S. cerevisiae

Mol Cell Biol, 1991 Aug, 11(8), 4045 - 52
The Saccharomyces cerevisiae YAK1 gene encodes a protein kinase that is induced by arrest early in the cell cycle; Garrett S et al.; Null mutations in the gene YAK1, which encodes a protein with sequence homology to known protein kinases, suppress the cell cycle arrest phenotype of mutants lacking the cyclic AMP-dependent protein kinase (A kinase) . That is, loss of the YAK1 protein specifically compensates for loss of the A kinase . Here, we show that the protein encoded by YAK1 has protein kinase activity . Yak1 kinase activity is low during exponential growth but is induced at least 50-fold by arrest of cells prior to the completion of S phase . Induction is not observed by arrest at stages later in the cell cycle . Depending on the arrest regimen, induction can occur either by an increase in Yak1 protein levels or by an increase in Yak1 specific activity . Finally, an increase in Yak1 protein levels causes growth arrest of cells with attenuated A kinase activity . These results suggest that Yak1 acts in a pathway parallel to that of the A kinase to negatively regulate cell proliferation.

Mol Cell Biol, 1991 Aug, 11(8), 3868 - 78
Viability of clathrin heavy-chain-deficient Saccharomyces cerevisiae is compromised by mutations at numerous loci: implications for the suppression hypothesis; Munn AL et al.; The gene encoding clathrin heavy chain in Saccharomyces cerevisiae (CHC1) is not essential for growth in most laboratory strains tested . However, in certain genetic backgrounds, a deletion of CHC1 (chc1) results in cell death . Lethality in these chc1 strains is determined by a locus designated SCD1 (suppressor of clathrin deficiency) which is unlinked to CHC1 (S . K . Lemmon and E . W . Jones, Science 238:504-509, 1987) . The lethal allele of SCD1 has no effect on cell growth when the wild-type version of CHC1 is present . This result led to the proposal that most yeast strains are viable in the absence of clathrin heavy chain because they possess the SCD1 suppressor . Discovery of another yeast strain that cannot grow without clathrin heavy chain has allowed us to perform a genetic test of the suppressor hypothesis . Genetic crosses show that clathrin-deficient lethality in the latter strain is conferred by a single genetic locus (termed CDL1, for clathrin-deficient lethality) . By constructing strains in which CHC1 expression is regulated by the GAL10 promoter, we demonstrate that the lethal alleles of SCD1 and CDL1 are recessive . In both cases, very low expression of CHC1 can allow cells to escape from lethality . Genetic complementation and segregation analyses indicate that CDL1 and SCD1 are distinct genes . The lethal CDL1 allele does not cause a defect in the secretory pathway of either wild-type or clathrin heavy-chain-deficient yeast . A systematic screen to identify mutants unable to grow in the absence of clathrin heavy chain uncovered numerous genes similar to SCD1 and CDL1 . These findings argue against the idea that viability of chc1 cells is due to genetic suppression, since this hypothesis would require the existence of a large number of unlinked genes, all of which are required for suppression . Instead, lethality appears to be a common, nonspecific occurrence when a second-site mutation arises in a strain whose cell growth is already severely compromised by the lack of clathrin heavy chain.

Appl Biochem Biotechnol, 1991 Aug, 30(2), 203 - 16
Continuous potable alcohol production by immobilized Saccharomyces cerevisiae on mineral kissiris; Koutinas AA et al.; A biocatalyst prepared by the immobilization of Saccharomyces cerevisiae on the surface of the mineral kissiris was used in the present study for continuous potable-alcohol production . An ethanol productivity (calculated on the basis of liquid volume) of 10.5 g/L/h was obtained at a 0.7/h dilution rate, 121 g/L sucrose content, and 29.6% conversion employing molasse as feed material . Glucose, raisin extracts, and molasse were successively used as feed materials without stopping the operation of the reactor for 6 mo . The ethanol productivity and yield remained constant during the operational-stability study of the reactor, carried out for 44 d . Biomass productivity, yield, and free-cell concentration in glucose, raisin extracts, and molasse were examined . Finally, a system with two continuous reactors joined successively was also studied in the present investigation.

Antimicrob Agents Chemother, 1991 Aug, 35(8), 1532 - 7
Physiological effects of fenpropimorph on wild-type Saccharomyces cerevisiae and fenpropimorph-resistant mutants; Lorenz RT et al.; Fenpropimorph-resistant mutants of Saccharomyces cerevisiae were isolated by a gradient selection procedure . The mutants were cross-resistant to other morpholines (fenpropidin, dodemorph, tridemorph) and 15-azasterol, but were susceptible to azoles (miconazole, clotrimazole, ketoconazole) and nystatin . In the absence of fenpropimorph, the major sterol produced by the mutants and the parental strain was ergosterol . In the presence of fenpropimorph, ignosterol (ergosta-8,14-dien-3 beta-ol) was the major sterol produced by the mutants and the parental strain . The resistance to fenpropimorph involves two recessive genes, each of which allows a semiresistance, when they are isolated apart from one another . Strain JR4 (erg3 erg11), which produces 14-methylfecosterol {14 alpha-methyl-ergosta-8,24(28)-dien- 3-beta-ol) as the major sterol in the presence or absence of fenpropimorph, was also found to be resistant to the drug . The growth inhibitory effect of fenpropimorph on wild-type cells appears to be linked to the production of ignosterol . The uptake of exogenous sterol by wild-type cells was greatly enhanced in the presence of fenpropimorph . The growth inhibition caused by fenpropimorph could only be overcome with bulk levels of exogenous C-5,6-unsaturated sterols.

Mol Gen Genet, 1991 Aug, 228(1-2), 270 - 80
The GAM1/SNF2 gene of Saccharomyces cerevisiae encodes a highly charged nuclear protein required for transcription of the STA1 gene; Yoshimoto H et al.; We have cloned and sequenced the GAM1 gene which is required for transcription of the STA1 gene encoding an extracellular glucoamylase in Saccharomyces cerevisiae var . diastaticus . Complementation tests indicated that GAM1 is the same gene as SNF2 which is required for derepression of the SUC2 gene encoding invertase . Accumulation of SNF2 RNA was not regulated by the GAM2 and GAM3 genes which are also required for STA1 expression . The SNF2 gene was predicted to encode a 194 kDa highly charged protein with a glutamine-rich tract . A bifunctional SNF2-lacZ fusion protein was shown by immunofluorescence microscopy to be localized to the nucleus, suggesting that the SNF2 protein is located in the nucleus.

Eur J Biochem, 1991 Aug 1, 199(3), 511 - 8
Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinases PI and PII; Rose M et al.; Genetic and biochemical analyses showed that hexokinase PII is mainly responsible for glucose repression in Saccharomyces cerevisiae, indicating a regulatory domain mediating glucose repression . Hexokinase PI/PII hybrids were constructed to identify the supposed regulatory domain and the repression behavior was observed in the respective transformants . The hybrid constructs allowed the identification of a domain (amino acid residues 102-246) associated with the fructose/glucose phosphorylation ratio . This ratio is characteristic of each isoenzyme, therefore this domain probably corresponds to the catalytic domain of hexokinases PI and PII . Glucose repression was associated with the C-terminal part of hexokinase PII, but only these constructs had high catalytic activity whereas opposite constructs were less active . Reduction of hexokinase PII activity by promoter deletion was inversely followed by a decrease in the glucose repression of invertase and maltase . These results did not support the hypothesis that a specific regulatory domain of hexokinase PII exists which is independent of the hexokinase PII catalytic domain . Gene disruptions of hexokinases further decreased repression when hexokinase PI was removed in addition to hexokinase PII . This proved that hexokinase PI also has some function in glucose repression . Stable hexokinase PI overproducers were nearly as effective for glucose repression as hexokinase PII . This showed that hexokinase PI is also capable of mediating glucose repression . All these results demonstrated that catalytically active hexokinases are indispensable for glucose repression . To rule out any further glycolytic reactions necessary for glucose repression, phosphoglucoisomerase activity was gradually reduced . Cells with residual phosphoglucoisomerase activities of less than 10% showed reduced growth on glucose . Even 1% residual activity was sufficient for normal glucose repression, which proved that additional glycolytic reactions are not necessary for glucose repression . To verify the role of hexokinases in glucose repression, the third glucose-phosphorylating enzyme, glucokinase, was stably overexpressed in a hexokinase PI/PII double-null mutant . No strong effect on glucose repression was observed, even in strains with 2.6 U/mg glucose-phosphorylating activity, which is threefold increased compared to wild-type cells . This result indicated that glucose repression is only associated with the activity of hexokinases PI and PII and not with that of glucokinase.

Proc Natl Acad Sci U S A, 1991 Aug 1, 88(15), 6878 - 82
Monitoring of intracellular calcium in Saccharomyces cerevisiae with an apoaequorin cDNA expression system; Nakajima-Shimada J et al.; A method is described for measuring cytosolic free Ca2+ and its time-dependent changes in the yeast Saccharomyces cerevisiae by using the luminescent protein aequorin as a Ca(2+)-specific indicator . This method with intact yeast cells is labeled "in vivo" to distinguish it from methods with cell extracts, labeled "in vitro." A plasmid in which the apoaequorin cDNA was joined downstream from the glyceraldehyde-3-phosphate dehydrogenase gene promoter was constructed and introduced into yeast cells . The intracellular concentration of apoaequorin expressed by the cDNA was approximately 1 microM, which was high enough to detect the cytosolic Ca2+ . Growth of the transformed cells was normal . In the in vitro method, apoaequorin in crude cell extracts was regenerated into aequorin by mixing with coelenterazine, the substrate for the luminescence reaction, whereas in the in vivo method, aequorin was regenerated by incubating intact cells with coelenterazine . Simultaneous addition of 10 mM CaCl2 and 10 microM A23187, a Ca2+ ionophore, to coelenterazine-incorporated cells generated luminescence . Coelenterazine-incorporated cells also responded to native extracellular stimuli . A mating pheromone, alpha-factor, added to cells of mating type a or alpha, generated extracellular Ca(2+)-dependent luminescence specifically in a mating type cells, with maximal intensity occurring 45-50 min after addition of alpha-factor . Glucose added to glucose-starved G0/G1 cells stimulated an increase in extracellular Ca(2+)-dependent luminescence with maximal intensity occurring 2 min after addition . These results show the usefulness of the aequorin system in monitoring {Ca2+}i response to extracellular stimuli in yeast cells.

Mutat Res, 1991 Aug, 263(4), 211 - 5
Transformation of Saccharomyces cerevisiae with UV-irradiated single-stranded plasmid; Zgaga Z; UV-irradiated single-stranded replicative plasmids were used to transform different yeast strains . The low doses of UV used in this study (10-75 J/m2) caused a significant decrease in the transforming efficiency of plasmid DNA in the Rad+ strain, while they had no effect on transformation with double-stranded plasmids of comparable size . Neither the rev3 mutation, nor the rad18 or rad52 mutations influenced the efficiency of transformation with irradiated single-stranded plasmid . However, it was found to be decreased in the double rev3 rad52 mutant . Extracellular irradiation of plasmid that contains both URA3 and LEU2 genes (psLU) gave rise to up to 5% Leu- transformants among selected Ura+ ones in the repair-proficient strain . Induction of Leu- transformants was dose-dependent and only partially depressed in the rev3 mutant . These results suggest that both mutagenic and recombinational repair processes operate on UV-damaged single-stranded DNA in yeast.

J Bacteriol, 1991 Aug, 173(16), 4977 - 82
Saturation mutagenesis of the UASNTR (GATAA) responsible for nitrogen catabolite repression-sensitive transcriptional activation of the allantoin pathway genes in Saccharomyces cerevisiae; Bysani N et al.; Saturation mutagenesis of the UASNTR element responsible for GLN3-dependent, nitrogen catabolite repression-sensitive transcriptional activation of allantoin pathway genes in yeast cells identified the dodecanucleotide sequence 5'-TTNCTGATAAGG-3' as the minimum required for UAS activity . There was significant flexibility in mutant sequences capable of supporting UAS activity, which correlates well with the high variation in UASNTR homologous sequences reported to be upstream of the DAL and DUR genes . Three of nine UASNTR-like sequences 5' of the DAL5 gene supported high-level transcriptional activation . The others, which contained nonpermissive substitutions, were not active.

Genetics, 1991 Aug, 128(4), 717 - 27
Genetic analysis of a meiotic recombination hotspot on chromosome III of Saccharomyces cerevisiae; Symington LS et al.; In a previous study, we analyzed meiotic recombination events that occurred in the 22-kb region (LEU2 to CEN3) of chromosome III of Saccharomyces cerevisiae . We found one region with an enhanced level of crossovers (a hotspot) and one region with a depressed level of crossovers . In this study, we show that about one-third of the crossovers that occur between LEU2 and CEN3 are initiated in a 1.3-kb region located approximately 6 kb from the centromere . Both crossovers and gene conversion events are initiated at this site . Events initiated at this position can be resolved as crossovers in regions located either centromere-distally or centromere-proximally from the initiation site.

Yeast, 1991 Aug-Sep, 7(6), 643 - 50
Cloning and sequencing of the yeast Saccharomyces cerevisiae SEC1 gene localized on chromosome IV; Aalto MK et al.; The SEC1 gene of yeast Saccharomyces cerevisiae was cloned by complementing the temperature-sensitive mutation of sec1-1 at 37 degrees C, and its nucleotide sequence was determined . SEC1 is a single copy gene and encodes a protein of 724 amino acids and 83,490 daltons with a predicted pI value of 6.11 . Hydrophobicity plotting showed no clearly hydrophobic regions suggesting a soluble nature for the protein . Amino acid sequence comparisons revealed no obvious homologies with the proteins in the SWISSPROT databank . Two consensus sequence for the cdc2 encoded protein kinase recognition site were revealed within Sec1p . The codon usage suggests a low expression level for SEC1 . The 5' non-translated region contains two TATA-like sequences at -52 and -215 nucleotides from the translation start site . Two potential regulatory sequences for DNA binding proteins were found in the non-coding 5' region: a HAP2/HAP3 consensus recognition sequence at nucleotide-154 and a BAF1 consensus recognition sequence at nucleotide-136 . The SEC1 specific probe detected a 2400 nucleotides long transcript, which was in reasonable agreement with the 2172 nucleotides long open reading frame.

Yeast, 1991 Aug-Sep, 7(6), 627 - 30
Cytochrome P450 lanosterol 14 alpha-demethylase (ERG11) and manganese superoxide dismutase (SOD1) are adjacent genes in Saccharomyces cerevisiae; Turi TG et al.; DNA sequencing and analysis of genomic DNA using the polymerase chain reaction were used to demonstrate that SOD1 and ERG11 are adjacent genes in Saccharomyces cerevisiae S288c and to establish the correct intergenic sequence of this segment on chromosome VIII.

Yeast, 1991 Aug-Sep, 7(6), 607 - 8
The use of proline as a nitrogen source causes hypersensitivity to, and allows more economical use of 5FOA in Saccharomyces cerevisiae; McCusker JH et al.; The use of proline as a nitrogen source causes hypersensitivity to 5-fluoro-orotic acid (5FOA) and allows up to 40-fold less of this drug to be used to select for the loss of URA3 function in Saccharomyces cerevisiae . 5FOA hypersensitivity is presumably due to the absence of nitrogen catabolite repression when proline is substituted for (NH4)2SO4 as a nitrogen source . There are two constraints to the use of the proline-5FOA combination: (1) S288c genetic background strains are hypersensitive to 5FOA when grown in proline as a nitrogen source but at least one other genetic background is resistant to low levels of 5FOA under these conditions . (2) The addition of some nutritional supplements confers phenotypic resistance to the 5FOA-proline combination.

Genetika, 1991 Aug, 27(8), 1342 - 9
{Genetic instability of colonies' morphologic characteristics in the yeast Saccharomyces cerevisiae . The influence of mutations of radiosensitivity}; Glazer VM et al.; The exposure to ionizing radiation of radiosensitive mutants of diploid yeast Saccharomyces cerevisiae deficient in double-strand break repair results in formation of morphologically unstable colonies . Some characteristics of this process were studied . The results obtained are consistent with the hypothesis on relationship between DNA double-strand breaks or their repair with the formation of unstable clones of diploid yeast cells.

Genetika, 1991 Aug, 27(8), 1316 - 23
{Cloning of segments of the Drosophila melanogaster genome using artificial chromosomes of the yeast Saccharomyces cerevisiae}; Kogan GL et al.; A partial genomic library from the Batumi L stock of Drosophila melanogaster was constructed using yeast artificial chromosomes as vectors . The DNA was restricted by Not1 and large fragments were inserted into the YAC5 vector . The size of cloned DNA varied from 90 to 500 kb . 48 random clones were characterized by in situ hybridization to the Batumi L polytene salivary gland chromosome . Single euchromatic sites of hybridization were detected for 27 clones; 11 clones revealed the main euchromatic hybridization site and several additional sites scattered along the chromosomes; 8 clones carried repeats which hybridized to chromocenter and other chromosomal sites; clones with 500 and 90 kb inserts originated from the Y chromosomes and nucleolus, respectively . The library is enriched by the repeated sequences related to the b-heterochromatin.

Yeast, 1991 Aug-Sep, 7(6), 589 - 98
Cyclic variations in the permeability of the cell wall of Saccharomyces cerevisiae; De Nobel JG et al.; To study cell-cycle-related variations in wall permeability of Saccharomyces cerevisiae, two approaches were used . First, an asynchronous culture was fractionated by centrifugal elutriation into subpopulations containing cells of increasing size . The subpopulations represented different stages of the cell cycle as judged by light microscopy . Cell wall porosity increased when these subpopulations became enriched with budded cells . Secondly, synchronous cultures were obtained by releasing MATa cells from alpha-factor induced G1-arrest . These cultures grew synchronously for at least two generations . The cell wall porosity increased sharply in these cultures, shortly before buds became visible and was maximal during the initial stages of bud growth . It decreased in cells which had completed nuclear migration and before abscission of the bud had occurred . The porosity reached its lowest value during abscission and in unbudded cells . We examined the incorporation of mannoproteins into the wall during the cell cycle . SDS-extractable mannoproteins were incorporated continuously . However, the incorporation of glucanase-extractable mannoproteins, which are known to affect cell wall porosity, showed cyclic oscillations and reached its maximum after nuclear migration . This coincided with a rapid decrease in cell wall porosity, indicating that glucanase-extractable mannoproteins might contribute to this decrease.

J Cell Biol, 1991 Aug, 114(4), 623 - 38
Characterization of SIS1, a Saccharomyces cerevisiae homologue of bacterial dnaJ proteins; Luke MM et al.; The Saccharomyces cerevisiae SIS1 gene was identified as a high copy number suppressor of the slow growth phenotype of strains containing mutations in the SIT4 gene, which encodes a predicted serine/threonine protein phosphatase . The SIS1 protein is similar to bacterial dnaJ proteins in the amino-terminal third and carboxyl-terminal third of the proteins . In contrast, the middle third of SIS1 is not similar to dnaJ proteins . This region of SIS1 contains a glycine/methionine-rich region which, along with more amino-terminal sequences, is required for SIS1 to associate with a protein of apparent molecular mass of 40 kD . The SIS1 gene is essential . Strains limited for the SIS1 protein accumulate cells that appear blocked for migration of the nucleus from the mother cell into the daughter cell . In addition, many of the cells become very large and contain a large vacuole . The SIS1 protein is localized throughout the cell but is more concentrated at the nucleus . About one-fourth of the SIS1 protein is released from a nuclear fraction upon treatment with RNase . We also show that overexpression of YDJ1, another yeast protein with similarity to bacterial dnaJ proteins, can not substitute for SIS1.

Yeast, 1991 Aug-Sep, 7(6), 575 - 87
The control of trehalose biosynthesis in Saccharomyces cerevisiae: evidence for a catabolite inactivation and repression of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase; Francois J et al.; During diauxic growth of yeast in glucose-rich medium, the accumulation of trehalose started well after complete exhaustion of glucose from the medium . The accumulation of the disaccharide was concomitant with a resumption of cell growth on the ethanol accumulated in the medium, but not with a degradation of glycogen which occurred as soon as glucose had been consumed . In contrast, in a mutant deficient in phosphoenolpyruvate carboxykinase, the synthesis of trehalose coincided exactly with the degradation of glycogen . Upon inoculation of stationary phase wild-type cells into a glucose medium, the activities of trehalose-6-phosphate (Tre6P) synthase and Tre6P phosphatase dropped in parallel to reach only 15% of their initial values after 3 h, and only recovered their original values as cells re-entered stationary phase . In the presence of cycloheximide, the decrease in Tre6P synthase and Tre6P phosphatase activities was restricted to 50-60%, the remaining decrease being inhibited by the drug . Furthermore, the reappearance of the enzyme activities following transfer of cells to an acetate medium was blocked by cycloheximide . It was also shown that loss of activity of these two enzymes required a combination of metabolizable sugars together with a nitrogen source . Low activities of Tre6P synthase and Tre6P phosphatase were measured in mutants with increased adenylate cyclase activity (RAS2ala18val19 mutants) . Moreover, derepression of these enzymes at the approach of stationary phase was prevented in a pde2 mutant when it was cultivated in the presence of exogenous cyclic nucleotide . The mechanism of this effect is not clear, but may involve a transcriptional regulation by cAMP of the genes encoding these proteins.

Mol Microbiol, 1991 Aug, 5(8), 1823 - 9
Regulation of retrotransposition in Saccharomyces cerevisiae; Curcio MJ et al.; Retrotransposons are a widely distributed group of eukaryotic mobile genetic elements that transpose through an RNA intermediate . The element Ty (Transposon yeast), found in the yeast Saccharomyces cerevisiae, is a model system for the study of retrotransposons because of the experimental tools that exist to manipulate and detect transposition . Ty transposition can be elevated to levels exceeding one transposition event per cell when an element is expressed from an inducible yeast promoter . In addition, individual genomic Ty elements can be tagged with a retrotransposition indicator gene that allows transposition events occurring at a rate of 10(-5) to 10(-7) per element per cell division to be detected phenotypically . These systems are being used to elucidate the mechanism of Ty transposition and clarify how Ty transposition is controlled.

Curr Genet, 1991 Aug, 20(3), 185 - 8
Heat shock and stationary phase induce transcription of the Saccharomyces cerevisiae iso-2 cytochrome c gene; Pillar TM et al.; Transcription of the iso-2 cytochrome c gene of Saccharomyces cerevisiae (CYC7) is strongly induced by stationary-growth phase, heat shock and low cAMP levels . CYC1, the iso-1 cytochrome c gene, is strongly repressed in stationary phase and unaffected by heat shock and cAMP levels . Heat shock-induced transcription of CYC7 occurs both aerobically (4-6 fold) and anaerobically (30 fold).

Genetics, 1991 Aug, 128(4), 729 - 38
Histone regulatory (hir) mutations suppress delta insertion alleles in Saccharomyces cerevisiae; Sherwood PW et al.; Changes in histone gene dosage as well as mutations within some histone genes suppress delta insertion mutations in the HIS4 and LYS2 loci of Saccharomyces cerevisiae by altering the site of transcription initiation . We have found that three histone regulatory (hir) mutations, identified by their effects on the regulation of histone gene expression, suppress the same insertion mutations . In addition, we have examined whether any previously identified spt (suppressor of Ty) mutations might suppress the delta insertion alleles because of effects on histone gene regulation . Our results demonstrate that mutations in the histone genes SPT11/HTA1 and SPT12/HTB1 and in three other SPT genes, SPT1, SPT10 and SPT21, confer Hir- phenotypes . The spt1 mutation was found to be an allele of HIR2 while the spt10 and spt21 mutations are not in any of the known HIR genes.

Genetics, 1991 Aug, 128(4), 703 - 16
Genetic analysis of the gyrase A-like domain of DNA topoisomerase II of Saccharomyces cerevisiae; Thomas W et al.; We have undertaken a genetic analysis of heat-sensitive and cold-sensitive mutations in TOP2, the gene encoding yeast DNA topoisomerase II . Deletion mapping was used to localize 14 heat-sensitive and four cold-sensitive top2 mutations created by a method biased toward mutations in the 3' two-thirds of the gene . The mutations all appear to be located in the region of DNA topoisomerase II that shows homology to the "A" subunit of bacterial DNA gyrase . The heat-sensitive mutations and one cold-sensitive mutation lie in the center of the gene near the sequence that encodes the active site tyrosine . The three other cold-sensitive mutations map farther toward the 3' end of the gene . The cold-sensitive mutations exhibit intragenic complementation, and the complementation groups correspond to the physical map . We sequenced nine top2 mutations and found that the mutations are usually single missense mutations, frequently involve proline, and affect conserved regions of the protein . Suppressor analysis yielded two intragenic suppressors and seven independent isolates of an allele-specific extragenic suppressor we named tos1; tos1 is not allelic to any genes predicted to encode type I topoisomerase-related proteins . The two intragenic suppressors were tested for allele-specificity; the results revealed a complex pattern of suppression between heat-sensitive and cold-sensitive top2 alleles . These top2 mutations may have compensatory effects on the general stability of the protein.

EMBO J, 1991 Aug, 10(8), 2165 - 70
The 3' to 5' exonuclease activity located in the DNA polymerase delta subunit of Saccharomyces cerevisiae is required for accurate replication; Simon M et al.; In Saccharomyces cerevisiae, DNA polymerase delta (POLIII), the product of the CDC2 (POL3) gene, possesses, in its N-terminal half, the well conserved 3-domain 3' to 5' exonuclease site . Strains selectively mutagenized in this site display a mutator phenotype detected as a drastically increased spontaneous forward mutation rate to canavanine resistance or as an elevated reversion rate to lysine prototrophy . Assays on a partially purified extract of the mutant giving the largest mutator effect indicate that the 3' to 5' exonuclease activity is reduced below the detection limit whereas the DNA polymerizing activity has wild-type level . Therefore, our results provide experimental support for the hypothesis that the exonucleolytic proofreading activity associated with DNA polymerase delta resides on the DNA polymerase delta subunit and enhances the fidelity of DNA replication in yeast.

Agric Biol Chem, 1991 Aug, 55(8), 1953 - 8
Cloning and nucleotide sequence of the KHS killer gene of Saccharomyces cerevisiae; Goto K et al.; A 5.3-kbp fragment of the KHS gene was cloned from a genomic bank of Saccharomyces cerevisiae No . 115 constructed with an E . coli as the host and YEp13 as the vector . A non-killer yeast strain was transformed to a killer strain with the multi-copy vector containing the KHS gene, and the transformant could secrete 3-4 times more killer toxin into culture media than the donor, strain No . 115 . The KHS toxin was purified 80-fold from the culture filtrate by gel filtration and column chromatography . The nucleotide sequence of a 2.8-kbp fragment of the KHS DNA that was enough for the expression of the killer activity was identified, and we found an open reading frame consisted of 2124 bp . Comparison of the open reading frame and N-terminal amino acid sequence of purified KHS toxin, suggested that the presumed peptide from the KHS gene might be processed between 36Gln and 37Ala before secretion.

Gene, 1991 Jul 31, 104(1), 99 - 102
New shuttle vectors for direct cloning in Saccharomyces cerevisiae; Silar P et al.; We have constructed new shuttle vectors to facilitate the screening of recombinant plasmids after direct transformation of yeast cells . The vectors are pBluescript-based shuttle vectors in which the lacZ marker has been replaced by an analogous system based on the Saccharomyces cerevisiae URA3 gene . DNA fragments are inserted in a polylinker located after the beginning of the URA3 coding sequence . Transformants are selected either by Trp or Leu prototrophy . Plasmids bearing an insert are selected by growth on 5-fluoro-orotic acid (5-FOA), a uracil analog toxic to cells containing a functional URA3+ gene (thus, this method requires the recipient strain to be ura3-); only cells containing a plasmid with an insert that disrupts the functional continuity of the URA3 gene can grow on medium containing 5-FOA . Using these plasmids, we were able to directly reclone the ACE1 gene from genomic DNA by directly transforming a strain deleted for ACE1 . These vectors can be used for a variety of purposes including rapid cloning of genes by complementation or expression of fusion genes driven from the URA3 promoter.

Gene, 1991 Jul 31, 104(1), 55 - 62
Sequences of two adjacent genes, one (DAL2) encoding allantoicase and another (DCG1) sensitive to nitrogen-catabolite repression in Saccharomyces cerevisiae; Yoo HS et al.; Reported are the nucleotide sequences of the yeast allantoicase-encoding gene (DAL2) and that of an unknown gene adjacent to it . Expression of the unidentified gene is sensitive to nitrogen catabolite repression (NCR) and regulated by the DAL80 product, a previously documented control element regulating allantoin pathway gene expression . Both genes possess multiple upstream activation sequences (UAS) homologous to the UASNTR element shown to be required for sensitivity to NCR . Also present upstream from DAL2 is a mutant form of the upstream induction sequence required for response of DAL7 to induction . Its occurrence in mutant form is consistent with the poor induction of DAL2 expression observed in vivo.

Gene, 1991 Jul 31, 104(1), 103 - 6
Sequence of RAD54, a Saccharomyces cerevisiae gene involved in recombination and repair; Emery HS et al.; The complete nucleotide sequence of the RAD54 gene of the yeast Saccharomyces cerevisiae has been determined . The sequenced region contains an open reading frame of 2694 bp, and the predicted RAD54 protein has a potential nucleotide-binding site and possible nuclear targeting sequences . Northern analysis reveals a transcript of approx . 3.0 kb which is induced following x-ray irradiation.

FEBS Lett, 1991 Jul 29, 286(1-2), 13 - 7
Osmoregulation in Saccharomyces cerevisiae . Studies on the osmotic induction of glycerol production and glycerol-3-phosphate dehydrogenase (NAD+)
Andre L, Hemming A, Adler L.
Production of glycerol and a key enzyme in glycerol production, glycerol 3-phosphate dehydrogenase (NAD+) (GPD), was studied in Saccharomyces cerevisiae cultured in basal media or media of high salinity, with glucose, raffinose or ethanol as the sole carbon source . At high salinity, glycerol production was stimulated with all carbon sources and glycerol was accumulated to high intracellular concentration in cells grown on glucose and raffinose . Cells grown on ethanol accumulated glycerol to a lower level but showed an increased content of trehalose at high salinity . However, the trehalose concentration corresponded only to about 20% of the glycerol level, and did not compensate for the shortfall in intracellular osmolyte content . Immunoblot analysis demonstrated an increased production of GPD at high salinity . This increase was osmotically mediated but was lower when glycerol was substituted for NaCl or sorbitol as the stress-solute . The enzyme also appeared to be subject to glucose repression; the specific activity of GPD was significantly lower in cells grown on glucose, than on raffinose or ethanol.

Biochem Pharmacol, 1991 Jul 25, 42(4), 753 - 8
The expression of human cytochrome P450IA1 in the yeast Saccharomyces cerevisiae; Ching MS et al.; Data from animal studies suggest that cytochrome P450IA1 catalyses the metabolic activation of several procarcinogenic compounds . In the present study, we have expressed human cytochrome P450IA1 in yeast cells . A 1.70 kb BclI/BamHI fragment containing a full-length human cytochrome P450IA1 cDNA was inserted into the BglII expression site of the yeast expression plasmid pMA91 thereby allowing the ATG initiation codon to be located adjacent to the PGK (phosphoglycerate kinase) promoter . The resulting recombinant plasmid, pCK-1, was introduced into Saccharomyces cerevisiae strains ATCC 44773 and AH22 . Microsomes prepared from yeast transformatants of strain ATCC 44773 contained undetectable levels of cytochrome P450 . In contrast, microsomes from strain AH22 contained cytochrome P450 with a specific content of 33.3 +/- 10.8 pmol/mg of microsomal protein and showed a reduced carbon monoxide difference spectrum with a peak at 448 nm . Control yeast cells transformed with pMA91 showed no cytochrome P450 . Western blots were carried out using an antibody that reacts against rat cytochrome P450IA1 and an antibody that reacts against a synthetic peptide representing a short sequence of human cytochrome P450IA1 . A band with a molecular weight of 54 kD was observed in microsomes of yeast transformed with pCK-1, but not with pMA91 . When microsomes from yeast transformed with pCK-1 were incubated with benzo(a)pyrene (10 min, 10-160 microM), an estimated Km value of 7 microM was obtained . The availability of yeast cells with functionally active human cytochrome P450IA1 will facilitate molecular structure-activity studies of procarcinogen and drug metabolism by this enzyme in man.

J Biol Chem, 1991 Jul 25, 266(21), 14046 - 54
Strand exchange protein 1 from Saccharomyces cerevisiae . A novel multifunctional protein that contains DNA strand exchange and exonuclease activities; Johnson AW et al.; Strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae catalyzes the formation of heteroduplex DNA molecules from single-stranded circles and homologous linear duplex DNA in vitro . Previously, Sep1 was purified as a 132,000-Da species; however, DNA sequence analysis indicates that the SEP1 gene is capable of encoding a 175,000-Da protein (Tishkoff, D.X., Johnson, A.W., and Kolodner, R.D . (1991) Mol . Cell . Biol . 11, 2593-2608) . The SEP1 gene was cloned into a GAL10 expression vector and expressed in a protease-deficient yeast strain . Intact Sep1, which migrated as a Mr-160,000 polypeptide during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was purified to apparent homogeneity and shown to have activities similar to those of the originally purified Mr = 132,000 fragment . We report here that, in addition to strand exchange activity, Sep1 contains an intrinsic exonuclease that is active on single- and double-stranded DNA with a severalfold preference for single-stranded DNA . The nuclease was induced in crude extracts upon induction with galactose, it co-purified with the strand exchange activity of Sep1, and the nuclease and strand exchange activities of Sep1 showed the same kinetics of heat inactivation . Sep1 nuclease, which requires Mg2+, can be functionally separated from the strand exchange activity by the substitution of Ca2+ for Mg2+ . Under these conditions, the nuclease is inactive, and strand exchange activity is dependent on prior resection of the DNA ends by an exogenous exonuclease . Thus, the nuclease is necessary for synapsis but not strand exchange . Electron microscopic analysis revealed that true strand exchange products, alpha molecules and nicked double-stranded circular molecules, were formed . In addition, strand transfer proceeded to similar extents on 5'-resected and 3'-resected DNA . This result suggests that the polarity of strand transfer by Sep1 is determined by the polarity of its intrinsic nuclease.

