDC25 C domain is lost upon coexpression of increasing amounts of Ha-Ras Asn-17 . In addition, coexpression of SDC25 C domain overcomes the inhibition of proliferation of NIH 3T3 cells caused by Ha-Ras Asn-17 . These results are consistent with the idea that the Ha-Ras Asn-17 mutant functions by titrating an upstream activator of cellular Ras proteins.

J Cell Biol, 1993 Jan, 120(1), 55 - 65
end3 and end4: two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae; Raths S et al.; alpha-factor, one of two peptide hormones responsible for synchronized mating between MATa and MAT alpha-cell types in Saccharomyces cerevisiae, binds to its cell surface receptor and is internalized in a time-, temperature-, and energy-dependent manner (Chvatchko, Y., I . Howald, and H . Riezman . 1986 . Cell . 46:355-364) . After internalization, alpha-factor is delivered to the vacuole via vesicular intermediates and degraded there consistent with an endocytic mechanism (Singer, B., and H . Riezman . 1990 . J . Cell Biol . 110:1911-1922; Chvatchko, Y., I . Howald, and H . Riezman . 1986 . Cell . 46:355-364) . We have isolated two mutants that are defective in the internalization process . Both mutations confer a recessive, temperature-sensitive growth phenotype upon cells that cosegregates with their endocytosis defect . Lucifer yellow, a marker for fluid-phase endocytosis, shows accumulation characteristics in the mutants that are similar to the uptake characteristics of 35S-alpha-factor . The endocytic defect in end4 cells appears immediately upon shift to restrictive temperature and is reversible at permissive temperature if new protein synthesis is allowed . Furthermore, the end4 mutation only affects alpha-factor internalization and not the later delivery of alpha-factor to the vacuole . Other vesicle-mediated processes seem to be normal in end3 and end4 mutants . END3 and END4 are the first genes shown to be necessary for the internalization step of receptor-borne and fluid-phase markers in yeast.

Folia Microbiol (Praha), 1993, 38(3), 225 - 8
Turnover of canavanine-containing proteins in Saccharomyces cerevisiae; Pazlarova J et al.; Saccharomyces cerevisiae grown for 2 h in the presence of 0.5 mmol/L canavanine in a synthetic medium with ethanol as the sole carbon source (OEC) exhibited a slowing down of protein synthesis for 3-4 h after a shift to fresh ethanol-based medium containing 1.0 mmol/L arginine (OEA) in comparison with untreated cells grown on OEA . The change of carbon source from ethanol to glucose (OGA) after growth in the OEC medium resulted in an even deeper decline of protein synthesis . The degradation of canavanine-containing proteins in cells pregrown and labelled in an OEC medium after transfer to OEA was more rapid than in the OGA medium . The initial rate of protein degradation during the first hour in the OGA medium was less than 1%/h whereas in the OEA medium it reached almost 10%/h . The fraction of proteins with high turnover (half-life 0.46 h) constituted 8.3% on OEA, while during subsequent growth on OGA it was only 0.75% with a half-life of 0.12 h.

Biochimie, 1993, 75(6), 467 - 72
Properties of Barrier, a novel Saccharomyces cerevisiae acid protease; Nath R; We have studied the specificity of Barrier, a protease secreted by Saccharomyces cerevisiae, towards its natural substrate alpha-factor, a tridecapeptide mating pheromone . Sub-fragments of alpha-factor synthesized or prepared by cyanogen bromide cleavage and a related pheromone from Saccharomyces kluveri were studied as potential substrates or competitive inhibitors . None of the tested peptides was a potent inhibitor or substrate for Barrier . Barrier shares extensive sequence similarity to the active site residues of aspartyl proteases but universal irreversible inhibitors of this class of enzymes failed to inactivate Barrier, suggesting that it is a novel fungal aspartyl protease.

Microbios, 1993, 74(299), 111 - 20
Effects of chlorhexidine diacetate and cetylpyridinium chloride on whole cells and protoplasts of Saccharomyces cerevisiae; Hiom SJ et al.; Chlorhexidine diacetate (CHA) and the quaternary ammonium compound, cetylpyridinium chloride (CPC), were fungicidal to Saccharomyces cerevisiae A364A and CHA to its mannoprotein mutant LB6-5D . Both CHA and CPC induced leakage of K+ and pentose material from both strains and both agents induced protoplast lysis as well as interacting with crude cell sap . Differences were observed between CHA and CPC in protoplast lysis and in cell sap interaction . Q25 values for A364A with CHA as test agent were 5.6 and 1.6 (fungicidal activity, depending on method of calculation), 1.46 (cell sap interaction) and 0.77 (leakage of pentoses) . A sub-lytic concentration of CHA reduced considerably protoplast regeneration . Although the plasma membrane is an important target site for both agents, interaction with other cellular components also contributes to fungal death.

Arch Microbiol, 1993, 160(5), 397 - 400
Characterization of acetyl-CoA: L-lysine N6-acetyltransferase, which catalyses the first step of carbon catabolism from lysine in Saccharomyces cerevisiae; Bode R et al.; The carbon catabolism of L-lysine starts in Saccharomyces cerevisiae with acetylation by an acetyl-CoA:L-lysine N6-acetyltransferase . The enzyme is strongly induced in cells grown on L-lysine as sole carbon source and has been purified about 530-fold . Its activity was specific for acetyl-CoA and, in addition to L-lysine, 5-hydroxylysine and thialysine act as acetyl acceptor . The following apparent Michaelis constants were determined: acetyl-CoA 0.8 mM, L-lysine 5.8 mM, DL-5-hydroxylysine, 2.8 mM, L-thialysine 100 mM . The enzyme had a maximum activity at pH 8.5 and 37 degrees C . Its molecular mass, estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was 52 kDa . Since the native molecular mass, determined by gel filtration, was 48 kDa, the enzyme is a monomer.

Arch Microbiol, 1993, 160(4), 324 - 8
Induction of pyruvate decarboxylase in glycolysis mutants of Saccharomyces cerevisiae correlates with the concentrations of three-carbon glycolytic metabolites; Boles E et al.; Pyruvate decarboxylase, PDCase, activity in wild-type yeast cells growing on ethanol is quite low but increases up to tenfold upon addition of glucose, less with galactose and only slightly with glycerol . PDCase levels in glycolysis mutant strains growing on ethanol or acetate were higher than in the wild-type strain . These levels correlated with the sum of the concentrations of three-carbon glycolytic metabolites . The highest accumulation was observed in a fructose bisphosphate aldolase deletion mutant concomitant with the highest PDCase activity ever observed under gluconeogenic conditions . Glucose addition induced an increase in PDCase activity in all mutants . However, the enzyme activities never reached wild-type level . On the other hand, the PDCase levels in the different mutants again correlated with the sum of the concentrations of the three-carbon glycolytic metabolites . This was interpreted to mean that full induction of PDCase activity requires the accumulation of hexose- and triosephosphates.

Folia Microbiol (Praha), 1993, 38(6), 479 - 85
Biochemical, morphological and cytochemical studies of enhanced autolysis of Saccharomyces cerevisiae . 2 . Morphological and cytochemical studies; Kollar R et al.; Morphological and cytochemical observation of Saccharomyces cerevisiae undergoing of induced autolysis were done in response to various chemical inducers of autolysis (NaCl, ethanol, fresh autolyzate) . Changes in the inner structure of yeast cells were monitored by transmission electron microscopy and the surface of the cell wall was observed by scanning electron microscopy during autolysis . Cytochemical characterization of autolyzed cells was performed using four synthetic substrates for determination of proteinase activities but only carboxypeptidase Y could be detected in the vacuolar membranes . The morphological studies supported the data obtained from biochemical studies and confirmed that optimized conditions of autolysis have a significant effect on the structural changes of autolyzed yeast.

Folia Microbiol (Praha), 1993, 38(6), 473 - 8
Biochemical, morphological and cytochemical studies of enhanced autolysis of Saccharomyces cerevisiae . 1 . Biochemical studies; Kollar R et al.; Biochemical aspects of induced autolysis of Saccharomyces cerevisiae were observed in the presence of various physical and chemical enhancers of autolysis (increased temperature, changes of pH, NaCl, ethanol, fresh autolyzate) . Direct assays of proteinases, nucleases, glucanases and acid phosphatases in homogenized autolyzed cells were performed . In addition, the degradation products of proteins, nucleic acids, polysaccharides and phosphate from phosphorylated compounds were determined in the supernatant of autolyzate after centrifugation . These results suggested that the inducers affected transport processes and that they had mostly negative effects on the activities of the above-mentioned enzymes.

Genetica, 1993, 91(1-3), 35 - 51
The genetics of aging in the yeast Saccharomyces cerevisiae; Jazwinski SM; The yeast Saccharomyces cerevisiae possesses a finite life span similar in many attributes and implications to that of higher eukaryotes . Here, the measure of the life span is the number of generations or divisions the yeast cell has undergone . The yeast cell is the organism, simplifying many aspects of aging research . Most importantly, the genetics of yeast is highly-developed and readily applicable to the dissection of longevity . Two candidate longevity genes have already been identified and are being characterized . Others will follow through the utilization of both the primary phenotype and the secondary phenotypes associated with aging in yeast . An ontogenetic theory of longevity that follows from the evolutionary biology of aging is put forward in this article . This theory has at its foundation the asymmetric reproduction of cells and organisms, and it makes specific predictions regarding the genetics, molecular mechanisms, and phenotypic features of longevity and senescence, including these: GTP-binding proteins will frequently be involved in determining longevity, asymmetric cell division will be often encountered during embryogenesis while binary fission will be more characteristic of somatic cell division, tumor cells of somatic origin will not be totipotent, and organisms that reproduce symmetrically will not have intrinsic limits to their longevity.

Drug Metab Dispos, 1993 Jan-Feb, 21(1), 43 - 9
Caffeine, estradiol, and progesterone interact with human CYP1A1 and CYP1A2 . Evidence from cDNA-directed expression in Saccharomyces cerevisiae; Eugster HP et al.; Heterologous expression of cytochrome P-450 cDNAs in yeast is a potent instrument for the study of enzyme-specific parameters and can be used to answer questions with regard to substrate specificity as well as drug interaction in a background with no interfering activities . Two cDNAs of human CYP1A1 and CYP1A2 were expressed in yeast Saccharomyces cerevisiae, and microsomes of transformed strains contained substantial amounts of functional heterologous enzymes . Enzyme kinetics with 7-ethoxyresorufin as substrate resulted in KM values of 0.017 and 1.67 microM and Vmax values of 840 and 387 pmol/mg/min for CYP1A1 and CYP1A2, respectively . Both heterologous enzymes showed an overlapping substrate specificity pattern assayed with different phenoxazone ethers and caffeine . Caffeine was shown to be metabolized by CYP1A2 and CYP1A1 . Both enzymes formed paraxanthine and minor amounts of theobromine; however, trimethyluric acid was exclusively formed by CYP1A1 . The fact that theophylline was not formed by either enzyme anticipates the involvement of additional enzyme(s) in the primary metabolism of caffeine . Inhibition studies with caffeine, phenacetin, 17 beta-estradiol, and progesterone as inhibitors of the CYP1A1 and CYP1A2 catalyzed O-deethylation of 7-ethoxyresorufin suggest all compounds as possible substrates of CYP1A enzymes . 17 beta-estradiol inhibited CYP1A1-catalyzed paraxanthine and trimethyluric acid formation . In contrast 17 beta-estradiol did not inhibit CYP1A2-catalyzed formation of primary caffeine metabolites . These data clearly demonstrate the capacity of human CYP1A1 and CYP1A2 to metabolize caffeine . Furthermore, possible consequences of CYP1A enzyme inhibition by caffeine, phenacetin, 17 beta-estradiol, and progesterone will be discussed.

Plant Cell Physiol, 1993 Jan, 34(1), 151 - 6
Preparation of a monoclonal antibody specific for a 48-kDa protein from mitochondrial nucleoids of the yeast, Saccharomyces cerevisiae; Miyakawa I et al.; Monoclonal antibodies (mAbs) were raised against yeast mitochondrial nucleoids (mt-nucleoids) . In an analysis by a combination of immunofluorescence microscopy and staining with 4',6-diamidino-2-phenylindole (DAPI), one of them, designated YMN-1, distinctly stained mt-nucleoids, which were visible as dots, in spheroplasts and in isolated mitochondria . However, staining of isolated mt-nucleoids was rather weak . YMN-1 mAb recognized a 48-kDa protein in immunoblots of both mitochondrial and mt-nucleoid proteins . The 48-kDa protein was a minor component of mt-nucleoid proteins and was separated from extract of both mitochondria and mt-nucleoids by immunoaffinity chromatography . The affinity-purified 48-kDa protein reassociated with mt-nucleoids when mixed with isolated mt-nucleoids, as monitored by immunofluorescence microscopy . The results suggest that a large amount of 48-kDa protein is associated with mt-nucleoids in vivo, and that lysis of mitochondria by the treatment with detergent releases a considerable amount of this protein from mt-nucleoids during the isolation of mt-nucleoids.

Gene Expr, 1993, 3(3), 237 - 51
Role of Saccharomyces cerevisiae Rap1 protein in Ty1 and Ty1-mediated transcription; Gray WM et al.; Binding sites for the transcription factor Rap1 are widespread in the yeast genome . With respect to many, but not all, genes, Rap1p has an apparent activation function . Whether Rap1 is itself a transcriptional activator, or whether it is in some way required for activation by additional factors, is not clear . We have identified a previously unrecognized Rap1p binding site in the internal regulatory region of Ty1 elements . We demonstrate that this site is capable of binding Rap1 in vitro and that, in vivo, Rap1p plays an important regulatory role in Ty1 and Ty1-mediated adjacent gene expression . Our data suggest that in Ty1 elements, maximal levels of RAP1-mediated activation depend on the formation of a complex with Mcm1, an independent DNA-binding protein that functions in transcription as well as in DNA replication, and with a third factor, IBF, previously identified as a binding activity with a site situated between the Rap1p and Mcm1p binding sites in this region of Ty1 elements.

Oncogene, 1993 Jan, 8(1), 215 - 8
The Saccharomyces cerevisiae gene product SDC25 C-domain functions as an oncoprotein in NIH3T3 cells; Barlat I et al.; Ras proteins in mammalian cells cycle between a GTP-bound 'on' state and a GDP-bound 'off' state . Activation of Ras p21 results from the dissociation of tightly bound GDP and the exchange of bound GDP for GTP . A guanine nucleotide exchange factor is required for this activation . Activation promotes interaction with effector molecules and allows the signal to be transduced . In Saccharomyces cerevisiae, the function of guanine nucleotide exchange has been ascribed to the product of the CDC25 gene . The C-terminus domain of SDC25, a homologue of CDC25, can substitute for the CDC25 protein in yeast . We have demonstrated that the SDC25 C-terminus domain promotes GTP binding to Ras p21 in CHO cells . In the present study, we found that the stable expression of the SDC25 C-terminus domain induced transformation of NIH3T3 cells . Ras proteins in these tumorigenic cells were GTP bound . In addition, the coexpression of wild-type Ha-Ras protein with the SDC25 C-terminus was found to enhance the tumorigenic properties of the NIH3T3 cells . These results imply that, in subsets of human tumours, cellular Ras p21 might be found in its GTP-bound active form as a consequence of an oncogenic activation of a mammalian Ras guanine nucleotide exchange factor.

Acta Microbiol Pol, 1993, 42(1), 101 - 4
Physiological suppression of superoxide dismutase deficiency in yeast Saccharomyces cerevisiae; Bilinski T et al.; Deficiency in superoxide dismutases results in pleiotropic effects including hypersensitivity to oxygen and amino acid auxotrophy . Various types of physiological suppressors of deficiency in cytosolic superoxide dismutase were isolated, whereas attempts to isolate suppressors of mitochondrial enzyme deficiency proved a failure . General characteristics of isolated suppressors are presented.

Biochim Biophys Acta, 1992 Dec 29, 1171(2), 211 - 4
AAT1, a gene encoding a mitochondrial aspartate aminotransferase in Saccharomyces cerevisiae; Morin PJ et al.; We have isolated a gene, AAT1, encoding an aspartate aminotransferase (AspAT) from a Saccharomyces cerevisiae genomic library . AAT1 encodes a 451 amino acid protein with a predicted molecular weight of 51,687, which is likely to be the yeast mitochondrial AspAT . Sequence comparison of this yeast AspAT with AspATs from other organisms shows a high degree of homology in regions previously shown to be important for catalysis . However, the yeast mitochondrial AspAT contains four obvious insertions with respect to all other known AspATs, suggesting that the AAT1-encoded protein represents a distinct AspAT.

J Mol Biol, 1992 Dec 20, 228(4), 1063 - 77
Topography of transcription factor complexes on the Saccharomyces cerevisiae 5 S RNA gene; Braun BR et al.; Locations of component proteins of yeast RNA polymerase III transcription factors (TFIII) A, C and B on a 5 S rRNA gene have been determined by site-specific DNA-protein photo-crosslinking . Comparison with a previously analyzed tRNA gene shows that similar nucleoprotein structures assemble on these two genes despite their differently located internal promoter elements . A principal signature of this homology is the placement of the 95 kDA subunit of TFIIIC, which associates with the box A promoter element of the tRNA gene . On the 5 S rRNA gene, the 95 kDa subunit occupies the same space in the absence of a box A sequence, and despite the presence of a box A-like sequence 30 base-pairs further downstream . A 90 kDa component that was not previously recognized as an integral part of TFIIIC has been specifically located at the 3' end of the 5 S rRNA gene.

Eur J Biochem, 1992 Dec 15, 210(3), 989 - 97
Expression and secretion of wheat germ agglutinin by Saccharomyces cerevisiae; Nagahora H et al.; Genes encoding pre-protein and prepro-protein of wheat germ agglutinin isolectin 2 (WGA2) were chemically synthesized and expressed in the yeast Saccharomyces cerevisiae under the control of the ENO1 promoter . Yeast harboring either a pre-WGA2 or a prepro-WGA2 gene expression plasmid secreted a mature form of WGA2 into the culture medium . The amount of WGA2 secreted by the strain KS58-2Ddel, which has a ssl1 mutation causing a supersecretion of human lysozyme {Suzuki, K., Ichikawa, K . & Jigami, Y . (1989) Mol . Gen . Genet . 219, 58-64}, was 20-fold greater than that secreted by the wild-type strain KK4 . The recombinant WGA2 from the cells containing the prepro-WGA2 gene expression plasmid was purified to homogeneity by a three-step ion-exchange chromatography scheme . As in wheat, the N-terminal signal peptide of recombinant WGA2 purified from yeast culture was processed to form an N-terminal 5-oxoprolyl (pyroglutamyl) residue . Likewise, we found that the C-terminal pro-region of recombinant WGA2 had also been processed in yeast . Using electrospray ionization mass spectrometry, we found the processed C-terminus to be heterogeneous in both recombinant WGA2 purified from yeast and in authentic WGA2 . The major component of the recombinant WGA2 contained two additional amino acids at its C-terminus compared to that of authentic WGA2 . In spite of this difference in the C-terminus, the recombinant WGA2 exhibited a sugar binding activity that was indistinguishable from that of authentic WGA2.

Proc Natl Acad Sci U S A, 1992 Dec 15, 89(24), 12068 - 72
The Saccharomyces cerevisiae ARG4 initiator of meiotic gene conversion and its associated double-strand DNA breaks can be inhibited by transcriptional interference; Rocco V et al.; In the yeast Saccharomyces cerevisiae, as in other eukaryotes, some regions of the genome have a much higher level of meiotic gene conversion than others . Previous deletion analysis indicated that the sequence necessary for the high level of gene conversion within the ARG4 region defined an initiation site located between positions -316 and -37 {relative to the first base pair (+1) of the ARG4 coding sequence} of the ARG4 promoter . To test whether this sequence is sufficient to promote gene conversion in a novel chromosomal context, we inverted on the chromosome various DNA fragments including the implicated region and the ARG4 coding sequence . Surprisingly, these inversions resulted in the loss of the normal recombination properties and double-strand-break formation associated with this process . By Northern analysis, we found that a transcript traverses the ARG4 initiation site in these inversion mutants but not in the wild type . When transcription through this region was prevented by a transcription terminator, the activity of the initiation site and the formation of double-strand breaks were restored . From these results and from complementary deletion analysis in the normal ARG4 orientation, we conclude that the activity of the ARG4 initiation site requires protection from transcriptional interference.

J Biol Chem, 1992 Dec 15, 267(35), 25321 - 7
A Saccharomyces cerevisiae DNA helicase associated with replication factor C; Li X et al.; A novel DNA helicase has been isolated from Saccharomyces cerevisiae . This DNA helicase co-purified with replication factor C (RF-C) during chromatography on S-Sepharose, DEAE-silica gel high performance liquid chromatography (HPLC), Affi-Gel Blue-agarose, heparin-agarose, single-stranded DNA-cellulose, fast protein liquid chromatography MonoS, and hydroxyapatite HPLC . Surprisingly, the helicase could be separated from RF-C by sedimentation on a glycerol gradient in the presence of 200 mM NaCl . The helicase is probably a homodimer of a 60-kDa polypeptide, which by UV cross-linking has been shown to bind ATP . It has a single-stranded DNA-dependent ATPase activity, with a Km for ATP of 60 microM . The DNA helicase activity depends on the hydrolysis of NTP (dNTP), with ATP and dATP the most efficient cofactors, followed by CTP and dCTP . The DNA helicase has a 5' to 3' directionality and is only marginally stimulated by coating the single-stranded DNA with the yeast single-stranded DNA-binding protein RF-A.

Biochem J, 1992 Dec 15, 288 ( Pt 3), 859 - 64
On the mechanism by which a heat shock induces trehalose accumulation in Saccharomyces cerevisiae; Neves MJ et al.; When the temperature of exponential-phase cultures of Saccharomyces cerevisiae was abruptly raised from 28 to 40 degrees C, trehalose immediately accumulated, whereas the activities of trehalase and trehalose-6-phosphate synthase/trehalose-6-phosphate phosphatase complex increased after a lag period of about 10 min . Heat shock also induced a sudden rise in intracellular glucose, simultaneously with a decrease in the concentration of hexose phosphate and fructose 2,6-bisphosphate . The increase of trehalose-metabolizing enzymes, but not the accumulation of glucose and trehalose, was prevented by cycloheximide . Investigation of the kinetic properties of partially purified enzymes showed that both non-activated and cyclic AMP-dependent-protein-kinase-activated forms of trehalase are almost inactive in the absence of Ca2+ and that the concentration of free Ca2+ required for half-maximal activity increased with increasing temperature, being approx . 1 microM at 30 degrees C and 20 microM at 40 degrees C for the activated form of trehalase . In contrast, trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase were three times more active at 40 degrees C . It is proposed that the rapid accumulation of trehalose induced by heat shock may be in part explained by changes in the kinetic properties of trehalase and trehalose-6-phosphate synthase/trehalose-6-phosphate phosphatase.

J Biol Chem, 1992 Dec 15, 267(35), 25337 - 46
The cAMP-binding ectoprotein from Saccharomyces cerevisiae is membrane-anchored by glycosyl-phosphatidylinositol; Muller G et al.; Saccharomyces cerevisiae contains an amphiphilic cAMP-binding glycoprotein at the outer face of the plasma membrane (M(r) = 54,000) . It is converted to a hydrophilic form by treatment with glycosyl-phosphatidylinositol-specific phospholipases C and D (GPI-PLC/D), suggesting membrane anchorage by a covalently bound glycolipid . Determination of the constituents of the purified anchor by gas-liquid chromatography and amino acid analysis reveals the presence of glycerol, myo-inositol, glucosamine, galactose, mannose, ethanolamine, and asparagine (as the carboxyl-terminal amino acid of the Pronase-digested protein to which the anchor is attached) . Complementary results are obtained by metabolic labeling, indicating that fatty acids and phosphorus are additional anchor constituents . The phosphorus is resistant to alkaline phosphatase, whereas approximately half is lost from the protein after treatment with GPI-PLD or nitrous acid, and all is removed by aqueous HF indicating the presence of two phosphodiester bonds . Inhibition of N-glycosylation by tunicamycin or removal of protein-bound glycan chains by N-glycanase or Pronase does not abolish radiolabeling of the anchor structure by any of the above compounds . Analysis of the products obtained after sequential enzymic and chemical degradation of the anchor agrees with the arrangement of constituents in GPIs from higher eucaryotes . Evidence for anchorage of the yeast cAMP-binding protein by a GPI anchor is strengthened additionally by the reactivity of the GPI-PLC-cleaved anchor with antibodies directed against the cross-reacting determinant of trypanosomal variant surface glycoproteins.

Proc Natl Acad Sci U S A, 1992 Dec 1, 89(23), 11131 - 5
A putative ATP-dependent RNA helicase involved in Saccharomyces cerevisiae ribosome assembly; Ripmaster TL et al.; We have isolated a cold-sensitive mutant of Saccharomyces cerevisiae in which there is a deficit of 60S ribosomal subunits . Cold sensitivity and the assembly defect are recessive and cosegregate, defining a single essential gene that we designated DRS1 (deficiency of ribosomal subunits) . The wild-type DRS1 gene was cloned by complementation of the cold-sensitive phenotype of drs1 . Sequence analysis reveals a high degree of similarity to a family of proteins that are thought to function as ATP-dependent RNA helicases . Pulse-chase analysis of ribosomal RNA synthesis and processing indicates that the drs1 mutant accumulates the 27S precursor of the mature 25S rRNA . These results suggest that, as in pre-mRNA splicing, RNA helicase activities are involved in ribosomal RNA processing.

J Biol Chem, 1992 Dec 5, 267(34), 24441 - 5
Human and Giardia ADP-ribosylation factors (ARFs) complement ARF function in Saccharomyces cerevisiae; Lee FJ et al.; ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that stimulate the ADP-ribosyltransferase activity of cholera toxin in vitro . ARFs are highly conserved, ubiquitously expressed in eukaryotic cells and appear to be involved in vesicular protein transport . The two yeast ARFs are > 60% identical to mammalian ARFs and are essential for cell viability (Stearns, T., Kahn, R . A., Botstein, D., and Hoyt, M . A . (1990) Mol . Cell . Biol . 10, 6690-6699) . Although the two yeast ARF proteins are 96% identical in amino acid sequence, the yeast ARF1 gene is constitutively expressed, whereas the ARF2 gene is repressed by glucose . Human ARF5 and ARF6 and a Giardia ARF differ substantially in size and amino acid identity from other mammalian and eukaryotic ARFs but will, as befits their designation, activate cholera toxin . Expression of human ARF5, ARF6, or Giardia ARF cDNA rescued the lethal yeast ARF double mutant (arf1, arf2) . Strains rescued by human ARF5, ARF6, or Giardia ARF grew much more slowly than wild-type yeast or strains rescued with yeast ARF1 . We infer from the impaired growth of these rescued strains that the homologous ARFs may have specific targeting information that does not interact effectively or efficiently with the yeast protein membrane trafficking system.

J Biol Chem, 1992 Dec 5, 267(34), 24181 - 3
A mouse CDC25-like product enhances the formation of the active GTP complex of human ras p21 and Saccharomyces cerevisiae RAS2 proteins; Jacquet E et al.; GDP-dissociation stimulators (GDSs) are the key element for the regeneration of the active state of ras proteins, but despite intensive investigations, little is so far known about their functional and structural properties, particularly in mammals . A growing number of genes from various organisms have been postulated to encode GDSs on the basis of sequence similarity with the Saccharomyces cerevisiae CDC25 gene, whose product acts as a GDS of RAS proteins . However, except for CDC25 and the related SDC25 C-domain, no biochemical evidence of ras GDS activity for these CDC25-like proteins has yet been available . We show that the product of a recently isolated mouse CDC25-like gene (CDC25Mm) can strongly enhance (more than 1000 times) the GDP release from both human c-Ha-ras p21 and yeast RAS2 in vitro . As a consequence, the CDC25Mm induces a rapid formation of the biologically active Ras.GTP complex . This GDS is much more active on the GDP than on the GTP complex and has a narrow substrate specificity, since it was found to be inactive on several ras-like proteins . The mouse GDS can efficiently substitute for yeast CDC25 in an in vitro adenylylcyclase assay on RAS2 cdc25 yeast membranes . Our results show that a cloned GDP to GTP exchange factor of mammalian ras belongs to the novel family of CDC25-like proteins.

J Biol Chem, 1992 Dec 5, 267(34), 24769 - 75
Three-dimensional structure of the truncated core of the Saccharomyces cerevisiae pyruvate dehydrogenase complex determined from negative stain and cryoelectron microscopy images; Stoops JK et al.; Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components are attached . We have imaged by negative stain and cryoelectron microscopy the truncated dihydrolipoamide acetyltransferase core (60 subunits; M(r) = 2.7 x 10(6)) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex . Using icosahedral particle reconstruction techniques, we determined its structure to 25 A resolution . Although the model derived from the negative stain reconstruction was approximately 20% smaller than the model derived from the frozen-hydrated data, when corrected for the effects of the electron microscope contrast transfer functions, the reconstructions showed excellent correspondence . The pentagonal dodecahedron-shaped macromolecule has a maximum diameter, as measured along the 3-fold axis, of approximately 226 A (frozen-hydrated value), and 12 large openings (approximately 63 A in diameter) on the 5-fold axes that lead into a large solvent-accessible cavity (approximately 76-140 A diameter) . The 20 vertices consist of cone-shaped trimers, each with a flattened base on the outside of the structure and an apex directed toward the center . The trimers are interconnected by 20 A thick "bridges" on the 2-fold axes . These studies also show that the highest resolution features apparent in the frozen-hydrated reconstruction are revealed in a filtered reconstruction of the stained molecule.

Mol Gen Genet, 1992 Dec, 236(1), 8 - 16
Differential repair and recombination of psoralen damaged plasmid DNA in Saccharomyces cerevisiae; Han EK et al.; Psoralen photoreaction with DNA produces interstrand crosslinks, which require the activity of excision and recombinational pathways for repair . Yeast replicating plasmids, carrying the HIS3, TRP1, and URA3 genes, were photoreacted with psoralen in vitro and transfected into Saccharomyces cerevisiae cells . Repair was assayed as the relative transformation efficiency . A recombination-deficient rad52 strain was the least efficient in the repair of psoralen-damaged plasmids; excision repair-deficient rad1 and rad3 strains had repair efficiencies intermediate between those of rad52 and RAD cells . The level of repair also depended on the conditions of transformant selection; repair was more efficient in medium lacking tryptophan than in medium from which either histidine or uracil was omitted . The plasmid repair differential between these selective media was greatest in rad1 cells, and depended on RAD52 . Plasmid-chromosome recombination was stimulated by psoralen damage, and required RAD52 function . Chromosome to plasmid gene conversion was seen most frequently at the HIS3 locus . In RAD and rad3 cells, the majority of the conversions were associated with plasmid integration, while in rad1 cells most were non-crossover events . Plasmid to chromosome gene conversion was observed most frequently at the TRP1 locus, and was accompanied by plasmid loss.

Mol Gen Genet, 1992 Dec, 236(1), 17 - 24
The REV3 gene of Saccharomyces cerevisiae is transcriptionally regulated more like a repair gene than one encoding a DNA polymerase; Singhal RK et al.; We measured the relative steady-state levels of the mRNA transcribed from the Saccharomyces cerevisiae REV3 gene in cells at different stages of the mitotic and meiotic cycles, and after UV irradiation . This gene is thought to encode a DNA polymerase concerned only with a specific recovery function, the replication on mutagen-damaged templates that produces damaged-induced mutations . In keeping with this proposed function, the REV3 gene showed no evidence of the periodic transcription at the G1/S boundary of the mitotic and meiotic cycle that occurs with genes encoding replication enzymes . However, levels of REV3 mRNA were much increased in late meiotic cells, like those of transcripts of some other DNA repair-related genes . Steady-state levels of REV3 transcript were increased only slightly in response to UV irradiation.

Mol Gen Genet, 1992 Dec, 236(1), 145 - 54
STE50, a novel gene required for activation of conjugation at an early step in mating in Saccharomyces cerevisiae; Rad MR et al.; A new gene, STE50, which plays an essential role in cell differentiation in Saccharomyces cerevisiae was detected and analysed . STE50 expression is not cell type-specific and its expression in MATa and MAT alpha cells is unaffected by pheromones . When present on a high copy number plasmid, STE50 causes supersensitivity to alpha-pheromone, and increases the level of alpha-pheromone-induced transcription of FUS1 in haploid a cells . Mutants bearing either of the two gene disruptions, ste50-1 or ste50-2, are sterile and have a modulated sensitivity to alpha-pheromone . The overexpression of STE4 (G beta) in wild-type cells elicits a constitutive growth arrest signal, however this phenotype is suppressed by a C-terminal truncation mutation in STE50 (ste50-2) . In contrast, the constitutive activation of the pheromone response pathway caused by disruption of GPA1 (G alpha) is not suppressed in ste50-2 mutants . The ste50-2 mutation partially suppresses the desensitisation defect of the sst2-1 mutation, and the resulting ste50-2 sst2-1 mutants restore fertility . Our results indicate that the ste50-2 mutant may have a defect in adaptation (hyperadaptation), and suggest a possible interaction of STE50-2 with the G alpha subunit of the G protein.