Biochemistry, 1991 Jul 16, 30(28), 7033 - 40
Proton nuclear magnetic resonance as a probe of differences in structure between the C102T and F82S,C102T variants of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae; Gao Y et al.; Differences in chemical shifts and in nuclear Overhauser effects between the C102T and F82S,C102T variants of Saccharomyces cerevisiae iso-1-cytochrome c in both the reduced and oxidized forms are reported and analyzed . There is evidence for small conformational differences in both oxidation states of the double variant near position 82 . Differences in structure are more evident in the oxidized forms of the variants . These differences extend to distant parts of the protein . It is concluded that the oxidized double variant has undergone a small rearrangement of several regions of the protein that are linked by a hydrogen-bond network . It is shown that the rearrangement involves hydrogen bonds associated with the two heme propionates and associated water molecules . The deductions from nuclear magnetic resonance data are compared with the differences in the crystal structures of the reduced forms of wild-type protein and the F82S variant {Louie, G . V., Pielak, G . J., Smith, M., & Brayer, G . D . (1988) Biochemistry 27, 7870-7876}.

J Biol Chem, 1991 Jul 15, 266(20), 13423 - 30
Purification and functional characterization of membranes derived from the rough endoplasmic reticulum of Saccharomyces cerevisiae; Sanderson CM et al.; Isolation and biochemical analysis of the components involved in protein translocation into the rough endoplasmic reticulum (ER) requires starting material highly enriched in membranes derived from this organelle . We have chosen to study the yeast Saccharomyces cerevisiae in order to profit from the ease of genetic manipulation . To date, however, no efficient scheme has been devised that allows the purification of functional rough ER-derived membranes from yeast, largely because proteins have yet to be identified that are rough ER-specific . In the experiments described here, we expressed the human rough ER marker ribophorin I to facilitate the analysis of subcellular fractionation . We found that the endoplasmic reticulum of yeast could be separated into two distinct domains by fractionation on continuous sucrose gradients . This procedure revealed a bimodal distribution of ER markers . The yeast homologue of the heavy chain-binding protein, BiP (encoded by the KAR2 gene), and the product of the SEC62 gene were present in two fractions having equilibrium densities of 1.146 and 1.192 g/ml, respectively . In contrast, our analysis showed that preprotein translocation activity and retention of the rough ER-specific protein ribophorin I were specific only to the membrane fraction with an equilibrium density of 1.192 g/ml . To prepare fractions highly enriched in translocation competent rough ER-derived membranes for analysis, we developed a density shift fractionation scheme that optimizes the purity of membranes containing human ribophorin I . Membranes obtained by this method were found to possess the majority of the appropriate functional markers, including ATP-independent preprotein binding, ribosome binding, and post-translational translocation . Mitochondria, the major contaminant of the 1.192 g/ml fraction, were significantly depleted in density-shifted membrane populations.

Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 6058 - 62
A cell cycle-responsive transcriptional control element and a negative control element in the gene encoding DNA polymerase alpha in Saccharomyces cerevisiae; Gordon CB et al.; Transcription of the POL1 gene of Saccharomyces cerevisiae, which encodes DNA polymerase alpha, the DNA polymerase required for the initiation of DNA replication, has previously been shown to be cell cycle regulated . To understand how the POL1 gene senses cell cycle position, we have investigated the cis-acting elements that respond to the factors that govern cell cycle progression . In this report we demonstrate that a region of 54 nucleotides containing the repeated element ACGCGT, which conforms to an Mlu I restriction endonuclease recognition site, contains all information necessary for transcriptional activation and cell cycle responsiveness . Although oligonucleotides lacking either one or both of the repeated Mlu I sites can function as an upstream activating sequence, the presence of at least one Mlu I site stimulates expression and, moreover, is absolutely essential for cell cycle regulation . A synthetic oligonucleotide corresponding to a 19-base-pair sequence in the POL1 promoter containing one Mlu I site can function as an autonomous cell cycle-responsive upstream element (upstream activation sequence) with temporal regulation indistinguishable from that previously described for the POL1 gene . Thus, the Mlu I site is an essential part of a cis-acting element responsible for the observed periodic activation . This sequence differs from previously defined cell cycle-responsive transcriptional control elements in the yeast HO endonuclease and histone genes . We also present evidence for a negative regulatory element in the 5' flanking region of the Mlu I upstream activation sequence.

J Biol Chem, 1991 Jul 15, 266(20), 13203 - 9
Heterologous expression of peptide hormone precursors in the yeast Saccharomyces cerevisiae . Evidence for a novel prohormone endoprotease with specificity for monobasic amino acids; Bourbonnais Y et al.; The peptide somatostatin (SRIF) exists as two different molecular species . In addition to the most common form, which is a 14-residue peptide, there is also a 14-amino acid amino-terminally extended form of the tetradecapeptide, SRIF-28 . Both peptides are synthesized as larger precursors containing paired basic and monobasic amino acids at their processing sites, which, upon cleavage, generate either SRIF-14 or -28, respectively . In mammals a single prepro-SRIF molecule undergoes tissue-specific processing to generate the mature hormone whereas in some species of fish separate genes encode two distinct but homologous precursors prepro-SRIF-I and -II that give rise to SRIF-14 and -28, respectively . To investigate the molecular basis for differential processing of the prohormones we introduce their cDNAs into yeast cells (Saccharomyces cerevisiae) . The signal peptides of both precursors were poorly recognized by the yeast endoplasmic reticulum translocation apparatus, consequently only low levels of SRIF peptides were synthesized . To circumvent this problem a chimeric precursor consisting of the alpha-factor signal peptide plus 30 residues of the proregion was fused to pro-SRIF-II . This fusion protein was efficiently transported through the yeast secretory pathway and processed to SRIF-28 exclusively, which is identical to the processing of the native precursor in pancreatic islet D-cells . Most significantly, cleavage of the precursor to SRIF-28 was independent of the Kex 2 endoprotease since processing occurred efficiently in a kex 2 mutant strain . We conclude that in addition to the Kex 2 protease, yeast possess a distinct prohormone converting enzyme with specificity toward monobasic processing sites.

Eur J Biochem, 1991 Jul 15, 199(2), 325 - 35
Determination of amino acid sequences involved in the processing of the ARG5/ARG6 precursor in Saccharomyces cerevisiae; Boonchird C et al.; In Saccharomyces cerevisiae, the ARG5/ARG6 locus encodes both acetylglutamate kinase and acetylglutamyl-phosphate reductase, localized in the mitochondria . Genetic analysis, determination of the nucleotide sequence of the ARG5/ARG6 gene and identification of the transcript indicate that it encodes a single translation product containing two enzyme activities . However, analysis of cellular extracts revealed that the activities are completely separable . In this work, we define different domains in the ARG5/ARG6 polypeptide; a mitochondrial target sequence and the acetylglutamate-kinase and acetylglutamyl-phosphate-reductase domains . We show that deletions in the N-terminal end of the protein and point mutations in the junction region between the acetylglutamate-kinase and acetylglutamyl-phosphate-reductase domains lead to the accumulation of large precursor . Our data support the idea that import of the ARG5/ARG6 precursor into the mitochondria is required for its processing into two mature enzymes.

Proc Natl Acad Sci U S A, 1991 Jul 1, 88(13), 5872 - 6
Spermidine or spermine is essential for the aerobic growth of Saccharomyces cerevisiae; Balasundaram D et al.; A null mutation in the SPE2 gene of Saccharomyces cerevisiae, encoding S-adenosylmethionine decarboxylase, results in cells with no detectable S-adenosylmethionine decarboxylase, spermidine, and spermine . This mutant has an absolute requirement for spermidine or spermine for growth; this requirement is not satisfied by putrescine . Polyamine-depleted cells show a number of microscopic abnormalities that are similar to those reported for several cell division cycle (cdc) and actin mutants . These include a striking increase in cell size, a marked decrease in budding, accumulation of vesicle-like bodies, absence of specific localization of chitin-like material, and abnormal distribution of actin-like material . The absolute requirement for polyamines for growth and the microscopic abnormalities are not seen if the cultures are grown under anaerobic conditions.

J Cell Biol, 1991 Jul, 114(1), 111 - 23
The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle; Shaw JA et al.; The morphology of three Saccharomyces cerevisiae strains, all lacking chitin synthase 1 (Chs1) and two of them deficient in either Chs3 (calR1 mutation) or Chs2 was observed by light and electron microscopy . Cells deficient in Chs2 showed clumpy growth and aberrant shape and size . Their septa were very thick; the primary septum was absent . Staining with WGA-gold complexes revealed a diffuse distribution of chitin in the septum, whereas chitin was normally located at the neck between mother cell and bud and in the wall of mother cells . Strains deficient in Chs3 exhibited minor abnormalities in budding pattern and shape . Their septa were thin and trilaminar . Staining for chitin revealed a thin line of the polysaccharide along the primary septum; no chitin was present elsewhere in the wall . Therefore, Chs2 is specific for primary septum formation, whereas Chs3 is responsible for chitin in the ring at bud emergence and in the cell wall . Chs3 is also required for chitin synthesized in the presence of alpha-pheromone or deposited in the cell wall of cdc mutants at nonpermissive temperature, and for chitosan in spore walls . Genetic evidence indicated that a mutant lacking all three chitin synthases was inviable; this was confirmed by constructing a triple mutant rescued by a plasmid carrying a CHS2 gene under control of a GAL1 promoter . Transfer of the mutant from galactose to glucose resulted in cell division arrest followed by cell death . We conclude that some chitin synthesis is essential for viability of yeast cells.

EMBO J, 1991 Jul, 10(7), 1843 - 52
Requirement for acidic amino acid residues immediately N-terminal to the conserved domain of Saccharomyces cerevisiae TFIID; Zhou QA et al.; TFIID binds to TATA boxes and initiates the assembly of general transcription factors and pol II on promoters . TFIID proteins from various species consist of a highly conserved carboxy terminal domain and very divergent amino terminal domains . We investigated the function of the non-conserved amino terminal domain (residues 1-60) of Saccharomyces cerevisiae TFIID (YIID, 240 residues) by testing the ability of a series of YIID amino terminal deletion mutants to complement a YIID deficient yeast strain . Mutants with deletions up to amino acid 48 restored the YIID deficient yeast strain to an apparently wild type phenotype . However, deletion up to position 57 or 60 produced yeast strains which formed extremely small colonies . Moreover, overexpression of YIID delta 2-57 or YIID delta 3-60 protein in the presence of wild type YIID resulted in a dominant-negative inhibition of growth . No difference between the basal transcriptional activity of wild type YIID and these amino terminal deletion mutants was observed in vitro . However, transcriptional activation in vivo of promoter-lacZ fusions showed that the YIID delta 2-57 deletion affects the ability of certain promoters (CUP1 and an HSP UAS-CYC1 promoter hybrid promoter) to respond to upstream factor stimulation . At least one inducible promoter, PHO5, was not affected by this deletion . The defect produced by YIID delta 2-57 was due to the deletion of several acidic residues present between residues 48 and 57 . The results show that the conserved carboxy terminal domain of YIID is sufficient for cell viability . However, an acidic region just amino terminal to the conserved domain is required for normal growth and transcription control in most yeast strains.

EMBO J, 1991 Jul, 10(7), 1699 - 709
The Saccharomyces cerevisiae STE14 gene encodes a methyltransferase that mediates C-terminal methylation of a-factor and RAS proteins; Hrycyna CA et al.; Post-translational processing of a distinct group of proteins and polypeptides, including the a-factor mating pheromone and RAS proteins of Saccharomyces cerevisiae, results in the formation of a modified C-terminal cysteine that is S-isoprenylated and alpha-methyl esterified . We have shown previously that a membrane-associated enzymatic activity in yeast can mediate in vitro methylation of an isoprenylated peptide substrate and that this methyltransferase activity is absent in ste14 mutants . We demonstrate here that STE14 is the structural gene for this enzyme by expression of its product as a fusion protein in Escherichia coli, an organism in which this activity is lacking . We also show that a-factor, RAS1 and RAS2 are physiological methyl-accepting substrates for this enzyme by demonstrating that these proteins are not methylated in a ste14 null mutant . It is notable that cells lacking STE14 methyltransferase activity exhibit no detectable impairment of RAS function or cell viability . However, we did observe a kinetic delay in the rate of RAS2 maturation and a slight decrease in the amount of membrane localized RAS2 . Thus, methylation does not appear to be essential for RAS2 maturation or localization, but the lack of methylation can have subtle effects on the efficiency of these processes.

Mol Cell Biol, 1991 Jul, 11(7), 3804 - 13
The HXT1 gene product of Saccharomyces cerevisiae is a new member of the family of hexose transporters; Lewis DA et al.; Two novel genes affecting hexose transport in the yeast Saccharomyces cerevisiae have been identified . The gene HXT1 (hexose transport), isolated from plasmid pSC7, was sequenced and found to encode a hydrophobic protein which is highly homologous to the large family of sugar transporter proteins from eucaryotes and procaryotes . Multicopy expression of the HXT1 gene restored high-affinity glucose transport to the snf3 mutant, which is deficient in a significant proportion of high-affinity glucose transport . HXT1 was unable to complement the snf3 growth defect in low copy number . The HXT1 protein was found to contain 12 putative membrane-spanning domains with a central hydrophilic domain and hydrophilic N- and C-terminal domains . The HXT1 protein is 69% identical to GAL2 and 66% identical to HXT2, and all three proteins were found to have a putative leucine zipper motif at a consensus location in membrane-spanning domain 2 . Disruption of the HXT1 gene resulted in loss of a portion of high-affinity glucose and mannose transport, and wild-type levels of transport required both the HXT1 and SNF3 genes . Unexpectedly, expression of beta-galactosidase activity by using a fusion of the lacZ gene to the HXT1 promoter in a multicopy plasmid was maximal during lag and early exponential phases of growth, decreasing approximately 100-fold upon further entry into exponential growth . Deletion analysis of pSC7 revealed the presence of another gene (called ORF2) capable of suppressing the snf3 null mutant phenotype by restoring high-affinity glucose transport and increased low-affinity transport.

Mol Cell Biol, 1991 Jul, 11(7), 3691 - 8
Protein synthesis requirements for nuclear division, cytokinesis, and cell separation in Saccharomyces cerevisiae; Burke DJ et al.; Protein synthesis inhibitors have often been used to identify regulatory steps in cell division . We used cell division cycle mutants of the yeast Saccharomyces cerevisiae and two chemical inhibitors of translation to investigate the requirements for protein synthesis for completing landmark events after the G1 phase of the cell cycle . We show, using cdc2, cdc6, cdc7, cdc8, cdc17 (38 degrees C), and cdc21 (also named tmp1) mutants, that cells arrested in S phase complete DNA synthesis but cannot complete nuclear division if protein synthesis is inhibited . In contrast, we show, using cdc16, cdc17 (36 degrees C), cdc20, cdc23, and nocodazole treatment, that cells that arrest in the G2 stage complete nuclear division in the absence of protein synthesis . Protein synthesis is required late in the cell cycle to complete cytokinesis and cell separation . These studies show that there are requirements for protein synthesis in the cell cycle, after G1, that are restricted to two discrete intervals.

Mol Cell Biol, 1991 Jul, 11(7), 3603 - 12
Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone; Marcus S et al.; We have undertaken total synthesis of the Saccharomyces cerevisiae a-factor (NH2-YIIKGVFWDPAC{S-farnesyl}-COOCH3) and several Cys-12 analogs to determine the significance of S-farnesylation and carboxy-terminal methyl esterification to the biological activity of this lipopeptide mating pheromone . Replacement of either the farnesyl group or the carboxy-terminal methyl ester by a hydrogen atom resulted in marked reduction but not total loss of bioactivity as measured by a variety of assays . Moreover, both the farnesyl and methyl ester groups could be replaced by other substituents to produce biologically active analogs . The bioactivity of a-factor decreased as the number of prenyl units on the cysteine sulfur decreased from three to one, and an a-factor analog having the S-farnesyl group replaced by an S-hexadecanyl group was more active than an S-methyl a-factor analog . Thus, with two types of modifications, a-factor activity increased as the S-alkyl group became bulkier and more hydrophobic . MATa cells having deletions of the a-factor structural genes (mfal1 mfa2 mutants) were capable of mating with either sst2 or wild-type MAT alpha cells in the presence of exogenous a-factor, indicating that it is not absolutely essential for MATa cells to actively produce a-factor in order to mate . Various a-factor analogs were found to partially restore mating to these strains as well, and their relative activities in the mating restoration assay were similar to their activities in the other assays used in this study . Mating was not restored by addition of exogenous a-factor to a cross of a wild-type MAT alpha strain and a MATaste6 mutant, indicating a role of the STE6 gene product in mating in addition to its secretion of a-factor.

Mol Cell Biol, 1991 Jul, 11(7), 3545 - 53
In vivo characterization of the Saccharomyces cerevisiae centromere DNA element I, a binding site for the helix-loop-helix protein CPF1; Niedenthal R et al.; The centromere DNA element I (CDEI) is an important component of Saccharomyces cerevisiae centromere DNA and carries the palindromic sequence CACRTG (R = purine) as a characteristic feature . In vivo, CDEI is bound by the helix-loop-helix protein CPF1 . This article describes the in vivo analysis of all single-base-pair substitutions in CDEI in the centromere of an artificial chromosome and demonstrates the importance of the palindromic sequence for faithful chromosome segregation, supporting the notion that CPF1 binds as a dimer to this binding site . Mutational analysis of two conserved base pairs on the left and two nonconserved base pairs on the right of the CDEI palindrome revealed that these are also relevant for mitotic CEN function . Symmetrical mutations in either half-site of the palindrome affect centromere activity to a different extent, indicating nonidentical sequence requirements for binding by the CPF1 homodimer . Analysis of double point mutations in CDEI and in CDEIII, an additional centromere element, indicate synergistic effects between the DNA-protein complexes at these sites.

Mol Cell Biol, 1991 Jul, 11(7), 3463 - 71
Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases; Schmid SR et al.; The eukaryotic translation initiation factor 4A (eIF-4A) possesses an in vitro helicase activity that allows the unwinding of double-stranded RNA . This activity is dependent on ATP hydrolysis and the presence of another translation initiation factor, eIF-4B . These two initiation factors are thought to unwind mRNA secondary structures in preparation for ribosome binding and initiation of translation . To further characterize the function of eIF-4A in cellular translation and its interaction with other elements of the translation machinery, we have isolated mutations in the TIF1 and TIF2 genes encoding eIF-4A in Saccharomyces cerevisiae . We show that three highly conserved domains of the D-E-A-D protein family, encoding eIF-4A and other RNA helicases, are essential for protein function . Only in rare cases could we make a conservative substitution without affecting cell growth . The mutants show a clear correlation between their growth and in vivo translation rates . One mutation that results in a temperature-sensitive phenotype reveals an immediate decrease in translation activity following a shift to the nonpermissive temperature . These in vivo results confirm previous in vitro data demonstrating an absolute dependence of translation on the TIF1 and TIF2 gene products.

Mol Microbiol, 1991 Jul, 5(7), 1615 - 20
The repair of double-strand breaks and S1 nuclease-sensitive sites can be monitored chromosome-specifically in Saccharomyces cerevisiae using pulse-field gel electrophoresis; Geigl EM et al.; Repair under non-growth conditions of DNA double-stranded breaks (DSBs) and S1 nuclease-sensitive sites (SSSs; e.g . DNA damage which is processed by in vitro treatment with S1 nuclease to DSBs) induced by {60Co}-gamma-rays (200 Gy; anoxic conditions) was monitored in a diploid repair-competent strain of Saccharomyces cerevisiae . We used pulsed-field gel electrophoresis (PFGE), which allows the separation of chromosome-sized yeast DNA molecules, to determine the number of DSBs and SSSs in individual chromosome species of yeast . Our results indicate that SSSs which have been regarded as clusters of base damage in opposite DNA strands are repaired efficiently in a repair-proficient diploid strain of yeast . The time course of SSS repair is comparable to the one of DSB repair, indicating similarities in the molecular mechanism . Both types of repair kinetics are different for different chromosome species.

J Biochem (Tokyo), 1991 Jul, 110(1), 103 - 10
Secretory expression of the human serum albumin gene in the yeast, Saccharomyces cerevisiae; Okabayashi K et al.; We have fused a cDNA gene encoding mature human serum albumin (HSA) to several secretory leader-encoding sequences . The hybrid genes were cloned into an episomal vector under the control of several yeast promoters and then introduced into yeast cells . The GAL1 promoter in combination with either the native HSA pre-sequence or a modified HSA pre-sequence gave the highest production of immunoreactive HSA, 90 mg/liter being reached in a shake flask culture . The invertase pre-sequence, the mating factor alpha 1 prepro-sequence, and the modified HSA pre-sequence directed accurate processing . In contrast, the chicken lysozyme pre-sequence and the native HSA pre-sequence directed incorrect processing . Episomal vectors were unstable within the host cells under non-selective culture conditions . To improve the plasmid stability, the hybrid genes were incorporated into an integrative vector . Transformants carrying multicopies of the plasmid integrated at the LEU2 locus stably secreted HSA . The highest yield of 65 mg/liter in a shake flask culture was obtained with the combination of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter and the modified HSA pre-sequence . By constructing transformed strains containing multicopies of plasmids integrated at both the chromosome LEU2 and HIS4 loci, we have obtained a stable strain that continuously secretes as much as 85 mg HSA per liter of culture medium.

J Bacteriol, 1991 Jul, 173(14), 4533 - 6
A dominant interfering mutation (CYR3) of the Saccharomyces cerevisiae RAS2 gene; Morishita T et al.; The dominant cyclic AMP-requiring mutation CYR3 had been previously reported as a mutation in the regulatory subunit of cyclic AMP-dependent protein kinase . However, recharacterization revealed that the CYR3 mutation was a nonconditional dominant lethal mutation and was a missense allele of RAS2 which results from the substitution of aspartic acid for glycine at amino acid 22.

Mol Cell Biol, 1991 Jul, 11(7), 3773 - 9
AAR1/TUP1 protein, with a structure similar to that of the beta subunit of G proteins, is required for a1-alpha 2 and alpha 2 repression in cell type control of Saccharomyces cerevisiae; Mukai Y et al.; We have cloned a DNA fragment complementing the aar1 mutation defective in the a1-alpha 2 repression of the alpha 1 cistron and haploid-specific genes in Saccharomyces cerevisiae . Nucleotide sequence and mapping data indicated that the AAR1 gene is identical with TUP1, which is allelic to the SFL2, FLK1, CYC9, UMR7, AMM1, and AER2 genes, whose mutations are known to confer a variety of phenotypes, such as thymidine uptake, flocculation, insensitivity to glucose repression, a defect in UV-induced mutagenesis, and a defect in ARS plasmid maintenance . The TUP1/AER2 protein is known to have significant similarity with the beta subunits of G proteins in the C-terminal half, in two glutamine-rich domains in the N-terminal half, and in a central region rich in serine and threonine residues . Disruption of the chromosomal AAR1 gene in alpha and a/alpha cells conferred the nonmating phenotype, and the a/alpha diploids could not sporulate . The AAR1/TUP1 gene is transcribed into a 2.5-kb mRNA independently of the mating-type information of the cell . These observations and mRNA analysis of cell-type-specific genes indicated that the AAR1/TUP1 protein is also indispensable for a1-alpha 2 repression of RME1 and for alpha 2 repression of a-specific genes.

Mol Cell Biol, 1991 Jul, 11(7), 3537 - 44
Mutational analysis of CDC42Sc, a Saccharomyces cerevisiae gene that encodes a putative GTP-binding protein involved in the control of cell polarity; Ziman M et al.; The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity . We have analyzed the effects of three CDC42 mutations (Gly to Val-12, Gln to Leu-61, and Asp to Ala-118) in the putative GTP-binding and hydrolysis domains and one mutation (Cys to Ser-188) in the putative isoprenylation site . The first three mutations resulted in either a dominant-lethal or dose-dependent dominant-lethal phenotype when present on plasmids in haploid cdc42-1ts or wild-type strains . Both wild-type and cdc42-1ts cells carrying plasmids (pGAL) with either the CDC42Val-12 or CDC42Leu-61 alleles under the control of a GAL promoter were arrested with a novel phenotype of large cells with elongated or multiple buds . Cells carrying pGAL-CDC42Ala-118 were arrested as large, round, unbudded cells reminiscent of cdc42-1ts arrested cells . The different phenotype of the CDC42Ala-118 mutant versus the CDC42Val-12 and CDC42Leu-61 mutants was unexpected since the phenotypes of all three analogous ras mutants were similar to each other . This suggests that aspects of the biochemical properties of the Cdc42 protein differ from those of the Ras protein . The cdc42Ser-188 mutant gene was incapable of complementing the cdc42-1ts mutation and was recessive to both wild-type and cdc42-1ts . In double-mutant alleles, the cdc42Ser-188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.

Yeast, 1991 Jul, 7(5), 489 - 94
Incorporation of unsaturated fatty acids by Saccharomyces cerevisiae: conservation of fatty-acyl saturation in phosphatidylinositol; Pilkington BJ et al.; Saccharomyces cerevisiae was grown anaerobically in media supplemented with myristoleic 14:1(9c), palmitoleic 16:1(9c), oleic 18:1(9c), linoleic 18:2(9,12c), gamma-linolenic 18:3(9,12,15c) or eicosenoic 20:1(11c) acid . Cells from exponential-phase cultures contained approximately the same proportions of the major phospholipid classes, namely phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine, the greatest differences being detected in cells grown in the presence of 14:1(9c) or 20:1(11c) acids . The extent to which phospholipids from cells were enriched with residues of the exogenously supplied acid varied from 52% in cells grown in the presence of 14:1(9c) acid to 13% in cells grown in media supplemented with 20:1(11c) acid . Analysis of the fatty-acyl composition of the four major phospholipid classes revealed that the degree of unsaturation varied considerably in three of the classes, while phosphatidylinositol conserved a high degree of saturation . The possible significance of the latter finding in relation to the physiological role of phosphatidylinositol in the plasma membrane is discussed.

Yeast, 1991 Jul, 7(5), 475 - 7
The YDp plasmids: a uniform set of vectors bearing versatile gene disruption cassettes for Saccharomyces cerevisiae; Berben G et al.; The YDp plasmids (Yeast Disruption plasmids) are pUC9 vectors bearing a set of yeast gene disruption cassettes, all uniform in structure and differing only in the selectable marker used (HIS3, LEU2, LYS2, TRP1 or URA3) . The markers, surrounded by translational termination codons, are embedded in the slightly modified sequence of the pUC9 multiple cloning sites.

Yeast, 1991 Jul, 7(5), 455 - 61
Glucan structure in a fragile mutant of Saccharomyces cerevisiae; Blagoeva J et al.; The phenotype of VY1160 fragile Saccharomyces cerevisiae mutant is characterized by cell lysis upon transfer to hypotonic solutions and increased permeability of cells growing in osmotically stabilized media . Two mutations, srb1 and ts1, have been identified in VY1160 cells and previous studies have shown that the increased permeability is due to the ts1 mutation which causes a shortening of mannan side-chains . Here we report that the srb1 mutation, which is the genetic determinant of cell lysis, is responsible for quantitative and structural changes of glucans . Experiments with isogenic single mutation strains, genetic studies coupled with quantitative measurements of glucan content per cell, and methylation analysis of glucans provide evidence that srb1 mutation leads to i) formation of mechanically unstable cell wall network made of insoluble glucan fibrils which are shorter and contain beta(1-6) inter-residue linkages and ii) insufficient filling of the space between the fibrils due to a shortage of the alkali-soluble glucan . Although growing exponentially in osmotically stabilized media, the srb1 cells cannot resist an osmotic shock and, hence, burst immediately.

Appl Environ Microbiol, 1991 Jul, 57(7), 2052 - 6
The secretion leader of Mucor pusillus rennin which possesses an artificial Lys-Arg sequence directs the secretion of mature human growth hormone by Saccharomyces cerevisiae; Hiramatsu R et al.; The prepro-peptide of fungal aspartic proteinase, Mucor pusillus rennin, is useful as a secretion leader for efficient secretion of human growth hormone (HGH) from Saccharomyces cerevisiae . For secretion by yeast cells of HGH with the same NH2 terminus as native HGH, an artificial Lys-Arg linker, which is one of the potential KEX2 recognition sequences, was introduced at the junction between the M . pusillus rennin secretion leader and mature HGH . The HGH directed by this construction was the same size as native HGH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino acid sequencing of its NH2 terminus revealed that the secretion leader peptide was removed correctly at the COOH-terminal side of the Lys-Arg linker . On the other hand, when the same plasmid was expressed in a kex2 mutant strain, unprocessed HGH of a higher molecular weight was secreted, indicating that no proteolytic cleavage at the Lys-Arg site occurred . These results clearly showed that the leader peptide with the Lys-Arg linker was recognized and specifically cleaved by the yeast KEX2 protease . The mature HGH purified from yeast culture medium was indistinguishable from native HGH in biological activity, determined by the adipocyte conversion assay, and in secondary structure, determined by circular dichroism spectroscopy.

Anal Biochem, 1991 Jul, 196(1), 156 - 60
Analysis of mutant tRNA gene transcripts in vivo in Saccharomyces cerevisiae by abortive primer extension; Wilhelm ML et al.; When the primer extension of a synthetic oligonucleotide hybridized to a complementary region of RNA is made in the presence of only three deoxyribonucleosides triphosphates, elongation of the primer stops as soon as the missing nucleotide is needed . This abortive primer extension assay has been adapted to analyse tRNA gene transcripts and has two main advantages . First it is specific and allows the identification of particular tRNA gene products in an homologous system provided the gene bears a point mutation . Second, it is highly sensitive and can be used to complement and confirm results of Northern blot hybridization . This assay should be a useful tool in the further in vivo study of the transcription and processing of particular tRNA genes in the homologous system . In this report the expression of wild-type and mutant yeast Sup4- tyrosine inserting suppressor gene was studied.

Mol Gen Genet, 1991 Jul, 227(3), 473 - 80
The Escherichia coli recA gene increases resistance of the yeast Saccharomyces cerevisiae to ionizing and ultraviolet radiation; Brozmanova J et al.; The Escherichia coli recA protein coding region was ligated into an extrachromosomally replicating yeast expression vector downstream of the yeast alcohol dehydrogenase promoter region to produce plasmid pADHrecA . Transformation of the wild-type yeast strains YNN-27 and 7799-4B, as well as the recombination-deficient rad52-1 C5-6 mutant, with this shuttle plasmid resulted in the expression of the bacterial 38 kDa RecA protein in exponential phase cells . The wild-type YNN27 and 7799-4B transformants expressing the bacterial recA gene showed increased resistance to the toxic effects of both ionizing and ultraviolet radiation . RecA moderately stimulated the UV-induced mutagenic response of 7799-4B cells . Transformation of the rad52-1 mutant with plasmid pADHrecA did not result in the complementation of sensitivity to ionizing radiation . Thus, the RecA protein endows the yeast cells with additional activities, which were shown to be error-prone and dependent on the RAD52 gene.

Mol Gen Genet, 1991 Jul, 227(3), 452 - 7
CDC7 protein kinase activity is required for mitosis and meiosis in Saccharomyces cerevisiae; Buck V et al.; The product of the CDC7 gene of Saccharomyces cerevisiae has multiple cellular functions, being needed for the initiation of DNA synthesis during mitosis as well as for synaptonemal complex formation and commitment to recombination during meiosis . The CDC7 protein has protein kinase activity and contains the conserved residues characteristic of the protein kinase catalytic domain . To determine which of the cellular functions of CDC7 require this protein kinase activity, we have mutated some of the conserved residues within the CDC7 catalytic domain and have examined the ability of the mutant proteins to support mitosis and meiosis . The results indicate that the protein kinase activity of the CDC7 gene product is essential for its function in both mitosis and meiosis and that this activity is potentially regulated by phosphorylation of the CDC7 protein.

Curr Genet, 1991 Jul, 20(1-2), 25 - 31
The TSM1 gene of Saccharomyces cerevisiae overlaps the MAT locus; Ray BL et al.; We have cloned the region from MAT to THR4 on chromosome III of Saccharomyces cerevisiae . Although the region is only 15 kb, the two loci are genetically separated by 22 cM . This is in sharp contrast to the very low level of recombination (2 cM in 22 kb) that is observed in the adjacent CRY1-MAT interval, and suggests that there may be a "hot spot" for recombination in the MAT-THR4 region . The DNA sequence of the first 4.4 kb distal to MAT reveals an open reading frame that we have identified as the essential gene, TSM1 . Surprisingly, the TSM1 open reading frame of 1,410 amino acids extends into the MAT locus, such that the 3'-end of the MAT alpha 1 transcript ends 15 bp from the 3'-end of the TSM1 open reading frame.

Res Virol, 1991 Jul-Aug, 142(4), 297 - 302
Expression of cauliflower mosaic virus gene I in Saccharomyces cerevisiae; Kirchherr D et al.; Cauliflower mosaic virus (CaMV) gene I encodes a 40-kDa protein, P1, which is thought to be involved in the cell-to-cell movement of the virus . In order to investigate its functioning, P1 was expressed in Saccharomyces cerevisiae transformed by an expression vector containing CaMV gene I . When produced in yeast, PI was 40 kDa in size and not N-glycosylated.

Genetika, 1991 Jul, 27(7), 1143 - 51
{Effect of ploidy and the status of the MAT locus in Saccharomyces cerevisiae yeast cells on the mitotic stability of episomal plasmids and the level of expression of the HBsAg gene of hepatitis B virus}; Voropaeva LA; The frequency of the YEp13, pEF91, YEp13 + HBs, pNMVG3954 plasmids' elimination in a series of isogenic strains n . 2n and 3n was studied . Plasmid stability and the level of expression of the gene of hepatitis B surface antigen (HBsAg) increased in polyploid transformants as a result of increase in plasmid copy number . Heterozygotes MATa/MAT differed from the homozygotes in higher stability of YEp13, pEF91, and YEp13 + HBs plasmids having the same quantity of the HBsAg antigen . The appearance of negative properties--destabilization of episomal plasmids or decrease in synthesis of heterologous protein have been discovered in certain diploid cell . These results point to limitations in constructing a polyploid producer on the basis of the similar type of genome.

Mol Gen Mikrobiol Virusol, 1991 Jul, (7), 8 - 12
{Saccharomyces cerevisiae mutants with increased mitotic stability of plasmids isolated during long-term culturing of transformants under selective conditions}; Voropaeva LA; Analysis of the plasmid containing clones of transformants of Saccharomyces cerevisiae in the population cultivated under the nonselective conditions has shown their vast heterogeneity in the mitotic stability of the plasmids Yep13 and Yep91 . For instance, the clones were obtained with the different types of the hereditary plasmid stabilization: integration with the chromosome and genotype or plasmid mutations increasing the vector copy number . The increased expression level was registered in the mutants for the heterologous genes AmpR of Escherichia coli and HBsAg of hepatitis B . The clones were found with the considerably varying mitotic stability of the plasmids of the modification type variability, the latter expressing the fluctuations of plasmid copy number at the change of cultivation conditions.

Mikrobiologiia, 1991 Jul-Aug, 60(4), 680 - 5
{Influence of plant extracts on the metabolic activity of the yeast Saccharomyces cerevisiae 39}; Krallish IL et al.; Addition to the nutrient medium of the extracts of camomile, nettle, mint, or Saint-John's-wort to a concentration of 0.5-4% was established to activate the yeast growth . The maximum effect was recorded on the addition of the extracts to the poor "water" medium . The extracts of camomile and Saint-John's-wort were shown to inhibit the alcohol dehydrogenase activity of the yeast cells, whereas the extracts of nettle and mint increased the activity of this enzyme by 62-70%.

Mol Microbiol, 1991 Jul, 5(7), 1769 - 77
Deletion of the COX7 gene in Saccharomyces cerevisiae reveals a role for cytochrome c oxidase subunit VII in assembly of remaining subunits; Calder KM et al.; Cytochrome c oxidase from Saccharomyces cerevisiae is composed of nine subunits . Subunits I, II and III are products of mitochondrial genes, while subunits IV, V, VI, VII, VIIa and VIII are products of nuclear genes . To investigate the role of cytochrome c oxidase subunit VII in biogenesis or functioning of the active enzyme complex, a null mutation in the COX7 gene, which encodes subunit VII, was generated, and the resulting cox7 mutant strain was characterized . The strain lacked cytochrome c oxidase activity and haem a/a3 spectra . The strain also lacked subunit VII, which should not be synthesized owing to the nature of the cox7 mutation generated in this strain . The amounts of remaining cytochrome c oxidase subunits in the cox7 mutant were examined . Accumulation of subunit I, which is the product of the mitochondrial COX1 gene, was found to be decreased relative to other mitochondrial translation products . Results of pulse-chase analysis of mitochondrial translation products are consistent with either a decreased rate of translation of COX1 mRNA or a very rapid rate of degradation of nascent subunit I . The synthesis, stability or mitochondrial localization of the remaining nuclear-encoded cytochrome c oxidase subunits were not substantially affected by the absence of subunit VII . To investigate whether assembly of any of the remaining cytochrome c oxidase subunits is impaired in the mutant strain, the association of the mitochondrial-encoded subunits I, II and III with the nuclear-encoded subunit IV was investigated.(ABSTRACT TRUNCATED AT 250 WORDS)

Curr Genet, 1991 Jul, 20(1-2), 39 - 44
Evidence that an endo-exonuclease controlled by the NUC2 gene functions in the induction of 'petite' mutations in Saccharomyces cerevisiae; Chow TY et al.; Defects in the RAD52 gene of the yeast Saccharomyces cerevisiae reduce the levels of the NUC2 endo-exonuclease by approximately 90% compared to the levels in wild-type strains . To examine the potential role of this nuclease in the induction of mitochondrial 'petite' mutations, congenic RAD52 and rad52-1 haploids were subjected to treatment with ethidium bromide, a well-known inducer of these mutations . The rad52 strain showed a much higher resistance to ethidium bromide-induced petite formation than the corresponding wild-type strain . Two approaches were taken to confirm that this finding reflected the nuclease deficiency, and not some other effect attributable to the rad52-1 mutation . First, a multicopy plasmid (YEp213-10) carrying NUC2 was transformed into a RAD52 strain . This resulted in an increased fraction of spontaneous petite mutations relative to that seen for the same strain without the plasmid and sensitized the strain carrying the plasmid to petite induction by ethidium bromide treatment . Second, a strain having a nuc2 allele that encodes a temperature-sensitive nuclease was treated with ethidium bromide at the restrictive and permissive temperatures . Petite induction was reduced under restrictive conditions . Enzyme assays revealed that the RAD52 (YEp213-10) strain had the highest level of antibody-precipitable NUC2 endo-exonuclease whereas the nuc2 and rad52 mutants had the lowest levels . Furthermore, addition of ethidium bromide to the reaction mixture stimulated the activity of the nuclease on double-stranded DNA . Petite induction by antifolate-mediated thymine nucleotide depletion was also inhibited by inactivation of RAD52 indicating that the effect of reduced NUC2 endo-exonuclease was not restricted to ethidium bromide treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1991 Jul 1, 88(13), 5724 - 8
Glucose induces cAMP-independent growth-related changes in stationary-phase cells of Saccharomyces cerevisiae; Granot D et al.; Nutrients play a critical role in the decision to initiate a new cell cycle . Addition of nutrients to arrested cells such as stationary-phase cells and spores induces them to begin growth . We have analyzed the nutrients required to induce early cellular events in yeast . When stationary-phase cells or spores are incubated in the presence of only glucose, morphological and physiological changes characteristic of mitotically growing cells are induced and, in the absence of additional nutrients to support growth, the cells rapidly lose viability . Preincubation of stationary-phase cells in the presence of glucose decreases the time required to reach bud emergence upon the subsequent addition of rich medium . These processes are specifically induced by D-glucose and not by other components such as nitrogen source or L-glucose . The glucose-induced events are independent of the adenylate cyclase pathway, since strains with a temperature-sensitive mutation in either the adenylate cyclase gene (CDC35) or its regulator (CDC25) undergo glucose-induced cellular changes when incubated at the restrictive temperature . We suggest that glucose triggers events in the induction of a new mitotic cell cycle and that these events are either prior to the adenylate cyclase pathway or are in an alternative pathway.

Biotechnol Prog, 1991 Jul-Aug, 7(4), 291 - 8
Bromodeoxyuridine labeling and flow cytometric identification of replicating Saccharomyces cerevisiae cells: lengths of cell cycle phases and population variability at specific cell cycle positions; Dien BS et al.; An immunofluorescent staining procedure has been developed to identify, with flow cytometry, replicating cells of Saccharomyces cerevisiae after incorporation of bromodeoxyuridine (BrdUrd) into the DNA . Incorporation of BrdUrd is made possible by using yeast strains with a cloned thymidine kinase gene from the herpes simplex virus . An exposure time of 4 min to BrdUrd results in detectable labeling of the DNA . The BrdUrd/DNA double staining procedure has been optimized and the flow cytometry measurements yield histograms comparable to data typically obtained for mammalian cells . On the basis of the accurate assessment of cell fractions in individual cell cycle phases of the asynchronously growing cell population, the average duration of the cell cycle phases has been evaluated . For a population doubling time of 100 min it was found that cells spend in average 41 min in the replicating phase and 24 min in the G2+M cell cycle period . Assuming that mother cells immediately reenter the S phase after cell division, daughter cells spend 65 min in the G1 cell cycle phase . Together with the single cell fluorescence parameters, the forward-angle light scattering intensity (FALS) has been determined as an indicator of cell size . Comparing different temporal positions within the cell cycle, the determined FALS distributions show the lowest variability at the beginning of the S phase . The developed procedure in combination with multiparameter flow cytometry should be useful for studying the kinetics and regulation of the budding yeast cell cycle.

J Biol Chem, 1991 Jun 25, 266(18), 12021 - 8
Cloning and functional analysis of the ubiquitin-specific protease gene UBP1 of Saccharomyces cerevisiae; Tobias JW et al.; In eukaryotes, both natural and engineered fusions of ubiquitin to itself or other proteins are cleaved by processing proteases after the last (Gly76) residue of ubiquitin . Using the method of sib selection, and taking advantage of the fact that bacteria such as Escherichia coli lack ubiquitin-specific enzymes, we have cloned a gene, named UBP1, of the yeast Saccharomyces cerevisiae that encodes a ubiquitin-specific processing protease . With the exception of polyubiquitin, the UBP1 protease cleaves at the carboxyl terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their size or the presence of an amino-terminal ubiquitin extension . These properties of UBP1 distinguish it from the previously cloned yeast protease YUH1, which deubiquitinates relatively short ubiquitin fusions but is virtually inactive with longer fusions such as ubiquitin-beta-galactosidase . The amino acid sequence of the 809-residue UBP1 lacks significant similarities to other known proteins, including the 236-residue YUH1 protease . Null ubp1 mutants are viable, and retain the ability to deubiquitinate ubiquitin-beta-galactosidase, indicating that the family of ubiquitin-specific proteases in yeast is not limited to UBP1 and YUH1.

J Biol Chem, 1991 Jun 25, 266(18), 11986 - 92
An enzyme from Saccharomyces cerevisiae uses NAD+ to transfer the splice junction 2'-phosphate from ligated tRNA to an acceptor molecule; McCraith SM et al.; An enzyme from Saccharomyces cerevisiae which removes the splice junction 2'-phosphate from ligated tRNA appears to require NAD+ . This two-component enzyme has been previously implicated in tRNA splicing because of its specificity for substrates bearing an internal 2'-phosphate and because of the absence of other observed proteins that can efficiently catalyze the same activity after fractionation of the extracts . We show here that component I of this enzyme is heat-stable, chromatographs as a small molecule, can be substituted efficiently by NAD+, and comigrates with NAD+ on a reversed-phase column . Dephosphorylation of ligated tRNA in the presence of component I or NAD+ is accompanied by stoichiometric transfer of the splice junction 2'-phosphate to an unidentified acceptor molecule.

Eur J Biochem, 1991 Jun 15, 198(3), 705 - 11
Ribosome-bound EF-1 alpha-like protein of yeast Saccharomyces cerevisiae; Didichenko SA et al.; The SUP2 (SUP35) omnipotent suppressor gene encodes the EF-1 alpha-like polypeptide, intimately involved in the control of translational ambiguity in the yeast Saccharomyces cerevisiae . The present study is devoted to the immunological characterization of the Sup2 protein . The SUP2 gene was fused to the Escherichia coli lacZ gene and a polyclonal antibody against the corresponding Sup2--beta-galactosidase hybrid protein was obtained . This antibody identified a 79-kDa protein that was absent in those cells where the SUP2 gene was disrupted, and an abundance of this protein was observed in cells overexpressing the SUP2 gene . The localization of this protein was studied in subcellular fractionation experiments . The SUP2 gene product proved to be uniformly distributed throughout ribosome-enriched samples, i.e . free polysomes, crude microsomes and rough endoplasmic reticulum . It was not found in the cytoplasm and smooth endoplasmic reticulum . The SUP2-encoded protein was fully ribosome associated and less abundant than the ribosomal protein L3 . Also, in a sucrose gradient, Sup2 preferentially cosedimented with the 40S ribosomal subunit, but not with the 60S subunit . The functional significance of this association is discussed.

J Biol Chem, 1991 Jun 15, 266(17), 11184 - 91
Isolation and characterization of two distinct myo-inositol transporter genes of Saccharomyces cerevisiae; Nikawa J et al.; By the complementation of a yeast mutant defective in myo-inositol transport (Nikawa, J., Nagumo, T., and Yamashita, S . (1982) J . Bacteriol . 150, 441-446), we isolated two myo-inositol transporter genes, ITR1 and ITR2, from a yeast gene library . The ITR1 and ITR2 genes contained long open reading frames capable of encoding 584 and 612 amino acids with calculated relative molecular masses of 63,605 and 67,041, respectively . The sequence similarity between the ITR1 and ITR2 products was extremely high, suggesting that the two genes arose from a common ancestor . Both gene products show significant sequence homology with a superfamily of sugar transporters, including human HepG2 hepatoma/erythrocyte glucose transporter and Escherichia coli xylose transporter . Hydropathy analysis indicated that the ITR1 and ITR2 products are both hydrophobic and contain 12 putative membrane-spanning regions . Thus, yeast myo-inositol transporters could be classified into the sugar transporter superfamily . Gene disruption and tetrad analysis showed that yeast cells contain two separate myoinositol transporters . The ITR1 product was the major transporter and the ITR2 product the minor one in cells grown in minimum medium containing glucose . Northern blot analysis showed that ITR1 mRNA was much more abundant than ITR2 mRNA . The previously isolated myo-inositol transport mutant was determined to be defective in ITR1.

J Biol Chem, 1991 Jun 15, 266(17), 10925 - 32
Identification of a glycogen synthase phosphatase from yeast Saccharomyces cerevisiae as protein phosphatase 2A; Peng ZY et al.; A glycogen synthase phosphatase was purified from the yeast Saccharomyces cerevisiae . The purified yeast phosphatase displayed one major protein band which coincided with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis . This phosphatase had a molecular mass of about 160,000 Da determined by gel filtration and was comprised of three subunits, termed A, B, and C . The subunit molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 60,000 (A), 53,000 (B), and 37,000 (C), indicating that this yeast glycogen synthase phosphatase is a heterotrimer . On ethanol treatment, the enzyme was dissociated to an active species with a molecular weight of 37,000 estimated by gel filtration . The yeast phosphatase dephosphorylated yeast glycogen synthase, rabbit muscle glycogen phosphorylase, casein, and the alpha subunit of rabbit muscle phosphorylase kinase, was not sensitive to heat-stable protein phosphatase inhibitor 2, and was inhibited 90% by 1 nM okadaic acid . Dephosphorylation of glycogen synthase, phosphorylase, and phosphorylase kinase by this yeast enzyme could be stimulated by histone H1 and polylysines . Divalent cations (Mg2+ and Ca2+) and chelators (EDTA and EGTA) had no effect on dephosphorylation of glycogen synthase or phosphorylase while Mn2+ stimulated enzyme activity by approximately 50% . The specific activity and kinetics for phosphorylase resembled those of mammalian phosphatase 2A . An antibody against a synthetic peptide corresponding to the carboxyl terminus of the catalytic subunit of rabbit skeletal muscle protein phosphatase 2A reacted with subunit C of purified yeast phosphatase on immunoblots, whereas the analogous peptide antibody against phosphatase 1 did not . These data show that this yeast glycogen synthase phosphatase has structural and catalytic similarity to protein phosphatase 2A found in mammalian tissues.

Mol Gen Genet, 1991 Jun, 227(2), 197 - 204
Relative contributions of MCM1 and STE12 to transcriptional activation of a- and alpha-specific genes from Saccharomyces cerevisiae; Hwang-Shum JJ et al.; We have examined the relative contributions of MCM1 and STE12 to the transcription of the a-specific STE2 gene by using a 367 bp fragment from the STE2 5'-noncoding region to drive expression of a reporter lacZ gene . Mutation of the MCM1 binding site destroyed MCM1.alpha 2-mediated repression in alpha cells and dramatically reduced expression in a cells . The residual expression was highly stimulated by exposure of cells to pheromone . Likewise, the loss of STE12 function reduced lacZ expression driven by the wild-type STE2 fragment . In the absence of both MCM1 and STE12 functions, no residual expression was observed . Thus, the STE2 fragment appears to contain two distinct upstream activation sequences (UASs), one that is responsible for the majority of expression in cells not stimulated by pheromone, and one that is responsible for increased expression upon pheromone stimulation . In further support of this idea, a chemically synthesized version of the STE2 MCM1 binding site had UAS activity, but the activity was neither stimulated by pheromone nor reduced in ste12 mutants . Although transcription of alpha-specific genes also requires both MCM1 and STE12, these genes differ from a-specific genes in that they have a single, MCM1-dependent UAS system . The activity of the minimal 26 bp UAS from the alpha-specific STE3 gene was both stimulated by pheromone and reduced in ste12 mutants . These data suggest that at alpha-specific genes STE12 and MCM1 exert their effects through a single UAS.

Mol Cell Biol, 1991 Jun, 11(6), 2952 - 61
Pheromone response elements are necessary and sufficient for basal and pheromone-induced transcription of the FUS1 gene of Saccharomyces cerevisiae; Hagen DC et al.; The FUS1 gene of Saccharomyces cerevisiae is transcribed in a and alpha cells, not in a/alpha diploids, and its transcription increases dramatically when haploid cells are exposed to the appropriate mating pheromone . In addition, FUS1 transcription is absolutely dependent on STE4, STE5, STE7, STE11, and STE12, genes thought to encode components of the pheromone response pathway . We now have determined that the pheromone response element (PRE), which occurs in four copies within the FUS1 upstream region, functions as the FUS1 upstream activation sequence (UAS) and is responsible for all known aspects of FUS1 regulation . In particular, deletion of 55 bp that includes the PREs abolished all transcription, and a 139-bp fragment that includes the PREs conferred FUS1-like expression to a CYC1-lacZ reporter gene . Moreover, three or four copies of a synthetic PRE closely mimicked the activity conferred by the 139-bp fragment, and even a single copy of PRE conferred a trace of activity that was haploid specific and pheromone inducible . In the FUS1 promoter context, four copies of the synthetic PRE inserted at the site of the 55-bp deletion restored full FUS1 transcription . Sequences upstream and downstream from the PRE cluster were important for maximal PRE-directed expression but, by themselves, did not have UAS activity . Other yeast genes with PREs, e.g., STE2 and BAR1, are more modestly inducible and have additional UAS elements contributing to the overall activity . In the FUS1 promoter, the PREs apparently act alone to confer activity that is highly stimulated by pheromone.

J Biol Chem, 1991 Jun 5, 266(16), 10093 - 8
Reconstitution of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex in vitro . The 86-kDa subunit facilitates but is not required for complex formation; Brooke RG et al.; The immunoaffinity-purified subunits of the yeast DNA primase-DNA polymerase protein complex and subunit-specific monoclonal antibodies were used to explore the structural relationships of the subunits in the complex . The reconstituted four-subunit complex (180-, 86-, 58-, and 49-kDa polypeptides) behaved as a single species, exhibiting a Stokes radius of 80 A and a sedimentation coefficient of 8.9 S . The calculated molecular weight of the reconstituted complex is 312,000 . We infer that the stoichiometry of the complex is one of each subunit per complex . The complex has a prolate ellipsoid shape with an axial ratio of approximately 16 . When the 180-kDa and DNA primase subunits were recombined in the absence of the 86-kDa subunit, a physical complex formed, as judged by immunoprecipitation of DNA primase activity and polypeptides with an anti-180-kDa monoclonal antibody . While the 86-kDa subunit readily forms a physical complex with the 180-kDa DNA polymerase catalytic subunit, we have not detected a complex containing 86-kDa and the DNA primase subcomplex (49- and 58-kDa subunits) . The 86-kDa subunit was not required for DNA primase-DNA polymerase complex formation; the 180-kDa subunit and DNA primase heterodimer directly interact . However, the presence of the 86-kDa subunit increased the rate at which the DNA primase and 180-kDa polypeptides formed a complex and increased the total fraction of DNA primase activity that was associated with DNA polymerase activity . The observations demonstrate that the DNA primase p49.p58 heterodimer and the DNA polymerase p86.p180 heterodimer interact via the 180-kDa subunit . The four-subunit reconstituted complex was sufficient to catalyze the DNA chain extension coupled to RNA primer synthesis on a single-stranded DNA template, as previously observed in the conventionally purified complex isolated from wild type cells.

Proc Natl Acad Sci U S A, 1991 Jun 1, 88(11), 4601 - 5
DPB2, the gene encoding DNA polymerase II subunit B, is required for chromosome replication in Saccharomyces cerevisiae; Araki H et al.; The Saccharomyces cerevisiae DNA polymerase II holoenzyme consists of five polypeptides . The largest is the catalytic subunit, whose gene (POL2) has been cloned and sequenced . Herein we describe the cloning and sequencing of DPB2, the gene for the second largest subunit of DNA polymerase II, and the isolation of temperature-sensitive dpb2 mutations . The DNA sequence revealed an open reading frame encoding a protein of Mr 79,461 and lacking significant sequence similarity to any protein in data bases . Disruption of DPB2 was lethal for the cell and the temperature-sensitive dpb2-1 mutant was partially defective in DNA synthesis at the restrictive temperature, indicating that the DPB2 protein is required for normal yeast chromosomal replication . Furthermore, the DNA polymerase II complex was difficult to obtain from dpb2-1 mutant cells, suggesting that a stable DNA polymerase II complex requires DPB2 and is essential for chromosomal replication . The DPB2 transcript periodically fluctuated during the cell cycle and, like those of other genes encoding DNA replication proteins, peaked at the G1/S phase boundary.

Eur J Biochem, 1991 Jun 1, 198(2), 485 - 91
Functionally important regions of glucose-6-phosphate dehydrogenase defined by the Saccharomyces cerevisiae enzyme and its differences from the mammalian and insect forms; Persson B et al.; The primary structure of Saccharomyces cerevisiae glucose-6-phosphate dehydrogenase has been determined . It consists of 503 amino acid residues, with an acetyl-blocked N-terminus . The structure shows equally extensive differences from the corresponding mammalian and fruit fly enzymes (52% residues non-identical) . Residues conserved in all the forms constitute about 40% of the structures and include two histidines . One of these (His200 in the numbering of the rat enzyme) occurs in a 10-residue conserved segment, including the reactive Lys204, probably related to substrate binding . Two segments with conserved Gly-Xaa-Xaa-Gly-Xaa-Xaa-Gly/Ala pattern constitute possibilities for the coenzyme-binding site . One is N-terminally located (positions 37-43) with two conserved arginine residues nearby (positions 56 and 71), of interest for phosphate binding . The other (positions 241-247) is in a middle region, with many residue identities, containing the conserved residues Arg256 and His264.

Mol Cell Biol, 1991 Jun, 11(6), 3307 - 16
The CYC8 and TUP1 proteins involved in glucose repression in Saccharomyces cerevisiae are associated in a protein complex; Williams FE et al.; Mutations of yeast CYC8 or TUP1 genes greatly reduce the degree of glucose repression of many genes and affect other regulatory pathways, including mating type . The predicted CYC8 protein contains 10 copies of the 34-amino-acid tetratricopeptide repeat unit, and the predicted TUP1 protein has six repeated regions found in the beta subunit of heterotrimeric G proteins . The absence of DNA-binding motifs and the presence of these repeated domains suggest that the CYC8 and TUP1 proteins function via protein-protein interaction with transcriptional regulatory proteins . We raised polyclonal antibodies against TrpE-CYC8 and TrpE-TUP1 fusion proteins expressed in Escherichia coli . The CYC8 and TUP1 proteins from yeast cells were detected as closely spaced doublets on Western immunoblots of sodium dodecyl sulfate-polyacrylamide gels . Western blots of nondenaturing gels revealed that both proteins are associated in a high-molecular-weight complex with an apparent size of 1,200 kDa . In extracts from delta cyc8 strains, the size of the complex is reduced to 830 kDa . The CYC8 and TUP1 proteins were coprecipitated by either antiserum, further supporting the conclusion that they are associated with each other . The complex could be reconstituted in vitro by mixing extracts from strains with complementary mutations in the CYC8 and TUP1 genes.

Mol Cell Biol, 1991 Jun, 11(6), 3229 - 38
The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter; Bun-Ya M et al.; The PHO84 gene specifies Pi-transport in Saccharomyces cerevisiae . A DNA fragment bearing the PHO84 gene was cloned by its ability to complement constitutive synthesis of repressible acid phosphatase of pho84 mutant cells . Its nucleotide sequence predicted a protein of 596 amino acids with a sequence homologous to that of a superfamily of sugar transporters . Hydropathy analysis suggested that the secondary structure of the PHO84 protein consists of two blocks of six transmembrane domains separated by 74 amino acid residues . The cloned PH084 DNA restored the Pi transport activity of pho84 mutant cells . The PHO84 transcription was regulated by Pi like those of the PHO5, PHO8, and PHO81 genes . A PHO84-lacZ fusion gene produced beta-galactosidase activity under the regulation of Pi, and the activity was suggested to be bound to a membrane fraction . Gene disruption of PHO84 was not lethal . By comparison of nucleotide sequences and by tetrad analysis with GAL80 as a standard, the PHO84 locus was mapped at a site beside the TUB3 locus on the left arm of chromosome XIII.

Mol Cell Biol, 1991 Jun, 11(6), 3203 - 16
GCD2, a translational repressor of the GCN4 gene, has a general function in the initiation of protein synthesis in Saccharomyces cerevisiae; Foiani M et al.; The GCD2 protein is a translational repressor of GCN4, the transcriptional activator of multiple amino acid biosynthetic genes in Saccharomyces cerevisiae . We present evidence that GCD2 has a general function in the initiation of protein synthesis in addition to its gene-specific role in translational control of GCN4 expression . Two temperature-sensitive lethal gcd2 mutations result in sensitivity to inhibitors of protein synthesis at the permissive temperature, and the gcd2-503 mutation leads to reduced incorporation of labeled leucine into total protein following a shift to the restrictive temperature of 36 degrees C . The gcd2-503 mutation also results in polysome runoff, accumulation of inactive 80S ribosomal couples, and accumulation of at least one of the subunits of the general translation initiation factor 2 (eIF-2 alpha) in 43S-48S particles following a shift to the restrictive temperature . The gcd2-502 mutation causes accumulation of 40S subunits in polysomes, known as halfmers, that are indicative of reduced 40S-60S subunit joining at the initiation codon . These phenotypes suggest that GCD2 functions in the translation initiation pathway at a step following the binding of eIF-2.GTP.Met-tRNA(iMet) to 40S ribosomal subunits . consistent with this hypothesis, we found that inhibiting 40S-60S subunit joining by deleting one copy (RPL16B) of the duplicated gene encoding the 60S ribosomal protein L16 qualitatively mimics the phenotype of gcd2 mutations in causing derepression of GCN4 expression under nonstarvation conditions . However, deletion of RPL16B also prevents efficient derepression of GCN4 under starvation conditions, indicating that lowering the concentration of 60S subunits and reducing GCD2 function affect translation initiation at GCN4 in different ways . This distinction is in accord with a recently proposed model for GCN4 translational control in which ribosomal reinitiation at short upstream open reading frames in the leader of GCN4 mRNA is suppressed under amino acid starvation conditions to allow for increased reinitiation at the GCN4 start codon.

Mol Cell Biol, 1991 Jun, 11(6), 3060 - 9
Different classes of polyadenylation sites in the yeast Saccharomyces cerevisiae; Irniger S et al.; This report provides an analysis of the function of polyadenylation sites from six different genes of the yeast Saccharomyces cerevisiae . These sites were tested for their ability to turn off read-through transcription into the URA3 gene in vivo when inserted into an ACT-URA3 fusion gene . The 3' ends of all polyadenylation sites inserted into the test system in their natural configuration are identical to the 3' ends of the chromosomal genes . We identified two classes of polyadenylation sites: (i) efficient sites (originating from the genes GCN4 and PHO5) that were functional in a strict orientation-dependent manner and (ii) bidirectional sites (derived from ARO4, TRP1, and TRP4) that had a distinctly reduced efficiency . The ADH1 polyadenylation site was efficient and bidirectional and was shown to be a combination of two polyadenylation sites of two convergently transcribed genes . Sequence comparison revealed that all efficient unidirectional polyadenylation sites contain the sequence TTTTTAT, whereas all bidirectional sites have the tripartite sequence TAG...TA (T)GT...TTT . Both sequence elements have previously been proposed to be involved in 3' end formation . Site-directed point mutagenesis of the TTTTTAT sequence had no effect, whereas mutations within the tripartite sequence caused a reduced efficiency for 3' end formation . The tripartite sequence alone, however, is not sufficient for 3' end formation, but it might be part of a signal sequence in the bidirectional class of yeast polyadenylation sites . Our findings support the assumption that there are at least two different mechanisms with different sequence elements directing 3' end formation in yeast.

Mol Cell Biol, 1991 Jun, 11(6), 3027 - 36
Ribosome association of GCN2 protein kinase, a translational activator of the GCN4 gene of Saccharomyces cerevisiae; Ramirez M et al.; The GCN4 gene of the yeast Saccharomyces cerevisiae encodes a transcriptional activator of amino acid biosynthetic genes that is regulated at the translational level according to the availability of amino acids . GCN2 is a protein kinase required for increased translation of GCN4 mRNA in amino acid-starved cells . Centrifugation of cell extracts in sucrose gradients indicated that GCN2 comigrates with ribosomal subunits and polysomes . The fraction of GCN2 cosedimenting with polysomes was reduced under conditions in which polysomes were dissociated, suggesting that GCN2 is physically bound to these structures . When the association of 40S and 60S subunits was prevented by omitting Mg2+ from the gradient, almost all of the GCN2 comigrated with 60S ribosomal subunits, and it remained bound to these particles during gel electrophoresis under nondenaturing conditions . GCN2 could be dissociated from 60S subunits by 0.5 M KCl, suggesting that it is loosely associated with ribosomes rather than being an integral ribosomal protein . Accumulation of GCN2 on free 43S-48S particles and 60S subunits occurred during polysome runoff in vitro and under conditions of reduced growth rate in vivo . These observations, plus the fact that GCN2 shows preferential association with free ribosomal subunits during exponential growth, suggest that GCN2 interacts with ribosomes during the translation initiation cycle . The extreme carboxyl-terminal segment of GCN2 is essential for its interaction with ribosomes . These sequences are also required for the ability of GCN2 to stimulate GCN4 translation in vivo, leading us to propose that ribosome association by GCN2 is important for its access to substrates in the translational machinery or for detecting uncharged tRNA in amino acid-starved cells.

Mol Gen Mikrobiol Virusol, 1991 Jun, (6), 16 - 8
{Localization of acid phosphatase in Saccharomyces cerevisiae and its export into culture media depends on the type of the N-terminal signal peptide}; Lupashin VV et al.; The aim of this work was to study the character of intracellular distribution and efficiency of yeast acid phosphatase export depending on the type of the N-terminal signal peptide used . A number of plasmids carrying the acid phosphatase genes with different signal peptides sequences was constructed . The main site of the enzyme accumulation for the variant containing its own acid phosphatase signal peptide was the periplasm . Approximately the same pattern was observed when the hybrid signal peptide consisting of acid phosphatase signal peptide and alpha-factor preprosegment tandem was used . Unlike the above-mentioned systems the strain carrying acid phosphatase under the control of alpha factor preprosegment was able to export the enzyme into the culture medium . The experiments have shown the possibility of changing the final localization of secretory proteins by replacing the N-terminal signal peptide.

J Gen Microbiol, 1991 Jun, 137 ( Pt 6), 1263 - 70
Cell surface structures in osmotically fragile mutants of Saccharomyces cerevisiae; Kopecka M et al.; Mutants of Saccharomyces cerevisiae characterized by osmotic fragility showed a marked fibrillar structure on the inner wall surface when studied by two electron microscopic techniques, i.e . freeze-etching of whole native cells and metal shadowing of isolated cell walls . The walls of the mutant cells were more permeable to macromolecules than were those of the wild-type parental strain . The synthesis and assembly of (1----3)-beta-D-glucan wall microfibrils studied in protoplasts of mutant cells were not impaired . It is suggested that the osmotic fragility of the mutant cells is related to the deficiency of the wall structure as a consequence of the srb1 mutation affecting biogenesis of the amorphous (glucan) component.

Int J Pept Protein Res, 1991 Jun, 37(6), 476 - 86
Synthesis of S-alkyl and C-terminal analogs of the Saccharomyces cerevisiae a-factor . Influence of temperature on the stability of Fmoc and OFm groups toward HF; Xue CB et al.; The a-mating factor of Saccharomyces cerevisiae Tyr-Ile-Ile-Lys-Gly-Val-Phe-Trp-Asp-Pro-Ala-Cys(farnesyl)OCH3, and 10 analogs modified at the cysteine side chain and/or the terminal carboxyl were synthesized using a combination of solid phase and solution phase methodologies . The strategy of synthesis involved the condensation of an amine terminal protected decapeptide with a carboxyl terminal S-alkylated dipeptide ester or amide using benzotriazol-l-yloxy-tris(methylamino)-phosphonium hexafluorophosphate as the coupling agent . The protected decapeptide was assembled on a PAM-resin using 9-fluorenylmethoxycarbonyl (Fmoc) for the protection of the Tyr alpha-amine and Lys epsilon-amine and 9-fluorenylmethyl ester (OFm) for the protection of the Asp beta-carboxyl . Premature loss of the OFm group from the HF cleavage was observed at 0-2 degrees, whereas no loss occurred when the cleavage reaction was conducted at -5 degrees . In contrast to these results, the OFm group in Asp(OFm) was partially removed by HF at -5 degrees and was completely stable to HF only at -20 degrees . The S-alkylated dipeptide esters were prepared, in yields from 64% to 88%, via thioalkylation of amine protected or unprotected dipeptide esters using potassium fluoride dihydrate as the base . The use of a tertiary amine as the base of thiohexadecanylation resulted in low reactivity.