Trends Genet, 1992 Dec, 8(12), 446 - 52
Mating-type gene switching in Saccharomyces cerevisiae; Haber JE; The study of yeast mating-type (MAT) gene switching has provided insights into several aspects of the regulation of gene expression . MAT switching is accomplished by a highly programmed site-specific homologous recombination event in which mating-type-specific sequences at MAT are replaced by alternative DNA sequences copied from one of two unexpressed donors . The mating-type system has also provided an opportunity to study both the genetic regulation of gene silencing by alterations in chromatin structure, and the basis of preferential recombination between a recipient of genetic information and one of several possible donors.

FEMS Microbiol Lett, 1992 Dec 1, 78(2-3), 193 - 7
Multiple transformation of Saccharomyces cerevisiae by protoplast fusion; Reeves E et al.; A technique for the multiple transformation of yeast by protoplast fusion is described . This involved the PEG-induced fusion of protoplasts from cells which had been treated with chromosome-fragmenting agents (in this case cupferron and hydroxylamine) with protoplasts of triply auxotrophic cells . The recovery of transformants was increased significantly if one of the amino acid requirements of the recipient strain was included in the selection medium . Transformants isolated on supplemented media remained auxotrophic for that requirement . Prototrophic, uninucleate transformants had a DNA content and cellular volume similar to that of the parental strains . Possible mechanisms of gene transfer are discussed . This technique offers the possibility of transferring desirable characteristics from one yeast strain to another without altering the ploidy level of the recipient strain.

J Steroid Biochem Mol Biol, 1992 Dec, 43(7), 741 - 3
A simple method for the isolation of zymosterol from a sterol mutant of Saccharomyces cerevisiae; Heiderpriem RW et al.; A simple method is described for the direct isolation of zymosterol (5 alpha-cholesta-8,24-dien-3 beta-ol) of high purity from a sterol mutant of Saccharomyces cerevisiae . This yeast strain, which is a double mutant of the ERG6 (sterol transmethylase) and ERG2 (C-8 sterol isomerase) genes, accumulates zymosterol as its major sterol component.

Genetics, 1992 Dec, 132(4), 987 - 1001
HTS1 encodes both the cytoplasmic and mitochondrial histidyl-tRNA synthetase of Saccharomyces cerevisiae: mutations alter the specificity of compartmentation; Chiu MI et al.; Genetic and biochemical evidence shows that a single nuclear gene HTS1 encodes both the mitochondrial and cytoplasmic histidyl-tRNA synthetases (Hts) . The gene specifies two messages, one with two in-frame ATGs (-60 and +1) and another with only the downstream ATG (+1) . We have made a new set of mutations that enables us to express only the mitochondrial or the cytoplasmic form and compared the subcellular distribution of the Hts1 protein in these mutants and wild type, using an antibody that interacts with both the mitochondrial and cytoplasmic Hts1 as well as Hts1::LacZ fusions . Mutations in the upstream ATG (-60) or frameshift mutations in the presequence affect only the mitochondrial enzyme and not the cytoplasmic enzyme . Mutations in the downstream ATG (+1 ATG to ATC) destroy the function of the cytosolic enzyme, but do not affect the function of the mitochondrial enzyme . Overexpression of this construct restores cytoplasmic function . Cells expressing a truncated form of Hts containing a deletion of the first 20 amino-terminal residues (Htsc) produce a functional cytoplasmic enzyme, which does not provide mitochondrial function . Overexpression of this truncated cytoplasmic protein provides mitochondrial function and produces detectable levels of the synthetase in the mitochondrion . These experiments suggest that Hts1 contains two domains that together allow efficient localization of Htsm to the mitochondrion: an amino-terminal presequence in the mitochondrial precursor that is likely cleaved upon delivery to the mitochondrion and a second amino-terminal sequence (residues 21-53) present in both the precursor and the cytoplasmic form . Neither one by itself is sufficient to act as an efficient mitochondrial targeting signal . Using our antibody we have been able to detect a protein of increased molecular mass that corresponds to that of the predicted precursor . Taken together these studies show that the specificity of compartmentation of the Hts protein depends upon both the primary sequence and the concentration of the protein in the cell.

Genetics, 1992 Dec, 132(4), 951 - 62
The CCR4 protein from Saccharomyces cerevisiae contains a leucine-rich repeat region which is required for its control of ADH2 gene expression; Malvar T et al.; The CCR4 gene from Saccharomyces cerevisiae is required for the transcription of the glucose-repressible alcohol dehydrogenase (ADH2) . Mutations in CCR4 also suppress the transcription at the ADH2 and his4-912delta loci caused by defects in the SPT10 (CRE1) and SPT6 (CRE2) genes . The CCR4 gene was mapped to the left arm of chromosome I and cloned by complementation of function using previously isolated segments of chromosome I . DNA sequence analysis of the cloned gene defined CCR4 as a 2511 bp open reading frame that would encode a polypeptide of 837 amino acids . The CCR4 mRNA was found to be 2.8 kb in size and Western analysis identified CCR4 as a 95,000 D protein . Disruption of the CCR4 gene resulted in reduced levels of ADH2 expression under both glucose and ethanol growth conditions and in temperature sensitive growth on nonfermentative medium, phenotypes essentially indistinguishable from previously identified mutations in CCR4 . The amino terminus of the CCR4 protein was found to be rich in glutamine residues similar to a number of genes which are required for transcription . More importantly, CCR4 showed similarity to a diverse set of proteins sharing a leucine-rich tandem repeat motif, the presence of which has been implicated in mediating protein-protein interactions . Deletions of several of the five leucine-rich repeats in CCR4 were shown to produce nonfunctional proteins indicating the importance of the repeats to CCR4 activity . This leucine-rich repeat region may mediate the contact CCR4 makes with another factor.

Genetics, 1992 Dec, 132(4), 943 - 50
Spontaneous amplification of the ADH4 gene in Saccharomyces cerevisiae; Dorsey M et al.; Five spontaneous amplifications of the ADH4 gene were identified among 1,894 antimycin A-resistant mutants isolated from a diploid strain after growth at 15 degrees . Four of these amplifications are approximately 40-kb linear extrachromosomal palindromes carrying telomere homologous sequences at each end similar to a previously isolated amplification . ADH4 is located at the extreme left end of chromosome VII, and the extrachromosomal fragments appear to be the fusion of two copies of the end of this chromosome . The fifth amplification is a chromosomal amplification carrying an extra copy of ADH4 on both homologs of chromosome VII . These results suggest that the ADH system can be used to study amplification in Saccharomyces cerevisiae.

Genetics, 1992 Dec, 132(4), 929 - 42
Donor locus selection during Saccharomyces cerevisiae mating type interconversion responds to distant regulatory signals; Weiler KS et al.; Mating type interconversion in homothallic strains of the yeast Saccharomyces cerevisiae results from directed transposition of a mating type allele from one of the two silent donor loci, HML and HMR, to the expressing locus, MAT . Cell type regulates the selection of the particular donor locus to be utilized during mating type interconversion: MATa cells preferentially select HML alpha and MAT alpha cells preferentially select HMRa . Such preferential selection indicates that the cell is able to distinguish between HML and HMR during mating type interconversion . Accordingly, we designed experiments to identify those features perceived by the cell to discriminate HML and HMR . We demonstrate that discrimination does not derive from the different structures of the HML and HMR loci, from the unique sequences flanking each donor locus nor from any of the DNA distal to the HM loci on chromosome III . Moreover, we find that the sequences flanking the MAT locus do not function in the preferential selection of one donor locus over the other . We propose that the positions of the donor loci on the left and right arms of chromosome III is the characteristic utilized by the cell to distinguish HML and HMR . This positional information is not generated by either CEN3 or the MAT locus, but probably derives from differences in the chromatin structure, chromosome folding or intranuclear localization of the two ends of chromosome III.

Gene, 1992 Dec 1, 122(1), 91 - 9
The mosaic organization of the mitochondrial introns of Saccharomyces cerevisiae: features and evolutionary origins; de Zamaroczy M et al.; The introns of three genes (oxi3, cob and 21S) from the mitochondrial (mt) genome of Saccharomyces cerevisiae contain closed reading frames (CRFs) . In the present work, we have analyzed these sequences in their oligodeoxyribonucleotide (oligo; isostich) patterns . We have shown that the relative amounts of di- to hexanucleotides, when compared to random sequences having the same sizes and compositions, exhibit the same deviations as the intergenic noncoding sequences of the mt genome (except for the CRFs from 21S intron) . In contrast, intronic open reading frames (ORFs) showed oligo patterns which were generally quite distinct from those of CRFs, although some similarities could be detected in some cases (especially for aI5 alpha) . The mt introns of yeast, therefore, are endowed with a mosaic structure, in which CRFs derive from mt intergenic sequences, whereas ORFs have a different origin (indicated as exogenous by other evidences) yet show, in some cases, the effects of 'sequence assimilation' with CRFs.

Gene, 1992 Dec 1, 122(1), 139 - 45
High-level production of a peroxisomal enzyme: Aspergillus flavus uricase accumulates intracellularly and is active in Saccharomyces cerevisiae; Leplatois P et al.; Strains of Saccharomyces cerevisiae producing Aspergillus flavus uricase (Uox) have been constructed . An artificial promoter which combined the upstream and downstream sequences of the GAL7 and ADH2 promoters, respectively, was found to be efficient in directing the synthesis of uaZ mRNAs encoding Uox . A good proportionality between the copy number of the uaZ expression cassette and the level of Uox production was found in the range of 1-10 copies . Transformants accumulated active and soluble Uox to a level exceeding 13% of total protein, as deduced from enzymatic assays . This relative level could be improved two- to threefold by using a recipient strain in which the wild-type GAL4 gene had been deleted and which expressed a GAL4 construct placed under the control of the ADH2 promoter.

Mol Cell Biol, 1992 Dec, 12(12), 5778 - 84
A mutation in the tRNA nucleotidyltransferase gene promotes stabilization of mRNAs in Saccharomyces cerevisiae; Peltz SW et al.; To identify trans-acting factors involved in mRNA decay in the yeast Saccharomyces cerevisiae, we have begun to characterize conditional lethal mutants that affect mRNA steady-state levels . A screen of a collection of temperature-sensitive mutants identified ts352, a mutant that accumulated moderately stable and unstable mRNAs after a shift from 23 to 37 degrees C (M . Aebi, G . Kirchner, J.-Y . Chen, U . Vijayraghavan, A . Jacobson, N.C . Martin, and J . Abelson, J . Biol . Chem . 265:16216-16220, 1990) . ts352 has a defect in the CCA1 gene, which codes for tRNA nucleotidyltransferase, the enzyme that adds 3' CCA termini to tRNAs (Aebi et al., J . Biol . Chem., 1990) . In a shift to the nonpermissive temperature, ts352 (cca1-1) cells rapidly cease protein synthesis, reduce the rates of degradation of the CDC4, TCM1, and PAB1 mRNAs three- to fivefold, and increase the relative number of ribosomes associated with mRNAs and the overall size of polysomes . These results were analogous to those observed for cycloheximide-treated cells and are generally consistent with models that invoke a role for translational elongation in the process of mRNA turnover.

Mol Cell Biol, 1992 Dec, 12(12), 5724 - 35
Evidence that POB1, a Saccharomyces cerevisiae protein that binds to DNA polymerase alpha, acts in DNA metabolism in vivo; Miles J et al.; Potential DNA replication accessory factors from the yeast Saccharomyces cerevisiae have previously been identified by their ability to bind to DNA polymerase alpha protein affinity matrices (J . Miles and T . Formosa, Proc . Natl . Acad . Sci . USA 89:1276-1280, 1992) . We have now used genetic methods to characterize the gene encoding one of these DNA polymerase alpha-binding proteins (POB1) to determine whether it plays a role in DNA replication in vivo . We find that yeast cells lacking POB1 are viable but display a constellation of phenotypes indicating defective DNA metabolism . Populations of cells lacking POB1 accumulate abnormally high numbers of enlarged large-budded cells with a single nucleus at the neck of the bud . The average DNA content in a population of cells lacking POB1 is shifted toward the G2 value . These two phenotypes indicate that while the bulk of DNA replication is completed without POB1, mitosis is delayed . Deleting POB1 also causes elevated levels of both chromosome loss and genetic recombination, enhances the temperature sensitivity of cells with mutant DNA polymerase alpha genes, causes increased sensitivity to UV radiation in cells lacking a functional RAD9 checkpoint gene, and causes an increased probability of death in cells carrying a mutation in the MEC1 checkpoint gene . The sequence of the POB1 gene indicates that it is identical to the CTF4 (CHL15) gene identified previously in screens for mutations that diminish the fidelity of chromosome transmission . These phenotypes are consistent with defective DNA metabolism in cells lacking POB1 and strongly suggest that this DNA polymerase alpha-binding protein plays a role in accurately duplicating the genome in vivo.

Mol Cell Biol, 1992 Dec, 12(12), 5640 - 51
Cloning and characterization of SRP1, a suppressor of temperature-sensitive RNA polymerase I mutations, in Saccharomyces cerevisiae; Yano R et al.; The SRP1-1 mutation is an allele-specific dominant suppressor of temperature-sensitive mutations in the zinc-binding domain of the A190 subunit of Saccharomyces cerevisiae RNA polymerase I (Pol I) . We found that it also suppresses temperature-sensitive mutations in the zinc-binding domain of the Pol I A135 subunit . This domain had been suggested to be in physical proximity to the A190 zinc-binding domain . We have cloned the SRP1 gene and determined its nucleotide sequence . The gene encodes a protein of 542 amino acids consisting of three domains: the central domain, which is composed of eight (degenerate) 42-amino-acid contiguous tandem repeats, and the surrounding N-terminal and C-terminal domains, both of which contain clusters of acidic and basic amino acids and are very hydrophilic . The mutational alteration (P219Q) responsible for the suppression was found to be in the central domain . Using antibody against the SRP1 protein, we have found that SRP1 is mainly localized at the periphery of the nucleus, apparently more concentrated in certain regions, as suggested by a punctate pattern in immunofluorescence microscopy . We suggest that SRP1 is a component of a larger macromolecular complex associated with the nuclear envelope and interacts with Pol I either directly or indirectly through other components in the structure containing SRP1.

Mol Cell Biol, 1992 Dec, 12(12), 5593 - 9
Alternative topogenic signals in peroxisomal citrate synthase of Saccharomyces cerevisiae; Singh KK et al.; The tripeptide serine-lysine-leucine (SKL) occurs at the carboxyl terminus of many peroxisomal proteins and serves as a peroxisomal targeting signal . Saccharomyces cerevisiae has two isozymes of citrate synthase . The peroxisomal form, encoded by CIT2, terminates in SKL, while the mitochondrial form, encoded by CIT1, begins with an amino-terminal mitochondrial signal sequence and ends in SKN . We analyzed the importance of SKL as a topogenic signal for citrate synthase, using oleate to induce peroxisomes and density gradients to fractionate organelles . Our experiments revealed that SKL was necessary for directing citrate synthase to peroxisomes . C-terminal SKL was also sufficient to target a leaderless version of mitochondrial citrate synthase to peroxisomes . Deleting this tripeptide from the CIT2 protein caused peroxisomal citrate synthase to be missorted to mitochondria . These experiments suggest that the CIT2 protein contains a cryptic mitochondrial targeting signal.

Mol Cell Biol, 1992 Dec, 12(12), 5455 - 63
Histone H3 transcription in Saccharomyces cerevisiae is controlled by multiple cell cycle activation sites and a constitutive negative regulatory element; Freeman KB et al.; The promoters of the Saccharomyces cerevisiae histone H3 and H4 genes were examined for cis-acting DNA sequence elements regulating transcription and cell division cycle control . Deletion and linker disruption mutations identified two classes of regulatory elements: multiple cell cycle activation (CCA) sites and a negative regulatory site (NRS) . Duplicate 19-bp CCA sites are present in both the copy I and copy II histone H3-H4 promoters arranged as inverted repeats separated by 45 and 68 bp . The CCA sites are both necessary and sufficient to activate transcription under cell division cycle control . A single CCA site provides cell cycle control but is a weak transcriptional activator, while an inverted repeat comprising two CCA sites provides both strong transcriptional activation and cell division cycle control . The NRS was identified in the copy I histone H3-H4 promoter . Deletion or disruption of the NRS increased the level of the histone H3 promoter activity but did not alter the cell division cycle periodicity of transcription . When the CCA sites were deleted from the histone promoter, the NRS element was unable to confer cell division cycle control on the remaining basal level of transcription . When the NRS element was inserted into the promoter of a foreign reporter gene, transcription was constitutively repressed and did not acquire cell cycle regulation.

J Virol, 1992 Dec, 66(12), 7253 - 61
Expression and characterization of the trans-activating protein Tax of human T-cell leukemia virus type I in Saccharomyces cerevisiae; Wu LC et al.; The trans-activator protein Tax of human T-cell leukemia virus type I (HTLV-I) stimulates transcription of the viral genome from the long terminal repeat . With a reporter HIS4TATA::lacZ fusion gene, the transcriptional activity of the Tax-responsive element in the long terminal repeat was tested in Saccharomyces cerevisiae . We found that fragments containing the 21-bp repeat of the HTLV-I enhancer stimulate synthesis of beta-galactosidase activity 15- to 20-fold . To test the ability of the Tax protein to trans activate the HTLV-I enhancer in yeast cells, the pX region of HTLV-I, encoding the Tax protein, was cloned under the control of the yeast GAL1 promoter . The expressed Tax protein is localized in the nucleus and associated with the yeast nuclear matrix fraction . In yeast cells that contained the integrated tax gene, two- to sixfold stimulation of expression from the HTLV-I enhancer was detected at the early stages of tax induction . This in vivo reconstitution system provides a new approach for examining the host factor(s), the signal transduction mechanism(s), and the role of nuclear architecture involved in Tax-mediated trans activation.

Biochimie, 1992 Dec, 74(12), 1103 - 15
Effects of 2-deoxy-D-glucose on the glucose metabolism in Saccharomyces cerevisiae studied by multinuclear-NMR spectroscopy and biochemical methods; Herve M et al.; The effects of various concentrations of deoxyglucose (DG) on the aerobic metabolism of glucose in glucose-grown repressed Saccharomyces cerevisiae cells were studied at 30 degrees C in a standard pyrophosphate medium containing 4.5 10(7) cells/ml . 31P-nuclear magnetic resonance (NMR) spectroscopy was used to monitor DG phosphorylation and the formation of polyphosphates . The production of soluble metabolites of glucose was evaluated by 13C- and 1H-NMR and biochemical techniques . The cells were aerobically incubated with 25 mM of glucose and various concentrations of DG (0, 5 and 10 mM) in order to determine the DG concentration leading to optimum of 2-deoxy-D-glucose 6-phosphate (DG6P) formation without over-inhibiting the synthesis of other metabolites . The production of DG6P increased by about 25% when the external DG concentration was doubled (from 5 to 10 mM) . The formation of polyphosphates (polyP), on the other hand, was found to be mainly conditioned by the DG concentration . The amount of polyP decreased by a factor of four upon addition of 5 mM DG and became undetectable in the presence of 10 mM DG . The glucose consumption and the production of soluble metabolites of {1-13C}glucose were then evaluated as a function of time in both the absence and presence of 5 mM DG . The effect of DG is to decrease the glucose consumption and the formation of polyphosphates, ethanol, glycerol, trehalose, glutamate, aspartate and succinate while stimulating the formation of arginine and citrate . Upon co-addition of 25 mM glucose and 5 mM DG, the ratio between the initial rates of glucose consumption (0.16 mM/min) and DG6P production (0.027 mM/min) is about (5.9 +/- 1.2), not very different from the ratio of the initial concentration of glucose and DG (= 5.0) . Therefore, hexokinase can phosphorylate deoxyglucose as well as glucose . However, after 100 min of incubation, the glucose concentration in the external medium decreased by about 64% while only 10% of DG was phosphorylated . DG6P was formed and quickly reached the limiting value about 30 min after co-addition of glucose and DG . Nevertheless, when the maximum quantity of DG6P was obtained, the DG consumption became negligible . By contrast, the glucose consumption and the production of ethanol and glycerol, although substantially reduced by about 42%, varied linearly with time up to 80 min of incubation . Thus even in the presence of an excess of DG, glycolysis is only slowed but not gradually or completely inhibited by DG.(ABSTRACT TRUNCATED AT 400 WORDS)

Mol Cell Biol, 1992 Dec, 12(12), 5736 - 47
CTF4 (CHL15) mutants exhibit defective DNA metabolism in the yeast Saccharomyces cerevisiae; Kouprina N et al.; We have analyzed the CTF4 (CHL15) gene, earlier identified in two screens for yeast mutants with increased rates of mitotic loss of chromosome III and artificial circular and linear chromosomes . Analysis of the segregation properties of circular minichromosomes and chromosome fragments indicated that sister chromatid loss (1:0 segregation) is the predominant mode of chromosome destabilization in ctf4 mutants, though nondisjunction events (2:0 segregation) also occur at an increased rate . Both inter- and intrachromosomal mitotic recombination levels are elevated in ctf4 mutants, whereas spontaneous mutation to canavanine resistance was not elevated . A genomic clone of CTF4 was isolated and used to map its physical and genetic positions on chromosome XVI . Nucleotide sequence analysis of CTF4 revealed a 2.8-kb open reading frame with a 105-kDa predicted protein sequence . The CTF4 DNA sequence is identical to that of POB1, characterized as a gene encoding a protein that associates in vitro with DNA polymerase alpha . At the N-terminal region of the protein sequence, zinc finger motifs which define potential DNA-binding domains were found . The C-terminal region of the predicted protein displayed similarity to sequences of regulatory proteins known as the helix-loop-helix proteins . Data on the effects of a frameshift mutation suggest that the helix-loop-helix domain is essential for CTF4 function . Analysis of sequences upstream of the CTF4 open reading frame revealed the presence of a hexamer element, ACGCGT, a sequence associated with many DNA metabolism genes in budding yeasts . Disruption of the coding sequence of CTF4 did not result in inviability, indicating that the CTF4 gene is nonessential for mitotic cell division . However, ctf4 mutants exhibit an accumulation of large budded cells with the nucleus in the neck . ctf4 rad52 double mutants grew very slowly and produced extremely high levels (50%) of inviable cell division products compared with either single mutant alone, which is consistent with a role for CTF4 in DNA metabolism.

J Gen Microbiol, 1992 Dec, 138 ( Pt 12), 2551 - 7
TPK gene products mediate cAMP-independent thermotolerance in Saccharomyces cerevisiae; Coote PJ et al.; Incubation of Saccharomyces cerevisiae with the plant cytokinin N6-(delta 2-isopentenyl)adenine (2iP) resulted in an induction of thermotolerance similar to that induced by sublethal temperatures . Intracellular cAMP levels did not change significantly either during incubation at a sublethal temperature or in the presence of 2iP or ethanol . This suggested that stress-induced thermotolerance is triggered by a mechanism independent of cAMP activation . However, measurement of stress-induced thermotolerance in two mutant strains (tpk1, tpk2, TPK3; tpk1, TPK2, tpk3) each deficient in two of the catalytic subunits of the cAMP-dependent protein kinase (cAPK), revealed that sublethal heat induces thermotolerance by a mechanism part-mediated by the catalytic subunits of cAPK . In contrast, 2iP and ethanol induced thermotolerance by a mechanism fully dependent on the catalytic subunits of cAPK for expression . Therefore, this implies there must be an alternative novel mechanism, other than cAMP, for activating cAPK during stress . Sublethal heating resulted in large increases in intracellular trehalose levels which correlated with the induction of thermotolerance . However, incubation in 2iP or ethanol had no significant effect . This suggests trehalose synthesis is either coincidental with heat stress or that different stress factors induce thermotolerance by alternative mechanisms . Incubation with protein synthesis inhibitors reduced the levels of trehalose synthesized during sublethal heating, suggesting that synthesis of trehalose-6-phosphate synthase during heat stress could be accounting for the increased trehalose levels.

Protein Expr Purif, 1992 Dec, 3(6), 441 - 52
Expression of recombinant cytochromes c from various species in Saccharomyces cerevisiae: post-translational modifications; Koshy TI et al.; A complete protocol for the expression of recombinant cytochrome c genes from yeast, Drosophila melanogaster, and rat in a yeast strain, GM-3C-2, which does not express its own cytochromes c is described . The construction of the expression vectors, transformation and large-scale growth of the yeast, and preparation and purification of the recombinant cytochromes c are described . It was found that, contrary to the way yeast modifies its own cytochromes c, the recombinant proteins were partially acetylated at their N-terminus, except for the drosophila protein, which remained entirely unblocked . Furthermore, the yeast and rat proteins were close to fully trimethylated at lysine 72, while the drosophila protein could be separated chromatographically into forms containing tri-, di-, mono-, and unmethylated lysine 72 showing corresponding resonances in the NMR spectrum . These observations emphasize that, in employing expression procedures to obtain native or mutant forms of cytochrome c, it is essential to identify the variety and extent of post-translational modifications and to separate the preparation into pure monomolecular species . Otherwise, it may become impossible to distinguish between the influence of a site-directed mutation and unexamined post-translational modifications.

J Cell Biol, 1992 Dec, 119(6), 1469 - 79
In vitro reactions of vacuole inheritance in Saccharomyces cerevisiae; Conradt B et al.; Vacuole inheritance is temporally coordinated with the cell cycle and is restricted spatially to an axis between the maternal vacuole and the bud . The new bud vacuole is founded by a stream of vacuole-derived membranous vesicles and tubules which are transported from the mother cell into the bud to form the daughter organelle . We now report in vitro formation of vacuole-derived tubules and vesicles . In semi-intact cells, formation of tubulovesicular structures requires ATP and the proteins encoded by VAC1 and VAC2, two genes which are required for vacuole inheritance in vivo . Isolation of vacuoles from cell lysates before in vitro incubation reveals that formation of tubulovesicular structures requires cytosol as well as ATP . After forming tubulovesicular structures, isolated vacuoles subsequently increase in size . Biochemical assays reveal that this increase results from vacuole to vacuole fusion, leading to mixing of organellar contents . Intervacuolar fusion is sensitive to the phosphatase inhibitors microcystin-LR and okadaic acid, suggesting that protein phosphorylation/dephosphorylation reactions play a role in this event.

Yeast, 1992 Dec, 8(12), 997 - 1006
The allantoin and uracil permease gene sequences of Saccharomyces cerevisiae are nearly identical; Yoo HS et al.; We have determined the structure of the allantoin permease (DAL4) gene of Saccharomyces cerevisiae . The gene putatively encodes a hydrophobic protein with a M(r) of 71,755 . It possesses the alternating hydrophobic-hydrophilic regions similar to those found in many other integral membrane proteins . The most striking feature of the allantoin permease component encoded by DAL4 is its striking similarity to the uracil permease component encoded by FUR4 . Although data available indicate that these proteins do not share any overlap of function, their predicted protein sequences are 68% identical, 81% similar, and their DNA sequences are 70% identical . The upstream regulatory region of DAL4 contains all of the characterized cis-acting elements previously reported for inducible allantoin pathway genes: six sequences homologous to UASNTR, the element responsible for nitrogen catabolite repression-sensitive activation of allantoin pathway gene expression, and two sequences homologous to the cis-acting element responsible for inducer-responsiveness of the allantoin pathway genes, UIS . The finding of these homologous sequences predicted to exist on the basis of DAL4's expression characteristics, supports and strengthens the suggestion that these elements mediate the functions we have previously ascribed to them.

Yeast, 1992 Dec, 8(12), 1033 - 41
Polyamines and cell wall organization in Saccharomyces cerevisiae; Miret JJ et al.; Cells of Saccharomyces cerevisiae 179-5, an ornithine decarboxylase mutant (spe-1), showed several ultrastructural abnormalities when cultivated in the absence of polyamines . Besides the appearance of microvacuole-like spaces in the cytoplasm and of deformed nuclei, the most important alterations seemed to be located in the cell wall, which was thicker and of heterogeneous texture, and in the cell membrane, of irregular contour . These modifications could not be evoked by general stress conditions elicited by lack of nutrients . The relative levels of cell wall polysaccharides were altered in polyamine-deprived organisms, giving an envelope with increased mannan and decreased glucan content; this cell wall was incompletely attacked by the lytic enzyme zymolyase . Polyamine depletion led also to some abnormalities in the budding pattern . The above observations suggest the involvement of polyamines in the correct structure and organization of the yeast cell.

Yeast, 1992 Dec, 8(12), 1015 - 24
Effect of sterol alterations on conjugation in Saccharomyces cerevisiae; Tomeo ME et al.; Sterol auxotrophic strains of Saccharomyces cerevisiae were grown and allowed to conjugate on media supplemented with various sterols . The mating efficiency of the auxotrophs is perturbed by the replacement of the normal yeast sterol, ergosterol, with other sterols . After 4 h of mating, cells grown on ergosterol exhibited a 30-fold higher productive mating efficiency than those cells grown in stigmasterol . Aberrant budding by the conjugants was enhanced following incubation on stigmasterol and other non-ergosterol sterols . Using light and electron microscopy, we demonstrated that there is a reduced ability for stigmasterol-grown cells to undergo cytoplasmic fusion during conjugation . Many of the mated pairs remained adherent but prezygotic even after 12 h of incubation . The addition of ergosterol to cells previously grown on stigmasterol rescued the organisms, allowing for zygote formation and normal budding.

Can J Microbiol, 1992 Dec, 38(12), 1298 - 306
Flocculation of Saccharomyces cerevisiae: inhibition by sugars; Masy CL et al.; Flocculation is governed by the competition between electrostatic repulsion (nonspecific interactions) and polysaccharide-protein bonds (specific interactions) . In our study, the inhibition of flocculation by sugars for 12 strains of Saccharomyces cerevisiae leads us to extend the classification described in the literature and to define three groups of yeasts: flocculation mannose sensitive (MS), flocculation glucose-mannose sensitive (GMS), and flocculation mannose insensitive (MI) . Only the first two groups showed specific interactions between proteins and mannans . n the MI group, the sugars tested did not inhibit flocculation . To characterize the particularities of the stereochemistry of the cell-wall proteic receptors of strains belonging to the MS and GMS groups, 31 sugars were used as inhibitor probes on two representative strains . The results show that the lectin specificity of strains belonging to the GMS group is less restricted regarding C-1 and C-2 hydroxyl groups than the lectin from strains belonging to the MS group, which interacts with all of the hydroxyl groups of mannopyranose . The two groups also differ with respect to inhibition by sugars: strains belonging to the MS group are partially inhibited whereas strains of the GMS group are completely inhibited . We observed that the presence of ethanol increases sugar fixation by strains from the MS group, but not from the GMS group . Moreover, both receptors interact with disaccharides, provided the two monomers are linked by an alpha(1-4), alpha(1-3), or alpha(1-2) bond.

Yeast, 1992 Dec, 8(12), 1007 - 14
RNA delivery in Saccharomyces cerevisiae using electroporation; Everett JG et al.; An efficient delivery method for introducing in vitro synthesized RNA into yeast has been developed using electroporation . Spheroplast preparation, electroporation, and subsequent expression analysis can be accomplished within a single day . The use of introduced mRNA constructs avoids any complications due to nuclear regulation and is particularly suited for cytoplasmic regulatory studies . Moreover, this technique is useful for introducing those RNAs that cannot be made in vivo, such as poly(A)- mRNAs or RNAs with base modifications . We demonstrate that the Escherichia coli GUS gene and the firefly Luc gene are both excellent reporter genes for RNA electroporation.

Mutat Res, 1992 Dec 1, 284(1), 111 - 23
DNA damage induced mating type switching in Saccharomyces cerevisiae; Schiestl RH et al.; Haploid cells of the yeast Saccharomyces cerevisiae are able to undergo a differentiation-like process: they can switch their mating type between the a and the alpha state . The molecular mechanism of this interconversion of mating types is intrachromosomal gene conversion . It has been shown in a variety of studies that mating type switching in heterothallic strains can be induced by DNA damaging agents, and that different DNA damaging agents differ in the length of incubation after treatment required for induction . Because X-rays induce switching immediately after irradiation and because the DNA double-strand break repair pathway is required for switching, the event initiating heterothallic mating type switching is likely to be a DNA double-strand break . Therefore the assay for heterothallic mating type switching may screen for the induction of DNA double-strand breaks . Several aspects indicating a relationship of mating type switching to mechanisms associated with carcinogenesis are discussed.