Mol Cell Biol, 1991 Jun, 11(6), 3105 - 14
Translation initiation factor 5A and its hypusine modification are essential for cell viability in the yeast Saccharomyces cerevisiae; Schnier J et al.; Translation intitiation factor eIF-5A (previously named eIF-4D) is a highly conserved protein that promotes formation of the first peptide bond . One of its lysine residues is modified by spermidine to form hypusine, a posttranslational modification unique to eIF-5A . To elucidate the function of eIF-5A and determine the role of its hypusine modification, the cDNA encoding human eIF-5A was used as a probe to identify and clone the corresponding genes from the yeast Saccharomyces cerevisiae . Two genes named TIF51A and TIF51B were cloned and sequenced . The two yeast proteins are closely related, sharing 90% sequence identity, and each is ca . 63% identical to the human protein . The purified protein expressed from the TIF51A gene substitutes for HeLa eIF-5A in the mammalian methionyl-puromycin synthesis assay . Strains lacking the A form of eIF-5A, constructed by disruption of TIF51A with LEU2, grow slowly, whereas strains lacking the B form, in which HIS3 was used to disrupt TIF51B, show no growth rate phenotype . However, strains with both TIF51A and TIF51B disrupted are not viable, indicating that eIF-5a is essential for cell growth in yeast cells . Northern (RNA) blot analysis shows two mRNA species, a larger mRNA (0.9 kb) transcribed from TIF51A and a smaller mRNA (0.8 kb) encoded by TIF51B . Under the aerobic growth conditions of this study, the 0.8-kb TIF51B transcript is not detected in the wild-type strain and is expressed only when TIF51A is disrupted . The TIF51A gene was altered by site-directed mutagenesis at the site of hypusination by changing the Lys codon to that for Arg, thereby producing a stable protein that retains the positive charge but is not modified to the hypusine derivative . The plasmid shuffle technique was used to replace the wild-type gene with the mutant form, resulting in failure of the yeast cells to grow . This result indicates that hypusine very likely is required for the vital in vivo function of eIF-5A and suggests a precise, essential role for the polyamine spermidine in cell metabolism.

Curr Genet, 1991 Jun, 19(6), 429 - 33
The SNQ3 gene of Saccharomyces cerevisiae confers hyper-resistance to several functionally unrelated chemicals; Hertle K et al.; A multi-copy plasmid containing the SNQ3 gene confers hyper-resistance to 4-nitroquinoline-N-oxide (4NQO), Trenimon, MNNG, cycloheximide, and to sulfometuron methyl in yeast transformants . Restriction analysis, subcloning, and DNA sequencing revealed an open reading frame of 1,950 bp on the SNQ3-containing insert DNA . Gene disruption and transplacement into chromosomal DNA yielded 4NQO-sensitive null mutants which were also more sensitive than the wild-type to Trenimon, cycloheximide, sulfometuron methyl, and MNNG . Hydropathic analysis showed that the SNQ3-encoded protein is most likely not membrane-bound, while the codon bias index points to low expression of the gene.

Virology, 1991 Jun, 182(2), 513 - 21
Expression of human papillomavirus proteins in yeast Saccharomyces cerevisiae; Carter JJ et al.; The L1 and L2 proteins of human papillomavirus (HPV) types 1, 6, and 16 and the E6 and E7 proteins of HPV 16 were expressed in Saccharomyces cerevisiae . The yeast expressed proteins were readily detected by immune blotting and were generally intact . The HPV 1 L1 and L2 proteins expressed in yeast were indistinguishable from the major and minor capsid proteins purified from HPV 1 virions as judged by gel electrophoresis and immunoblotting . The HPV 6 and HPV 16 L2 proteins and HPV 16 E7 proteins were secreted from yeast by fusion to the yeast pre-pro-alpha-factor leader sequence . Following secretion of the HPV 16 E7 protein a rapid method of purification was developed . The yeast expressed proteins were used as antigen targets to study the human immune response in Western blot assay, ELISA, and immune precipitation . One human serum reacted with intact, but not denatured HPV 16 L2 proteins, suggesting that the yeast expressed proteins will be useful to detect antibodies reactive with conformational epitopes.

Mol Microbiol, 1991 Jun, 5(6), 1539 - 48
A new system for amplifying 2 microns plasmid copy number in Saccharomyces cerevisiae; Unternahrer S et al.; The yeast 2 microns plasmid is found in the nucleus of almost all Saccharomyces cerevisiae strains . Its replication is very similar to that of chromosomal DNA . Although the plasmid does not encode essential genes it is stably maintained in the yeast population and exhibits only a small, though detectable, loss rate . This stability is achieved by a plasmid-encoded copy-number control system which ensures constant plasmid levels . For the investigation of 2 microns replication, a yeast strain that is absolutely dependent on this plasmid was constructed . This was achieved by disruption of the chromosomal CDC9 gene, coding for DNA ligase and providing this essential gene on a 2 microns-derived plasmid . This plasmid is absolutely stable under all growth conditions tested . Using the temperature-sensitive mutant allele cdc9-1 we have developed an artificial control system which allows one to change the copy number of 2 microns-derived plasmids solely by changing the incubation temperature.

J Bacteriol, 1991 Jun, 173(11), 3605 - 8
Further characterizations of bleomycin-sensitive (blm) mutants of Saccharomyces cerevisiae with implications for a radiomimetic model; Moore CW; Direct selection for 12 mutations (blm) conferring hypersensitivities to lethal effects of bleomycins in Saccharomyces cerevisiae resulted in mutants exhibiting cross-hypersensitivity to ionizing radiation and hydrogen peroxide . Remaining mutations did not confer cross-hypersensitivity to radiation . All blm mutations were recessive, except codominant blm3-1, and were assigned to seven complementation groups.

Cell Struct Funct, 1991 Jun, 16(3), 195 - 201
Estrogen delays entry of the yeast Saccharomyces cerevisiae into meiosis; Hasegawa S et al.; Estrogen has been suggested to influence the cell cycle of haploid yeast cells in the early G1 phase of mitosis, its effect possibly being mediated by control of the level of cAMP (TANAKA, S . et al . (1989) . Cell, 57: 675-681) . Therefore, we were interested in whether estrogen also affects the meiotic phase of diploid yeast cells . Accordingly, we measured the amounts of adenylate cyclase mRNA and intracellular cAMP, the proportions of dividing cells and 4n cells and the doubling times of diploid yeast cells during the presporulation stage in the presence and absence of estrogen . The amount of adenylate cyclase mRNA was found to decrease rapidly within 24 hours after inoculation of cells onto sporulation-promoting plates (YPA plates) . The cAMP level of these cells also decreased rapidly . Mitotic cell division continued for 18 hours after cell inoculation, but about 24 hours after inoculation, the amount of cAMP per cell had decreased to a minimum and the cells began to enter meiosis . By contrast, when the cells were inoculated onto YPA plates in the presence of estrogen, their intracellular cAMP and adenylate cyclase mRNA levels became higher than those in control cultures without estrogen and cell division continued for 24 hours . But after 30 hours their intracellular cAMP level decreased to a minimum and they began to enter meiosis . These results show that estrogen delayed the entry of diploid yeast cells into meiosis on sporulation-promoting plates and suggest that its effect may be mediated by control of the level of cAMP.

Bioessays, 1991 Jun, 13(6), 295 - 302
Eukaryotic DNA repair: glimpses through the yeast Saccharomyces cerevisiae; Friedberg EC; Eukaryotic cells are able to mount several genetically complex cellular responses to DNA damage . The yeast Saccharomyces cerevisiae is a genetically well characterized organism that is also amenable to molecular and biochemical studies . Hence, this organism has provided a useful and informative model for dissecting the biochemistry and molecular biology of DNA repair in eukaryotes.

Protein Eng, 1991 Jun, 4(5), 575 - 8
The function of the Saccharomyces cerevisiae iso-1-cytochrome c gene is independent of the codon at invariant residue Phe82 when the gene is present on a low-copy-number vector; Hilgen SE et al.; Phe82 is the most studied invariant residue of cytochrome c . However, the physiological relevance of amino acid substitutions at this position is unclear because previous studies were either performed in vitro (i.e . using purified protein) or in yeast where the gene for the protein is present on a multi-copy vector . Multi-copy vectors yield a level of cytochrome c in yeast that is greater than the wild-type level . Oligodeoxyribonucleotide-directed mutagenesis was used to change the codon for Phe82 to that of the other 19 naturally occurring amino acids as well as the amber stop codon . The alleles are present on a yeast shuttle phagemid containing the CEN6 gene which ensures a vector copy number of one to two in yeast . All the missense alleles support growth under conditions requiring a functional iso-1-cytochrome c . However the F82C, F82P, and F82R variants grow at a significantly lower rate . After selection for function, phagemids were rescued from the transformants and the identity of the mutation verified . It is concluded that all 20 amino acids are capable of supporting function . Reasons for the evolutionary invariance of Phe82 are discussed.

Cell Regul, 1991 Jun, 2(6), 439 - 52
Genetic fine-structural analysis of the Saccharomyces cerevisiae alpha-pheromone receptor; Konopka JB et al.; The alpha-pheromone receptor encoded by the STE2 gene contains seven potential transmembrane domains . Its ability to transduce the pheromone signal is thought to require the action of a G protein . As an initial step toward defining the structural features of the receptor required for its activity, we examined the phenotypic consequences of linker insertion mutations (12 bp) at 10 different sites in the STE2 gene . Three mutant classes, which correspond to three different regions of the receptor protein, were observed . 1) The two mutants affecting the C-terminal region (C-terminal mutants) were essentially wild type for mating efficiency, pheromone binding, and pheromone sensitivity . 2) The three mutants in the N-terminus mated with reduced efficiency, showed reduced pheromone binding capacity, and were partially defective in pheromone induction of agglutinin production and cell division arrest . Increased gene dosage of these N-terminal alleles suppressed their mutant phenotypes, whereas the sst2-1 mutation, which blocks adaptation to pheromone, did not result in suppression . Thus, the N-terminal mutants were apparently limited by receptor production, but not by the adaptation function SST2 . 3) The five mutants in the central region containing the seven transmembrane segments (central mutants) were completely defective for mating and did not respond to pheromone, but could be distinguished by their ability to bind pheromone . Inserts in or near transmembrane domains 2 and 4 blocked pheromone binding, whereas inserts into transmembrane domains 1, 5, and 6 retained partial pheromone binding activity even though they failed to transduce a signal . The central mutants were not suppressed by increased gene dosage, and one mutant (ste2-/101) was partially suppressed by sst2-1 . Furthermore, the central core mutants were also distinguished from one another in that three of the five mutants were able to partially complement the temperature sensitivity of ste2-3.

Nucleic Acids Res, 1991 May 25, 19(10), 2603 - 8
Yeast ribosomal proteins: XII . YS11 of Saccharomyces cerevisiae is a homologue to E . coli S4 according to the gene analysis; Mizuta K et al.; We isolated and sequenced a gene, YS11A, encoding ribosomal protein YS11 of Saccharomyces cerevisiae . YS11A is one of two functional copies of the YS11 gene, located on chromosome XVI and transcribed in a lower amount than the other copy which is located on chromosome II . The disruption of YS11A has no effect on the growth of yeast . The 5'-flanking region contains a similar sequence to consensus UASrpg and the T-rich region . The open reading frame is interrupted with an intron located near the 5'-end . The predicted amino acid sequence reveals that yeast YS11 is a homologue to E . coli S4, one of the ram proteins, three chloroplast S4s and others out of the ribosomal protein sequences currently available.

J Biol Chem, 1991 May 25, 266(15), 9732 - 9
Kinetic and structural evidence for a sequential ordered Bi Bi mechanism of catalysis by Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase; Rudnick DA et al.; The mechanism of catalysis of Escherichia coli-derived Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase (NMT) has been characterized . Previous studies indicated that a high affinity reaction intermediate forms between NMT and myristoyl-CoA in the absence of a peptide substrate . This complex has been further characterized using S-(2-oxo)pentadecyl-CoA, a nonhydrolyzable myristoyl-CoA analog . Binding studies involving this analog, as well as myristoylpeptide and CoA, have indicated that the CoA moiety of the acyl substrate is retained in the acyl-NMT complex prior to peptide addition . These structural data, along with kinetic studies of myristoylpeptide and CoA product inhibition, indicate that the mechanism of catalysis of NMT is ordered Bi Bi, with myristoyl-CoA binding to NMT occurring prior to peptide binding and CoA release taking place before release of acyl peptide . Further analyses of the interactions between NMT, acyl peptide, and CoA demonstrate that NMT is able to deacylate a myristoylpeptide in the presence of CoA.

Eur J Biochem, 1991 May 23, 198(1), 217 - 22
Production and purification of recombinant human interleukin-6 secreted by the yeast Saccharomyces cerevisiae; Guisez Y et al.; The coding region of the human interleukin-6 (hIL6) gene was fused to the prepro secretion signal of the alpha-mating factor gene in several yeast host strains . It was found that the KEX-2 protease was unable to cleave the prepro-Lys-Arg-Pro-IL6 sequence, but that unspecific cleavage of the precursor protein had occurred . The prepro-Lys-Arg-Ala-Pro-IL6 sequence, however, was correctly recognized and cleaved by the KEX-2 protease, and IL6 was efficiently secreted into the culture medium . The N-terminal Ala-Pro peptide was removed during processing by wild-type yeast strains, but was retained in a ste13 mutant . IL6 as well as the aberrant proteins were not glycosylated . The transformed cells could secrete up to 30 micrograms/ml IL6 . The protein was purified from the medium to homogeneity by ion-exchange chromatography and gel filtration, and had a specific activity of about 2 x 10(8) IU/mg in a proliferation assay.

Gene, 1991 May 15, 101(1), 149 - 52
Characterization of PDR4, a Saccharomyces cerevisiae gene that confers pleiotropic drug resistance in high-copy number: identity with YAP1, encoding a transcriptional activator {corrected}; Hussain M et al.; PDR4 is a gene that confers pleiotropic drug resistance (pdr) to the yeast Saccharomyces cerevisiae when present in high copy number {Leppert et al., Genetics 125 (1990) 13-20} . Transposon insertion mutations had identified the active region of the gene as a 3.7-kb SalI-EcoRI restriction fragment of the 8-kb cloned fragment . We have confirmed this by showing that this fragment is sufficient to confer pdr, and have sequenced its entire 3761 bp . It contains a single complete open reading frame (ORF) extending from nucleotide (nt) position 1631-3580, coding for a protein of 650 amino acids (aa) . A 2.7-kb fragment containing this ORF is also sufficient to confer pdr . The aa sequence contains no recognizable homologies or consensus sequences, so it is a novel protein of unknown function . It is apparently soluble, since no transmembrane-type sequences were predicted . A second, partial ORF was also found, on the opposite strand, extending from nt position 774 to past the SalI site, which is apparently unrelated to pdr.

Proc Natl Acad Sci U S A, 1991 May 15, 88(10), 4448 - 52
Protein geranylgeranyltransferase of Saccharomyces cerevisiae is specific for Cys-Xaa-Xaa-Leu motif proteins and requires the CDC43 gene product but not the DPR1 gene product; Finegold AA et al.; Protein prenylation occurs by modification of proteins with one of at least two isoprenoids, the farnesyl group and the geranylgeranyl group . Protein farnesyltransferases have been identified, but no such enzyme has been identified for geranylgeranylation . We report the identification of an activity in crude soluble yeast extracts that catalyzes the transfer of a geranylgeranyl moiety from geranylgeranyl pyrophosphate to proteins having the C-terminal sequence Cys-Ile-Ile-Leu or Cys-Val-Leu-Leu but not to a similar protein ending with Cys-Ile-Ile-Ser . This activity is dependent upon the CDC43/CAL1 gene, which is involved in budding and the control of cell polarity, but does not require the DPR1/RAM1 gene, which is known to be required for the farnesylation of Ras proteins . These results indicate that the protein geranylgeranyltransferase activity is distinct from the protein farnesyltransferase activity and that its specificity depends in part on the extreme C-terminal leucine in the protein to be prenylated.

Proc Natl Acad Sci U S A, 1991 May 15, 88(10), 4089 - 93
The multifunctional protein OBF1 is phosphorylated at serine and threonine residues in Saccharomyces cerevisiae; Francesconi SC et al.; We have purified a DNA replication enhancer-binding protein, OBF1, from yeast cells grown in a medium containing 32P-labeled orthophosphate . The purified 32P-labeled protein comigrated on polyacrylamide gels with OBF1 bands identified by immunoblotting with anti-OBF1 antibodies . Furthermore, trypsin treatment of the 32P-labeled OBF1 revealed several phosphorylated peptides, suggesting that OBF1 is multiply phosphorylated in vivo . Incubation of phosphorylated peptides with calf intestinal phosphatase liberated the radiolabel as free phosphate, indicating a phosphoester linkage . Acid hydrolysis of the tryptic peptides revealed 32P-label label comigrating with phosphoserine; some of it, however, was also identified as phosphothreonine . Using anti-OBF1 antibodies, we cloned the OBF1 gene from a lambda gt11 yeast expression library . The DNA sequence of the isolated gene and its over-expression in yeast indicated that OBF1 is identical to ABF-1 and BAF1 proteins, believed to have a role in transcriptional repression and activation . Therefore, we suggest that OBF1 is a multifunctional protein, acting in transcription and replication, and that these activities are regulated by phosphorylation.

J Biol Chem, 1991 May 15, 266(14), 9023 - 31
Purification of a protein histidine kinase from the yeast Saccharomyces cerevisiae . The first member of this class of protein kinases; Huang JM et al.; An enzyme of molecular weight 32,000 comprising a single subunit has been isolated from whole cell extracts of the yeast Saccharomyces cerevisiae . In vitro, the enzyme transfers the gamma phosphate of ATP to a protein substrate, histone H4, to produce an alkali-stable phosphorylation . Modification of the substrate histidine with diethylpyrocarbonate prevented phosphorylation . Phosphoamino acid analysis of the phosphorylated substrate showed the presence of 1-phosphohistidine . Hence, the isolated enzyme is a protein histidine kinase . A novel assay for acid-labile alkali-stable protein phosphorylation was used in the purification of the kinase activity to a final specific activity of 2,700 nmol/15 min/mg . The purified enzyme phosphorylates specifically histidine 75 in histone H4 and does not phosphorylate histidine 18 nor histidine residues in any other core histone . Steady state kinetic data are consistent with an ordered sequential reaction with Km values for Mg-ATP and histone H4 of 60 and 17 microM, respectively . The protein histidine kinase requires a divalent cation such as Mg2+, Co2+, or Mn2+ but will not use Ca2+, Zn2+, Cu2+, Fe2+, spermine, or spermidine . This is the first purification of an enzyme that catalyzes N-linked phosphorylation in proteins.

J Biol Chem, 1991 May 15, 266(14), 8741 - 6
Purification and characterization of poly(A) polymerase from Saccharomyces cerevisiae; Lingner J et al.; Poly(A) polymerase was purified 22,000-fold to homogeneity from a whole cell extract of Saccharomyces cerevisiae with a yield of 22% . The enzyme is a monomeric polypeptide with a denatured molecular weight of 63,000 . Incorporation of labeled ATP into acid-precipitable material by the purified enzyme proceeds faster with manganese than with magnesium ions . Various RNA homopolymers as well as Escherichia coli tRNA or rRNA can serve as primers . An RNA that terminates at the natural poly(A) site of the CYC1 gene is not more efficiently elongated than several nonspecific substrates, indicating the requirement for additional factors to provide specificity . Elongation of the primer is distributive . Covering of a poly(A) primer with poly(A)-binding protein reduces the enzyme's activity more than 10-fold.

Gene, 1991 May 15, 101(1), 89 - 96
Cloning and characterisation of the Saccharomyces cerevisiae glycerol-3-phosphate dehydrogenase (GUT2) promoter; Sleep D et al.; The Saccharomyces cerevisiae glycerol-3-phosphate dehydrogenase (GUT2) promoter and part of the protein-coding region have been isolated on a 6.3-kb genomic DNA fragment . Nucleotide sequence analysis shows that the promoter has many structural features in common with yeast glycolytic enzyme promoters . Chromosomal mapping indicates that this genomic fragment is located on chromosome XII . The GUT2 promoter has been used to construct a recombinant human albumin (reHA) secretion vector; yeast transformed with this vector secrete reHA into the culture supernatant.

Mol Cell Biochem, 1991 May 29-Jun 12, 104(1-2), 163 - 8
Termination of transcription of ribosomal RNA in Saccharomyces cerevisiae; Johnson SP et al.; We have attempted to determine the site of termination of transcription of ribosomal RNA in the yeast, Saccharomyces cerevisiae . While a quantitative description of the termination sites of RNA polymerase I is not possible using presently available methods, we conclude that transcription of most molecules continues through a large portion of the adjacent enhancer region . There are two potential termination sites within the enhancer, one of which is near the binding site of the DNA binding protein REBI . In addition there is an apparently fail-safe termination site approximately 950 nucleotides beyond the 3' end of 35S ribosomal precursor RNA . Processing at the end of 35S RNA influences the choice of downstream termination site . Conversely downstream sequences also influence the site of termination.

Biochemistry, 1991 May 7, 30(18), 4612 - 9
Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization; Macheroux P et al.; Glycolate oxidase from spinach has been expressed in Saccharomyces cerevisiae . The active enzyme was purified to near-homogeneity (purification factor approximately 1400-fold) by means of hydroxyapatite and anion-exchange chromatography . The purified glycolate oxidase is nonfluorescent and has absorbance peaks at 448 (epsilon = 9200 M-1 cm-1) and 346 nm in 0.1 M phosphate buffer, pH 8.3 . The large bathochromic shift of the near-UV band indicates that the N(3) position is deprotonated at pH 8.3 . A pH titration revealed that the pK of the N(3) is shifted from 10.3 in free flavin to 6.4 in glycolate oxidase . Glycolate oxidase is competitively inhibited by oxalate with a Kd of 0.24 mM at 4 degrees C in 0.1 M phosphate buffer, pH 8.3 . Three pieces of evidence demonstrate that glycolate oxidase stabilizes a negative charge at the N(1)-C(2 = O) locus: the enzyme forms a tight sulfite complex with a Kd of 2.7 x 10(-7) M and stabilizes the anionic flavosemiquinone and the benzoquinoid form of 8-mercapto-FMN . Steady-state analysis at pH 8.3, 4 degrees C, yielded a Km = 1 x 10(-3) M for glycolate and Km = 2.1 x 10(-4) M for oxygen . The turnover number has been determined to be 20 s-1 . Stopped-flow studies of the reductive (k = 25 s-1) and oxidative (k = 8.5 x 10(4) M-1 s-1) half-reactions have identified the reduction of glycolate oxidase to be the rate-limiting step.

J Biol Chem, 1991 May 5, 266(13), 8255 - 61
Separation and characterization of two alpha 1,2-mannosyltransferase activities from Saccharomyces cerevisiae; Lewis MS et al.; Two GDP-mannose-dependent mannosyltransferase activities (designated M1MT-I and M2MT-I) from Triton X-100 extracts of Saccharomyces cerevisiae mnn1 microsomes were separated by concanavalin A lectin chromatography and partially purified . The two transferases were distinguished by differences in concanavalin A affinity and in carbohydrate acceptor specificity . Analyses of the reaction products indicate that both enzymes are alpha 1,2-mannosyltransferases . M1MT-I utilizes mannose or methyl-alpha-mannoside as acceptor while M2MT-I catalyzes the transfer of mannose from GDP-mannose to unsubstituted nonreducing alpha 1,6-linked mannose residues in the acceptor molecule . M2MT-I activity correlates with the presence of a single alpha 1,2-linked mannose residue at the nonreducing terminus of mnn2mnn9 and mnn2mnn10 outer chain oligosaccharides, and the enzyme may be involved in regulating outer chain elongation.

Biochim Biophys Acta, 1991 May 2, 1089(1), 47 - 53
Posttranscriptional regulation of the expression of MET2 gene of Saccharomyces cerevisiae; Forlani N et al.; The first step of the specific pathway for methionine biosynthesis in the yeast Saccharomyces cerevisiae is catalyzed by the enzyme L-homoserine-O-acetyltransferase (HSTase) (EC 2.3.1.31), encoded by the MET2 gene . In order to ascertain whether there is a posttranscriptional control on the MET2 gene expression, as suggested by previous results on the expression of the cloned gene, systems for high inducible expression of MET2 gene were constructed . In these constructs the MET2 gene was cloned in yeast expression vectors under the control of an inducible yeast GAL promoter element so that the MET2 was transcribed at very high levels under induced conditions . Measurements of the specific mRNA levels showed a strong stimulation of MET2 gene transcription in yeast transformants grown on galactose as carbon source, corresponding to 50-100-fold the repressed conditions, while only a 2-fold increase of the enzymatic activity was observed . In addition, no evidence of a strong induced polypeptide of appropriate size on two dimensional gel electrophoresis was obtained . To understand the functional role of the non-coding 5' region of MET2 mRNA, we performed either a partial and a complete deletion of the 5' leader sequence, but even with these constructs an elevated mRNA level was not associated to a marked increase of the HSTase activity . These data support the idea of a posttranscriptional regulation of MET2 gene expression and show that the untranslated region of the specific mRNA is not involved in this regulatory mechanism.

Genetics, 1991 May, 128(1), 79 - 88
Isolation of mutants defective in early steps of meiotic recombination in the yeast Saccharomyces cerevisiae; Malone RE et al.; Using a selection based upon the ability of early Rec- mutations (e.g., rad50) to rescue the meiotic lethality of a rad52 spo13 strain, we have isolated 177 mutants . Analysis of 56 of these has generated alleles of the known Rec genes SPO11, ME14 and MER1, as well as defining five new genes: REC102, REC104, REC107, REC113 and REC114 . Mutations in all of the new genes appear to specifically affect meiosis; they do not have any detectable mitotic phenotype . Mutations in REC102, REC104 and REC107 reduce meiotic recombination several hundred fold . No alleles of RED1 or HOP1 were isolated, consistent with the proposal that these genes may be primarily involved with chromosome pairing and not exchange.

Mutat Res, 1991 May, 254(3), 247 - 53
A similar defect in UV-induced mutagenesis conferred by the rad6 and rad18 mutations of Saccharomyces cerevisiae; Cassier-Chauvat C et al.; The single rad6 and rad18 yeast mutants share a number of physiological and biochemical properties related to DNA repair, suggesting that they affect closely related steps . However, it has been reported that UV-induced mutagenesis is considerably more depressed in rad6 than it is in rad18 cells . In an attempt to better understand the role of these genes, a genetic system believed to differentiate between targeted and untargeted events was used . The data are interpreted to mean that both mutations prevent the occurrence of targeted events, as if they prevent error-prone replication in front of pyrimidine dimers . The number of non-targeted mutants per survivor in each mutant was increased by UV irradiation . This may correspond to a stimulation of the error-prone replication.

Mol Gen Genet, 1991 May, 227(1), 149 - 54
A PMR2 tandem repeat with a modified C-terminus is located downstream from the KRS1 gene encoding lysyl-tRNA synthetase in Saccharomyces cerevisiae; Martinez R et al.; The KRS1 gene encodes the cytoplasmic form of Saccharomyces cerevisiae lysyl-tRNA synthetase . The KRS1 locus has been characterized . The lysyl-tRNA synthetase gene is unique in the yeast genome . The gene is located on the right arm of chromosome IV and disruption of the open reading frame leads to lethality . These results contrast with the situation encountered in Escherichia coli where lysyl-tRNA synthetase is coded by two distinct genes, lysS and lysU, and further address the possible biological significance of this gene duplication . The nucleotide sequence of the 3'-flanking region has been established . It encodes a long open reading frame whose nucleotide and amino acid structures are almost identical to PMR2, a cluster of tandemly repeated genes coding for P-type ion pumps . The sequence alterations relative to PMR2 are mainly located at the C-terminus of the protein.

Mol Gen Genet, 1991 May, 226(3), 383 - 92
Positive and negative elements upstream of the meiosis-specific glucoamylase gene in Saccharomyces cerevisiae; Kihara K et al.; The SGA1 gene encoding glucoamylase is specifically expressed late in meiotic development of the yeast Saccharomyces cerevisiae . We found that accumulation of both enzyme activity and transcripts was regulated negatively by both nutritional signals and a haploid-specific negative regulator gene of meiosis . RME1, and positively by the inducer genes for meiosis, IME1 and IME2 . To study the role of sequences upstream of the SGA1 gene in its expression and regulation, we generated internal deletions in the 5' non-coding region of the gene and chimeric genes with portions of the upstream sequence inserted into a reporter gene . By analyzing the expression of these genes, we have identified both a 19 bp upstream activation sequence (UAS) and a 49 bp negatively regulating element (NRE) . The UAS activated transcription with no requirement for heterozygosity at the mating-type locus, but this activation was still under negative control by nutrients . The NRE showed no UAS-like activity but conferred IME2-dependent (or meiosis-specific) expression on a heterologous promoter . These results suggest that meiosis-specific expression of the SGA1 gene is established by a regulatory hierarchy including positive and negative factors, the actions of which are mediated through the two separate upstream regulatory elements, UAS and NRE, respectively . Also, that two independently acting cascades exist for the regulation of SGA1 expression: one transduces both the mating-type and nutritional signals and includes the IME2 product, which acts to relieve the repression through NRE; and another transduces only the nutritional signal independently of the above pathway and inhibits positive factors acting on UAS.

Proc Natl Acad Sci U S A, 1991 May 1, 88(9), 3962 - 6
Synthesis of large rRNAs by RNA polymerase II in mutants of Saccharomyces cerevisiae defective in RNA polymerase I; Nogi Y et al.; The 35S rRNA gene of the yeast Saccharomyces cerevisiae was fused to the GAL7 promoter . This hybrid gene, when present on a multicopy plasmid and induced by galactose, suppressed the growth defects of a temperature-sensitive RNA polymerase I (pol I) mutant and those of a mutant in which the gene for the second largest subunit of pol I was deleted . Analysis of pulse-labeled RNA directly demonstrated that rRNA synthesis in this deletion mutant is from the GAL7 promoter . These experiments show that the sole essential function of pol I is the transcription of the rRNA genes, that pol I is not absolutely required for the synthesis of rRNA and ribosomes or cell growth if 35S rRNA synthesis is achieved by some other means, and that the tandemly repeated structure of the chromosomal rRNA genes is also not absolutely required for the synthesis of rRNA and ribosomes.

Proc Natl Acad Sci U S A, 1991 May 1, 88(9), 3574 - 8
The CDC7 protein of Saccharomyces cerevisiae is a phosphoprotein that contains protein kinase activity; Yoon HJ et al.; The CDC7 protein of Saccharomyces cerevisiae may be involved in the G1/S-phase transition and/or in the initiation of mitotic DNA synthesis . The CDC7 gene has two in-frame AUG codons as possible translation start sites, which would produce 58- and 56-kDa proteins, respectively . Both p58 and p56 derived from recombinant plasmids complement the temperature-sensitive growth defect of the cdc7-1 allele . To determine the biochemical function of the CDC7 protein, the CDC7 gene was cloned and polyclonal antibodies were produced against the CDC7 protein . CDC7 immune complexes prepared from yeast with these antibodies phosphorylate histone H1 . Kinase activity is thermolabile in strains carrying the cdc7-1 temperature-sensitive mutant allele and is elevated greater than 10-fold in strains carrying plasmids overexpressing either p56 or p58, confirming that the kinase in the immunoprecipitates is the CDC7 gene product . In addition, we show that CDC7 is a phosphoprotein itself . Indirect immunofluorescence and biochemical fractionation show that the CDC7 protein is present at relatively high concentrations in the nucleus compared with the cytoplasm, suggesting that nuclear proteins may be substrates for the CDC7 protein.

Mol Cell Biol, 1991 May, 11(5), 2641 - 6
The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to ras; Jones S et al.; The product of the CDC25 gene of Saccharomyces cerevisiae, in its capacity as an activator of the RAS/cyclic AMP pathway, is required for initiation of the cell cycle . In this report, we provide an identification of Cdc25p, the product of the CDC25 gene, and evidence that it promotes exchange of guanine nucleotides bound to Ras in vitro . Extracts of strains containing high levels of Cdc25p catalyze both removal of GDP from and the concurrent binding of GTP to Ras . This same activity is also obtained with an immunopurified Cdc25p-beta-galactosidase fusion protein, suggesting that Cdc25p participates directly in the exchange reaction . This biochemical activity is consistent with previous genetic analysis of CDC25 function.

J Cell Biol, 1991 May, 113(3), 539 - 51
The Saccharomyces cerevisiae MYO2 gene encodes an essential myosin for vectorial transport of vesicles; Johnston GC et al.; After the initiation of bud formation, cells of the yeast Saccharomyces cerevisiae direct new growth to the developing bud . We show here that this vectorial growth is facilitated by activity of the MYO2 gene . The wild-type MYO2 gene encodes an essential form of myosin composed of an NH2-terminal domain typical of the globular, actin-binding domain of other myosins . This NH2-terminal domain is linked by what appears to be a short alpha-helical domain to a novel COOH-terminal region . At the restrictive temperature the myo2-66 mutation does not impair DNA, RNA, or protein biosynthetic activity, but produces unbudded, enlarged cells . This phenotype suggests a defect in localization of cell growth . Measurements of cell size demonstrated that the continued development of initiated buds, as well as bud initiation itself, is inhibited . Bulk secretion continues in mutant cells, although secretory vesicles accumulate . The MYO2 myosin thus may function as the molecular motor to transport secretory vesicles along actin cables to the site of bud development.

J Cell Biol, 1991 May, 113(3), 527 - 38
Immunolocalization of Kex2 protease identifies a putative late Golgi compartment in the yeast Saccharomyces cerevisiae; Redding K et al.; The Kex2 protein of the yeast Saccharomyces cerevisiae is a membrane-bound, Ca2(+)-dependent serine protease that cleaves the precursors of the mating pheromone alpha-factor and the M1 killer toxin at pairs of basic residues during their transport through the secretory pathway . To begin to characterize the intracellular locus of Kex2-dependent proteolytic processing, we have examined the subcellular distribution of Kex2 protein in yeast by indirect immunofluorescence . Kex2 protein is located at multiple, discrete sites within wild-type yeast cells (average, 3.0 +/- 1.7/mother cell) . Qualitatively similar fluorescence patterns are observed at elevated levels of expression, but no signal is found in cells lacking the KEX2 gene . Structures containing Kex2 protein are not concentrated at a perinuclear location, but are distributed throughout the cytoplasm at all phases of the cell cycle . Kex2-containing structures appear in the bud at an early, premitotic stage . Analysis of conditional secretory (sec) mutants demonstrates that Kex2 protein ordinarily progresses from the ER to the Golgi but is not incorporated into secretory vesicles, consistent with the proposed localization of Kex2 protein to the yeast Golgi complex.

J Cell Biol, 1991 May, 113(3), 515 - 25
Biosynthesis of mannosylinositolphosphoceramide in Saccharomyces cerevisiae is dependent on genes controlling the flow of secretory vesicles from the endoplasmic reticulum to the Golgi; Puoti A et al.; Saccharomyces cerevisiae contains several abundant phosphoinositol-containing sphingolipids, namely inositolphosphoceramides (IPCs), mannosyl-inositolphosphoceramide (MIPC), which is substituted on the headgroup with an additional mannose, and M(IP)2C, a ceramide substituted with one mannose and two phosphoinositol groups . Using well-defined temperature-sensitive secretion mutants we demonstrate that the biosynthesis of MIPC, M(IP)2C, and a subclass if IPCs is dependent on genes that are required for the vesicular transport of proteins from the ER to the Golgi . Synthesis of these lipids in intact cells is dependent on metabolic energy . A likely but tentative interpretation of the data is that the biosynthesis of these sphingolipids is restricted to the Golgi apparatus, and that one or more substrates for the biosynthesis of these sphingolipids (phosphatidylinositol, IPCs, or MIPC) are delivered to the Golgi apparatus by an obligatory vesicular transport step . Alternative models to explain the data are also discussed.

J Mol Evol, 1991 May, 32(5), 439 - 42
Sequence rearrangements at the ori 7 region of Saccharomyces cerevisiae mitochondrial DNA; Skelly PJ et al.; Three ori elements (ori 2, ori 5, and ori 7) have been sequenced in Saccharomyces cerevisiae strain Dip 2 and compared to the equivalent ori elements of a second strain (B) . Both ori 2 and ori 5 exhibit 98% base matching between strains Dip 2 and B . In contrast, the third ori element (ori 7) exhibits extensive sequence rearrangements whereby a segment located downstream in the consensus strain occurs within the ori structure in Dip 2 . This represents a novel polymorphic form of the yeast mitochondrial genome.

New Biol, 1991 May, 3(5), 511 - 24
A quantitative model for translational control of the GCN4 gene of Saccharomyces cerevisiae; Abastado JP et al.; Expression of the GCN4 gene of Saccharomyces cerevisiae is regulated at the translational level by short open reading frames (uORFs) present in the leader sequence of its mRNA . Under conditions of amino acid sufficiency, these sequences restrict the flow of initiating ribosomes to the GCN4 AUG start codon . Mutational analysis of GCN4 has led to a model in which ribosomes must translate the 5'-proximal uORF1 and reassemble an initiation complex in order to translate GCN4 . This reassembly process is thought to be rapid when amino acids are abundant, such that reinitiation occurs at uORF2, uORF3, or uORF4 . Reinitiation at these sites prevents translation of GCN4, presumably because ribosomes dissociate from the mRNA following termination at uORFs 2 to 4 . Because of reduced initiation factor activity under starvation conditions, a substantial fraction of ribosomal subunits scanning downstream from uORF1 are not ready to reinitiate when they reach uORFs 2 to 4, but become competent to do so while scanning the additional sequences between uORF4 and GCN4 . Examination of the effects of point mutations in the ATG codons of the different uORFs suggests a quantitative model for this control mechanism that describes the probability of reinitiation as a function of the distance scanned downstream from uORF1 . This model accounts for the phenotypes of a number of deletion and insertion mutations that alter the intercistronic spacing between the uORFs and GCN4 . The correspondence between observed and predicted results implies that the differential rates of reinitiation at GCN4 versus uORFs 2 to 4 are determined largely by the different scanning times required to reach each of these start sites following translation of uORF1 . In addition, it supports the notion that an increased scanning-time requirement for reinitiation in amino acid-starved cells forms the basis for translational derepression of GCN4 expression.

Can J Microbiol, 1991 May, 37(5), 397 - 403
Role of cations in the flocculation of Saccharomyces cerevisiae and discrimination of the corresponding proteins; Kuriyama H et al.; Asexual yeast flocculation was studied using strong flocculents of Saccharomyces cerevisiae . The inhibitory effect of cations on flocculation is considered to be caused by competition between those cations and Ca2+ at the binding site of the Ca(2+)-requiring protein that is involved in flocculation . Inhibition of flocculation by various cations occurred in the following order: La3+, Sr2+, Ba2+, Mn2+, Al3+, and Na+ . Cations such as Mg2+, Co2+, and K+ promoted flocculation . This promoting effect may be based on the reduction of electrostatic repulsive force between cells caused by binding of these cations anionic groups present on the cell surface . In flocculation induced by these cations, trace amounts of Ca2+ excreted on the cell surface may activate the corresponding protein . The ratio of Sr2+/Ca2+ below which cells flocculated varied among strains: for strains having the FLO5 gene, it was 400 to 500; for strains having the FLO1 gene, about 150; and for two alcohol yeast strains, 40 to 50 . This suggests that there are several different types of cell surface proteins involved in flocculation in different yeast strains.

Yeast, 1991 May-Jun, 7(4), 425 - 9
Sequence of the CDC10 region at chromosome III of Saccharomyces cerevisiae; Steensma HY et al.; A 4.74 kb DNA fragment from the right arm of chromosome III of Saccharomyces cerevisiae, adjacent to the centromere region was sequenced . Four open reading frames with an ATG initiation codon and larger than 200 bp were found in this fragment . The largest open reading frame of 966 bp was identified as the CDC10 gene.

Yeast, 1991 May-Jun, 7(4), 367 - 78
Induction of a heat-shock-type response in Saccharomyces cerevisiae following glucose limitation; Bataille N et al.; The protein pattern of yeast cells which have arrested proliferation in response to glucose exhaustion is drastically different from that of exponentially growing cells (Boucherie, 1985) . In this study, we used two-dimensional gel electrophoresis to characterize the protein events responsible for these alterations . We found that the induction of heat-shock proteins is one of the major events responsible for these changes . This induction accounts for the synthesis of 18 of the 35 novel polypeptides observed in glucose-limited cells . It was shown to occur in combination with two other protein events: the derepression of carbon catabolite repressed proteins, which accounts for the synthesis of the other novel polypeptides, and an arrest of the synthesis of almost all the proteins present in exponentially growing cells . The time course of each of these events was determined by carrying out a detailed analysis of the pattern of proteins synthesized at various stages of a culture exhausting its glucose supply, and by the measurement of the rate of synthesis of individual polypeptides . The results showed in particular that the synthesis of most of the heat-shock proteins synthesized in glucose-limited cells was induced closely before glucose exhaustion, and that this synthesis was transient, climaxing by the time glucose was exhausted . Under the culture condition investigated, the entry into stationary phase associated with glucose limitation began several hours before glucose exhaustion . It was thus concluded that the observed induction of heat-shock proteins is directly related to the nutritional limitation and is independent from the arrest of cell proliferation.

Yeast, 1991 May-Jun, 7(4), 347 - 56
DNA insertions in the 'silent' regions of the 2 microns plasmid of Saccharomyces cerevisiae influence plasmid stability; Bijovet JF et al.; The 2 microns plasmid of the yeast Saccharomyces cerevisiae is in principle a suitable vector for expression of foreign genes, due to its high copy number and extreme stability . However, the cloning of genes into 2 microns often results in a reduced copy number and/or reduced stability . One reason for this observed instability could be that the inserts in general were made in one of the several open reading frames (ORFs) of the plasmid . Therefore we studied the effect on stability of insertions in the silent regions of 2 microns without interrupting any known essential regions or ORFs . Using the SnaBI site, a yeast-integrating plasmid (Yip5) was introduced into the region between the ARS and STB locus in two possible orientations . The resulting plasmids could be stably maintained in the cells without the need for complementation by the wild-type 2 microns plasmid . However, the stability of these plasmids in a cir . host was still one to two orders of magnitude lower (0.2% and 0.8% respectively) as reported for the wild-type 2 microns (0.01%) . Removal of 2 kb of the bacterial sequences from Yip5 did not increase stability . The stability was dependent on the orientation of the insert . We found that in the less stable orientation, transcription originating from the insert was running into the STB region . DNA inserted in the XmaIII site located outside the ORFs in the REP2/FLP intergenic region influenced both stability and copy number of the plasmid . These effects are strongly dependent on the size of the insert . Insertion of a 2 kb DNA fragment increased the copy number, probably through an effect on FLP expression.

J Gen Microbiol, 1991 May, 137 ( Pt 5), 1039 - 44
Catabolite repression by galactose in overexpressed GAL4 strains of Saccharomyces cerevisiae; Lodi T et al.; Catabolite repression by galactose was investigated in several strains of Saccharomyces cerevisiae grown on different carbon sources . Galactose repressed as much as glucose; raffinose was less effective . Full derepression was achieved with lactate . The functions tested were L-lactate ferricytochrome c oxidoreductase, NAD-glutamate dehydrogenase, and respiration . Galactose repression was observed only in the GAL4 but not in the gal4 strain . The presence of multiple copies of the GAL4 gene enhanced the repression by galactose . Different alleles of the GAL4 gene and the copy number did not affect glucose repression.

Mol Cell Biol, 1991 May, 11(5), 2736 - 43
Inhibition of Ty1 transposition by mating pheromones in Saccharomyces cerevisiae; Xu H et al.; The Ty1 elements in the yeast Saccharomyces cerevisiae are a family of retrotransposons which transpose via a process similar to that of retroviral replication . We report here that the Ty1 transposition process can be blocked posttranscriptionally by treatment of cells with mating pheromones . When haploid yeast cells are treated with appropriate mating pheromones, the transposition frequency of a marked Ty1 element driven by the GAL1 promoter is greatly diminished . Ty1 viruslike particles (VLPs), the putative intermediates for transposition, can be isolated from mating pheromone-treated cells . These VLPs accumulate to normal levels but are aberrant in that they produce very few reverse transcripts of Ty1 RNA both in vivo and in vitro and contain subnormal amounts of p90-TYB and related proteins . In addition, a TYA phosphoprotein product accumulates in treated cells, and some species of TYB proteins have decreased stability . We also show that decreased transposition in mating pheromone-treated cells is not a consequence of simply blocking cell division, since Ty1 transposes at a nearly normal rate in yeast cells arrested in G2 by the drug nocodazole.

Mol Cell Biol, 1991 May, 11(5), 2583 - 92
Cloning and characterization of DST2, the gene for DNA strand transfer protein beta from Saccharomyces cerevisiae; Dykstra CC et al.; The gene encoding the 180-kDa DNA strand transfer protein beta from the yeast Saccharomyces cerevisiae was identified and sequenced . This gene, DST2 (DNA strand transferase 2), was located on chromosome VII . dst2 gene disruption mutants exhibited temperature-sensitive sporulation and a 50% longer generation time during vegetative growth than did the wild type . Spontaneous mitotic recombination in the mutants was reduced severalfold for both intrachromosomal recombination and intragenic gene conversion . The mutants also had reduced levels of the intragenic recombination that is induced during meiosis . Meiotic recombinants were, however, somewhat unstable in the mutants, with a decrease in recombinants and survival upon prolonged incubation in sporulation media . spo13 or spo13 rad50 mutations did not relieve the sporulation defect of dst2 mutations . A dst1 dst2 double mutant has the same phenotype as a dst2 single mutant . All phenotypes associated with the dst2 mutations could be complemented by a plasmid containing DST2.

Mol Cell Biol, 1991 May, 11(5), 2399 - 405
Genetic evidence that different functional domains of the PET54 gene product facilitate expression of the mitochondrial genes COX1 and COX3 in Saccharomyces cerevisiae; Valencik ML et al.; Expression of the yeast mitochondrial genes COX1 and COX3, which encode subunits I and III of cytochrome oxidase, respectively, is controlled by a common nuclear-encoded trans-acting factor . This protein, encoded by the PET54 gene, controls expression of COX1 at the level of RNA splicing and COX3 at the level of mRNA translation . While the steps of COX1 and COX3 gene expression affected by the PET54 gene product are different, it is possible that the PET54 protein is monofunctional and affects expression of each gene by a single mechanism, such as modulation of RNA secondary structure . The goal of this study was to address whether the PET54 protein is monofunctional or multifunctional with respect to its role in COX1 and COX3 gene expression . Ten insertion mutations, which each resulted in the in-frame addition of four amino acids within the PET54 polypeptide, were generated, and the resulting mutants were characterized for respiration phenotype and mitochondrial gene expression . Five of the ten mutants were respiration deficient . Two of these five mutants were defective in expression of COX3 but not in expression of COX1, while two other mutants had the opposite phenotype (primarily defective in expression of COX1) . The fifth mutant was equally defective in expression of both genes . These results demonstrate that the two functions of PET54 are genetically separable and support the idea that the PET54 protein is multifunctional.

Mol Cell Biol, 1991 May, 11(5), 2593 - 608
Molecular and genetic analysis of the gene encoding the Saccharomyces cerevisiae strand exchange protein Sep1; Tishkoff DX et al.; Vegetatively grown Saccharomyces cerevisiae cells contain an activity that promotes a number of homologous pairing reactions . A major portion of this activity is due to strand exchange protein 1 (Sep1), which was originally purified as a 132,000-Mr species (R . Kolodner, D . H . Evans, and P . T . Morrison, Proc . Natl . Acad . Sci . USA 84:5560-5564, 1987) . The gene encoding Sep1 was cloned, and analysis of the cloned gene revealed a 4,587-bp open reading frame capable of encoding a 175,000-Mr protein . The protein encoded by this open reading frame was overproduced and purified and had a relative molecular weight of approximately 160,000 . The 160,000-Mr protein was at least as active in promoting homologous pairing as the original 132,000-Mr species, which has been shown to be a fragment of the intact 160,000-Mr Sep1 protein . The SEP1 gene mapped to chromosome VII within 20 kbp of RAD54 . Three Tn10LUK insertion mutations in the SEP1 gene were characterized . sep1 mutants grew more slowly than wild-type cells, showed a two- to fivefold decrease in the rate of spontaneous mitotic recombination between his4 heteroalleles, and were delayed in their ability to return to growth after UV or gamma irradiation . Sporulation of sep1/sep1 diploids was defective, as indicated by both a 10- to 40-fold reduction in spore formation and reduced spore viability of approximately 50% . The majority of sep1/sep1 diploid cells arrested in meiosis after commitment to recombination but prior to the meiosis I cell division . Return-to-growth experiments showed that sep1/sep1 his4X/his4B diploids exhibited a five- to sixfold greater meiotic induction of His+ recombinants than did isogenic SEP1/SEP1 strains . sep1/sep1 mutants also showed an increased frequency of exchange between HIS4, LEU2, and MAT and a lack of positive interference between these markers compared with wild-type controls . The interaction between sep1, rad50, and spo13 mutations suggested that SEP1 acts in meiosis in a pathway that is parallel to the RAD50 pathway.

Curr Genet, 1991 May, 19(5), 343 - 51
Properties of two nuclear pet mutants affecting expression of the mitochondrial oli1 gene of Saccharomyces cerevisiae; Payne MJ et al.; This study details the characteristics of two temperature-conditional pet mutants of yeast, strains ts1860 and ts379, which at the non-permissive temperature show deficiencies in the formation of three mitochondrially encoded subunits of the ATP synthase complex . By analysis of mitochondrial translation products, and of mitochondrial transcription in temperature shift experiments from the permissive (22 degrees C) to the non-permissive (36 degrees C) temperature, it was concluded that the nuclear mutations in both mutants primarily inhibit synthesis of ATP synthase subunit 9, and that reductions in subunit 8 and 6 synthesis are secondary pleiotropic effects . Following transfer to 36 degrees C, cells of mutant ts379 display a near complete inhibition of subunit 9 synthesis within 1 h, coincident with a marked reduction in the level of the cognate oli1 mRNA . On the other hand, near complete inhibition of subunit 9 synthesis in strain ts1860 occurs after 3 h at 36 degrees C, at which time there is little change in the level of subunit 9 mRNA . In both mutants the mRNA levels for subunits 6 and 8 are not significantly affected at the time of inhibition of subunit 9 synthesis . Provision of an alternative source of subunit 8, translated extra-mitochondrially for import into the organelle, does not overcome the mutant phenotype of either mutant at 36 degrees C, confirming that subunit 8 is not the sole or primary deficiency in each mutant . The mutants indicate that the products of a least two nuclear genes (designated AEP1 and AEP2) are required for the expression of the mitochondrial oli1 gene and the synthesis of subunit 9 . (ABSTRACT TRUNCATED AT 250 WORDS)

Gene Expr, 1991 May, 1(2), 149 - 67
CTD kinase large subunit is encoded by CTK1, a gene required for normal growth of Saccharomyces cerevisiae; Lee JM et al.; We previously purified a yeast protein kinase that specifically hyperphosphorylates the carboxyl-terminal repeat domain (CTD) of RNA polymerase II largest subunit and showed that this CTD kinase consists of three subunits of 58, 38, and 32 kDa . We have now cloned, sequenced, and characterized CTK1, the gene encoding the 58 kDa alpha subunit . The CTK1 gene product contains a central domain homologous to catalytic subunits of other protein kinases, notably yeast CDC28, suggesting that the 58 kDa subunit is catalytic . Cells that carry a disrupted version of the CTK1 gene lack the characterized CTD kinase activity, grow slowly and are cold-sensitive, demonstrating that the CTK1 gene product is essential for CTD kinase activity and normal growth . While ctk1 mutant cells do contain phosphorylated forms of the RNA polymerase II largest subunit, these forms differ from those found in wild type cells, implicating CTK1 as a component of the physiologically significant CTD phosphorylating machinery . As befitting an enzyme with a nuclear function, the N-terminal region of the CTK1 protein contains a nuclear targeting signal.

Mutagenesis, 1991 May, 6(3), 229 - 36
Comparison of three forward mutation systems in Saccharomyces cerevisiae for sensitivity to polycyclic and heterocyclic compounds; Mitchell I et al.; Forward mutation to cycloheximide resistance, L-canavanine resistance and DL-alpha-aminoadipic acid resistance in Saccharomyces cerevisiae wild-type strain S7a was tested for sensitivity to nine mutagens in treat-and-plate assays . Eight of these agents, 2-aminofluorene, 2-acetylaminofluorene, benzo{a}pyrene, benzidine, cyclophosphamide, acriflavine, 7,12-dimethylbenz{a}anthracene and 2-aminoanthracene were known or suspected to be difficult to detect whilst one, methyl methanesulphonate, was known to be very active in yeast . Forward mutation to cycloheximide resistance was, overall, the most sensitive system, detecting all nine agents under optimal conditions, although neither benzidine nor benzo{a}pyrene were consistently positive . Mutation to adipic acid resistance occasionally gave responses superior to those at the cycloheximide loci, but mutation to canavanine resistance was never more sensitive than the cycloheximide resistance system . We conclude that forward mutation in strain S7a using both cycloheximide and adipic acid resistance loci is capable of detecting the genetic effects of a range of polycyclic and heterocyclic compounds with greater sensitivity than is seen in other published gene mutation assays with yeast . Although sensitivity is much lower than in bacterial assays, such yeast assays provide a reasonable alternative to bacterial genotoxicity screening for agents such as potent bactericides.

J Gen Microbiol, 1991 May, 137 ( Pt 5), 1033 - 7
13C NMR analysis of a developmental pathway mutation in Saccharomyces cerevisiae reveals a cell derepressed for succinate dehydrogenase; Dickinson JR et al.; 13C nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of {2-13C}acetate in a diploid strain of Saccharomyces cerevisiae homozygous for the spo50 mutation . This mutation results in failure to initiate sporulation and suppresses spd mutations (which cause derepressed sporulation) . By analysing the pattern of 13C-labelling in glutamate it was deduced that the glyoxylate cycle is responsible for most of the acetate utilization and that there is very little tricarboxylic acid cycle activity . The labelling of alpha,alpha'-trehalose indicated that gluconeogenesis and the hexose monophosphate pathway operate in a similar way to the wild-type . The mutant strain has higher levels of succinate dehydrogenase than the wild-type . All of the physiological alterations caused by the spo50 mutation can be explained by this difference.

Genetics, 1991 May, 128(1), 69 - 77
The SNF2, SNF5 and SNF6 genes are required for Ty transcription in Saccharomyces cerevisiae; Happel AM et al.; The Saccharomyces cerevisiae SNF2, SNF5 and SNF6 genes were initially identified as genes required for expression of SUC2 and other glucose repressible genes . The Suc- defect in all three of these classes of mutants is suppressed by mutations in the SPT6 gene . Since mutations in SPT6 had also been identified as suppressors of Ty and solo delta insertion mutations at the HIS4 and LYS2 loci, we have examined Ty transcription in snf2, snf5 and snf6 mutants and have found that Ty transcription is abolished or greatly reduced . The snf2, snf5 and snf6 defect for Ty transcription, like the defect for SUC2 transcription, is suppressed by spt6 mutations . In contrast to other mutations that abolish or greatly reduce Ty transcription (in the SPT3, SPT7 and SPT8 genes), mutations in these SNF genes do not cause suppression of insertion mutations . This result suggests that the SNF2, SNF5 and SNF6 gene products act by a distinct mechanism from the SPT3, SPT7 and SPT8 gene products to promote transcription of Ty elements . This result also suggests that a reduction of Ty transcription is not always sufficient for activation of adjacent gene expression.

Genetics, 1991 May, 128(1), 59 - 67
A tester system for detecting each of the six base-pair substitutions in Saccharomyces cerevisiae by selecting for an essential cysteine in iso-1-cytochrome c; Hampsey M; A collection of isogenic yeast strains that is specifically diagnostic for the six possible base-pair substitutions is described . Each strain contains a single, unique base-pair substitution at the Cys-22 codon of the CYC1 gene, which codes for iso-1-cytochrome c . These mutations encode replacements of the functionally critical Cys-22 and render each strain unable to grow on media containing nonfermentable carbon sources (Cyc-) . Specific base-pair substitutions, which restore the Cys-22 codon, can be monitored simply by scoring for reversion to the Cyc+ phenotype . These strains revert spontaneously at very low frequencies and exhibit specific patterns of reversion in response to different mutagens . Only true (CYC1+) revertants were recovered after 7 days on selection medium . The following mutagen specificities were observed: ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine, G.C----A.T; 4-nitroquinoline-1-oxide, G.C----T.A and G.C----A.T; diepoxybutane, A.T----T.A, A.T----G.C and G.C----T.A; 5-azacytidine, G.C----C.G . Methyl methanesulfonate induced all six mutations, albeit at relatively low frequencies, with preference for A.T----T.A and A.T----G.C . Ultraviolet light was the most inefficient mutagen used in this study, consistent with its preference for transition mutations at dipyrimidine sequences reported in other systems . This tester system is valuable as a simple and reliable assay for specific mutations without DNA sequence analysis.

Mol Gen Genet, 1991 May, 227(1), 52 - 9
The Saccharomyces cerevisiae genes (CMP1 and CMP2) encoding calmodulin-binding proteins homologous to the catalytic subunit of mammalian protein phosphatase 2B; Liu Y et al.; Saccharomyces cerevisiae genomic clones that encode calmodulin-binding proteins were isolated by screening a lambda gt11 expression library using 125I-labeled calmodulin as probe . Among the cloned yeast genes, we found two closely related genes (CMP1 and CMP2) that encode proteins homologous to the catalytic subunit of phosphoprotein phosphatase . The presumed CMP1 protein (62,999 Da) and CMP2 protein (68,496 Da) contain a 23 amino acid sequence very similar to those identified as calmodulin-binding sites in many calmodulin-regulated proteins . The yeast genes encode proteins especially homologous to the catalytic subunit of mammalian phosphoprotein phosphatase type 2B (calcineurin) . The products of the CMP1 and CMP2 genes were identified by immunoblot analysis of cell extracts as proteins of 62,000 and 64,000 Da, respectively . Gene disruption experiments demonstrated that elimination of either or both of these genes had no effect on cell viability, indicating that these genes are not essential for normal cell growth.

Biochemistry, 1991 Apr 30, 30(17), 4284 - 9
Identification of structurally and functionally important histidine residues in cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae; Gasparini S et al.; Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is an alpha 2 dimer (alpha, Mr 63,000), each alpha containing 12 histidines . The covalent incorporation of 6-7 mol of diethyl pyrocarbonate per monomer corresponded to complete enzyme inactivation . This inactivation was reversed by hydroxylamine hydrolysis which regenerates free histidine (and tyrosine) while leaving the carbethoxy group still attached to the epsilon-amino group of lysine . Three histidines, one tyrosine, and four lysines were the main targets of the reagent . Site-directed mutagenesis was also tried to replace each of these modified residues . Given the unstability of the carbethoxy-imidazole bond, the nine histidines that were not modified by diethyl pyrocarbonate were mutated too . For these experiments, the enzyme was expressed in Escherichia coli by using a vector bearing the structural gene in which the first 13 codons were replaced by the first 14 of the CII lambda gene . This substitution had no effect on the kinetic parameters . The combined results of chemical modification and site-directed mutagenesis show that one histidine seems to be part of the active site while two others play an important structural role . On the other hand, labeled lysines and tyrosine are nonessential residues . These results are discussed in light of two recent articles establishing the existence of a second family of aminoacyl-tRNA synthetases devoid of the HIGH and KMSKS consensus sequences and containing no Rossmann's domain in their three-dimensional structures.

J Biol Chem, 1991 Apr 15, 266(11), 7008 - 15
Half-calmodulin is sufficient for cell proliferation . Expressions of N- and C-terminal halves of calmodulin in the yeast Saccharomyces cerevisiae; Sun GH et al.; Calmodulin (CaM) has been shown to be an essential component for progression of nuclear division in the yeast Saccharomyces cerevisiae (Ohya, Y., and Anraku, Y . (1989) Curr . Genet . 15, 113-120) . To define the functional domain of the molecule required for cell proliferation, we constructed plasmids expressing a series of N- and C-terminal halves of the CaM under the control of the galactose-inducible GAL1 promoter . These plasmids were introduced into a cmd1-disrupted yeast haploid strain, and the growth properties of the cells depending on the half-CaMs were examined . Plasmids expressing the N-terminal half (Ser1-Leu76) and the C-terminal half (Leu85-Cys147), which each maintain two complete EF-hand structures, complemented the growth defect of the cmd1 null mutation, whereas those expressing shorter regions of C- and N-terminal CaM did not . The half-CaMs that complemented the cmd1 null mutation were found to be approximately 6-fold overexpressed relative to expression of native CaM by the wild-type CMD1 gene . The levels of expression of the half CaMs with the true CMD1 promoter were not sufficient for complementation . These results demonstrate that half-CaMs (either the N- or the C-terminal) are capable of supporting growth of yeast cells when they are suitably overproduced . Cells depending solely on half-CaMs all showed a temperature-sensitive growth phenotype, suggesting that half-CaMs cannot carry out all the cellular functions of the complete CaM molecule.

Nucleic Acids Res, 1991 Apr 11, 19(7), 1385 - 91
rar mutations which increase artificial chromosome stability in Saccharomyces cerevisiae identify transcription and recombination proteins; Kipling D et al.; In an attempt to identify trans-acting factors involved in replication origin function, we have characterized the RAR3 and RAR5 genes, identified by mutations which increase the mitotic stability of artificial chromosomes whose replication is dependent on the activity of weak ARS elements . Sequence analysis has shown that the RAR3 gene is identical to GAL11/SPT13, which encodes a putative transcription factor involved in the expression of a wide range of genes . Change-of-function mutations that truncate the RAR3 protein appear to be required to enhance chromosome stability . In contrast, loss of the RAR5 protein results in enhanced chromosome stability, as if the protein is an inhibitor of ARS function . The RAR5 gene encodes the 175 kDa DNA strand transfer protein beta, an activity that can promote the transfer of a strand from a double-stranded DNA molecule to a complementary single strand . This observation implies that a presumed recombination activity can affect eukaryotic chromosomal replication.

Eur J Biochem, 1991 Apr 10, 197(1), 271 - 9
Effects of amphotericin B on the glucose metabolism in Saccharomyces cerevisiae cells . Studies by 13C-, 1H-NMR and biochemical methods; Tran-Dinh S et al.; A new approach is proposed to investigate the metabolic perturbation induced by drugs in cells . The effects of various concentrations of amphotericin B on the aerobic {1-13C}glucose metabolism in glucose-grown repressed Saccharomyces cerevisiae cells were studied as a function of time using 13C-, 1H-NMR and biochemical methods . The 13C enrichment of different compounds such as ethanol, glycerol and trehalose were determined by 1H-NMR spectroscopy . In the absence of amphotericin B, glycerol diffuses slowly from the internal to the external medium, whereas in its presence this diffusion is greatly facilitated by the formation of pores in the cell membrane . Amphotericin B has been found to exert a marked influence on the glucose consumption and the production of all metabolites; for example, at 1 microM, the glucose consumption and the production of ethanol decrease while the production of glycerol and trehalose increases . The 13C relative enrichments of ethanol, glycerol and trehalose are almost the same with and without the drug . Thus it can be concluded that amphotericin B induces a large effect on the production of these compounds in the cytosol but shows no significant influence on the mechanism of their formation . Upon addition of glucose, all the amino acid concentrations decrease continuously with time; this effect is more pronounced in the presence of the drug . The ratio of the integrated resonances of glutamate (C2 + C3)/C4 reflects the activity of pyruvate carboxylase relative to citrate synthase rather than to pyruvate dehydrogenase . Without amphotericin B, this ratio (approximately 1.0) is practically constant upon addition of glucose which suggests that the activities of pyruvate carboxylase and citrate synthase are equivalent . By contrast, upon coaddition of 25 mM glucose and 1 microM amphotericin B, the glutamate C4 resonance remains virtually unchanged while that of glutamate C2 is much smaller than in its absence and continuously decreases with time . It seems likely that amphotericin B induces a reduction in the activity of pyruvate carboxylase in the mitochondria.

Biochim Biophys Acta, 1991 Apr 9, 1073(3), 502 - 8
Yeast proteinase yscB inactivates the leucyl tRNA synthetase in extracts of Saccharomyces cerevisiae; Larrinoa IF et al.; The aminoacyl-tRNA synthetases are inactivated in extracts of Saccharomyces cerevisiae preferentially to other yeast enzymes and the rate of inactivation greatly increases in extracts of nitrogen-starved cells . The intensity of inactivation varies for the different synthetases . Under conditions in which more than 80 per cent of the leucyl and isoleucyl-tRNA synthetases are inactivated, the activities of the synthetases for serine and arginine remain unchanged and the synthetases for other amino acids are inactivated to different extents . We have analyzed the characteristics of inactivation of the leucyl-tRNA synthetase, and identified the inactivating agent as the yeast proteinase yscB by the following criteria: co-induction of both activities by nitrogen starvation; same pattern of sensitivity to yeast proteinase inhibitors; co-purification through a procedure designed to purify the proteinase yscB and lack of inactivating activity in extracts of a nitrogen-starved yeast mutant lacking proteinase yscB.

J Biol Chem, 1991 Apr 5, 266(10), 6174 - 80
Inhibition of glycogen synthesis in Saccharomyces cerevisiae by the mating pheromone alpha-factor; Francois J et al.; Treatment of the yeast Saccharomyces cerevisiae with the pheromone alpha-factor caused an inhibition of glycogen synthesis in MATa haploid cells but not in MAT alpha cells or MATa/MAT alpha diploid cells . The concentration of alpha-factor required for a half-maximal inhibition was comparable to that required for the induction of the FUS1 gene . Strains containing a disruption in ste2 or ste12 or temperature-sensitive mutations in ste4, ste7, or ste11 continued to divide and to accumulate glycogen in the presence of alpha-factor . In contrast, inhibition of glycogen occurred upon exposure to mating pheromone of far1 mutants which, under this condition, fail to arrest in G1 and continue to divide while simultaneously undergoing the transcriptional induction and morphological changes typical of mating cells . The inhibition of glycogen accumulation by alpha-factor persisted in a strain lacking glycogen phosphorylase (EC 2.4.1.1), which ruled out the participation of this enzyme in the pheromone response . Glycogen synthase (EC 2.4.1.11) from a cells treated with alpha-factor was found primarily in the glucose 6-phosphate-dependent (inactive) form whereas the total activity was unaltered . This indicates that the action of mating pheromone is mainly to inhibit the interconversion of the inactive glucose 6-phosphate-dependent form to the active glucose 6-phosphate-independent form of glycogen synthase without affecting the concentration of the enzyme.

Biotechnol Appl Biochem, 1991 Apr, 13(2), 269 - 76
Identification and characterization of a thermolabile antigen (TLAb, glyceraldehyde-3-phosphate dehydrogenase) in Saccharomyces cerevisiae; Watanabe Y et al.; Five thermolabile antigens (TLAa, TLAb, TLAc, TLAd, and TLAe) have been purified from Saccharomyces cerevisiae . Recently, we reported that TLAa was identical with yeast enolase (EC 4.2.1.11) . In this paper, TLAb was identified as yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) on the following bases: (1) Mr, N-terminal amino acid sequence, and isomer number of TLAb were the same as those of GAPDH; (2) anti-TLAb serum was reactive to GAPDH in the Ouchterlony test and in sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting; and (3) TLAb possessed GAPDH enzyme activity which was inhibited by anti-TLAb serum . The effect of various growth conditions on the proportion of three TLAb isoproteins (TLAb-1, TLAb-2, and TLAb-3) was examined . The proportion of two TLAb isoproteins (TLAb-1 and TLAb-2) changed depending on the cell growth phase the carbon sources, and sodium chloride shock . It is concluded that environmental stress has a differential effect on the biosynthesis of TLAb isoproteins.