Biochem Biophys Res Commun, 1992 Nov 30, 189(1), 85 - 91
Ergosterol depletion and 4-methyl sterols accumulation in the yeast Saccharomyces cerevisiae treated with an antifungal, 6-amino-2-n-pentylthiobenzothiazole; Kuchta T et al.; In Saccharomyces cerevisiae treated with an antifungal agent, 6-amino-2-n-pentylthiobenzothiazole, levels of ergosterol and other 4-desmethylsterols were found to be significantly reduced . Major sterols in treated yeast were lanosterol, 4,4-dimethylzymosterol, 4-methylzymosterol and 4-methylfecosterol . A hypothesis is stated that the antifungal agent inhibits sterol demethylation at C-4 and forces the biosynthesis to a blind pathway ending by 4-methylfecosterol.

Nucleic Acids Res, 1992 Nov 25, 20(22), 5955 - 61
Regulatory gene INO4 of yeast phospholipid biosynthesis is positively autoregulated and functions as a transactivator of fatty acid synthase genes FAS1 and FAS2 from Saccharomyces cerevisiae; Schuller HJ et al.; The sequence motif 5' TYTTCACATGY 3' functions as an upstream activation site common to both yeast fatty acid synthase genes, FAS1 and FAS2 . In addition, this UASFAS element is shared by all so far characterized genes of yeast phospholipid biosynthesis . We have investigated the influence of a functional INO4 gene previously described as a regulator of inositol biosynthesis on the expression of FAS1 and FAS2 . In a delta ino4 null allele strain, both genes are expressed at only 50% of wild type level . Using individual UASFAS sequence motifs inserted into a heterologous test system, a drastic decrease of reporter gene expression to 2-10% of the wild type reference was observed in the delta ino4 mutant . In gel retardation assays, the protein-DNA complex involving the previously described FAS binding factor 1, Fbf1, was absent when using a protein extract from the delta ino4 mutant . On the other hand, this signal was enhanced with an extract from cells grown under conditions of inositol/choline derepression . Subsequent experiments demonstrated that INO4 expression is itself affected by phospholipid precursors, mediated by an UASFAS element in the INO4 upstream region . Thus, in addition of being an activator of phospholipid biosynthetic genes, INO4 is also subject to a positive autoregulatory loop in its own biosynthesis.

J Biol Chem, 1992 Nov 25, 267(33), 24117 - 25
Purification and characterization of a soluble phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae; Flanagan CA et al.; A phosphatidylinositol (PI) 4-kinase was purified 25,000-fold from the cytosolic fraction of extracts from the yeast Saccharomyces cerevisiae . The purification consisted of an ammonium sulfate fractionation followed by chromatography on sulfonated-agarose (S-Sepharose), phosphocellulose, threonine-agarose, and quaternary amino (Mono Q), and sulfonated (Mono S) beads . Major contaminants in the purification, Hsc82 and Hsp82 (yeast homologs of the mammalian heat shock protein Hsp90), were eliminated by using a combination of molecular genetics (to construct a null mutation in HSC82), altered growth conditions (to minimize expression from the inducible HSP82 gene), and high ionic strength fractionation conditions (to remove the residual Hsp82) . The purified enzyme had an apparent subunit molecular weight of 125,000, much larger than any other well characterized PI-4-kinase reported previously . Like mammalian PI-4-kinases, the yeast enzyme specifically phosphorylated PI on position 4 of the inositol ring and was stimulated by Triton X-100 . However, activity was not inhibited by adenosine, a potent inhibitor of certain (type II) mammalian PI-4-kinases . The enzyme displayed typical Michaelis-Menten kinetics with apparent Km values of 100 microM for ATP and 50 microM for PI . To date, this yeast enzyme is the first soluble PI-4-kinase purified from any source.

Biochemistry, 1992 Nov 24, 31(46), 11376 - 82
Mutation of the heme-binding crevice of flavocytochrome b2 from Saccharomyces cerevisiae: altered heme potential and absence of redox cooperativity between heme and FMN centers; Kay CJ et al.; Kinetic and thermodynamic properties of yeast flavocytochrome b2 (EC 1.1.2.3) are modified by the product pyruvate, which binds to the flavosemiquinone (FSQ) form of the prosthetic flavin and decreases the thermodynamic driving force for electron transfer from FSQ to heme . Pyruvate inhibits flavocytochrome b2, but the catalytic competence of pyruvate-ligated FSQ in intramolecular electron transfer to heme is unclear; one kinetic study suggested pyruvate prevented this reaction {Tegoni, M, Janot J.-M., & Labeyrie, F . (1990) Eur . J . Biochem . 190, 329-342}, while laser flash photolysis indicated pyruvate was essential {Walker, M . C., & Tollin, G . (1991) Biochemistry 30, 5546-5555} . To address this problem, wild-type (WT) and mutant (L36I) flavocytochromes b2 have been expressed in Escherichia coli . Both forms incorporated heme and FMN prosthetic groups and were catalytically active . The mutation L36I was a conservative substitution within the heme-binding crevice and was designed to alter the midpoint potential (Em) of the heme to alter the pyruvate-FSQ/heme equilibrium . Potentiometric titrations yielded Em values (pH 7.0, 25 degrees C) of +8 and -28 mV for WT and L36I forms, respectively . The FMN midpoint potentials in the absence of pyruvate (-58 mV, n = 2) were identical within experimental error in WT and L36I species and were also identical (+5 mV, n = 1) in the presence of pyruvate . These results indicated the absence of redox cooperativity between FMN and heme.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutat Res, 1992 Nov 16, 270(2), 151 - 66
Formaldehyde, glyoxal, urethane, methyl carbamate, 2,3-butanedione, 2,3-hexanedione, ethyl acrylate, dibromoacetonitrile and 2-hydroxypropionitrile induce chromosome loss in Saccharomyces cerevisiae; Zimmermann FK et al.; Induction of mitotic chromosome loss could be demonstrated for the dialdehyde glyoxal, the diketones 2,3-butanedione and 2,3-hexanedione, ethyl and methyl carbamate, ethyl acrylate, dibromoacetonitrile, 2-hydroxypropionitrile and formaldehyde, but only when they were combined with subacute concentrations of propionitrile, which is a strong inducer of chromosomal malsegregation . The same chemicals did not induce mitotic chromosome loss when applied in pure form . However, glyoxal, ethyl acrylate, dibromoacetonitrile and formaldehyde when applied in pure form also induced mitotic recombination . Respiratory deficiency was induced, in the absence of propionitrile, by these recombinogenic agents and also by 2,3-hexanedione and 2-hydroxypropionitrile which are not recombinogenic.

Eur J Biochem, 1992 Nov 15, 210(1), 343 - 9
Purification of assembly-competent tubulin from Saccharomyces cerevisiae; Bellocq C et al.; We have developed a straightforward, two-step procedure to isolate highly purified yeast tubulin that reproducibly assembles into microtubules . The starting extracts are obtained from cells genetically engineered to overproduce both the alpha and beta subunits of tubulin, under control of the galactose promoter, to approximately 10-times wild-type levels . The first step of purification is carried out with the high-speed supernatant of lysed cells loaded onto a DEAE-Sephadex column; after this step the tubulin preparation is approximately 30% pure . In the second step, the tubulin fractions are loaded onto an immunoaffinity column prepared by coupling the anti-(alpha-tubulin) monoclonal antibody YL 1/2 to Sepharose-4B . Following elution with 0.8 M KCl, the tubulin present in the peak is 90% pure . Upon addition of porcine brain microtubule-associated proteins or DEAE-dextran, this tubulin preparation is functionally active for assembly into microtubules, as visualized by electron microscopy on negatively stained samples . Virtually identical microtubule structures are produced in parallel experiments on the assembly of yeast or porcine brain tubulin, with differences observed only at acidic pH values . Overall, this relatively simple procedure provides a useful tool for the production of functional tubulin suitable both for structural studies and for investigations of the assembly process.

Eur J Biochem, 1992 Nov 15, 210(1), 125 - 32
The 70-kilodalton heat-shock proteins of the SSA subfamily negatively modulate heat-shock-induced accumulation of trehalose and promote recovery from heat stress in the yeast, Saccharomyces cerevisiae; Hottiger T et al.; In the yeast, Saccharomyces cerevisiae, the disaccharide trehalose is a stress-related metabolite that accumulates upon exposure of cells to heat shock or a variety of non-heat inducers of the stress response . Here, we describe the influence of mutations in individual heat-shock-protein genes on trehalose metabolism . A strain mutated in three proteins of the SSA subfamily of 70-kDa heat-shock proteins (hsp70) overproduced trehalose during heat shock at 37 degrees C or 40 degrees C and showed abnormally slow degradation of trehalose upon temperature decrease from 40 degrees C to 27 degrees C . The mutant cells were unimpaired in the induction of thermotolerance; however, the decay of thermotolerance during recovery at 27 degrees C was abnormally slow . Since both a high content of trehalose and induced thermotolerance are associated with the heat-stressed state of cells, the abnormally slow decline of trehalose levels and thermotolerance in the mutant cells indicated a defect in recovery from the heat-stressed state . A similar albeit minor defect, as judged from measurements of trehalose degradation during recovery, was detected in a delta hsp104 mutant, but not in a strain deleted in the polyubiquitin gene, UB14 . In all our experiments, trehalose levels were closely correlated with thermotolerance, suggesting a thermoprotective function of trehalose . In contrast, heat-shock proteins, in particular hsp70, appear to be involved in recovery from the heat-stressed state rather than in the acquisition of thermotolerance . Cells partially depleted of hsp70 displayed an abnormally low activity of neutral trehalase when shifted to 27 degrees C after heat shock at 40 degrees C . Trehalase activity is known to be under positive control by cAMP-dependent protein kinases, suggesting that hsp70 directly or indirectly stimulate these protein-kinase activities . Alternatively, hsp70 may physically interact with neutral trehalase, thereby protecting the enzyme from thermal denaturation.

J Biol Chem, 1992 Nov 15, 267(32), 23383 - 7
Purification and properties of Saccharomyces cerevisiae RNA polymerase II general initiation factor a; Sayre MH et al.; RNA polymerase II initiation factor a was purified to apparent homogeneity from yeast whole cell extract and consisted of two highly charged polypeptides with apparent masses of 66 and 43 kDa . Separation and renaturation of the subunits showed that both were required for transcription activity . The native mass of factor a was estimated to be 240-260 kDa by gel filtration, but its sedimentation rate in a glycerol gradient was similar to that of a much smaller globular protein, suggesting an extended conformation . Factor a was required for utilization of six different eukaryotic promoters in vitro, indicating a general role in promoter-directed transcription by yeast RNA polymerase II.

J Biol Chem, 1992 Nov 15, 267(32), 23099 - 107
Biochemical and genetic dissection of the Saccharomyces cerevisiae RNA polymerase C53 subunit through the analysis of a mitochondrially mis-sorted mutant construct; Chiannilkulchai N et al.; RPC53 has previously been shown to encode an essential subunit required for tRNA gene transcription by RNA polymerase C in vivo (Mann, C., Micouin, J.-Y., Chiannilkulchai, N., Treich, I., Buhler, J.-M., and Sentenac, A . (1992) Mol . Cell . Biol . 12, in press) . In this paper, we have determined that an unusual rho+ lethality associated with the rpc53::HIS3-1 disruption mutation is due to the inadvertent formation of a Pet56-C53 fusion protein . This fusion protein is missorted to mitochondria, thereby reducing the quantity of the C53 subunit available for RNA polymerase C assembly . We show that the carboxyl-terminal region of C53 contains the essential functional domain of the subunit and that a mutant RNA polymerase containing only this domain is thermolabile for its function in vivo and in vitro . The thermolability of the carboxyl-terminal C53 domain is suppressed by five different genes on multicopy plasmids, including RPC160, encoding the largest subunit of RNA polymerase C and SSD1/SRK1, which has been implicated in the activity of protein phosphatases.

J Biol Chem, 1992 Nov 15, 267(32), 23376 - 82
Reconstitution of transcription with five purified initiation factors and RNA polymerase II from Saccharomyces cerevisiae; Sayre MH et al.; Yeast RNA polymerase II initiation factors a, b, e, and g were isolated from whole cell extract and found to be sufficient, when combined with bacterially expressed yeast transcription factor (TF) IID, to enable RNA polymerase II to utilize nine different eukaryotic promoters in vitro, and to initiate transcription at sites used in vivo . The purified factors did not contain the previously described transcription factor IIA (TFIIA) . TFIIA failed to substitute for any purified factor or to stimulate transcription with the complete set of factors, indicating that its function in crude extracts is primarily as an anti-inhibitor.

J Mol Biol, 1992 Nov 5, 228(1), 298 - 9
Crystallization and preliminary X-ray investigation of phosphoribosylaminoimidazolesuccinocarboxamide synthase from the yeast Saccharomyces cerevisiae; Grebenko AI et al.; Crystals of phosphoribosylaminoimidazolesuccinocarboxamide synthase (EC 6.3.2.6) from the yeast Saccharomyces cerevisiae were grown by the vapor diffusion hanging-drop technique, using ammonium sulfate as the precipitant . The crystals had dimensions up to 1.2 mm . X-ray diffraction experiments indicated a space group of P2(1)2(1)2(1) and unit cell parameters of a = 62.3 A, b = 63.5 A and c = 80.9 A, with one molecule in the asymmetric unit . Native data have been collected to 2.5 A resolution.

J Biol Chem, 1992 Nov 5, 267(31), 22562 - 9
Bending of the Saccharomyces cerevisiae 5S rRNA gene in transcription factor complexes; Braun BR et al.; Bending of the yeast 5S rRNA gene by its transcription factors (TF) IIIA, IIIC, and IIIB has been investigated by two electrophoretic methods that exploit the anomalous mobility of bent DNA in tight gel networks . A minor bend is induced by TFIIIA, and a very strong bend, centered at its upstream DNA-binding site, is induced by TFIIIB . Despite binding to different DNA sequences on the 5S rRNA gene and the previously analyzed tRNA(Glu) gene, TFIIIB generates nearly identical bends in each site . Fully assembled transcription factor complexes bend the 5S rRNA gene in the same net direction as does TFIIIB alone.

Mol Cell Biochem, 1992 Nov 4, 117(1), 11 - 21
Electron microscopic visualisation of the 5S rRNA-YL3 complex from Saccharomyces cerevisiae; Kyle KM et al.; The complex comprising 5S ribosomal RNA and the ribosomal protein YL3 (5S rRNP) was isolated from yeast (Saccharomyces cerevisiae), and positively contrasted preparations were imaged by transmission electron microscopy . The overall dimensions of the 5S rRNP complex in the micrographs were 10 nm by 6 nm . Three predominant projections were selected from several hundred putative particles for digitisation and computer averaging to yield two-dimensional constructions with reproducible spatial resolutions exceeding 2 nm . The enhanced projection images were compatible with structural models of this complex based on biochemical studies.

Carbohydr Res, 1992 Nov 4, 235, 247 - 57
Development of a water-soluble, sulfated (1-->3)-beta-D-glucan biological response modifier derived from Saccharomyces cerevisiae; Williams DL et al.; This report describes a method for the solubilization of micro-particulate (1-->3)-beta-D-glucan . Insoluble glucan is dissolved in methyl sulfoxide and urea (8 M) and partially sulfated at 100 degrees . The resulting water-soluble product is called glucan sulfate . The conversion rate is 98%, and the preparation is endotoxin free as determined by the Limulus lysate procedure . Glucan sulfate is composed of 34.06% C, 6.15% H, 50.30% O, 5.69% S and 3.23% N, and has a repeating unit empirical formula of (C6H10O5)8.3 SO3NH4+.4 H2O, suggesting that, on the average, a sulfate group is substituted on every third glucose subunit along the polymer . Molecular weight averages, polydispersity, and intrinsic viscosity were determined by aqueous high-performance size-exclusion chromatography (HPSEC) . Two polymer peaks were resolved . Peak 1 (Mw = 1.25 x 10(6) g/mol) represents < 1% of the total polymer mass . Peak 2 (Mw = 1.45 x 10(4) g/mol) comprises > 99% of polymers . 13C NMR spectroscopy confirmed the beta-(1-->3) interchain linkage . In solution, glucan sulfate polymers self-associate in a triple helix . Glucan sulfate stimulates murine bone marrow proliferation following intravenous administration . The ability to prepare a immunologically active, water-soluble (1-->3)-beta-D-glucan preparation will greatly enhance the clinical utility of this class of compounds.

Genetics, 1992 Nov, 132(3), 651 - 64
XRS2, a DNA repair gene of Saccharomyces cerevisiae, is needed for meiotic recombination; Ivanov EL et al.; The XRS2 gene of Saccharomyces cerevisiae has been previously identified as a DNA repair gene . In this communication, we show that XRS2 also encodes an essential meiotic function . Spore inviability of xrs2 strains is rescued by a spo13 mutation, but meiotic recombination (both gene conversion and crossing over) is highly depressed in spo13 xrs2 diploids . The xrs2 mutation suppresses spore inviability of a spo13 rad52 strain suggesting that XRS2 acts prior to RAD52 in the meiotic recombination pathway . In agreement with the genetic data, meiosis-specific double-strand breaks at the ARG4 meiotic recombination hotspot are not detected in xrs2 strains . Despite its effects on meiotic recombination, the xrs2 mutation does not prevent mitotic recombination events, including homologous integration of linear DNA, mating-type switching and radiation-induced gene conversion . Moreover, xrs2 strains display a mitotic hyper-rec phenotype . Haploid xrs2 cells fail to carry out G2-repair of gamma-induced lesions, whereas xrs2 diploids are able to perform some diploid-specific repair of these lesions . Meiotic and mitotic phenotypes of xrs2 cells are very similar to those of rad50 cells suggesting that XRS2 is involved in homologous recombination in a way analogous to that of RAD50.

Mol Gen Genet, 1992 Nov, 235(2-3), 450 - 2
The DAC2/FUS3 protein kinase is not essential for transcriptional activation of the mating pheromone response pathway in Saccharomyces cerevisiae; Fujimura HA; The DAC2/FUS3 gene of Saccharomyces cerevisiae, which encodes a CDC28/cdc2-related protein kinase, is essential both for the arrest of cell division induced by mating pheromones and for cell fusion during conjugation . To elucidate the role of the DAC2 gene product in the pheromone response pathway, I determined the nucleotide sequence of the DAC2 gene and characterized two types of deletion mutants of the DAC2 gene . Here, I show that the DAC2 gene is identical to the FUS3 gene and that dac2/fus3 deletion mutants respond to mating pheromones by activating transcription . Therefore, the DAC2/FUS3 gene is not essential for transcriptional activation in the pheromone response pathway . The DAC2/FUS3 protein kinase has a positive role in cell fusion during sexual conjugation.

Mol Gen Genet, 1992 Nov, 235(2-3), 311 - 6
The pso4-1 mutation reduces spontaneous mitotic gene conversion and reciprocal recombination in Saccharomyces cerevisiae; Meira LB et al.; Spontaneous mitotic recombination was examined in the haploid pso4-1 mutant of Saccharomyces cerevisiae and in the corresponding wild-type strain . Using a genetic system involving a duplication of the his4 gene it was shown that the pso4-1 mutation decreases at least fourfold the spontaneous rate of mitotic recombination . The frequency of spontaneous recombination was reduced tenfold in pso4-1 strains, as previously observed in the rad52-1 mutant . However, whereas the rad52-1 mutation specifically reduces gene conversion, the pso4-1 mutation reduces both gene conversion and reciprocal recombination . Induced mitotic recombination was also studied in pso4-1 mutant and wild-type strains after treatment with 8-methoxypsoralen plus UVA and 254 nm UV irradiation . Consistent with previous results, the pso4-1 mutation was found strongly to affect recombination induction.

Radiat Res, 1992 Nov, 132(2), 141 - 3
Dual cell cycle checkpoints sensitive to chromosome replication and DNA damage in the budding yeast Saccharomyces cerevisiae; Weinert TA; In eucaryotic cells chromosomes must be fully replicated and repaired before mitosis begins . Genetic studies indicate that this dependence of mitosis on completion of DNA replication and DNA repair derives from a negative control called a checkpoint which somehow checks for replication and DNA damage and blocks cell entry into mitosis . Here we summarize our current understanding of the genetic components of the cell cycle checkpoint in budding yeast . Mutants were identified and their phase and signal specificity tested primarily through interactions of the arrest-defective mutants with cell division cycle mutants . The results indicate that dual checkpoint controls exist in budding yeast, one control sensitive to inhibition of DNA replication (S-phase checkpoint), and a distinct but overlapping control sensitive to DNA repair (G2 checkpoint) . Six genes are required for arrest in G2 phase after DNA damage (RAD9, RAD17, RAD24, MEC1, MEC2, and MEC3), and two of these are also essential for arrest in S phase when DNA replication is blocked (MEC1 and MEC2).

J Cell Biol, 1992 Nov, 119(4), 773 - 86
The VPS1 protein, a homolog of dynamin required for vacuolar protein sorting in Saccharomyces cerevisiae, is a GTPase with two functionally separable domains; Vater CA et al.; The product of the VPS1 gene, Vps1p, is required for the sorting of soluble vacuolar proteins in the yeast Saccharomyces cerevisiae . We demonstrate here that Vps1p, which contains a consensus tripartite motif for guanine nucleotide binding, is capable of binding and hydrolyzing GTP . Vps1p is a member of a subfamily of large GTP-binding proteins whose members include the vertebrate Mx proteins, the yeast MGM1 protein, the Drosophila melanogaster shibire protein, and dynamin, a bovine brain protein that bundles microtubules in vitro . Disruption of microtubules did not affect the fidelity or kinetics of vacuolar protein sorting, indicating that Vps1p function is not dependent on microtubules . Based on mutational analyses, we propose a two-domain model for Vps1p function . When VPS1 was treated with hydroxylamine, half of all mutations isolated were found to be dominant negative with respect to vacuolar protein sorting . All of the dominant-negative mutations analyzed further mapped to the amino-terminal half of Vps1p and gave rise to full-length protein products . In contrast, recessive mutations gave rise to truncated or unstable protein products . Two large deletion mutations in VPS1 were created to further investigate Vps1p function . A mutant form of Vps1p lacking the carboxy-terminal half of the protein retained the capacity to bind GTP and did not interfere with sorting in a wild-type background . A mutant form of Vps1p lacking the entire GTP-binding domain interfered with vacuolar protein sorting in wild-type cells . We suggest that the amino-terminal domain of Vps1p provides a GTP-binding and hydrolyzing activity required for vacuolar protein sorting, and the carboxy-terminal domain mediates Vps1p association with an as yet unidentified component of the sorting apparatus.

Curr Genet, 1992 Nov, 22(5), 371 - 6
Direct induction of tetraploids or homozygous diploids in the industrial yeast Saccharomyces cerevisiae by hydrostatic pressure; Hamada K et al.; Hydrostatic pressure and a dye plate method were used to investigate the direct induction of tetraploids or homozygous diploids from the industrial diploid or haploid yeast Saccharomyces cerevisiae . Above 200 MPa, hydrostatic pressure greatly inactivated the strains HF399s1 (alpha haploid), P-540 (a/alpha diploid), and P-544 (a/alpha diploid) . At the same time, when pressure-treated cells of these strains were spread on a dye plate, some of the visible colonies were stained red/blue or dark blue (variant colonies); the rest stained violet, similar to colonies originating from diploid cells or haploid cells that were not pressure-treated . In addition, above 100 MPa, the formation of variant colonies increased with increasing pressure, and maximized (1 x 10(-1)) at 200 and 250 MPa, respectively . The size of almost all variant cells from P-544, P-540, and HF399s1 was visibly increased compared with that of untreated cells and the measured cellular DNA content of P-540 and HF399s1 was double that of untreated cells . Furthermore, based on random spore analysis and mass-matings, induced variants in the diploid strains were found to be tetraploid with an a/a/alpha/alpha genotype at the mating-type locus or, in the haploid strains, homozygous diploid with an alpha/alpha genotype . From these results we conclude that pressure treatment in combination with a dye plate is a useful method for strain improvement by direct induction of tetraploids or homozygous diploids from industrial strains whether diploid or haploid.

Curr Genet, 1992 Nov, 22(5), 345 - 55
Complementation of Saccharomyces cerevisiae acid phosphatase mutation by a genomic sequence from the yeast Yarrowia lipolytica identifies a new phosphatase; Treton BY et al.; A Yarrowia lipolytica gene library was constructed in vector YRp7 and transformed into a Saccharomyces cerevisiae strain lacking both major acid phosphatase activities . A 2.18 kb genomic sequence restoring the ability to hydrolyze alpha-naphthyl phosphate was isolated . Its sequencing revealed an ORF encoding 358 amino acids without significant homology with any known phosphatase . A putative signal peptide and several possible sites for N-glycosylation were identified . Phosphate-regulated expression of the cloned gene was observed in Y . lipolytica . Disruption data favoured the hypothesis that it might encode a minor phosphatase species.

DNA Cell Biol, 1992 Nov, 11(9), 685 - 92
Cloning, sequencing, and disruption of the gene encoding sterol C-14 reductase in Saccharomyces cerevisiae; Lorenz RT et al.; A sterol C-14 reductase (erg24-1) mutant of Saccharomyces cerevisiae was selected in a fen1, fen2, suppressor background on the basis of nystatin resistance and ignosterol (ergosta-8,14-dienol) production . The erg24-1 allele segregated genetically as a single, recessive gene . The wild-type ERG24 gene was cloned by complementation onto a 12-kb fragment from a yeast genomic library, and subsequently subcloned onto a 2.4-kb fragment . This was sequenced and found to contain an open reading frame of 1,314 bp, predicting a polypeptide of 438 amino acids (M(r) 50,612) . A 1,088-bp internal region of the ERG24 gene was excised, replaced with a LEU2 gene, and integrated into the chromosome of the parental strain, FP13D (fen1, fen2) by gene replacement . The ERG24 null mutant produced ergosta-8,14-dienol as the major sterol, indicating that the delta 8-7 isomerase, delta 5-desaturase and the delta 22-desaturase were inactive on sterols with the C14 = 15 double bond.

Arch Biochem Biophys, 1992 Nov 1, 298(2), 318 - 24
Expression of mouse cathepsin L cDNA in Saccharomyces cerevisiae: evidence that cathepsin L is sorted for targeting to yeast vacuole; Nishimura Y et al.; To investigate the intracellular transport mechanism of lysosomal cathepsin L in yeast cells, we attempted to produce mouse cathepsin L in Saccharomyces cerevisiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene . Immunoblotting analysis by the use of an antibody specific for rat cathepsin L revealed that the yeast cells carrying the cathepsin L coding sequence produced 39- and 30-kDa products, which correspond to the rat procathepsin L and the single-chain form of mature cathepsin L, respectively . The precursor polypeptide showed sensitivity toward endoglycosidase H treatment . Cell fractionation experiments demonstrated that the processed form of 30-kDa cathepsin L was found to be colocalized to the yeast vacuole with the marker enzyme carboxypeptidase Y in a Ficoll step gradient . In the prepared vacuolar fraction, a considerable amount of cathepsin L was revealed to be cofractionated with the vacuolar membranes . Furthermore, the phase separation experiments with Triton X-114 provide the first evidence showing that the mature form of cathepsin L polypeptide is strongly associated with the vacuolar membranes . Therefore, the present results suggest that the mouse cathepsin L precursor polypeptide is initially synthesized as the proenzyme in the yeast cells and then correctly delivered to the vacuole . During the intracellular sorting pathway, the procathepsin L would undergo the post-translational proteolytic processing step to generate the mature enzyme . Based on these lines of evidence, we propose that cathepsin L is recognized by mechanisms similar to those for the intracellular sorting and processing of vacuolar proteins in the yeast cells.

Mol Cell Biol, 1992 Nov, 12(11), 5249 - 59
Identification of a new set of cell cycle-regulatory genes that regulate S-phase transcription of histone genes in Saccharomyces cerevisiae; Xu H et al.; Histone mRNA synthesis is tightly regulated to S phase of the yeast Saccharomyces cerevisiae cell cycle as a result of transcriptional and posttranscriptional controls . Moreover, histone gene transcription decreases rapidly if DNA replication is inhibited by hydroxyurea or if cells are arrested in G1 by the mating pheromone alpha-factor . To identify the transcriptional controls responsible for cycle-specific histone mRNA synthesis, we have developed a selection for mutations which disrupt this process . Using this approach, we have isolated five mutants (hpc1, hpc2, hpc3, hpc4, and hpc5) in which cell cycle regulation of histone gene transcription is altered . All of these mutations are recessive and belong to separate complementation groups . Of these, only one (hpc1) falls in one of the three complementation groups identified previously by other means (M . A . Osley and D . Lycan, Mol . Cell . Biol . 7:4204-4210, 1987), indicating that at least seven different genes are involved in the cell cycle-specific regulation of histone gene transcription . hpc4 is unique in that derepression occurs only in the presence of hydroxyurea but not alpha-factor, suggesting that at least one of the regulatory factors is specific to histone gene transcription after DNA replication is blocked . One of the hpc mutations (hpc2) suppresses delta insertion mutations in the HIS4 and LYS2 loci . This effect allowed the cloning and sequence analysis of HPC2, which encodes a 67.5-kDa, highly charged basic protein.

Mol Cell Biol, 1992 Nov, 12(11), 5159 - 73
C-terminal truncation of RAP1 results in the deregulation of telomere size, stability, and function in Saccharomyces cerevisiae; Kyrion G et al.; The Saccharomyces cerevisiae DNA-binding protein RAP1 is capable of binding in vitro to sequences from a wide variety of genomic loci, including upstream activating sequence elements, the HML and HMR silencer regions, and the poly(G1-3T) tracts of telomeres . Recent biochemical and genetic studies have suggested that RAP1 physically and functionally interacts with the yeast telomere . To further investigate the role of RAP1 at the telomere, we have identified and characterized three intragenic suppressors of a temperature-sensitive allele of RAP1, rap1-5 . These telomere deficiency (rap1t) alleles confer several novel phenotypes . First, telomere tract size elongates to up to 4 kb greater than sizes of wild-type or rap1-5 telomeres . Second, telomeres are highly unstable and are subject to rapid, but reversible, deletion of part or all of the increase in telomeric tract length . Telomeric deletion does not require the RAD52 or RAD1 gene product . Third, chromosome loss and nondisjunction rates are elevated 15- to 30-fold above wild-type levels . Sequencing analysis has shown that each rap1t allele contains a nonsense mutation within a discrete region between amino acids 663 and 684 . Mobility shift and Western immunoblot analyses indicate that each allele produces a truncated RAP1 protein, lacking the C-terminal 144 to 165 amino acids but capable of efficient DNA binding . These data suggest that RAP1 is a central regulator of both telomere and chromosome stability and define a C-terminal domain that, while dispensable for viability, is required for these telomeric functions.

Mol Cell Biol, 1992 Nov, 12(11), 4937 - 45
The 70-kilodalton adenylyl cyclase-associated protein is not essential for interaction of Saccharomyces cerevisiae adenylyl cyclase with RAS proteins; Wang J et al.; In the yeast Saccharomyces cerevisiae, adenylyl cyclase is regulated by RAS proteins . We show here that the yeast adenylyl cyclase forms at least two high-molecular-weight complexes, one with the RAS protein-dependent adenylyl cyclase activity and the other with the Mn(2+)-dependent activity, which are separable by their size difference . The 70-kDa adenylyl cyclase-associated protein (CAP) existed in the former complex but not in the latter . Missense mutations in conserved motifs of the leucine-rich repeats of the catalytic subunit of adenylyl cyclase abolished the RAS-dependent activity, which was accompanied by formation of a very high molecular weight complex having the Mn(2+)-dependent activity . Contrary to previous results, disruption of the gene encoding CAP did not alter the extent of RAS protein-dependent activation of adenylyl cyclase, while a concomitant decrease in the size of the RAS-responsive complex was observed . These results indicate that CAP is not essential for interaction of the yeast adenylyl cyclase with RAS proteins even though it is an inherent component of the RAS-responsive adenylyl cyclase complex.