Mol Gen Genet, 1991 Apr, 226(1-2), 277 - 82
Genetic studies of the PRP11 gene of Saccharomyces cerevisiae; Schappert K et al.; PRP11 is a gene that encodes an essential function for pre-messenger RNA (mRNA) processing in Saccharomyces cerevisiae . We have carried out a mutational study to locate essential and non-essential regions of the PRP11 protein . The existing temperature-sensitive (ts) mutation (prp11-1) was isolated from the chromosome of the original mutant and its position in the gene was determined . When the prp11-1 gene was transcribed from the GAL1 promoter, the overproduced protein was able to reverse the ts prp11-1 phenotype; this is compatible with the possibility that the defect in the prp11-1 gene product affects its binding to the spliceosome . Thirteen linker-insertion mutations were constructed . Only five (prp11-4, 11-6, 11-10, -13 and -14) resulted in a null phenotype . One of these became temperature-sensitive when the insertion was reduced in size from four (prp11-10) to two (prp11-15) amino acids . A sequence of ten amino acids of which also occurs in the human U1 small nuclear ribonucleoprotein particle (snRNP) A protein and the U2 snRNP B" protein, when deleted from PRP11, had no phenotype and thus appears to be nonessential for PRP11 function . However, a linker-insertion mutation (prp11-10) immediately adjacent to this region resulted in a null phenotype.

Genetics, 1991 Apr, 127(4), 681 - 98
Physical map of the Saccharomyces cerevisiae genome at 110-kilobase resolution; Link AJ et al.; A physical map of the Saccharomyces cerevisiae genome is presented . It was derived by mapping the sites for two restriction endonucleases, SfiI and NotI, each of which recognizes an 8-bp sequence . DNA-DNA hybridization probes for genetically mapped genes and probes that span particular SfiI and NotI sites were used to construct a map that contains 131 physical landmarks--32 chromosome ends, 61 SfiI sites and 38 NotI sites . These landmarks are distributed throughout the non-rDNA component of the yeast genome, which comprises 12.5 Mbp of DNA . The physical map suggests that those genes that can be detected and mapped by standard genetic methods are distributed rather uniformly over the full physical extent of the yeast genome . The map has immediate applications to the mapping of genes for which single-copy DNA-DNA hybridization probes are available.

J Bacteriol, 1991 Apr, 173(8), 2556 - 61
The RSF1 gene regulates septum formation in Saccharomyces cerevisiae; Veinot-Drebot LM et al.; Septum formation in the mitotic cell cycle of the budding yeast Saccharomyces cerevisiae occurs by conversion of the chitin ring, laid down at bud formation, into the primary septum . We show here that under certain conditions this septation is dependent on the newly identified RSF1 gene . However, cells harboring the rsf1-1 mutation accumulated in a postcytokinesis state, with delayed conversion of the chitin-rich annulus into the primary septum . This rsf1-1-mediated inhibition of septum formation only occurred under conditions of biosynthetic stress and was correlated with biosynthetically mediated inhibition of the cell-cycle regulatory step START . The RSF1 gene is distinct from the CHS2 chitin synthase gene that is responsible for septation, and thus RSF1 most likely encodes a regulator of chitin synthesis . We hypothesize that RSF1 activity facilitates septum formation during times of biosynthetic stress, to allow efficient septation even under these conditions.

J Cell Biol, 1991 Apr, 113(2), 245 - 60
A cytoskeleton-related gene, uso1, is required for intracellular protein transport in Saccharomyces cerevisiae; Nakajima H et al.; The Saccharomyces cerevisiae mutant strains blocked in the protein secretion pathway are not able to induce sexual aggregation . We have utilized the defect of aggregation to concentrate the secretion-deficient cells and identified a new gene which functions in the process of intracellular protein transport . The new mutant, uso1, is temperature sensitive for growth and protein secretion . At the restrictive temperature (37 degrees C), uso1 mutant accumulated the core-glycosylated precursor form of the exported protein invertase in the cells . Ultrastructural study of the mutant fixed by the freeze-substitution method revealed expansion of the nuclear envelope lumen and accumulation of the ER at the restrictive temperature . Abnormally oriented bundles of microtubules were often found in the nucleus . The USO1 gene was cloned by complementation of the uso1 temperature-sensitive growth defect . DNA sequence analysis revealed a hydrophilic protein of 1790 amino acids with a COOH-terminal 1,100-amino acid-long alpha-helical structure characteristic of the coiled-coil rod region of the cytoskeleton-related proteins . These observations suggest that Uso1 protein plays a role as a cytoskeletal component in the protein transport from the ER to the later secretory compartments.

Genes Dev, 1991 Apr, 5(4), 605 - 15
Mutations in the HML E silencer of Saccharomyces cerevisiae yield metastable inheritance of transcriptional repression; Mahoney DJ et al.; Mating-type genes resident in the silent cassette HML at the left arm of chromosome III are repressed by the action of four SIR gene products, mediated independently through two cis-acting sites, termed the E and I silencers . We have found that in the absence of the I silencer, deletion of any one of three distinct elements within E yields partial derepression of the mating-type genes resident at HML, whereas deletion of any two yields full derepression . These elements correspond to a binding site for the abundant DNA-binding protein RAP1, an autonomous replicating sequence (ARS), and an as yet undistinguished region . From detailed deletion analysis of the E site we conclude that the ARS element contributes to silencer function in a capacity distinct from its role as an initiator of DNA replication . In addition, we find that strains deleted for any one of these elements comprise two genetically identical but phenotypically distinct types of cells: Those with HML apparently fully derepressed, and those with HML apparently completely repressed . These results reinforce the notion that epigenetic inheritance is an intrinsic characteristic of silencer action.

EMBO J, 1991 Apr, 10(4), 987 - 96
Double-strand-break repair and recombination catalyzed by a nuclear extract of Saccharomyces cerevisiae; Symington LS; An in vitro system for double-strand-break repair and recombination of plasmid substrates catalyzed by extracts prepared from yeast nuclei has been developed . Recombination events that generate crossover products were detected amongst reaction products by Southern blot hybridization, or by the polymerase chain reaction (PCR) . The recombination reaction was found to be stimulated by a double-strand break within homologous sequences and proceeded by a mechanism that involved branched DNA intermediates . In addition to pairing events that generate crossovers, the formation of inverted repeats (head-to-head and tail-to-tail joined products) was also detected . Two models are presented which propose that the formation of crossover products and inverted repeats occur by similar mechanisms.

Mol Cell Biol, 1991 Apr, 11(4), 2253 - 62
The SIR1 gene of Saccharomyces cerevisiae and its role as an extragenic suppressor of several mating-defective mutants; Stone EM et al.; The SIR1 gene product of Saccharomyces cerevisiae is one of several proteins involved in repressing transcription of the silent mating-type genes . Strains with mutations in the genes coding for these proteins are defective in mating due to derepression of the silent loci . We have found that overexpression of the SIR1 gene suppresses the mating defects of several of these mutants, including nat1 and ard1 mutants (the products of these two genes are responsible for N-terminal acetylation of a subset of yeast proteins), certain sir3 mutants, and a histone H4 mutant . The SIR1 gene has been sequenced and found to contain an open reading frame coding for a 678-amino-acid protein.

Mol Cell Biol, 1991 Apr, 11(4), 2013 - 7
Effects of controlled RAD52 expression on repair and recombination in Saccharomyces cerevisiae; Dornfeld KJ et al.; We have examined the effects of RAD52 overexpression on methyl methanesulfonate (MMS) sensitivity and spontaneous mitotic recombination rates . Cells expressing a 10-fold excess of RAD52 mRNA from the ENO1 promoter are no more resistant to MMS than are wild-type cells . Similarly, under the same conditions, the rate of mitotic recombination within a reporter plasmid does not exceed that measured in wild-type cells . This high level of expression is capable of correcting the defects of rad52 mutant cells in carrying out repair and recombination . From these observations, we conclude that wild-type amounts of Rad52 are not rate limiting for repair of MMS-induced lesions or plasmid recombination . By placing RAD52 under the control of the inducible GAL1 promoter, we find that induction results in a 12-fold increase in the fraction of recombinants within 4 h . After this time, the fraction increases less rapidly . When RAD52 expression is quickly repressed during induction, the amount of RAD52 mRNA decreases rapidly and no nascent recombinants are formed . This result suggests a short active half-life for the protein product . Induction of RAD52 in G1-arrested mutant cells also causes a rapid increase in recombinants, suggesting that replication is not necessary for plasmid recombination.

Mol Gen Genet, 1991 Apr, 226(1-2), 224 - 32
The synthesis of the two S-adenosyl-methionine synthetases is differently regulated in Saccharomyces cerevisiae; Thomas D et al.; S-adenosyl-L-methionine (AdoMet) is synthesized by transfer of the adenosyl moiety of ATP to the sulfur atom of methionine . This reaction is catalysed by AdoMet synthetase . In all eukaryotic organisms studied so far, multiple forms of AdoMet synthetases have been reported and from their recent study, it appears that AdoMet synthetase is an exceptionally well conserved enzyme through evolution . In Saccharomyces cerevisiae, we have demonstrated the existence of two AdoMet synthetases encoded by genes SAM1 and SAM2 . Yeast, which is able to concentrate exogenously added AdoMet, is thus a particularly useful biological system to understand the role and the physiological significance of the preservation of two almost identical AdoMet synthetases . The analysis of the expression of the two SAM genes in different genetic backgrounds during growth under different conditions shows that the expression of SAM1 and SAM2 is regulated differently . The regulation of SAM1 expression is identical to that of other genes implicated in AdoMet metabolism, whereas SAM2 shows a specific pattern of regulation . A careful analysis of the expression of the two genes and of the variations in the methionine and AdoMet intracellular pools during the growth of different strains lead us to postulate the existence of two different AdoMet pools, each one supplied by a different AdoMet synthetase but in equilibrium with each other . This could be a means of storing AdoMet whenever this metabolite is overproduced, thus avoiding the degradation of a metabolite the synthesis of which is energetically expensive.

Yeast, 1991 Apr, 7(3), 275 - 80
Physical, transcriptional and genetical mapping of a 24 kb DNA fragment located between the PMA1 and ATE1 loci on chromosome VII from Saccharomyces cerevisiae; Capieaux E et al.; A physical map of a contiguous DNA fragment of 60 kb, extending from the centromere to TRP5 on the left arm of the chromosome VII of Saccharomyces cerevisiae, strain IL125-2B, was established . Within a 31 kb region from PMA1 towards TRP5, a total of 12 transcription products ranging from 0.6 to 3.6 kb were identified in cells grown exponentially on rich medium . Near 87% of the DNA investigated was transcribed and on average one transcript, of 2.3 kb average length, was detected every 2.7 kb of DNA . The physical and genetical distances between the markers CEN7, pma1, leu1, pdr1 and trp5 were compared . A recombination frequency of 1 cM corresponds to an average distance of 3.3 kb between alleles in this region of chromosome VII.

Yeast, 1991 Apr, 7(3), 265 - 73
CDC15, an essential cell cycle gene in Saccharomyces cerevisiae, encodes a protein kinase domain; Schweitzer B et al.; The cell division cycle gene CDC15 is essential for the late nuclear division in the yeast Saccharomyces cerevisiae . The amino acid sequence of the 974 amino acids/110 kDa CDC15 gene product, as deduced from the nucleotide sequence, includes an aminoterminal protein kinase domain which contains a primary sequence mosaic showing patterns specific for protein serine/threonine kinases besides those for protein tyrosine kinases . Many protein kinases non-essential for growth are known . CDC15 represents an essential protein kinase like CDC7 and CDC28 . A carboxyterminal deletion of 32 amino acids renders the protein inactive.

Yeast, 1991 Apr, 7(3), 229 - 44
Calcium-dependent secretory vesicle-binding and lipid-binding proteins of Saccharomyces cerevisiae; Creutz CE et al.; Yeast (Saccharomyces cerevisiae) cytosol was examined for the presence of calcium-dependent membrane- or lipid-binding proteins that might play fundamental roles in membrane-associated phenomena in stimulated cells . A complex group of proteins was isolated from late log phase cultures of yeast strain YP3 on the basis of calcium-dependent association with yeast secretory vesicles isolated from the temperature-sensitive sec6-4 secretory mutant . The masses of the major proteins in this group were 32, 35, 47, 51, 55, 60 and 120 kDa . A similar group of proteins was isolated by calcium-dependent association with bovine brain lipids enriched in the predominant acidic phospholipids of the yeast secretory vesicles . The 47 kDa protein was highly purified when commercial yeast cake was used as the source of yeast cytosol . The 32 kDa and 60 kDa proteins were demonstrated to reassociate with lipids at calcium concentrations of 100 microM or higher, while no association was promoted by 2 mM-magnesium . The 47 kDa protein could be removed from lipids by reducing the calcium concentration to between 1 and 32 microM . The sequences of peptides isolated from digests of several of these proteins indicate that they are novel proteins but are insufficient to judge the possible homology of these proteins with mammalian membrane-binding proteins . The sequence data may be adequate to permit isolation and modification of the corresponding genes in order to assess the possible function of this class of proteins in stimulated cells.

Yeast, 1991 Apr, 7(3), 211 - 7
Increased endocytosis in the Saccharomyces cerevisiae fragile mutant VY1160; Waltschewa L et al.; The VY1160 mutant is characterized by cell lysis in hypotonic solutions and generally increased permeability to substances for which Saccharomyces cerevisiae cells are not permeable . Two mutations, srb1 and ts1, have been identified in VY1160 mutant, and previous studies (Kozhina et al., 1979) have shown srb1 to be responsible for cell lysis . We now present evidence that the ts1 mutation leads to increased endocytosis in VY1160 cells . The internalization of lucifer yellow carbohydrazide in VY1160 cells is time-, temperature- and energy-dependent and consistent with a fluid-phase mechanism of endocytosis . The rate of steady-state accumulation of the dye at 37 degrees C is 145 ng/micrograms DNA per h for VY1160 mutant and 23 ng/micrograms DNA per h for S288C parental strain . Studies with isogenic strains having either the srb1 or the ts1 mutation, or SRB1 TS1 wild-type alleles have shown that only ts1 strains possess increased endocytosis . Quantitation of endocytosis in cells grown at 24 degrees C and shifted at 38 degrees C shows that ts1 strains, but not srb1 and wild-type strains, increase ten-fold the internalization of lucifer yellow 2 h after the shift at 38 degrees C . The analysis of ts1 x wild-type crosses provides evidence that the temperature-sensitive phenotype segregates together with the enhanced endocytosis . It is concluded that the increased endocytosis might explain the generally increased permeability of VY1160 mutant cells.

Genetika, 1991 Apr, 27(4), 644 - 8
{Genetic mapping of genes regulating synthesis of acid phosphatases in the yeast Saccharomyces cerevisiae of the Peterhoff yeast collection}; Sambuk EV et al.; Genetic mapping of yeast genes ACP5, acp83 and ACP82 was performed . According to the results obtained, ACP5 and acp83 are located in the chromosome VI and ACP82--in the chromosome XVI . Based on identity of chromosome location and phenotypes of mutations arising, it was proposed that ACP5 and acp83 are homologous to PHO4 and pho82 genes, respectively, and ACP82--to PHO85 in the yeasts of the same line.

J Biochem (Tokyo), 1991 Apr, 109(4), 566 - 9
Subsite structure of Saccharomycopsis alpha-amylase secreted from Saccharomyces cerevisiae; Matsui I et al.; The kinetic parameters (kcat/Km) and the cleaved-bond distributions for the hydrolysis of linear maltooligosaccharides Gn (3 less than or equal to n less than or equal to 9) by Saccharomycopsis alpha-amylase (Sfamy) secreted from Saccharomyces cerevisiae were determined at pH 5.25 and 25 degrees C . The subsite affinities of Sfamy were also evaluated from these data . The subsite structure of Sfamy is characteristic of the active site of an endo-cleavage type enzyme, consisting of internal repulsive sites with the catalytic residues and external attractive sites . Moreover, the pKa values of the catalytic residues were calculated from the pH dependence plot of the kinetic parameter (kcat/Km) . The amino acid residues which contribute to the subsite affinities and the catalytic activity of Sfamy are proposed and compared with those of Taka-amylase A.

J Gen Microbiol, 1991 Apr, 137 ( Pt 4), 765 - 70
Biochemical and genetic studies on the function of, and relationship between, the PGI1- and CDC30-encoded phosphoglucose isomerases in Saccharomyces cerevisiae; Dickinson JR; Isoelectric focusing was used to compare the complement of phosphoglucose isomerase isoenzymes in a wild-type strain of Saccharomyces cerevisiae and in a strain with a deletion in the PGI1 structural gene . Deletion of the PGI1 gene did not result in the absence of the high-Km isoenzyme I but the low-Km isoenzyme II was absent . Hence, the isoenzymes must be the products of two genes . If PGI1 were the sole structural gene its deletion would result in the disappearance of both isoenzymes . After a temperature shift-up a cdc30-bearing strain had cell cycle arrested and contained only 8% of the polysaccharide in the wild-type . Phosphoglucose isomerase is required for the synthesis of fructose 6-phosphate (F6-P), a precursor of the cell wall components chitin and mannoprotein ('mannan'), which are a polysaccharide and contain polysaccharide, respectively . Since the cdc30 mutation confers a temperature-sensitive phosphoglucose isomerase, the likely explanation for cell cycle arrest caused by this mutation is that the defective phosphoglucose isomerase results in a reduction of F6-P and hence an inability to synthesize the mannan and chitin needed for cytokinesis and cell separation . Revertants of a pgi1-1 bearing strain were selected for their ability to grow on glucose at 25 degrees C and this yielded a number of different phenotypes . Amongst the isolates was a strain which had undergone an intragenic reversion at the pgi1 locus, designated pgi1-1,100 . This mutation permits growth and cell division at 25 degrees C but results in cell cycle arrest at 36 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1991 Apr, 226(1-2), 145 - 53
Characterization of products of TY1-mediated reverse transcription in Saccharomyces cerevisiae; Muller F et al.; Transposition of the yeast transposable element, Ty, has been shown to require a reverse transcription process . By analysing the extrachromosomal Ty-specific nucleic acid molecules associated with overproduced Ty virus-like particles (Ty-VLPs), we identified several reverse transcribed cDNA strands . Most of them resemble the characteristic intermediates of the reverse transcription process described for authentic retroviruses: a (-) strong-stop DNA strand covalently bound to an RNA primer, two elongated (-) strands with one or two long terminal repeat (LTR) sequences and a (+) strong-stop DNA . Surprisingly, complete (+) strands and full-length linear duplex Ty DNA could not be detected . The structural features of two additional (+) strands may indicate some differences between the mechanisms of (+) strand synthesis in Ty and other retrotransposons or retroviruses.

J Bacteriol, 1991 Apr, 173(8), 2716 - 9
A constitutive thiamine metabolism mutation, thi80, causing reduced thiamine pyrophosphokinase activity in Saccharomyces cerevisiae; Nishimura H et al.; We identified a strain carrying a recessive constitutive mutation (thi80-1) with an altered thiamine transport system, thiamine-repressible acid phosphatase, and several enzymes of thiamine synthesis from 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-beta-hydroxyethylthiazole . The mutant shows markedly reduced activity of thiamine pyrophosphokinase (EC 2.7.6.2) and high resistance to oxythiamine, a thiamine antagonist whose potency depends on thiamine pyrophosphokinase activity . The intracellular thiamine pyrophosphate content of the mutant cells grown with exogenous thiamine (2 x 10(-7) M) was found to be about half that of the wild-type strain under the same conditions . These results suggest that the utilization and synthesis of thiamine in Saccharomyces cerevisiae is controlled negatively by the intracellular thiamine pyrophosphate level.

Proc Natl Acad Sci U S A, 1991 Apr 1, 88(7), 2913 - 7
Expression of three mammalian cDNAs that interfere with RAS function in Saccharomyces cerevisiae; Colicelli J et al.; Saccharomyces cerevisiae strains expressing the activated RAS2Val19 gene or lacking both cAMP phosphodiesterase genes, PDE1 and PDE2, have impaired growth control and display an acute sensitivity to heat shock . We have isolated two classes of mammalian cDNAs from yeast expression libraries that suppress the heat shock-sensitive phenotype of RAS2Val19 strain . Members of the first class of cDNAs also suppress the heat shock-sensitive phenotype of pde1- pde2- strains and encode cAMP phosphodiesterases . Members of the second class fail to suppress the phenotype of pde1- pde2- strains and therefore are candidate cDNAs encoding proteins that interact with RAS proteins . We report the nucleotide sequence of three members of this class . Two of these cDNAs share considerable sequence similarity, but none are clearly similar to previously isolated genes.

DNA Cell Biol, 1991 Apr, 10(3), 201 - 9
Expression and functional study of wild-type and mutant human cytochrome P450c21 in Saccharomyces cerevisiae; Wu DA et al.; The most common cause of congenital adrenal hyperplasia is deficiency of cytochrome P450c21 (21-hydroxylase), which catalyzes the synthesis of adrenal steroids . We have cloned the human P450c21 cDNA into yeast expression vectors under the control of either the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) promoter or the aldehyde-dehydrogenase (ADH) promoter . P450c21 RNA, protein, and enzyme activity can be detected, indicating that both promoters drive the synthesis of P450c21 . The expressed P450c21 catalyzes the conversion of both of its substrates, with Km and Vmax values of 0.33 microM and 280 nmoles/hr.nmole of P450c21 protein for progesterone, and 0.23 microM and 450 nmoles/hr.nmole for 17-hydroxyprogesterone . These kinetic properties are similar to those of human P450c21 expressed in COS-1 cells . The microsomal fraction containing P450c21 exhibited an absorption peak at 450 nm upon binding to CO, demonstrating its hemoprotein nature . The CO-difference spectra indicated that there were about 0.08 nmole P450c21 hemoprotein/mg microsomal protein . Coupling this expression system with site-directed mutagenesis, the Asn-172 mutant of P450c21 had about 20-100 lower Vmax values; yet it retained normal affinity toward both substrates . This mutant protein also exhibited an altered absorbance with a peak at 420 nm rather than at 450 nm.

Eur J Cell Biol, 1991 Apr, 54(2), 305 - 12
Vector-mediated overexpression of catalase A in the yeast Saccharomyces cerevisiae induces inclusion body formation; Binder M et al.; To study the morphological effects of overexpression of catalase A in yeast, the gene coding for catalase A was introduced into Saccharomyces cerevisiae on a multicopy vector . After induction of microbody biogenesis and catalase A expression by growth on oleic acid as sole carbon source, cells were analyzed by immunofluorescence and immunoelectron microscopy . In addition, overexpression of catalase A was studied by quantitative immunoblotting and by activity measurement . Quantitative immunoblotting resulted in a 16-fold difference between immunoreactive material from transformed and non-transformed cells . An 18-fold increase of enzyme activity was measured in transformed cells due to overexpression of catalase A from plasmid pAH521 . Immunofluorescent staining of semithin sections of Lowicryl HM20-embedded cells with anti-catalase localized peroxisomes and--at a low percentage--larger particles . By immunoelectron microscopy, these larger structures could be identified as agranular, electron-dense aggregates which are morphologically clearly distinct from the cytoplasm and not bounded by a membrane . These structures, which have been named inclusion bodies, contain catalase A but not other peroxisomal enzymes like thiolase . These findings suggest that cells are capable of compensating for overproduced proteins by formation of particular types of structures.

Yeast, 1991 Apr, 7(3), 219 - 28
The product of the KIN1 locus in Saccharomyces cerevisiae is a serine/threonine-specific protein kinase; Lamb A et al.; The catalytic domain (30 kDa) of all protein kinases can be aligned for maximum homology, thereby revealing both invariant and highly conserved residues . The KIN1 locus from Saccharomyces cerevisiae was isolated by hybridization to a degenerate oligonucleotide encoding the conserved protein kinase domain, DVWSFG . The predicted amino acid sequence revealed significant homology to the catalytic domain of protein kinases . Using antibodies raised against a bacterial LacZ/KIN1 fusion protein, we have identified by immunoprecipitation the yeast KIN1 gene product as a 145,000 dalton protein (p145KIN1) . In exponentially growing yeast cells, the KIN1 protein is phosphorylated primarily on serine residues . The gene product of KIN1 was shown to be a serine/threonine-specific protein kinase in immune complexes, as determined by the transfer of label from {gamma-32P}ATP to either pp145KIN1 or to an exogenously added substrate, alpha-casein . The optimal metal ion concentration in this assay was 20 mM-MnCl2 . Subsequent phosphoamino acid analysis of the radiolabelled product, pp145KIN1, demonstrated that this autophosphorylation was specific for serine/threonine residues . There is no apparent difference between wild-type cells and cells containing a disrupted KIN1 gene . The biochemical characterization of protein kinases in simple eukaryotes such as yeast will aid us in determining the role of phosphorylation in cellular growth and physiology.

J Muscle Res Cell Motil, 1991 Apr, 12(2), 201 - 7
Production, isolation and characterization of human profilin from Saccharomyces cerevisiae; Aspenstrom P et al.; The yeast Saccharomyces cerevisiae was used to express human profilin cDNA . The recombinant protein, isolated by affinity chromatography on poly(L-proline)-Sepharose followed by ion exchange chromatography, associates with non-muscle actin and phosphatidylinositol-(4,5)-bisphosphate as authentic profilin.

Agric Biol Chem, 1991 Apr, 55(4), 941 - 9
The glucoamylase cDNA from Aspergillus oryzae: its cloning, nucleotide sequence, and expression in Saccharomyces cerevisiae; Hata Y et al.; A cDNA for Aspergillus oryzae glucoamylase was cloned, using oligodeoxyribonucleotide probes derived from amino sequences of peptide fragments of the enzyme . The glucoamylase cDNA, when introduced into Saccharomyces cerevisiae, directed the secretion of active glucoamylase into the culture medium . The complete nucleotide sequence of the cDNA contained an open reading frame encoding 612 amino acid residues . Comparative studies with other fungal glucoamylases showed homologies of 67% with A . niger and 30% with Rhizopus oryzae of the deduced amino acid sequences . In the five conserved regions reported in other fungal glucoamylases, the levels of homologies between those regions of A . oryzae and A . niger enzymes were much higher (78-94%) . A . oryzae glucoamylase contained no peptide region abundant in threonine and serine residue (TS-region), like that proposed to adsorb onto raw starch in A . awamori var . kawachii glucoamylase.

Appl Microbiol Biotechnol, 1991 Apr, 35(1), 46 - 30
Growth rate control in fed-batch cultures of recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg); Gu MB et al.; A recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg) exhibited growth-associated product formation . By controlling the medium feed rate, based on the calculated amount of medium required for 1 h, a constant specific growth rate was obtained in the range of 0.12-0.18 h-1 . In order to prolong the exponential growth phase, the medium feed rate was increased exponentially . A fed-batch cultivation method based on the production kinetics of batch culture enhanced HBsAg production ten times more than in batch culture . The reason for the increase can be explained by the fact that the production of HBsAg is expressed as an exponential function of time when the specific growth rate is controlled to a constant value in growth-associated product formation kinetics . In the scale-up of this culture to 91, the specific growth rate could also be maintained constant and the HBsAg production trend was similar to that in a 1-1 culture . However, ethanol accumulation occurred at a late stage in fed-bach culture . Ethanol produced was not reutilized and inhibited further cell growth.

Eur J Biochem, 1991 Mar 28, 196(3), 545 - 9
Activation and induction by copper of Cu/Zn superoxide dismutase in Saccharomyces cerevisiae . Presence of an inactive proenzyme in anaerobic yeast; Galiazzo F et al.; The Cu/Zn superoxide dismutase activity of Saccharomyces cerevisiae was found to be strictly related to the extent of oxygen metabolism, since cells grown under anaerobic or repressed conditions were found to contain 10% and 40% the activity of derepressed cells, respectively . The dependence of Cu/Zn superoxide dismutase on oxygen was found to be related to the availability of copper to the cells since the enzyme activity and immunoreactive protein measured under the various conditions was roughly proportional to the copper content of cells and in anaerobic cells a large fraction of the enzyme was found to be in the form of an inactive proenzyme which was activated by the addition of copper to cell extracts . The Cu/Zn superoxide dismutase mRNA did not parallel the dependence of the enzyme concentration on oxygen metabolism, suggesting that the gene expression was affected by copper also at the post-transcriptional level . However, under conditions of copper overloading, a more direct effect on transcription was observed and the presence of the inactive proenzyme in anaerobic cultures was associated with the over-expression of metallothionein.

FEBS Lett, 1991 Mar 25, 280(2), 367 - 70
Expression of rat liver vitamin D3 25-hydroxylase cDNA in Saccharomyces cerevisiae; Akiyoshi-Shibata M et al.; The cDNA coding for the precursor protein of rat liver mitochondrial vitamin D3 25-hydroxylase, cytochrome P450LMT25, was expressed under the control of the yeast alcohol dehydrogenase I promoter and terminator in Saccharomyces cerevisiae AH22 cells . The transformed yeast cells produced a P450LMT25 protein with an almost similar apparent molecular weight as compared with that of the authentic mature enzyme . The expression level of the P450LMT25 hemoprotein was about 5 x 10(4) molecules per cell as determined by reduced CO-difference spectra . The mitochondrial fraction prepared from the transformed yeast cells exhibited both 25-hydroxylase activity toward 1 alpha-hydroxyvitamin D3 and 27-hydroxylase activity toward 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol in a reconstituted system containing bovine adrenodoxin and NADPH-adrenodoxin reductase.

J Biol Chem, 1991 Mar 25, 266(9), 5547 - 51
Structure of Saccharomyces cerevisiae alg3, sec18 mutant oligosaccharides; Verostek MF et al.; Asparagine-linked oligosaccharides are synthesized by transfer of Glc3Man9GlcNAc2 from dolichol pyrophosphate to nascent polypeptides . Assembly of the precursor proceeds by highly ordered sequential addition of mannose and glucose to form Glc3Man9GlcNAc2-P-P-dolichol . Yeast mutants in asparagine-linked glycosylation (alg), generated by an 3H-Man suicide technique, were assigned to eight complementation groups which define steps in oligosaccharide-lipid synthesis (Huffaker, T.C., and Robbins, P.W . (1982) J . Biol . Chem . 257, 3203-3210) . Alg3 invertase oligosaccharides are resistant to endo-beta-N-acetylglucosaminidase H, and the lipid-oligosaccharide pool yields Man5Glc-NAc2, suggesting its structure may be that from mammalian cells lacking Man-P-dolichol (Chapman, A., et al . (1980) J . Biol . Chem . 255, 4441-4446) . To test this supposition, the endoplasmic reticulum form of invertase derepressed in alg3,sec18 yeast at 37 degrees C was isolated as a source of oligosaccharides whose processing beyond glucose and/or mannose trimming, if involved, would be prevented . Man8GlcNAc2 and Man5GlcNAc2 were released by peptide-N-glycosidase F from alg3,sec18 invertase in a 1:5 molar ratio . 1H NMR spectroscopy revealed Man8GlcNAc2 to be the alpha 1,2-mannosidase-trimming product described earlier (Byrd, J . C., Tarentino, A . L., Maley, F., Atkinson, P . H., and Trimble, R . B . (1982) J . Biol . Chem . 257, 14657-14666), while Man5GlcNAc2 was Man alpha 1, 2Man alpha 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc beta 1, 4GlcNAc . This provides a structural proof for the lipid-linked Man5GlcNAc2 originally proposed from enzymatic and chemical analyses of the radiolabeled mammalian precursor . Experimental evidence indicates that, unlike the mammalian cell mutants which are unable to synthesize Man-P-dolichol, alg3 yeast accumulate Man5GlcNAc2-P-P-dolichol due to a defective alpha 1,3-mannosyltransferase required for the next step in oligosaccharide-lipid elongation.

Biochemistry, 1991 Mar 19, 30(11), 2834 - 9
Disruption and mutagenesis of the Saccharomyces cerevisiae PDX1 gene encoding the protein X component of the pyruvate dehydrogenase complex; Lawson JE et al.; Disruption of the PDX1 gene encoding the protein X component of the mitochondrial pyruvate dehydrogenase (PDH) complex in Saccharomyces cerevisiae did not affect viability of the cells . However, extracts of mitochondria from the mutant, in contrast to extracts of wild-type mitochondria, did not catalyze a CoA- and NAD(+)-linked oxidation of pyruvate . The PDH complex isolated from the mutant cells contained pyruvate dehydrogenase (E1 alpha + E1 beta) and dihydrolipoamide acetyltransferase (E2) but lacked protein X and dihydrolipoamide dehydrogenase (E3) . Mutant cells transformed with the gene for protein X on a unit-copy plasmid produced a PDH complex that contained protein X and E3, as well as E1 alpha, E1 beta, and E2, and exhibited overall activity similar to that of the wild-type PDH complex . These observations indicate that protein X is not involved in assembly of the E2 core nor is it an integral part of the E2 core . Rather, protein X apparently plays a structural role in the PDH complex; i.e., it binds and positions E3 to the E2 core, and this specific binding is essential for a functional PDH complex . Additional evidence for this conclusion was obtained with deletion mutations . Deletion of most of the lipoyl domain (residues 6-80) of protein X had little effect on the overall activity of the PDH complex . This observation indicates that the lipoyl domain, and its covalently bound lipoyl moiety, is not essential for protein X function . However, deletion of the putative subunit binding domain (residues approximately 144-180) of protein X resulted in loss of high-affinity binding of E3 and concomitant loss of overall activity of the PDH complex.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1991 Mar 15, 266(8), 5342 - 7
DNA sequence recognition by a eukaryotic sequence-specific endonuclease, Endo.SceI, from Saccharomyces cerevisiae; Kawasaki K et al.; A eukaryotic sequence-specific endonuclease, Endo.SceI, causes sequence-specific double-stranded scission of double-stranded DNA to produce cohesive ends with four bases protruding at the 3' termini . Unlike in the case of restriction enzymes, an asymmetric 26-base pair consensus sequence was found around the cleavage site for Endo.SceI instead of a common sequence . We analyzed the base pairs that interacted with Endo.SceI on the recognition of its cleavage sites . A region comprising -10 through +16 base pairs from the center of the cleavage site was shown to be essential and sufficient for the sequence-specific cutting with Endo.SceI by experiments involving synthesized DNAs . Methylation interference experiments indicate that bases in the region comprising the +7 through +14 base pairs is involved in close contact with Endo.SceI in its recognition of the cleavage site . This +7 through +14-base pair region overlaps the most stringently conserved sequence in the consensus sequence for the cleavage site, suggesting that this region constitutes the core for the recognition by Endo.SceI.