J Cell Biol, 1992 Nov, 119(3), 583 - 93
Role of astral microtubules and actin in spindle orientation and migration in the budding yeast, Saccharomyces cerevisiae; Palmer RE et al.; In the yeast Saccharomyces cerevisiae, before the onset of anaphase, the spindle apparatus is always positioned with one spindle pole at, or through, the neck between the mother cell and the growing bud . This spindle orientation enables proper chromosome segregation to occur during anaphase, allowing one replicated genome to be segregated into the bud and the other to remain in the mother cell . In this study, we synchronized a population of cells before the onset of anaphase such that > 90% of the cells in the population had spindles with the correct orientation, and then disrupted specific cytoskeletal elements using temperature-sensitive mutations . Disruption of either the astral microtubules or actin function resulted in improper spindle orientation in approximately 40-50% of the cells . When cells with disrupted astral microtubules or actin function entered into anaphase, there was a 100-200-fold increase in the frequency of binucleated cell bodies . Thus, the maintenance of proper spindle orientation by these cytoskeletal elements was essential for proper chromosome segregation . These data are consistent with the model that proper spindle orientation is maintained by directly or indirectly tethering the astral microtubules to the actin cytoskeleton . After nuclear migration, but before anaphase, bulk chromosome movement occurs within the nucleus apparently because the chromosomes are attached to a mobile spindle . The frequency and magnitude of bulk chromosome movement is greatly diminished by disruption of the astral microtubules but not by disruption of the nonkinetochore spindle microtubules . These results suggest that astral microtubules are not only important for spindle orientation before anaphase, but they also mediate force on the spindle, generating spindle displacement and in turn chromosome movement . Potential roles for this force in spindle assembly and orientation are discussed.

J Bacteriol, 1992 Nov, 174(21), 6831 - 9
Tripartite structure of the Saccharomyces cerevisiae arginase (CAR1) gene inducer-responsive upstream activation sequence; Viljoen M et al.; Arginase (CAR1) gene expression in Saccharomyces cerevisiae is induced by arginine . The 5' regulatory region of CAR1 contains four separable regulatory elements--two inducer-independent upstream activation sequences (UASs) (UASC1 and UASC2), an inducer-dependent UAS (UASI), and an upstream repression sequence (URS1) which negatively regulates CAR1 and many other yeast genes . Here we demonstrate that three homologous DNA sequences originally reported to be present in the inducer-responsive UASI are in fact three exchangeable elements (UASI-A, UASI-B, and UASI-C) . Although two of these elements, either the same or different ones, are required for transcriptional activation to occur, all three are required for maximal levels of induction . The elements operate in all orientations relative to one another and to the TATA sequence . All three UASI elements bind protein(s); protein binding does not require arginine or overproduction of any of the putative arginine pathway regulatory proteins . The UASI-protein complex was also observed even when extracts were derived from arg80/argRI or arg81/argRII deletion mutants . Similar sequences situated upstream of ARG5,6 and ARG3 and reported to negatively regulate their expression are able to functionally substitute for the CAR1 UASI elements and mediate reporter gene expression.

EMBO J, 1992 Nov, 11(11), 4007 - 15
Isolation of multiple mouse cDNAs with coding homology to Saccharomyces cerevisiae CDC25: identification of a region related to Bcr, Vav, Dbl and CDC24; Cen H et al.; In Saccharomyces cerevisiae, the product of the CDC25 gene is an essential Ras activator that appears to function by stimulating guanine nucleotide exchange on Ras . Using the ability of a mouse cDNA expression library to complement yeast cells lacking functional CDC25, Martegani et al . have identified a 1.7 kb partial cDNA from a gene, designated CDC25Mm, with homology to CDC25 . We have now screened a mouse brain cDNA library to identify full-length clones of CDC25Mm . This cloning has led to the isolation of six distinct full-length cDNAs, each of which appear to be derived from the CDC25Mm gene, since their 3' 2 kb appear to be identical and to encode the same 661 C-terminal amino acids . Three cDNAs are predicted to encode protein products of 666 or 667 amino acids . The other three cDNAs encode products that are 836, 1120 and 1260 amino acids, respectively . A 241 amino acid region near the N-terminus of the two largest products was found to have homology to a domain shared by Bcr, Vav, Dbl and CDC24 . Polyclonal antibodies raised to a peptide encoded by all the cDNAs have identified at least two protein products in NIH3T3 fibroblasts . Their apparent molecular weights are 75 and 95 kDa, which correspond closely to those predicted to be encoded, respectively, by the two shorter classes of cDNAs . In NIH3T3, the 95 kDa form is much more abundant than the 75 kDa form, while PC-12 pheochromocytoma cells contain relatively high levels of the 75 kDa form . We conclude that CDC25Mm is a complex gene whose protein products are regulated in a tissue-specific manner.

Proc Natl Acad Sci U S A, 1992 Nov 1, 89(21), 10232 - 6
Coordinated regulation and inositol-mediated and fatty acid-mediated repression of fatty acid synthase genes in Saccharomyces cerevisiae; Chirala SS; In Saccharomyces cerevisiae, FAS1, FAS2, and FAS3 are the genes involved in saturated fatty acid biosynthesis . The enzymatic activities of both fatty acid synthase (FAS) and acetyl-CoA carboxylase are reduced 2- to 3-fold when yeast cells are grown in the presence of exogenous fatty acids . The mRNA levels of the FAS genes are correspondingly lower under repressive conditions . Expression of the FAS-lacZ reporter gene is also regulated by fatty acids . When a FAS2 multicopy plasmid is present in the cells, expression of both FAS1 and FAS3 increases . Thus, the FAS genes are coordinately regulated . Deletion analyses of the regulatory regions of FAS1 and FAS2 revealed common regulatory sequences . These include the GGCCAAAAAC and AGCCAAGCA sequences that have a common GCCAA core sequence and the UASINO (upstream activation sequence) . Derepression of the FAS genes in the absence of exogenous inositol is not observed when UASINO is mutated, indicating that this cis element is a positive regulator of these genes . The GCCAA elements and UASINO act synergistically for optimal expression of the FAS genes.

Mol Gen Genet, 1992 Nov, 235(2-3), 365 - 72
The cauliflower mosaic virus 35S promoter is regulated by cAMP in Saccharomyces cerevisiae; Ruth J et al.; The cauliflower mosaic virus 35S promoter confers strong gene expression in plants, animals and fission yeast, but not in budding yeast . On investigating this paradox, we found that in budding yeast the promoter acts through two domains . Whereas the upstream domain acts as a silencer, the downstream domain couples expression to the nutritional state of the cells via the RAS/cAMP pathway . Point mutations indicate that two boxes with similarity to the cAMP regulated element (CRE) of mammalian cells mediate this response . Gel retardation assays show that, in both yeast and plant protein extracts, factors bind to this promoter element . Therefore, transcriptional activation appears to be highly conserved at the level of transcription factors and specific DNA target elements in eukaryotes . This offers new ways to investigate gene regulation mechanisms of higher eukaryotes, which are not as amenable to genetic analysis as yeast.

Curr Genet, 1992 Nov, 22(5), 363 - 70
Identification of UAS elements and binding proteins necessary for derepression of Saccharomyces cerevisiae fructose-1,6-bisphosphatase; Niederacher D et al.; Fructose-1,6-bisphosphatase is a key enzyme in gluconeogenesis and the FBP1 gene is not transcribed during growth with glucose . Genetic analysis indicated a positive regulation of FBP1 expression after exhaustion of glucose . By linker-deletion analysis, two upstream activation sites (UAS1 and UAS2) were localized and the respective UAS-binding factors (DAP I and DAP II for derepression activating protein) were identified by gel retardation . UAS1 and UAS2 span about 30 bp each, and are separated by approximately 30 bp . Both UAS sites act synergistically . Although UAS1 showed some similarities to the DNA-binding consensus for the general yeast activator Rap1, competition experiments and DEAE-chromatography proved that DAP I and Rap1 correspond to different proteins . Gel retardation by DAP I depended on carbon sources and did not occur in cells growing logarithmically with glucose, whereas a strong retardation signal was obtained with ethanol-grown cells . The present results suggest that DAP I and DAP II are the final regulatory elements for glucose derepression.

Mol Cell Biol, 1992 Nov, 12(11), 4946 - 59
Inactivation of the protein phosphatase 2A regulatory subunit A results in morphological and transcriptional defects in Saccharomyces cerevisiae; van Zyl W et al.; We have determined that TPD3, a gene previously identified in a screen for mutants defective in tRNA biosynthesis, most likely encodes the A regulatory subunit of the major protein phosphatase 2A species in the yeast Saccharomyces cerevisiae . The predicted amino acid sequence of the product of TPD3 is highly homologous to the sequence of the mammalian A subunit of protein phosphatase 2A . In addition, antibodies raised against Tpd3p specifically precipitate a significant fraction of the protein phosphatase 2A activity in the cell, and extracts of tpd3 strains yield a different chromatographic profile of protein phosphatase 2A than do extracts of isogenic TPD3 strains . tpd3 deletion strains generally grow poorly and have at least two distinct phenotypes . At reduced temperatures, tpd3 strains appear to be defective in cytokinesis, since most cells become multibudded and multinucleate following a shift to 13 degrees C . This is similar to the phenotype obtained by overexpression of the protein phosphatase 2A catalytic subunit or by loss of CDC55, a gene that encodes a protein with homology to a second regulatory subunit of protein phosphatase 2A . At elevated temperatures, tpd3 strains are defective in transcription by RNA polymerase III . Consistent with this in vivo phenotype, extracts of tpd3 strains fail to support in vitro transcription of tRNA genes, a defect that can be reversed by addition of either purified RNA polymerase III or TFIIIB . These results reinforce the notion that protein phosphatase 2A affects a variety of biological processes in the cell and provide an initial identification of critical substrates for this phosphatase.

EMBO J, 1992 Nov, 11(11), 3821 - 9
BCS1, a novel gene required for the expression of functional Rieske iron-sulfur protein in Saccharomyces cerevisiae; Nobrega FG et al.; Respiratory deficient pet mutants of Saccharomyces cerevisiae assigned to complementation group G2 define a new gene, named BCS1, whose product is shown to be necessary for the expression of functional ubiquinol-cytochrome c reductase (bc1) complex . Immunological assays indicate a gross reduction in the Rieske iron-sulfur subunit in bcs1 mutants, while other subunits of the ubiquinol-cytochrome c reductase complex are present at concentrations comparable to the wild type . Transformation of bcs1 mutants with the iron-sulfur protein gene on a multicopy plasmid led to elevated mitochondrial concentrations of Rieske protein, but did not correct the enzymatic defect, indicating that BCS1 is involved either in forming the active site iron-sulfur cluster or providing a chaperone-like function in assembling the Rieske protein with the other subunits of the complex . Both postulated functions are consistent with the localization of BCS1 in mitochondria . To facilitate further studies on this novel protein, BCS1 was cloned by transformation of a bcs1 mutant and its structure determined . The primary structure of the encoded BCS1 protein bears similarity to a group of proteins that have been implicated in intracellular protein sorting, membrane fusion and regulation of transcription . The region of BCS1 homologous to this diverse group of proteins is approximately 200 amino acids long and includes several signature sequences commonly found in ATPases and nucleotide binding proteins.

J Exp Biol, 1992 Nov, 172, 271 - 87
Complex modulation of cation channels in the tonoplast and plasma membrane of Saccharomyces cerevisiae: single-channel studies; Bertl A et al.; Detailed patch-clamp studies have been made of ion channels in the plasma membrane and tonoplast of the yeast Saccharomyces cerevisiae . The predominant tonoplast channel is a high-conductance cation-selective inward rectifier (passing ions easily into the cytoplasm from the vacuole), with its open probability (Po) peaking at about -80 mV (cytoplasm negative) and falling to near zero at +80 mV . It has a maximal slope conductance of approximately 150 pS in 100 mmol l-1 KCl, and conducts Na+, K+ and Ca2+ . Elevated cytoplasmic Ca2+ concentration, alkaline pH and reducing agents can activate the channel, its likely physiological function being to adjust cytoplasmic Ca2+ concentration from the vacuolar reservoir . The predominant plasma-membrane channel is a strongly outward rectifying K+ channel (passing K+ easily out of the cytoplasm to the extracellular medium), which is activated by positive-going membrane voltages as well as by elevated cytoplasmic Ca2+ concentration and alkaline pH . Interaction between membrane voltage and {Ca2+}cyt is complex and defines three parallel closed states for the channel: a Ca(2+)-independent brief closure (I), a calcium-inhibited long closure (G) and, at large positive voltages, a calcium-induced brief blockade (B) . This channel is likely to function in steady-state turgor regulation and in charge balancing during proton-coupled substrate uptake.

Philos Trans R Soc Lond B Biol Sci, 1992 Oct 29, 338(1283), 63 - 72
Calcium- and voltage-dependent ion channels in Saccharomyces cerevisiae; Bertl A et al.; Ion channels in both the tonoplast and the plasma membrane of Saccharomyces cerevisiae have been characterized at the single channel level by patch-clamp techniques . The predominant tonoplast channel is cation selective, has an open-channel conductance of 120 pS in 100 mM KCl, and conducts Na+ or K+ equally well, and Ca2+ to a lesser extent . Its open probability (Po) is voltage-dependent, peaking at about -80 mV (cytoplasm negative), and falling to near zero at +80 mV . Elevated cytoplasmic Ca2+, alkaline cytoplasmic pH, and reducing agents activate the channel . The predominant plasma membrane channel is highly selective for K+ over anions and other cations, and shows strong outward rectification of the time-averaged current-voltage curves in cell-attached experiments . In isolated inside-out patches with micromolar cytoplasmic Ca2+, this channel is activated by positive going membrane voltages: mean Po is zero at negative membrane voltages and near unity at 100 mV . At moderate positive membrane voltages (20-40 mV), elevating cytoplasmic Ca2+ activates the channel to open in bursts of several hundred milliseconds duration . At higher positive membrane voltages, however, elevating cytoplasmic Ca2+ blocks the channel in a voltage-dependent fashion for periods of 2-3 ms . The frequency of these blocking events depends on cytoplasmic Ca2+ and membrane voltage according to second-order kinetics . Alternative cations, such as Mg2+ or Na+, block the yeast plasma-membrane K+ channel in a similar but less pronounced manner.

J Biol Chem, 1992 Oct 25, 267(30), 21570 - 6
Cloning and characterization of a cysteine proteinase from Saccharomyces cerevisiae; Kambouris NG et al.; We have isolated a gene from Saccharomyces cerevisiae that encodes a protein homologous to the mammalian cysteine proteinase bleomycin hydrolase . Sequence comparison between the yeast and rabbit proteins indicates an amino acid identity of 41.5% over 277 residues and a similarity of 78.3% when conservative substitutions are included . The apparent mass of the yeast protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 47 kDa, although sequence analysis indicates two potential initiator methionines that suggest calculated masses of either 51 or 55 kDa . The protein is nonessential in yeast as haploid mutants disrupted at several positions along the open reading frame remain viable . Furthermore, these mutants do not exhibit any readily observable growth defects under varying conditions of temperature, nutrients, osmotic strength, or exogenous bleomycin . However, the purified protein does exhibit marked hydrolytic activity toward the substrate arginine 4-methyl-7-coumarylamide (Km = 12.8 microM, Vmax = 2.56 mumol mg-1 h-1), and yeast cells engineered to express this protein at higher levels maintain increased resistance to bleomycin compared to wild-type cells . Because this protein represents the first example of a cysteine proteinase identified in yeast, we have named it Ycp1 (yeast cysteine proteinase).

J Biol Chem, 1992 Oct 25, 267(30), 21390 - 5
An essential and specific subunit of RNA polymerase III (C) is encoded by gene RPC34 in Saccharomyces cerevisiae; Stettler S et al.; The RPC34 gene of Saccharomyces cerevisiae was cloned by immunological screening, using antibodies raised against the C34 polypeptide of the RNA polymerase III (C) . This single copy gene was located near the centromere of chromosome XIV . It included a coding sequence of 317 amino acids that strictly matched two internal oligopeptides of C34 . This polypeptide is a specific component of RNA polymerase III, with no significant homology to any other RNA polymerase subunit known so far . It is an essential subunit, since inactivation by deletion or nonsense mutations led to a recessive lethal phenotype . Moreover, a partially blocked mutant, rpc34-F297, had a reduced tRNA synthesis in vivo but no detectable effect on 5 S RNA synthesis . The latter phenotype was observed for all conditionally defective RNA polymerase III mutants isolated so far.