J Biol Chem, 1991 Mar 15, 266(8), 5162 - 8
Saccharomyces cerevisiae transcription factors IIIB and IIIC bend the DNA of a tRNA(Gln) gene; Leveillard T et al.; The ability of Saccharomyces cerevisiae transcription factors IIIC and IIIB to bend DNA at their binding sites in a tRNA(Gln) gene has been analyzed by two methods that are based on the anomalous electrophoretic mobility of bent DNA in a tight gel network . Both transcription factors bend DNA, TFIIIB more than TFIIIC . The effective centers of the transcription factor IIIB- and IIIC-induced bends are not in helical phase.

J Biol Chem, 1991 Mar 15, 266(8), 5094 - 103
sn-1,2-diacylglycerol choline- and ethanolaminephosphotransferases in Saccharomyces cerevisiae . Nucleotide sequence of the EPT1 gene and comparison of the CPT1 and EPT1 gene products; Hjelmstad RH et al.; The complete nucleotide sequence of the Saccharomyces cerevisiae EPT1 gene, a structural gene encoding an sn-1,2-diacylglycerol ethanolamine- and cholinephosphotransferase (Hjelmstad, R . H., and Bell, R . M . (1988) J . Biol . Chem . 263, 19748-19757), was determined . The 2123-nucleotide extent of DNA sequenced contained an open reading frame encoding 391 amino acids interrupted by an intron near its 5' end . Northern hybridization analysis detected a single 1.4-kilobase transcript . The inferred 44,525-dalton EPT1 gene product exhibited 54% amino acid sequence homology to the cholinephosphotransferase product of the yeast CPT1 gene . Predictive structural analysis of the EPT1 gene product revealed close structural similarity to the CPT1 gene product with respect to membrane topography, features of secondary structure, and transmembrane asymmetry . Regional protein homologies were identified between the EPT1 gene product and several enzymes as well as the nicotinic acetylcholine receptor . Comparative analysis of this set of protein homologies and the related set of protein homologies to the CPT1 gene product permitted identification of a presumptive active site region which contains highly conserved and divergent subregions and a common mononucleotide binding site.

Mol Cell Biochem, 1991 Mar 13, 101(2), 175 - 87
Metals are directly involved in the redox interconversion of Saccharomyces cerevisiae glutathione reductase; Peinado J et al.; Redox inactivation of glutathione reductase involves metal cations, since chelators protected against NADPH-inactivation, 3 microM EDTA or 10 microM DETAPAC yielding full protection . Ag+, Zn2+ and Cd2+ potentiated the redox inactivation promoted by NADPH alone, while Cr3+, Fe2+, Fe3+, Cu+, and Cu2+ protected the enzyme . The Zn2+ and Cd2+ effect was time-dependent, unlike conventional inhibition . Glutathione reductase interconversion did not require dioxygen, excluding participation of active oxygen species produced by NADPH and metal cations . One Zn2+ ion was required per enzyme subunit to yield full NADPH-inactivation, the enzyme being reactivated by EDTA . Redox inactivation of glutathione reductase could arise from the blocking of the dithiol formed at the active site of the reduced enzyme by metal cations, like Zn2+ or Cd2+ . The glutathione reductase activity of yeast cell-free extracts was rapidly inactivated by low NADPH or moderate NADH concentrations; NADP+ also promoted rapid inactivation in fresh extracts, probably after reduction to NADPH . Full inactivation was obtained in cell-free extracts incubated with glucose-6-phosphate or 6-phosphogluconate; the inactivating efficiency of several oxidizable substrates was directly proportional to the specific activities of the corresponding dehydrogenases, confirming that redox inactivation derives from NADPH formed in vitro.

J Biol Chem, 1991 Mar 5, 266(7), 4417 - 24
Genetic and biochemical characterization of Cu,Zn superoxide dismutase mutants in Saccharomyces cerevisiae; Chang EC et al.; The allele scd 1 is a recessive chromosomal mutation in Saccharomyces cerevisiae that eliminates Cu,Zn superoxide dismutase (SOD-1) activity . SOD-1- strains are unable to grow in 100% O2 in rich medium and are methionine and lysine auxotrophic when grown in air (Bilinski, T., Krawiec, Z., Liczmanski, A., and Litwinska, J . (1985) Biochem . Biophys . Res . Commun . 130, 533-539) . In this report, scd1 was genetically mapped to the right arm of chromosome X, 11 centimorgans proximal to cdc11 . The gene for SOD-1 (SOD1) was physically mapped by Southern blot to restriction fragments containing CDC11 . scd1 failed to complement a complete deletion of SOD1 . Thus, scd1 maps to the SOD1 locus and is designated sod1-1 . The molecular basis for the lack of SOD-1 activity in sodl-1 carrying strains has also been established . The size and amount of SOD-1 mRNA in the mutant were essentially the same as in wild type cells . Western blot analysis showed that the SOD-1 dimer and 16-kilodalton subunit that co-migrated electrophoretically with wild type yeast SOD-1 were abundant in mutant cell extracts . However, two additional SOD-1 immunoreactive polypeptides were detected in these extracts in both denaturing and nondenaturing gels . None of the SOD-1 immunoreactive species in the mutant extracts exhibited superoxide dismutase activity . Transformants of the mutant strain carrying episomal, wild type SOD1 expressed wild type, active SOD-1 protein, indicating that the mutant allele had no discernible effect on the correct synthesis and activation of apoSOD-1 . Size exclusion chromatography of soluble cell extracts derived from wild type and SOD1 deletion strains identified a copper binding peak that corresponded to SOD-1 . This copper-binding fraction was absent in cell extracts from the sod1-1-containing strain although Western blot analysis of the corresponding chromatographic fractions showed that SOD-1 polypeptide was present in these fractions . Sequence data derived from the cloned genes showed that sod1-1 differed from SOD1 only in the adjacent 5'-noncoding region . The biochemical data indicate that this genetic alteration results in the synthesis of a collection of SOD-1 polypeptides that fail to bind copper and may also fail to completely self-associate . Both phenotypes could be due to the inability of these polypeptides to adopt the native SOD-1 conformation.

J Biol Chem, 1991 Mar 5, 266(7), 4357 - 65
sn-1,2-diacylglycerol choline- and ethanolaminephosphotransferases in Saccharomyces cerevisiae . Mixed micellar analysis of the CPT1 and EPT1 gene products; Hjelmstad RH et al.; The Saccharomyces cerevisiae CPT1 and EPT1 genes are structural genes encoding distinct sn-1,2-diacylglycerol choline- and ethanolaminephosphotransferases . A haploid cpt1 ept1 double null mutant lacked detectable choline- and ethanolaminephosphotransferase activity but was viable for growth, establishing that these enzymes are nonessential . The activities of the CPT1 and EPT1 gene products were independently studied in membranes prepared from strains mutant in the cognate locus using mixed micellar assays . Both enzymes absolutely required phospholipid cofactors; half-maximal activation was observed at low mole fractions, suggesting that a small number of phospholipid molecules are required . The activities of the CPT1 and EPT1 gene products were compared with respect to dioleoylglycerol dependence, CDP-aminoalcohol specificity, phospholipid activation, and inhibition by CMP . The EPT1 gene product utilized CDP-ethanolamine, -monomethylethanolamine, -dimethylethanolamine, and -choline to significant extents, while the CPT1 gene product manifested relative specificity for CDP-choline and -dimethylethanolamine . The CPT1 and EPT1 gene products exhibited differing properties with respect to phospholipid activation, but this difference was dependent on the CDP-aminoalcohol substrate . In contrast, the two enzymes could be distinguished on the basis of their dioleoylglycerol dependencies, activation by Mg2+, and CMP inhibition profiles regardless of the CDP-aminoalcohol substrate employed . These studies provide the first definitive kinetic properties of individual choline- and ethanolaminephosphotransferases.

Gene, 1991 Mar 1, 99(1), 137 - 8
Copy number and chromosomal location of Saccharomyces cerevisiae tRNA(Trp)-encoding genes; Yesland K et al.; Yeast tRNA(Trp)-encoding genes have been identified by Southern-blot analysis of chromosomal DNA . Seven copies of this gene are detected in blots of both restriction endonuclease digests and intact chromosomal DNA . Electrophoretic mapping of tDNA(Trp) indicates one copy is located on each of chromosomes X, XI, XIII, and XVI . The remaining three copies are localized to chromosomes VII and/or XV . Three different yeast strains gave identical results indicating this multi-gene family is relatively stable.

Mol Gen Genet, 1991 Mar, 225(3), 483 - 91
Mitochondrial protein import: isolation and characterization of the Saccharomyces cerevisiae MFT1 gene; Garrett JM et al.; Mitochondrial targeting of an Atp2-LacZ fusion protein confers a respiration-defective phenotype on yeast cells . This effect has been utilized to select strains that grow on nonfermentable carbon sources, some of which have decreased levels of hybrid protein localized to the organelle . Many of the mutants obtained were also temperature-sensitive for growth on all media . The recessive mft (mitochondrial fusion targeting) mutants have been assigned to three complementation groups . MFT1 was cloned and sequenced: it encodes a 255 amino acid protein that is highly basic and has no predicted membrane-spanning domains or organelle-targeting sequences . The MFT1 gene is 91% identical to an open reading frame 3' of the SIR3 gene . Evidence is presented that these two closely related genes could represent a recent gene duplication.

Genetics, 1991 Mar, 127(3), 463 - 73
Random segregation of chromatids at mitosis in Saccharomyces cerevisiae; Neff MW et al.; Previous experiments suggest that mitotic chromosome segregation in some fungi is a nonrandom process in which chromatids of the same replicative age are destined for cosegregation . We have investigated the pattern of chromatid segregation in Saccharomyces cerevisiae by labeling the DNA of a strain auxotrophic for thymidine with 5-bromodeoxyuridine . The fate of DNA strands was followed qualitatively by immunofluorescence microscopy and quantitatively by microphotometry using an anti-5-bromodeoxyuridine monoclonal antibody . Chromatids of the same replicative age were distributed randomly to daughter cells at mitosis . Quantitative measurements showed that the amount of fluorescence in the daughter nuclei derived from parents with hemilabeled chromosomes diminished in intensity by one half . The concentration of 5-bromodeoxyuridine used in the experiments had little effect on the frequency of either homologous or sister chromatid exchanges . We infer that the 5-bromodeoxyuridine was distributed randomly due to mitotic segregation of chromatids and not via sister chromatid exchanges.

J Bacteriol, 1991 Mar, 173(6), 2026 - 34
Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae; Zinser E et al.; Subcellular membranes of Saccharomyces cerevisiae, including mitochondria, microsomes, plasma membranes, secretory vesicles, vacuoles, nuclear membranes, peroxisomes, and lipid particles, were isolated by improved procedures and analyzed for their lipid composition and their capacity to synthesize phospholipids and to catalyze sterol delta 24-methylation . The microsomal fraction is heterogeneous in terms of density and classical microsomal marker proteins and also with respect to the distribution of phospholipid-synthesizing enzymes . The specific activity of phosphatidylserine synthase was highest in a microsomal subfraction which was distinct from heavier microsomes harboring phosphatidylinositol synthase and the phospholipid N-methyltransferases . The exclusive location of phosphatidylserine decarboxylase in mitochondria was confirmed . CDO-diacylglycerol synthase activity was found both in mitochondria and in microsomal membranes . Highest specific activities of glycerol-3-phosphate acyltransferase and sterol delta 24-methyltransferase were observed in the lipid particle fraction . Nuclear and plasma membranes, vacuoles, and peroxisomes contain only marginal activities of the lipid-synthesizing enzymes analyzed . The plasma membrane and secretory vesicles are enriched in ergosterol and in phosphatidylserine . Lipid particles are characterized by their high content of ergosteryl esters . The rigidity of the plasma membrane and of secretory vesicles, determined by measuring fluorescence anisotropy by using trimethylammonium diphenylhexatriene as a probe, can be attributed to the high content of ergosterol.

EMBO J, 1991 Mar, 10(3), 593 - 8
Proofreading in vivo: editing of homocysteine by methionyl-tRNA synthetase in the yeast Saccharomyces cerevisiae; Jakubowski H; Homocysteine thiolactone is a product of an error-editing reaction, catalyzed by Escherichia coli methionyl-tRNA synthetase, which prevents incorporation of homocysteine into tRNA and protein, both in vitro and in vivo . Here, the thiolactone is also shown to occur in cultures of the yeast Saccharomyces cerevisiae . In yeast, the thiolactone is made from homocysteine in a reaction catalyzed by methionyl-tRNA synthetase . One molecule of homocysteine is edited as thiolactone per 500 molecules of methionine incorporated into protein . Homocysteine, added exogenously to the medium or overproduced by some yeast mutants, is detrimental to cell growth . The cost of homocysteine editing in yeast is minimized by the presence of a pathway leading from homocysteine to cysteine, which keeps intracellular homocysteine at low levels . These results not only directly demonstrate that editing of errors in amino acid selection by methionyl-tRNA synthetase operates in vivo in yeast but also establish the importance of proofreading mechanisms in a eukaryotic organism.

Mol Cell Biol, 1991 Mar, 11(3), 1718 - 23
Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein; Koltin Y et al.; Rapamycin is a macrolide antifungal agent with structural similarity to FK506 . It exhibits potent immunosuppressive properties analogous to those of both FK506 and cyclosporin A (CsA) . Unlike FK506 and CsA, however, rapamycin does not inhibit the transcription of early T-cell activation genes, including interleukin-2, but instead appears to block downstream events leading to T-cell activation . FK506 and CsA receptor proteins (FKBP and cyclophilin, respectively) have been identified and shown to be distinct members of a class of enzymes that possess peptidyl-prolyl cis-trans isomerase (PPIase) activity . Despite the apparent differences in their mode of action, rapamycin and FK506 act as reciprocal antagonists in vivo and compete for binding to FKBP . As a means of rapidly identifying a target protein for rapamycin in vivo, we selected and genetically characterized rapamycin-resistant mutants of Saccharomyces cerevisiae and isolated a yeast genomic fragment that confers drug sensitivity . We demonstrate that the resonse to rapamycin in yeast cells is mediated by a gene encoding a 114-amino-acid, approximately 13-kDa protein which has a high degree of sequence homology with human FKBP; we designated this gene RBP1 (for rapamycin-binding protein) . The RBP1 protein (RBP) was expressed in Escherichia coli, purified to homogeneity, and shown to catalyze peptidyl-prolyl isomerization of a synthetic peptide substrate . PPIase activity was completely inhibited by rapamycin and FK506 but not by CsA, indicating that both macrolides bind to the recombinant protein . Expression of human FKBP in rapamycin-resistant mutants restored rapamycin sensitivity, indicating a functional equivalence between the yeast and human enzymes.

Mol Cell Biol, 1991 Mar, 11(3), 1295 - 305
Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes involved in morphogenesis in Saccharomyces cerevisiae; Bender A et al.; Genes CDC24 and CDC42 are required for the establishment of cell polarity and for bud formation in Saccharomyces cerevisiae . Temperature-sensitive (Ts-) mutations in either of these genes cause arrest as large, unbudded cells in which the nuclear cycle continues . MSB1 was identified previously as a multicopy suppressor of Ts- cdc24 and cdc42 mutations . We have now sequenced MSB1 and constructed a deletion of this gene . The predicted amino acid sequence does not closely resemble any other in the available data bases, and the deletion does not produce any readily detectable phenotype . However, we have used a colony-sectoring assay to identify additional genes that appear to interact with MSB1 and play a role in bud emergence . Starting with a strain deleted for the chromosomal copy of MSB1 but containing MSB1 on a high-copy-number plasmid, mutants were identified in which MSB1 had become essential for viability . The new mutations defined two genes, BEM1 and BEM2; both the bem1 and bem2 mutations are temperature sensitive and are only partially suppressed by MSB1 . In bem1 cells, a single copy of MSB1 is necessary and sufficient for viability at 23 or 30 degrees C, but even multiple copies of MSB1 do not fully suppress the growth defect at 37 degrees C . In bem2 cells, a single copy of MSB1 is necessary and sufficient for viability at 23 degrees C, multiple copies are necessary for viability at 30 degrees C, and even multiple copies of MSB1 do not suppress the growth defect at 37 degrees C . In a wild-type background (i.e., a single chromosomal copy of MSB1), both bem1 and bem2 mutations cause cells to become large and multinucleate even during growth at 23 degrees C, suggesting that these genes are involved in bud emergence . This suggestion is supported for BEM1 by other evidence obtained in a parallel study (J . Chant, K . Corrado, J . Pringle, and I . Herskowitz, submitted for publication) . BEM1 maps centromere distal to TYR1 on chromosome II, and BEM2 maps between SPT15 and STP2 on chromosome V.

Mol Cell Biol, 1991 Mar, 11(3), 1248 - 57
CAP is a bifunctional component of the Saccharomyces cerevisiae adenylyl cyclase complex; Gerst JE et al.; CAP, a protein from Saccharomyces cerevisiae that copurifies with adenylyl cyclase, appears to be required for yeast cells to be fully responsive to RAS proteins . CAP also appears to be required for normal cell morphology and responsiveness to nutrient deprivation and excess . We describe here a molecular and phenotypic analysis of the CAP protein . The N-terminal domain is necessary and sufficient for cellular response to activated RAS protein, while the C-terminal domain is necessary and sufficient for normal cellular morphology and responses to nutrient extremes . Thus, CAP is a novel example of a bifunctional component involved in the regulation of diverse signal transduction pathways.

J Bacteriol, 1991 Mar, 173(6), 2134 - 6
Expression in Escherichia coli of the Saccharomyces cerevisiae CCT gene encoding cholinephosphate cytidylyltransferase; Tsukagoshi Y et al.; The coding region of the CCT gene from the yeast Saccharomyces cerevisiae was cloned into the pUC18 expression vector . The plasmid directed the synthesis of an active cholinephosphate cytidylyltransferase in Escherichia coli, confirming that CCT is the structural gene for this enzyme . The enzyme produced in E . coli efficiently utilized cholinephosphate and N,N-dimethylethanolaminephosphate, but N-methylethanolamine-phosphate and ethanolaminephosphate were poor substrates . Consistently, disruption of the CCT locus in the wild-type yeast cells resulted in a drastic decrease in activities with respect to the former two substrates . When activity was expressed in E . coli, over 90% was recovered in the cytosol, whereas most of the activity of yeast cells was associated with membranes, suggesting that yeast cells possess a mechanism that promotes membrane association of cytidylyltransferase.

EMBO J, 1991 Mar, 10(3), 585 - 92
Negative regulation of transcription of the Saccharomyces cerevisiae catalase T (CTT1) gene by cAMP is mediated by a positive control element; Belazzi T et al.; Transcription of the CTT1 (catalase T) gene of Saccharomyces cerevisiae is controlled by oxygen via heme, by nutrients via cAMP and by heat shock . Nitrogen limitation triggers a rapid, cycloheximide-insensitive derepression of the gene . Residual derepression in a cAMP-nonresponsive mutant with attenuated protein kinase activity (bcy1 tpk1w tpk2 tpk3) demonstrates the existence of an alternative, cAMP-independent nutrient signaling mechanism . Deletion analysis using CTT1-lacZ fusion genes revealed the contribution of multiple control elements to derepression, not all of which respond to the cAMP signal . A positive promoter element responding to negative control by cAMP was inactivated by deletion of a DNA region between base pairs -340 and -364 . Upstream fragments including this element confer negative cAMP control to a LEU2-lacZ fusion gene . Northern analysis of CTT1 expression in the presence or absence of heme, 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 (37 degrees C) shows that CTT1 is only induced to an appreciable extent when at least two of the three factors contributing to its expression (oxidative stress signaled by heme, nutrient starvation (low cAMP) and heat stress) activate the CTT1 promoter.

EMBO J, 1991 Mar, 10(3), 563 - 71
Distinct cis-acting signals enhance 3' endpoint formation of CYC1 mRNA in the yeast Saccharomyces cerevisiae; Russo P et al.; The cyc1-512 mutant of the yeast Saccharomyces cerevisiae contains a 38 bp deletion in the 3' untranslated region of the CYC1 gene, resulting in CYC1 mRNAs that are elongated, presumably labile, and reduced to 10% of the normal level . Analysis with S1 nuclease and a novel PCR procedure revealed that the low amount of cyc1-512 mRNA contained many discrete 3' termini at certain sites, ranging from the wild-type position to over 2000 nucleotides (nt) downstream . The cyc1-512 mRNA deficiency was completely or almost completely restored in eight intragenic revertants that contained six different single and multiple base-pair changes within a 300 bp region downstream from the translation terminator codon . Two of the six different reversions formed the sequence TAG...TATGTA, whereas the other four reversions created the sequences TATATA or TACATA . The positions of these revertant sequences varied, even though they caused an increased use of specific major downstream mRNA 3' endpoints, apparently identical to those seen in the cyc1-512 mRNA . However, several revertants contained minor end points not corresponding to any of the cyc1-512 mRNAs . The capacity of these three signals to form 3' ends was confirmed with sequences constructed by site-directed mutagenesis . We therefore suggest that the production of 3' termini of yeast mRNA may involve at least two functionally distinct elements working in concert . One type of element determines the sites of preferred 3' mRNA termini, as represented by the cyc1-512 termini . The second type of element, which includes TAG...TATGTA and TATATA motifs, operates at a distance to enhance the use of the downstream 3' preferred sites.(ABSTRACT TRUNCATED AT 250 WORDS)

J Gen Microbiol, 1991 Mar, 137 ( Pt 3), 645 - 51
Activation of plasma membrane ATPase of Saccharomyces cerevisiae by octanoic acid; Viegas CA et al.; Plasma membrane ATPase activity of Saccharomyces cerevisiae IGC 3507III grown in the presence of the lipophilic acid octanoic acid {4-50 mg l-1 (0.03-0.35 mM), pH 4.0} was 1.5-fold higher than that in cells grown in its absence . The Km for ATP, the pH profile and the sensitivity to orthovanadate of the basal and the activated forms of the membrane ATPase were identical . This activation was closely associated with a decrease in the biomass yield and an increase in the ethanol yield, and was rapidly reversed in vivo after removal of the acid . However, the activated level was preserved when membranes were extracted and subjected to manipulations which eliminated or decreased octanoic acid incorporation in the plasma membrane . The activity of the basal plasma membrane ATPase in the total membrane fraction was slightly increased by incubation at pH 6.5 with octanoic acid at 100 mg l-1 or less (2.4 mg acid form plus 97.6 mg octanoate ion l-1) . However, destruction of the permeability barrier between the enzyme and its substrate could not explain the in vivo activation . A role for plasma membrane ATPase activation in the regulation of the intracellular pH (pHi) of cells grown with octanoic acid was not proven.

Proc Natl Acad Sci U S A, 1991 Mar 1, 88(5), 1948 - 52
FK 506-binding protein proline rotamase is a target for the immunosuppressive agent FK 506 in Saccharomyces cerevisiae; Heitman J et al.; FK 506 and cyclosporin A are potent immunosuppressive compounds that inhibit T-cell activation by interfering with signal transduction . In vitro, FK 506 binds and inhibits the activity of FK 506-binding protein (FKBP), a peptidylprolyl rotamase (cis-trans isomerase) . Cyclosporin A acts similarly on a different proline rotamase, cyclophilin . Experiments described here demonstrate genetically that FKBP is a target for FK 506 in vivo . We have isolated the gene encoding the FKBP proline rotamase (FPR1) from Saccharomyces cerevisiae . The encoded yeast protein is highly homologous with bovine and human FKBP and shares no homology with cyclophilin . Disruption of FPR1 and CPR1 (encoding cyclophilin) individually or in combination is not lethal; thus, either enzymatic proline rotamerization is not essential for life or an unknown proline rotamase can substitute for the missing enzymes . Overexpression or disruption of FPR1 confers resistance to growth inhibition by FK 506, suggesting that FKBP is a target for FK 506 in yeast . However, FKBP is only one of at least two targets because strains lacking FKBP are only partially resistant to FK 506.

J Gen Microbiol, 1991 Mar, 137 ( Pt 3), 637 - 44
Glutathione as an endogenous sulphur source in the yeast Saccharomyces cerevisiae; Elskens MT et al.; Glutathione-deficient mutants (gshA) of the yeast Saccharomyces cerevisiae, impaired in the first step of glutathione (GSH) biosynthesis were studied with respect to the regulation of enzymes involved in GSH catabolism and cysteine biosynthesis . Striking differences were observed in the content of the sulphur amino acids when gshA mutants were compared to wild-type strains growing on the same minimal medium . Furthermore, all mutants examined showed a derepression of gamma-glutamyltranspeptidase (gamm-GT), the enzyme initiating GSH degradation . However, gamma-cystathionase and cysteine synthase were unaffected by the GSH deficiency as long as the nutrient sulphate source was not exhausted . The results suggest that the mutants are probably not impaired in the sulphate assimilation pathway, but that the gamma-glutamyl cycle could play a leading role in the regulation of the sulphur fluxes . Studies of enzyme regulation showed that the derepression of gamma-GT observed in the gshA strains was most probably due to an alteration of the thiol status . The effectors governing the biosynthesis of cysteine synthase and gamma-cystathionase seemed different from those playing a role in gamma-GT regulation and it was only under conditions of total sulphate deprivation that all these enzymes were derepressed . As a consequence the endogenous pool of GSH was used in the synthesis of cysteine . GSH might, therefore, fulfil the role of a storage compound.

Appl Microbiol Biotechnol, 1991 Mar, 34(6), 756 - 64
Expression of the human blood coagulation protein factor XIIIa in Saccharomyces cerevisiae: dependence of the expression levels from host-vector systems and medium conditions; Broker M et al.; The human blood coagulation protein Factor XIIIa (FXIIIa) was expressed in Saccharomyces cerevisiae employing Escherichia coli-yeast shuttle vectors based on a 2-mu plasmid . Several factors affecting high production yield of recombinant FXIIIa were analysed . The use of the regulatable GAL-CYC1 hybrid promoter resulted in higher FXIIIa expression when compared with the constitutive ADCI promoter . Screening for suitable yeast strains for expression of FXIIIa under the transcriptional control of the GAL-CYC1 hybrid promoter revealed a broad spectrum of productivity . No obvious correlation between the expression rate and the genetic markers of the strains could be identified . The medium composition markedly influenced the FXIIIa expression rates . The expression of FXIIIa was strictly regulated by the carbon source . Glucose as the only sugar and energy source repressed the synthesis of FXIIIa, whereas addition of galactose induced FXIIIa expression . Special feeding schemes resulted in a productivity of up to 100 mg FXIIIa/l in shake flasks.

Eur J Biochem, 1991 Feb 26, 196(1), 185 - 90
Protein O-glycosylation in Saccharomyces cerevisiae . Purification and characterization of the dolichyl-phosphate-D-mannose-protein O-D-mannosyltransferase; Strahl-Bolsinger S et al.; The enzyme dolichyl-phosphate-D-mannose:protein O-D-mannosyltransferase has been solubilized from Saccharomyces cerevisiae membranes and its mannosyltransferase activity demonstrated using short peptides . The specific activity of the protein was enriched 130-fold before it was further purified by native and SDS gel chromatography . A 92-kDa band correlated well with the enzyme activity; an antibody raised against this protein precipitated the mannosyltransferase . The 92-kDa band was hydrolysed to 84 kDa after treatment with endoglycosidase F, indicating that the protein is a glycoprotein which may contain four carbohydrate chains . The purified mannosyltransferase is distinctly influenced in transfer specificity by amino acids next to serine and threonine within the acceptor peptides . Thus acidic amino acids strongly inhibit acceptor activity as do glycine and proline residues as amino-terminal and carboxy-terminal neighbours, respectively.

Nucleic Acids Res, 1991 Feb 25, 19(4), 893 - 8
Transcript levels of the Saccharomyces cerevisiae DNA repair gene RAD18 increase in UV irradiated cells and during meiosis but not during the mitotic cell cycle; Jones JS et al.; We have examined the transcript levels of the Saccharomyces cerevisiae DNA repair gene RAD18 in UV irradiated cells, in the mitotic cell cycle, and during meiosis . Levels of RAD18 mRNA increased upon UV irradiation, but remained constant during the mitotic cell cycle . During meiosis, RAD18 mRNA levels rose about 4 fold at a stage coincident with the period when high levels of genetic recombination occur . RAD18 resembles the other DNA repair genes, RAD2, RAD6, RAD7, RAD23, and RAD54, all of which also exhibit increased transcription in response to DNA damage and during meiosis . Comparisons of sequences in 5' flanking regions of RAD genes suggest that different upstream sequences are involved in regulating the expression of DNA repair genes belonging to different epistasis groups.

Proc R Soc Lond B Biol Sci, 1991 Feb 22, 243(1307), 165 - 71
The identification and purification of a mammalian-like protein kinase C in the yeast Saccharomyces cerevisiae; Simon AJ et al.; We have purified a yeast protein kinase that is phospholipid-dependent and activated by Diacylglycerol (DAG) in the presence of Ca2+ or by the tumour-promoting agent tetradecanoyl-phorbol acetate (TPA) . The properties of this enzyme are similar to those of the mammalian protein kinase C (PKC) . The enzyme was purified using chromatography on DEAE-cellulose followed by hydroxylapatite . The latter chromatography separated the activity to three distinguishable sub-species, analogous to the mammalian PKC isoenzymes . The fractions enriched in PKC activity contain proteins that specifically bind TPA, are specifically phosphorylated in the presence of DAG and recognized by anti-mammalian PKC antibodies.

Biochem J, 1991 Feb 15, 274 ( Pt 1), 199 - 205
Mitochondrial gene expression in Saccharomyces cerevisiae . Proteolysis of nascent chains in isolated yeast mitochondria optimized for protein synthesis; Black-Schaefer CL et al.; We demonstrate here that mitochondrial translation products synthesized by isolated yeast mitochondria are subject to rapid proteolysis . The loss of label from mitochondrial peptides synthesized in vitro comes from two distinct pools of peptides: one that is rapidly degraded (t1/2 of minutes) and one that is much more resistant to proteolysis (t1/2 of hours) . As the length of the incubation period increases, the percentage of labelled peptides in the rapidly-turning-over pool decreases and cannot be detected after 60 min of incubation . This proteolysis is inhibited by chloramphenicol and is dependent on the presence of ATP . The loss of label during the chase occurs from fully completed translation products . The proteolysis observed here markedly affects measurements of rates of mitochondrial protein synthesis in isolated yeast mitochondria . In earlier work, in which proteolysis was not considered, mitochondrial translation was thought to stop after 20-30 min of incubation . In the present study, by taking proteolysis into account, we demonstrate that the rate of translation in isolated mitochondria is actually constant for nearly 60 min and then decreases to near zero by 80 min of incorporation . These findings have allowed us to devise a procedure for measuring the 'true' rate of translation in isolated mitochondria . In addition, they suggest that mitochondrial translation products which normally assemble with nuclear-encoded gene products into multimeric enzyme complexes are unstable without their nuclear-encoded counterparts.

Proc Natl Acad Sci U S A, 1991 Feb 15, 88(4), 1565 - 9
Binding protein BiP is required for translocation of secretory proteins into the endoplasmic reticulum in Saccharomyces cerevisiae; Nguyen TH et al.; The endoplasmic reticulum of mammalian cells contains a heat shock protein of approximately 70 kDa (hsp70) termed binding protein BiP that is thought to promote the folding and subunit assembly of newly synthesized proteins . To study BiP function, we placed the BiP-encoding gene from Saccharomyces cerevisiae under the control of a regulated promoter and examined the effects of BiP depletion . Reduction of BiP protein to about 15% of normal levels led to a profound reduction in secretion of alpha factor and invertase . At the same time, unglycosylated precursors of these proteins accumulated intracellularly . The predominant form of the invertase precursor had undergone signal sequence cleavage but accumulated as a soluble species in the cytosol . In contrast, the alpha-factor precursor was exclusively in the signal-uncleaved form . It sedimented with microsomal membranes and was exposed at the cytoplasmic face in a protease-resistant form . These findings suggest that, in yeast, BiP function is required for translocation of soluble proteins into the endoplasmic reticulum at a stage beyond the initial nascent chain-membrane association.

Proc Natl Acad Sci U S A, 1991 Feb 15, 88(4), 1153 - 6
An Escherichia coli tyrosine transfer RNA is a leucine-specific transfer RNA in the yeast Saccharomyces cerevisiae; Edwards H et al.; While the Escherichia coli Su-3 (tyrT) tyrosine tRNA suppressor inserts only tyrosine at amber codons in E . coli, we show here that in Saccharomyces cerevisiae this tRNA inserts leucine and no significant amounts of any other amino acid . Thus, the E . coli tyrosine tRNA is functionally a leucine tRNA in yeast cytoplasm . This functional identity may correlate with a structural relationship between the E . coli tyrosine and yeast leucine tRNAs, which are both members of the uncommon type II class of tRNA structures . The results raise the possibility that in evolution a tRNA may be more closely related to a tRNA of different acceptor specificity, but of the same type class, than to one with the same amino acid specificity, but of a different type class.

Biochem Biophys Res Commun, 1991 Feb 14, 174(3), 1318 - 23
Bilateral hydrophobic zipper as a hypothetical structure which binds acidic ribosomal protein family together on ribosomes in yeast Saccharomyces cerevisiae; Tsurugi K et al.; Acidic ribosomal protein family of yeast Saccharomyces cerevisiae consists of four species of 13-kDa proteins and one species of 38-kDa protein . These proteins are thought to form a complex on ribosomes functioning in the translational elongation reaction, but the structural basis how to associate with one another is not known . In this communication, we show for the first time the presence of a structure equivalent to the leucine zipper on a putative alpha-helix in the 38-kDa acidic ribosomal protein, A0 . Then, all the 13-kDa acidic ribosomal proteins are shown to have two periodic arrays of hydrophobic amino acid residues arranged on the opposite sides of a putative alpha-helix, which is referred to as the "bilateral hydrophobic zipper" . Therefore, it is proposed that one of the 13-kDa acidic ribosomal proteins associates with 38-kDa protein A0 via the hydrophobic zipper and then the other 13-kDa proteins associate side by side via the bilateral hydrophobic zippers.