Biochim Biophys Acta, 1992 Oct 19, 1111(1), 120 - 6
Transport of phospholipids between subcellular membranes of wild-type yeast cells and of the phosphatidylinositol transfer protein-deficient strain Saccharomyces cerevisiae sec 14; Gnamusch E et al.; The transfer of glycerophospholipids between microsomes and mitochondria, and from internal membranes to the plasma membrane of Saccharomyces cerevisiae was characterized . Cellular energy production was found to be essential for intracellular translocation of phospholipids, but neither a membrane potential nor an intact cytoskeleton are required for this process . Using the temperature-sensitive mutant strain Saccharomyces cerevisiae sec 14, which is defective in the phosphatidylinositol transfer protein, it could be demonstrated that this protein is not involved in the transport of phosphatidylinositol and phosphatidylcholine from internal membranes to the plasma membrane . Our results also confirm earlier findings that phosphatidylinositol and phosphatidylcholine can be delivered to the plasma membrane in a process independent of the flux of vesicles competent for protein secretion.

J Biol Chem, 1992 Oct 15, 267(29), 21167 - 71
Interactions of the eIF-4F subunits in the yeast Saccharomyces cerevisiae; Lanker S et al.; Recognition of the cap structure at the 5' end of mRNA is one of the first events in initiation of eukaryotic translation . This step is mediated by the translation initiation factor 4F (eIF-4F) . In mammalian cells this factor is composed of the cap-binding protein eIF-4E, eIF-4A, and a 220-kDa polypeptide . In yeast Saccharomyces cerevisiae, eIF-4E is found associated with a 150-kDa protein (p150) and a 20-kDa protein (p20) . The resulting protein complex is proposed to represent yeast eIF-4F . To study the functions of p150 and p20 and their interaction with eIF-4E, we disrupted the genes encoding p150 and p20 and analyzed the effects on protein complex formation and cell viability . Yeast cells with single and double disruptions of the genes encoding p150 and p20 are viable, but p150 single and p150/p20 double disruptions show a slow growth phenotype . Gel chromatography and immunoadsorption experiments with a monoclonal anti-eIF-4E antibody coupled to protein G-Sepharose show that both p150 and p20 bind independently of each other to eIF-4E.

Eur J Biochem, 1992 Oct 15, 209(2), 697 - 705
Characterization of Saccharomyces cerevisiae mutants lacking the E1 alpha subunit of the pyruvate dehydrogenase complex; Wenzel TJ et al.; Pyruvate dehydrogenase mutants of Saccharomyces cerevisiae were isolated by disruption of the PDA1 gene . To this end, the PDA1 gene encoding the E1 alpha subunit of the pyruvate dehydrogenase complex was replaced by the dominant Tn5ble marker . Disruption of the PDA1 gene abolished production of the E1 alpha subunit and pyruvate dehydrogenase activity . Two additional phenotypes were observed in the Pdh-mutants: (a) a reduced growth rate in glucose medium which was partially complemented by the amino acid leucine; (b) an increase in formation of petites which lack mitochondrial DNA {rho0}, during growth on glucose . Both phenotypes were shown to be a result of inactivation of the PDA1 gene . Explanations for these phenotypes are discussed.

Biochemistry, 1992 Oct 13, 31(40), 9823 - 31
Oligosaccharide structures of the major exoglucanase secreted by Saccharomyces cerevisiae; Hernandez LM et al.; We have determined the structures of the N-linked carbohydrate chains, released by endo H, of exoglucanase II that are secreted by wild-type Saccharomyces cerevisiae and by the mnn1 mnn9 and mnn1 glycosylation mutants . The mnn9 mutation does not significantly affect N-linked oligosaccharides of exoglucanase II since we found almost identical structures in both mutant strains consisting of a slightly enlarged core with the basic structure shown in A (where M = mannose) . Most of the molecules (77%) were phosphorylated on one of the starred mannoses (34%) or on both (43%) with a diesterified (alpha M-->P-->) or monoesterified phosphate group . In addition, some of the molecules apparently escape normal processing and retain the alpha-(1-->2)-linked mannose (italicized) and/or the three glucoses that are characteristic of the lipid-linked precursor (structure B) . In the wild type, we found the same basic structure but more {formula; see text} than 90% of the molecules were modified with one to four alpha-(1-->3)-linked mannoses, which were absent in the strains bearing the mnn1 mutation (structure C) . The proportion of acidic components was similar to that found in the mutants (78%), although, in this case, the monophosphorylated forms were more abundant (50%) than the diphosphorylated ones (28%) . Most of the phosphate groups (69%) were diesterified by a disaccharide (alpha M-->3 alpha M-->P-->) instead of the single mannose found when the mnn1 mutation was present . In both mnn1 and wild type 10-15% of the oligosaccharides had an extra alpha-(1-->6)-linked mannose in the outer chain, a structure described in the recently isolated vrg1 mutant {Ballou, L., Hitzeman, R.A., Lewis, M . S., & Ballou, C . E . (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 3209-3212}.

Gene, 1992 Oct 12, 120(1), 59 - 65
Differential expression of the invertase-encoding SUC genes in Saccharomyces cerevisiae; del Castillo Agudo L et al.; Invertase (INV) is encoded in Saccharomyces cerevisiae by a family of genes, comprising SUC1-SUC5 and SUC7 . Production of INV is highly variable, dependent on the strain and SUC gene present in the cell . The differences in INV production derive from the structure of the genes or are dependent on the genetic background of the strain . Centromeric plasmids (based on YCp50) carrying one of the SUC genes (except SUC7) were introduced into a strain (SEY2101) lacking SUC genes . The INV produced by the transformants was dependent on the individual SUC genes, and correlated with INV mRNA levels . Plasmids in which SUC2 had been placed under control of promoters from the other SUC genes, were used to transform SEY2101 cells . The amounts of INV produced by cells carrying hybrid SUC genes were in agreement with the levels expected if the promoter controlled the expression of the SUC2 structural region . It is suggested that the differences in expression are a function of the transcription efficiency of the different SUC gene promoters, based on the divergence of 5' sequences.

Gene, 1992 Oct 12, 120(1), 43 - 9
The cell division cycle gene CDC60 encodes cytosolic leucyl-tRNA synthetase in Saccharomyces cerevisiae; Hohmann S et al.; The cdc60 mutation (for cell division cycle) of the yeast, Saccharomyces cerevisiae, confers arrest at the START point of the cell cycle upon shift to the restrictive temperature {Bedard et al., Curr . Genet . 4 (1981) 205-214} . We have cloned the CDC60 gene by complementation of the temperature-sensitive phenotype . Sequence analysis revealed a single open reading frame of 3270 bp and the deduced amino acid sequence showed 50.5% sequence identity to the cytosolic leucyl-tRNA synthetase (LeuRS) from Neurospora crassa, implying that CDC60 encodes the corresponding yeast protein . Thus, CDC60 does not appear to be involved directly in the regulation of the cell cycle . Rather, the cdc60 mutation leads to cell-cycle arrest at the nutrient control point START due to a deficiency of charged leucyl-tRNA . The CDC60 gene product also shows homology to LeuRSs from other organisms and to aminoacyl-RS for isoleucine, valine and methionine.

Nucleic Acids Res, 1992 Oct 11, 20(19), 5067 - 72
Identification of a DNA supercoiling activity in Saccharomyces cerevisiae; Koo HS et al.; A relaxed plasmid DNA is shown to become positively supercoiled in cell extracts from top1 strains of Saccharomyces cerevisiae . This positive supercoiling activity is dependent on the presence of bacterial DNA topoisomerase I and ATP (or dATP), and the positive supercoils generated in this reaction are not constrained by protein(s) . Non-hydrolyzable ATP analogs cannot substitute for ATP in this supercoiling reaction, and the supercoiling activity is not due to RNA synthesis . The presence of an ARS sequence in the DNA does not alter the activity . Furthermore, this activity is equally active against UV irradiated or intact DNA . Extracts prepared from rad50 and rad52 mutant cells exhibited the same activity . Partial purification of this activity suggests that a protein factor with a native molecular weight of approximately 150 kDa is primarily responsible for the activity . The possibility that this supercoiling activity may be due to tracking of a protein along the intact duplex DNA is discussed.

J Biol Chem, 1992 Oct 5, 267(28), 20270 - 6
Localized mutagenesis and evidence for post-transcriptional regulation of MAK3 . A putative N-acetyltransferase required for double-stranded RNA virus propagation in Saccharomyces cerevisiae; Tercero JC et al.; The MAK3 gene of Saccharomyces cerevisiae is necessary for the propagation of the L-A double-stranded RNA virus and its satellites, such as M1 that encodes a killer toxin . We cloned the MAK3 gene based on its genetic map position using physically mapped lambda-clones covering nearly all of the yeast genome . The minimal sequence necessary to complement the mak3-1 mutation contained 3 open reading frames (ORFs) . Only one (ORF3) was necessary to complement mak3-1 . A deletion insertion mutant of ORF3 grew slowly on nonfermentable carbon sources, an effect not due simply to its loss of L-A . Although ORF3 alone is sufficient for MAK3 activity when expressed from an expression vector, in its native context an additional 669 base pairs 3' to the ORF and complementary to the gene for a non-histone protein are necessary for expression, but not for normal steady state transcript levels . This suggests a post-transcriptional control of MAK3 expression by the 3' region . The MAK3 protein has substantial homology with several N-acetyltransferases with consensus patterns h..h.h . . . Y..{HK}GI{AG}{KR}.Lh . . .h and h.h{DE} . . . .N..A . . .Y . . .GF . . . . . . . .Y . . {DE}G, (h = hydrophobic) . Mutation of any of the underlined conserved residues (94GI----AA, 123N----A, 130Y----A, 134GF----SL, 144Y----A, and 149G----A) inactivated the gene, supporting the hypothesis that MAK3 encodes an N-acetyltransferase.

J Biol Chem, 1992 Oct 5, 267(28), 19798 - 805
Ty4, a new retrotransposon from Saccharomyces cerevisiae, flanked by tau-elements; Janetzky B et al.; We have isolated and sequenced a genomic clone from Saccharomyces cerevisiae that shows structural features of a novel retrotransposon, designated Ty4 . The element is 6.2 kilobases in length, and its genetic organization of the deduced functional domains is similar to Ty1 and Ty2 and thus different from Ty3 . In contrast to hitherto known Ty elements from yeast, Ty4 is flanked by long terminal tau-element repeats instead of delta or sigma sequences . Ty4 contains two overlapping open reading frames . The first open reading frame, TYA4, is 1230 base pairs long and encodes a protein with a motif found in the nucleic acid-binding gag-protein of retroviruses . The second 4395-base pair open reading frame, TYB4, encodes a polyprotein that has domains with significant homology to retroviral protease, integrase, reverse transcriptase, and RNase H, structurally arranged in that order . The deduced amino acid sequence shows the greatest similarity with Ty2 and Ty1 . The overall identity of the deduced functional protein domains is 28% with Ty2, 25% with Ty1, 19% with copia from Drosophila, and 18% with Ty3 . Examination of genomic DNA from several laboratory strains indicates that Ty4 is present in two to four copies . Ty4 mRNA is of low abundance as compared to other Ty retrotransposons . At the 3' end of Ty4, two "solo" delta-elements, a full length and an overlapping, truncated one, are associated.

Biochemistry, 1992 Sep 22, 31(37), 8713 - 9
13C NMR detection of folate-mediated serine and glycine synthesis in vivo in Saccharomyces cerevisiae; Pasternack LB et al.; Saccharomyces cerevisiae has both cytoplasmic and mitochondrial C1-tetrahydrofolate (THF) synthases . These trifunctional isozymes are central to single-carbon metabolism and are responsible for interconversion of the THF derivatives in the respective compartments . In the present work, we have used 13C NMR to study folate-mediated single-carbon metabolism in these two compartments, using glycine and serine synthesis as metabolic endpoints . The availability of yeast strains carrying deletions of cytoplasmic and/or mitochondrial C1-THF synthase allows a dissection of the role each compartment plays in this metabolism . When yeast are incubated with {13C}formate, 13C NMR spectra establish that production of {3-13C}serine is dependent on C1-THF synthase and occurs primarily in the cytosol . However, in a strain lacking cytoplasmic C1-THF synthase but possessing the mitochondrial isozyme, {13C}formate can be metabolized to {2-13C}glycine and {3-13C}serine . This provides in vivo evidence for the mitochondrial assimilation of formate, activation and conversion to {13C}CH2-THF via mitochondrial C1-THF synthase, and subsequent glycine synthesis via reversal of the glycine cleavage system . Additional supporting evidence of reversibility of GCV in vivo is the production of {2-13C}glycine and {2,3-13C}serine in yeast strains grown with {3-13C}serine . This metabolism is independent of C1-THF synthase since these products were observed in strains lacking both the cytoplasmic and mitochondrial isozymes . These results suggest that when formate is the one-carbon donor, assimilation is primarily cytoplasmic, whereas when serine serves as one-carbon donor, considerable metabolism occurs via mitochondrial pathways.

Mol Gen Genet, 1992 Oct, 235(1), 97 - 103
Effect of donor copy number on the rate of gene conversion in the yeast Saccharomyces cerevisiae; Melamed C et al.; Nonreciprocal recombination (gene conversion) between homologous sequences at nonhomologous locations in the genome occurs readily in the yeast Saccharomyces cerevisiae . In order to test whether the rate of gene conversion is dependent on the number of homologous copies available in the cell to act as donors of information, the level of conversion of a defined allele was measured in strains carrying plasmids containing homologous sequences . The level of recombination was elevated in a strain carrying multiple copies of the plasmid, compared with the same strain carrying a single copy of the homologous sequences either on a plasmid or integrated in the genome . Thus, the level of conversion is proportional to the number of copies of donor sequences present in the cell . We discuss these results within the framework of currently favoured models of recombination.

Genetics, 1992 Oct, 132(2), 387 - 402
Sister chromatids are preferred over homologs as substrates for recombinational repair in Saccharomyces cerevisiae; Kadyk LC et al.; A diploid Saccharomyces cerevisiae strain was constructed in which the products of both homolog recombination and unequal sister chromatid recombination events could be selected . This strain was synchronized in G1 or in G2, irradiated with X-rays to induce DNA damage, and monitored for levels of recombination . Cells irradiated in G1 were found to repair recombinogenic damage primarily by homolog recombination, whereas those irradiated in G2 repaired such damage preferentially by sister chromatid recombination . We found, as have others, that G1 diploids were much more sensitive to the lethal effects of X-ray damage than were G2 diploids, especially at higher doses of irradiation . The following possible explanations for this observation were tested: G2 cells have more potential templates for repair than G1 cells; G2 cells are protected by the RAD9-mediated delay in G2 following DNA damage; sister chromatids may share more homology than homologous chromosomes . All these possibilities were ruled out by appropriate tests . We propose that, due to a special relationship they share, sister chromatids are not only preferred over homologous chromatids as substrates for recombinational repair, but have the capacity to repair more DNA damage than do homologs.

Genetics, 1992 Oct, 132(2), 351 - 9
Identification and characterization of three genes that affect expression of ADH2 in Saccharomyces cerevisiae; Karnitz L et al.; Using a new selection protocol we have identified and preliminarily characterized three new loci (ADR7, ADR8 and ADR9) which affect ADH2 (alcohol dehydrogenase isozyme II) expression . Mutants were selected which activate ADH2 expression in the presence of an over-expressed, normally inactive ADR1 allele . The mutants had very similar phenotypes with the exception that one was temperature sensitive for growth . In the absence of any ADR1 allele, the mutants allowed ADH2 to partially escape glucose repression . However, unlike wildtype strains deleted for ADR1, the mutants were able to efficiently derepress ADH2 . The mutations allowed a small escape from glucose repression for secreted invertase, but had no effect on the glucose repression of isocitrate lyase or malate dehydrogenase . The mutations were shown to be nonallelic to a wide variety of previously characterized mutations, including mutations that affect other glucose-repressed enzymes.

Chromosoma, 1992 Oct, 101(10), 590 - 5
Meiotic chromosome condensation and pairing in Saccharomyces cerevisiae studied by chromosome painting; Scherthan H et al.; Non-isotopic high resolution in situ hybridization was applied to cytological preparations of sporulating yeast cells . Ribosomal DNA (rDNA) and chromosome V-specific recombinant lambda clones were used to tag individual chromosomes and chromosome subregions . This allowed the study of chromosome behaviour during early meiotic prophase . It was found that chromatin becomes condensed and homologous DNA sequences then appear to become aligned prior to synaptonemal complex formation.

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