Eur J Biochem, 1991 Feb 14, 195(3), 857 - 62
Isolation and inactivation of the nuclear gene encoding the rotenone-insensitive internal NADH: ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae; Marres CA et al.; We have recently described the isolation of a mitochondrial rotenone-insensitive NADH:ubiquinone oxidoreductase from Saccharomyces cerevisiae {de Vries, S . & Grivell, L . A . (1988) Eur . J . Biochem . 176, 377-384} . We now report the isolation of the nuclear gene encoding this single-subunit enzyme . Null mutants have been constructed by means of one-step gene disruption . Oxygen-uptake experiments, performed with mitochondria isolated from the mutant cells, showed that this NADH dehydrogenase catalyzes the oxidation of NADH generated inside the mitochondrion . Inactivation of this NADH dehydrogenase does not affect growth on glucose and ethanol, but growth on lactate, pyruvate and acetate is impaired or absent . This phenotype is discussed in terms of the interplay between different metabolic pathways in yeast.

J Biol Chem, 1991 Feb 5, 266(4), 2537 - 41
Purification and characterization of the Saccharomyces cerevisiae mitochondrial leucyl-tRNA synthetase; Zagorski W et al.; We have purified the product of the NAM2 gene, the mitochondrial leucyl-tRNA synthetase, from yeast mitochondria . The purified protein cross-reacts with antibodies raised against the product of a LacZ/NAM2 gene fusion and antibodies raised against the purified Escherichia coli leucyl-tRNA synthetase . The mass as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is about 100 kDa, consistent with the size predicted by the gene sequence (102 kDa) . The N-terminal sequence of the protein has been determined and shows that the first nine amino acids predicted by the gene sequence have been removed, probably during transport into the mitochondria.

J Biol Chem, 1991 Feb 5, 266(4), 2339 - 45
Isolation, nucleotide sequence, and disruption of the Saccharomyces cerevisiae gene encoding mitochondrial NADP(H)-specific isocitrate dehydrogenase; Haselbeck RJ et al.; Mitochondrial NADP(H)-specific isocitrate dehydrogenase (IDP1) was purified from yeast cells grown with acetate as a carbon source . IDP1 was shown to be a dimer with a subunit molecular weight of approximately 45,000 . Immunochemical levels of IDP1 were found to vary in inverse proportion with those of mitochondrial NAD(H)-specific isocitrate dehydrogenase in cells grown with glucose or with acetate as a carbon source . A 20-residue amino-terminal sequence was obtained for IDP1, and degenerate oligonucleotides were used to synthesize a 50-base pair polymerase chain reaction product corresponding to the coding region for a portion of the amino terminus . The 50-base pair DNA fragment was used as a hybridization probe to identify plasmids containing the IDP1 gene in a yeast genomic DNA library . The complete nucleotide sequence of the IDP1 coding region was determined and translated into a 412-residue amino acid sequence for the mature protein which is preceded by a putative 16-residue mitochondrial targeting presequence . A haploid yeast strain containing a chromosomal disruption of the IDP1 locus was constructed and found to be capable of growth with glucose but not with other carbon sources, suggesting that IDP1 provides a critical function and may be the primary source of NADPH in yeast mitochondria.

Acta Med Okayama, 1991 Feb, 45(1), 1 - 10
Expression of the hepatitis B surface antigen gene containing the preS2 region in Saccharomyces cerevisiae; Yoshida I et al.; We constructed a plasmid, pBH103-ME5, in which the region encoding the 10 preS2 amino acid residues and the S domain of the hepatitis B surface antigen (HBsAg) were regulated by the promoter of the yeast repressible acid phosphatase gene . Saccharomyces cerevisiae carrying pBH103-ME5 produced the HBs antigen (yHBsAg), when it was cultured in a medium containing a low concentration of phosphate . The antigen was purified to homogeneity . Its molecular weight was determined by Western blotting to be 24,000, and its amino acid composition agreed well with that deduced from the nucleotide sequence . The C-terminal amino acid sequence of yHBsAg was exactly the same as that predicted from the nucleotide sequence, while the N-terminal amino acid acetylserine, which was followed by 8 amino acid residues coded by the preS2 region . These results indicate that the recombinant yeast produced a single polypeptide consisting of the preS2 region and the subsequent S domain after being processed at the N-terminus.

Yeast, 1991 Feb, 7(2), 79 - 90
Sequence and genetic analysis of NHP2: a moderately abundant high mobility group-like nuclear protein with an essential function in Saccharomyces cerevisiae; Kolodrubetz D et al.; In order to determine the biological functions of moderately abundant, high mobility group (HMG)-like nuclear proteins, a genetic approach has been taken . The gene for one such protein, NHP2, has been cloned and characterized from Saccharomyces cerevisiae . NHP2 has been called 'HMG-like' because of the physical/chemical properties it shares with the HMG proteins from higher eukaryotic cells . However, nucleotide sequence analysis revealed that NHP2 could encode a 17.1 kilodalton basic protein which was not significantly homologous to any previously sequenced HMG proteins . Thus NHP2 defines a new member of the HMG class of proteins . A search of protein databases showed that the amino acid sequence of NHP2 shared significant identities with two ribosomal proteins; the acidic ribosomal protein S6 from Halobacterium marismorium and protein L7a from mammals . The biological relevance of these homologies is unclear since previous biochemical results indicated that NHP2 was not a ribosomal protein . S1 nuclease analysis indicated that the gene contained no introns but had multiple transcription initiation sites 20 to 40 bases before the ATG codon . Finally, NHP2 has been shown to have a critical role in the cell; when a diploid yeast strain deleted of one copy of the NHP2 gene was sporulated and dissected, only half of the spores grew into normal colonies . The rest of the spores germinated, but only formed microcolonies containing 12 to 40 cells . None of the spores which grew into normal-sized colonies contained the mutant NHP2 gene, thus demonstrating that the NHP2 protein has an essential physiological function.

Yeast, 1991 Feb, 7(2), 119 - 25
Production of the STA2-encoded glucoamylase in Saccharomyces cerevisiae is subject to feed-back control; Suntsov NI et al.; Three modes of production of the extracellular glucoamylase (GA) in Saccharomyces cerevisiae have been identified; repressed, basal and induced . The repressed mode is found with cells grown in rich media containing non-limiting concentrations of monosaccharides or disaccharides, including GA-hydrolysable maltose, as a sole carbon source . Both the basal and the induced modes (spanned by some seven-fold differences in the rate of GA production) can be displayed by either glucose-limited or glycerol-plus ethanol-consuming cultures; the induced mode is switched over to the basal one due to a feed-back inhibition by extracellularly accumulated GA . It is proposed that the feed-back control involved in GA production can be attenuated by starch which can thus 'induce' higher rates of GA production compared to the basal mode.

FEMS Microbiol Lett, 1991 Feb, 62(1), 89 - 93
The osmotic responses of Saccharomyces cerevisiae in K(+)-depleted medium; Meikle AJ et al.; Saccharomyces cerevisiae showed reduced growth in a K(+)-depleted medium but was still capable of synthesizing and accumulating glycerol in the presence of 2.28% (w/v) NaCl; glycerol levels were similar in both K(+)-replete and K(+)-depleted media . The activity of glycerol-3-phosphate dehydrogenase, a key enzyme of glycerol synthesis, was inhibited to a similar extent by exogenous K+ and Na+ although such effects may be modified in vivo due to ionic compartmentation within the vacuole . These results indicate that exogenous K+ does not play a significant role in the initiation of glycerol synthesis in osmotically-stressed yeast.

Genetics, 1991 Feb, 127(2), 279 - 85
Genetic analysis of Saccharomyces cerevisiae chromosome I: on the role of mutagen specificity in delimiting the set of genes identifiable using temperature-sensitive-lethal mutations; Harris SD et al.; In a previous attempt to identify as many as possible of the essential genes on Saccharomyces cerevisiae chromosome I, temperature-sensitive (Ts-) lethal mutations that had been induced by ethyl methane-sulfonate or nitrosoguanidine were analyzed . Thirty-two independently isolated mutations that mapped to chromosome I identified only three complementation groups, all of which had been known previously . In contrast, molecular analyses of segments of the chromosome have suggested the presence of numerous additional essential genes . In order to assess the degree to which problems of mutagen specificity had limited the set of genes detected using Ts- lethal mutations, we isolated a new set of such mutations after mutagenesis with UV or nitrogen mustard . Surprisingly, of 21 independently isolated mutations that mapped to chromosome I, 17 were again in the same three complementation groups as identified previously, and two of the remaining four mutations were apparently in a known gene involved in cysteine biosynthesis . Of the remaining two mutations, one was in one of the essential genes identified in the molecular analyses, and the other was too leaky to be mapped . These results suggest that only a minority of the essential genes in yeast can be identified using Ts- lethal mutations, regardless of the mutagen used, and thus emphasize the need to use multiple genetic strategies in the investigation of cellular processes.

Oncogene, 1991 Feb, 6(2), 347 - 9
The COOH-domain of the product of the Saccharomyces cerevisiae SCD25 gene elicits activation of p21-ras proteins in mammalian cells; Rey I et al.; The evidence presented here indicates that the domain containing the COOH-terminal part of the Saccharomyces cerevisiae SCD25 gene product (C-domain), which is homologous to the COOH-terminal part of CDC25 protein, can elicit activation of mammalian ras proteins in CHO cells . Transfection of expression vectors carrying the C-domain of SCD25, but not of CDC25, promotes the GTP-bound form of ras proteins as determined by analysis of the guanine nucleotides bound to ras proteins immunoprecipitated by Y13-259 mAb, and enhances transcription of a HIV-LTR-CAT construct . This is the first demonstration of the activation of ras proteins by transfection of a single heterologous gene.

J Cell Biol, 1991 Feb, 112(4), 535 - 44
Cellular morphogenesis in the Saccharomyces cerevisiae cell cycle: localization of the CDC3 gene product and the timing of events at the budding site; Kim HB et al.; Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud . Electron microscopic observations suggest that these filaments assemble at the budding site coincident with bud emergence and disassemble shortly before cytokinesis (Byers, B . and L . Goetsch . 1976 . J . Cell Biol . 69:717-721) . Mutants defective in any of four genes (CDC3, CDC10, CDC11, or CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and an inability to complete cytokinesis . We showed previously by immunofluorescence that the CDC12 gene product is probably a constituent of the ring of 10-nm filaments (Haarer, B . and J . Pringle . 1987 . Mol . Cell . Biol . 7:3678-3687) . We now report the use of fusion proteins to generate polyclonal antibodies specific for the CDC3 gene product . In immunofluorescence experiments, these antibodies decorated the neck regions of wild-type and mutant cells in patterns suggesting that the CDC3 gene product is also a constituent of the ring of 10-nm filaments . We also used the CDC3-specific and CDC12-specific antibodies to investigate the timing of localization of these proteins to the budding site . The results suggest that the CDC3 protein is organized into a ring at the budding site well before bud emergence and remains so organized for some time after cytokinesis . The CDC12 product appears to behave similarly, but may arrive at the budding site closer to the time of bud emergence, and disappear from that site more quickly after cytokinesis, than does the CDC3 product . Examination of mating cells and cells responding to purified mating pheromone revealed novel arrangements of the CDC3 and CDC12 products in the regions of cell wall reorganization . Both proteins were present in normal-looking ring structures at the bases of the first zygotic buds.

Mol Cell Biol, 1991 Feb, 11(2), 822 - 32
The URE2 gene product of Saccharomyces cerevisiae plays an important role in the cellular response to the nitrogen source and has homology to glutathione s-transferases; Coschigano PW et al.; The URE2 gene of Saccharomyces cerevisiae has been cloned and sequenced . It encodes a predicted polypeptide of 354 amino acids with a molecular weight of 40,226 . Deletion of the first 63 amino acids does not have any effect on the function of the protein . Studies with disruption alleles of the URE2 and GLN3 genes showed that both genes regulate GLN1 and GDH2, the structural genes for glutamine synthetase and NAD-linked glutamate dehydrogenase, respectively, at the transcriptional level, but expression of the regulatory genes does not appear to be regulated . Active URE2 gene product was required for the inactivation of glutamine synthetase upon addition of glutamine to cells growing with glutamate as the source of nitrogen . The predicted URE2 gene product has homology to glutathione S-transferases . The gene has been mapped to chromosome XIV, 5.9 map units from petX and 3.4 map units from kex2.

Mol Cell Biol, 1991 Feb, 11(2), 785 - 94
Specific cis-acting sequence for PHO8 expression interacts with PHO4 protein, a positive regulatory factor, in Saccharomyces cerevisiae; Hayashi N et al.; The PHO8 gene of Saccharomyces cerevisiae encodes repressible alkaline phosphatase (rALPase; EC 3.1.3.1) . The rALPase activity of the cells is two to three times higher in medium containing a low concentration of Pi than in high-Pi medium due to transcription of PHO8 . The Pi signals are conveyed to PHO8 by binding of PHO4 protein, a positive regulatory factor, to a promoter region of PHO8 (PHO8p) under the influence of the PHO regulatory circuit . Deletion analysis of PHO8p DNA revealed two separate regulatory regions required for derepression of rALPase located at nucleotide positions -704 to -661 (distal region) and -548 to -502 (proximal region) and an inhibitory region located at -421 to -289 relative to the translation initiation codon . Gel retardation experiments showed that a beta-galactosidase-PHO4 fusion protein binds to a 132-bp PHO8p fragment bearing the proximal region but not to a 226-bp PHO8 DNA bearing the distal region . The fusion protein also binds to a synthetic oligonucleotide having the same 12-bp nucleotide sequence as the PHO8p DNA from positions -536 to -525 . The 132-bp PHO8p fragment, connected at position -281 of the 5' upstream region of a HIS5'-'lacZ fused gene, could sense Pi signals in vivo, but a 20-bp synthetic oligonucleotide having the same sequence from -544 to -525 of the PHO8p DNA could not . Linker insertions in the PHO8p DNA indicated that the 5-bp sequence 5'-CACGT-3' from positions -535 to -531 is essential for binding the beta-galactosidase-PHO4 fusion protein and for derepression of rALPase.

Mol Cell Biol, 1991 Feb, 11(2), 737 - 45
Repair of specific base pair mismatches formed during meiotic recombination in the yeast Saccharomyces cerevisiae; Detloff P et al.; Heteroduplexes formed between DNA strands derived from different homologous chromosomes are an intermediate in meiotic crossing over in the yeast Saccharomyces cerevisiae and other eucaryotes . A heteroduplex formed between wild-type and mutant genes will contain a base pair mismatch; failure to repair this mismatch will lead to postmeiotic segregation (PMS) . By analyzing the frequency of PMS for various mutant alleles in the yeast HIS4 gene, we showed that C/C mismatches were inefficiently repaired relative to all other point mismatches . These other mismatches (G/G, G/A, T/T, A/A, T/C, C/A, A/A, and T/G) were repaired with approximately the same efficiency . We found that in spores with unrepaired mismatches in heteroduplexes, the nontranscribed strand of the HIS4 gene was more frequently donated than the transcribed strand . In addition, the direction of repair for certain mismatches was nonrandom.

Mol Cell Biol, 1991 Feb, 11(2), 721 - 30
Characterization of RPR1, an essential gene encoding the RNA component of Saccharomyces cerevisiae nuclear RNase P; Lee JY et al.; RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein . We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPR1 (RNase P ribonucleoprotein 1) . Gene disruption experiments showed RPR1 to be single copy and essential . Characterization of the gene region located RPR1 600 bp downstream of the URA3 coding region on chromosome V . We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPR1 RNA . The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPR1 transcript is subjected to multiple processing steps to obtain the 369-nucleotide form . Complementation of RPR1-disrupted haploids with one variant of RPR1 gave a slow-growth and temperature-sensitive phenotype . This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPR1 RNA is an essential component of this enzyme.

Mol Cell Biol, 1991 Feb, 11(2), 1161 - 6
The DAL81 gene product is required for induced expression of two differently regulated nitrogen catabolic genes in Saccharomyces cerevisiae; Bricmont PA et al.; We demonstrate that the DAL81 gene, previously thought to be specifically required for induced expression of the allantoin pathway genes in Saccharomyces cerevisiae, functions in a more global manner . The data presented show it to be required for utilization of 4-aminobutyrate as a nitrogen source and for 4-aminobutyrate-induced increases in the steady-state levels of UGA1 mRNA . The DAL81 gene encodes a 970-amino-acid protein containing sequences homologous to the Zn(II)2Cys6 motif and two stretches of polyglutamine residues . Deletion of sequences homologous to the Zn(II)2Cys6 motif did not result in a detectable loss of function . On the other hand, loss of one of the polyglutamine stretches, but not the other, resulted in a 50% loss of DAL81 function.

Mol Cell Biol, 1991 Feb, 11(2), 1080 - 91
A role for CDC7 in repression of transcription at the silent mating-type locus HMR in Saccharomyces cerevisiae; Axelrod A et al.; The mating-type genes at MAT in Saccharomyces cerevisiae are expressed, whereas the same genes located at HML and HMR are transcriptionally repressed . The DNA element responsible for repression at HMR has been termed a silencer and contains an autonomous replication sequence, a binding site for GRFI/RAPI, and a binding site for ABFI . A double-mutant HMR-E silencer that contains single nucleotide substitutions in both the GRFI/RAPI- and ABFI-binding sites no longer binds either factor in vitro, nor represses transcription at HMR in vivo . In MAT alpha cells, this derepression of a information results in a nonmating phenotype . Second-site suppressor mutations were isolated that restored the alpha mating phenotype to MAT alpha cells containing the double-mutant silencer . One of these suppressors, designated sas1-1, conferred a temperature-sensitive lethal phenotype to the cell . SAS1 was found to be identical to CDC7, a gene which encodes a protein kinase required for the initiation of DNA replication . This new allele of CDC7 was designated cdc7-90 . cdc7-90 restored the alpha mating phenotype by restoring silencing . The original allele of CDC7, isolated on the basis of the cell cycle phenotype it confers, also restored silencing, and overexpression of CDC7 interfered with silencing . cdc7-90 did not restore detectable binding of GRFI/RAPI or ABFI to the double-mutant silencer in vitro . These results indicate that a reduced level of CDC7 function restores silencing to a locus defective in binding two factors normally required for silencing.

Mol Cell Biol, 1991 Feb, 11(2), 1030 - 9
Degradation of a-factor by a Saccharomyces cerevisiae alpha-mating-type-specific endopeptidase: evidence for a role in recovery of cells from G1 arrest; Marcus S et al.; Mating response between opposite mating types of Saccharomyces cerevisiae is dependent upon alpha factor, a tridecapeptide, and a-factor, an isoprenylated, methyl esterified dodecapeptide whose interaction with the alpha target cell has not been characterized . We report on the first biochemical and physiological evidence of an alpha-mating-type-specific a-factor-degrading activity . Radioiodinated a-factor was used to identify the a-factor-degrading activity, which is cell associated, endoproteolytic, and not required for response to pheromone . a-factor degradation was not energy dependent, nor did it require pheromone internalization or interaction with its receptor . Phenylmethylsulfonyl fluoride and tosyl-L-arginyl-methyl ester inhibited degradation of a-factor and increased the time required by alpha cells to recover from a-factor-induced growth arrest and morphological alteration, providing evidence that a-factor degradation plays a role in the recovery of alpha cells from the pheromone response.

J Toxicol Sci, 1991 Feb, 16 Suppl 1, 75 - 82
Characterization and expression of RAD4 gene involved in nucleotide excision repair of UV-damaged Saccharomyces cerevisiae; Choi IS et al.; Saccharomyces cerevisiae express RAD4 gene for nucleotide excision repair of UV-induced DNA damages . Upon complementation with rad4-4 mutant, a 7.6 kb clone containing the RAD4 gene designated as pPC1 was isolated from a yeast genomic library . The pPC1 was further narrowed to 2.5 kb flanked with BglII and BamHI sites . The cloned RAD4 gene was found to propagate in E . coli without loss of its complementing activity . Pulse-field gel electrophoresis indicated that the cloned RAD4 gene was localized in the right arm of chromosome V . DNA-tRNA hybridization revealed that the cloned gene did not contain a suppressor tRNA gene . The rad4 mutants with various plasmids containing the cloned RAD4 gene, regardless of their copy number, had enhanced resistance against UV damages equivalent to that found in wild type . As determined by S1 nuclease digestion, the RAD4 transcript was found to be 2.3 kb in size and the S1 nuclease mapping revealed the production of a protected fragment of 760 nucleotides within the transcript . Transcriptional start point was found at 48 base pairs upstream from the first ATG codon of the translation initiation codon . The overexpressed Rad4 protein was estimated to be 89 kD and confirmed the expected size based on the actual length of RAD4 gene . Upon stationary phase culturing, E . coli cells transformed with the cloned RAD4 gene had a delayed entrance into exponential growth phase and produced reduced amount of host proteins . These results have indicated that the pPC1 is a functional RAD4 gene playing a unique role involved in the nucleotide excision repair of yeast without any genetic change during amplication in E . coli.

Yeast, 1991 Feb, 7(2), 157 - 65
Influence of the codon following the initiation codon on the expression of the lacZ gene in Saccharomyces cerevisiae; Looman AC et al.; A set of 32 different codons were introduced in a lacZ expression vector (pPTK400) immediately 3' from the AUG initiation codon . Expression of the lacZ gene was determined in Saccharomyces cerevisiae by measuring the amount of beta-galactosidase fusion protein using immuno-gel electrophoresis . A 5.3-fold difference in expression was found among the various constructs . It was found that there was no preference for a certain nucleotide in any position of the second codon and there was no distinct correlation between the level of tRNA corresponding to any particular second codon and expression . No correlation could be found between the local secondary structure and expression . When the overall codon usage in yeast and the codon usage in the second position of the mRNA is compared, there is no obvious significant difference in preference . This indicates that in yeast, in contrast to Escherichia coli, the codon choice at the beginning of the mRNA does not deviate from the one further downstream and is determined by the requirements for optimal translation elongation . Important determinants of the optimal context for an initiation codon in yeast therefore must be located mainly 5' from this codon.

Mol Cell Biol, 1991 Feb, 11(2), 987 - 1001
Spk1, a new kinase from Saccharomyces cerevisiae, phosphorylates proteins on serine, threonine, and tyrosine; Stern DF et al.; A Saccharomyces cerevisiae lambda gt11 library was screened with antiphosphotyrosine antibodies in an attempt to identify a gene encoding a tyrosine kinase . A subclone derived from one positive phage was sequenced and found to contain an 821-amino-acid open reading frame that encodes a protein with homology to protein kinases . We tested the activity of the putative kinase by constructing a vector encoding a glutathione-S-transferase fusion protein containing most of the predicted polypeptide . The fusion protein phosphorylated endogenous substrates and enolase primarily on serine and threonine . The gene was designated SPK1 for serine-protein kinase . Expression of the Spk1 fusion protein in bacteria stimulated serine, threonine, and tyrosine phosphorylation of bacterial proteins . These results, combined with the antiphosphotyrosine immunoreactivity induced by the kinase, indicate that Spk1 is capable of phosphorylating tyrosine as well as phosphorylating serine and threonine . In in vitro assays, the fusion protein kinase phosphorylated the synthetic substrate poly(Glu/Tyr) on tyrosine, but the activity was weak compared with serine and threonine phosphorylation of other substrates . To determine if other serine/threonine kinases would phosphorylate poly(Glu/Tyr), we tested calcium/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase . The two kinases had similar tyrosine-phosphorylating activities . These results establish that the functional difference between serine/threonine- and tyrosine-protein kinases is not absolute and suggest that there may be physiological circumstances in which tyrosine phosphorylation is mediated by serine/threonine kinases.

Mol Cell Biol, 1991 Feb, 11(2), 699 - 704
The Saccharomyces cerevisiae ADR1 gene is a positive regulator of transcription of genes encoding peroxisomal proteins; Simon M et al.; Expression of the CTA1 gene of Saccharomyces cerevisiae, encoding catalase A, the peroxisomal catalase of this yeast, is sensitive to glucose repression . A DNA fragment cloned as a multicopy plasmid suppressing the glucose repression of CTA1 transcription was demonstrated to contain the ADR1 gene . Multiple copies of ADR1 increased catalase A formation not only on 10% glucose, but also on ethanol medium and in the presence of oleic acid, an inducer of peroxisome proliferation . Compared with wild-type cells, adr1 null mutants produced by disruption of the gene exhibit reduced CTA1 expression . This demonstrates that ADR1 is a true positive regulator of CTA1 . Further experiments showed that it acts directly on CTA1 . Alcohol dehydrogenase II, which is under ADR1 control, was excluded as a mediator of the effect on CTA1; deletion of bases -123 to -168 of CTA1 reduces expression and eliminates the response to the ADR1 multicopy plasmid without eliminating fatty acid induction; and gel retardation experiments demonstrated that ADR1 binds to a CTA1 upstream fragment (-156 to -184) with limited similarity to the ADR1 binding site of ADH2 . Northern hybridization experiments further demonstrated that expression of two genes encoding enzymes of peroxisomal beta-oxidation (beta-ketothiolase, trifunctional enzyme) and of a gene involved in peroxisome assembly (PAS1) is also negatively affected by the adr1 null mutation . These findings demonstrate that the ADR1 protein has much broader regulatory functions than previously recognized.

Mol Cell Biol, 1991 Feb, 11(2), 1062 - 8
Heat shock proteins affect RNA processing during the heat shock response of Saccharomyces cerevisiae; Yost HJ et al.; In the yeast Saccharomyces cerevisiae, the splicing of mRNA precursors is disrupted by a severe heat shock . Mild heat treatments prior to severe heat shock protect splicing from disruption, as was previously reported for Drosophila melanogaster . In contrast to D . melanogaster, protein synthesis during the pretreatment is not required to protect splicing in yeast cells . However, protein synthesis is required for the rapid recovery of splicing once it has been disrupted by a sudden severe heat shock . Mutations in two classes of yeast hsp genes affect the pattern of RNA splicing during the heat shock response . First, certain hsp70 mutants, which overproduce other heat shock proteins at normal temperatures, show constitutive protection of splicing at high temperatures and do not require pretreatment . Second, in hsp104 mutants, the recovery of RNA splicing after a severe heat shock is delayed compared with wild-type cells . These results indicate a greater degree of specialization in the protective functions of hsps than has previously been suspected . Some of the proteins (e.g., members of the hsp70 and hsp82 gene families) help to maintain normal cellular processes at higher temperatures . The particular function of hsp104, at least in splicing, is to facilitate recovery of the process once it has been disrupted.

Biochimie, 1991 Feb-Mar, 73(2-3), 285 - 8
Expression of the Escherichia coli recA gene in the yeast Saccharomyces cerevisiae; Cernakova L et al.; The isolation of the protein coding region of the recA gene from Escherichia coli by extensive Bal31 digestion is described . The structural recA gene was ligated into an extrachromosomally replicating yeast expression vector, downstream of the yeast alcohol-dehydrogenase gene promoter region, to produce pADHrecA plasmid . The pADHrecA plasmid was transformed into the wild-type and the repair deficient strains of Saccharomyces cerevisiae . The crude protein samples were extracted from the individual yeast transformants . A 38 kDa protein was present in all transformants containing the recA gene on plasmid . Thus the recA gene from E coli was successfully expressed in cells from a lower eukaryote.

Biochem J, 1991 Feb 1, 273 ( Pt 3), 615 - 20
Partial purification and substrate specificity of a ubiquitin hydrolase from Saccharomyces cerevisiae; Agell N et al.; A ubiquitin hydrolase that removes ubiquitin from a multi-ubiquitinated protein has been purified 600-fold from Saccharomyces cerevisiae . Four different ubiquitin-protein conjugates were assayed as substrates during the purification procedure . Enzymic activities that removed ubiquitin from ubiquitinated histone H2A, a ubiquitin-ubiquitin dimer and a ubiquitin-ribosomal fusion protein were separated during the purification from an activity that removed a single ubiquitin molecule linked by an isopeptide bond to a ubiquitinated protein . The size of the native enzyme was 160 kDa, based on its sedimentation in a sucrose gradient, and the subunit molecular mass was estimated to be 160 kDa, based on a profile of proteins eluted in different fractions by thiol-affinity chromatography . The partially purified hydrolase was not inhibited by a variety of protease inhibitors, except for thiol-blocking reagents . The natural substrate for this enzyme may be the polyubiquitin chain containing ubiquitin molecules bound to each other in isopeptide bonds, with one of them linked to a lysine residue of a protein targeted for intracellular proteolysis.

J Bacteriol, 1991 Feb, 173(3), 1035 - 40
Induction and substrate specificity of the lanosterol 14 alpha-demethylase from Saccharomyces cerevisiae Y222; Wright GD et al.; The potential inducibility of the lanosterol 14 alpha-demethylase (P-45014DM) from Saccharomyces cerevisiae Y222 by xenobiotics was investigated . This enzyme and NADPH-cytochrome P-450 reductase were unaffected by a number of compounds known to induce mammalian and some yeast cytochrome P-450 monooxygenases . Furthermore, dibutyryl cyclic AMP did not affect P-45014DM or P-450 reductase levels, while growth at 37 degrees C resulted in a slight decrease . P-45014DM was found to be specific for lanosterol and did not metabolize a number of P-450 substrates including benzo{a}pyrene.

Mol Cell Biol, 1991 Feb, 11(2), 746 - 53
Suppressor analysis of temperature-sensitive RNA polymerase I mutations in Saccharomyces cerevisiae: suppression of mutations in a zinc-binding motif by transposed mutant genes; McCusker JH et al.; Starting with two temperature-sensitive mutants (rpa190-1 and rpa190-5) of Saccharomyces cerevisiae, both of which are amino acid substitutions in the putative zinc-binding domain of the largest subunit (A190) of RNA polymerase I, we have isolated many independent pseudorevertants carrying extragenic suppressors (SRP) of rpa190 mutations . All the SRP mutations were dominant over the corresponding wild-type genes . They were classified into at least seven different loci by crossing each suppressed mutant with all of the other suppressed mutants and analyzing segregants . SRP mutations representing each of the seven loci were studied for their effects on other known rpa190 mutations . All of the SRP mutations were able to suppress both rpa190-1 and rpa190-5 . In addition, one particular suppressor, SRP5, was found to suppress two other rpa190 mutations as well as an rpa190 deletion . Southern blot analysis combined with genetic crosses demonstrated that SRP5 maps to a region on chromosome XV loosely linked to rpa190 and represents a transposed mutant gene in two copies . Analysis of the A190 subunit by using anti-A190 antiserum indicated that the cellular concentration of A190 and hence of RNA polymerase I decreases in rpa190-1 mutants after a shift to 37 degrees C and that in the mutant strain carrying SRP5 this decrease is partially alleviated, presumably because of increased synthesis caused by increased gene dosage . These results suggest that the zinc-binding domain plays an important role in protein-protein interaction essential for the assembly and/or stability of the enzyme, regardless of whether it also participates directly in the interaction of the assembled enzyme with DNA.

Mol Cell Biol, 1991 Feb, 11(2), 666 - 76
Two types of TATA elements for the CYC1 gene of the yeast Saccharomyces cerevisiae; Li WZ et al.; Functional TATA elements in the 5' untranslated region of the CYC1 gene in the yeast Saccharomyces cerevisiae have been defined by transcriptional analysis of site-directed mutations . Five sites previously suggested to contain functional TATA elements were altered individually and in all possible combinations . The results indicated that only two elements are required for transcription at the normal level and the normal start sites . The two functional TATA elements are located at sites -178 and -123, where the A of the ATG start codon is assigned nucleotide position +1 . They direct initiation within windows encompassing -70 to -46 and -46 to -28, respectively . Only when both of the upstream TATA sites were rendered nonfunctional were the third and fourth downstream TATA-like sequences activated, as indicated by the presence of low levels of transcription starting at -28 . The two upstream functional TATA elements differed in sequence . The sequence of the most 5' one at site 1, denoted beta-type, was ATATATATAT, whereas that of the second one at site 2, denoted alpha-type, was TATATAAAA . The following rearrangements of the beta-type and alpha-type elements at two sites (1 and 2) were examined: site1 beta-site2 alpha; site 1 alpha-site 2 beta; site1 alpha-site2 alpha; and site1 beta-site2 beta . When different types were at different sites (site1 beta-site2 alpha and site1 alpha-site2 beta), both were used equally . In contrast, when the same type was present at both sites (site1 alpha-site2 alpha and site1 beta-site2 beta), only the upstream element was used . We suggest that the two TATA elements are recognized by different factors of the transcription apparatus.

Genetics, 1991 Feb, 127(2), 287 - 98
Molecular analysis of Saccharomyces cerevisiae chromosome I: identification of additional transcribed regions and demonstration that some encode essential functions; Diehl BE et al.; Saccharomyces cerevisiae chromosome I has provided a vivid example of the "gene-number paradox." Although molecular studies have suggested that there are greater than 100 transcribed regions on the chromosome, classical genetic studies have identified only about 15 genes, including just 6 identified in intensive studies using Ts- lethal mutations . To help elucidate the reasons for this disparity, we have undertaken a detailed molecular analysis of a 34-kb segment of the left arm of the chromosome . This segment contains the four known genes CDC24, WHI1, CYC3 and PYK1 plus at least seven transcribed regions of unknown function . The 11 identified transcripts have a total length of approximately 25.9 kb, suggesting that greater than or equal to 75% of the DNA in this region is transcribed . Of the transcribed regions of unknown function, three are essential for viability on rich medium and three appear to be nonessential, as judged by the lethality or nonlethality of deletions constructed using integrative transformation methods . No obvious phenotypes were associated with the deletions in the apparently nonessential genes . However, two of these genes may have homologs elsewhere in the genome, as judged from the appearance of additional bands when DNA-DNA blot hybridizations were performed at reduced stringency . Taken together, the results provide further evidence that the limitations of classical genetic studies of chromosome I cannot be explained solely by a lack of genes, or even a lack of essential genes, on the chromosome.

Virology, 1991 Feb, 180(2), 837 - 41
The cauliflower mosaic virus reverse transcriptase is not produced by the mechanism of ribosomal frameshifting in Saccharomyces cerevisiae; Wurch T et al.; The capsid protein and the reverse transcriptase of cauliflower mosaic virus (CaMV) are encoded by two genes (ORF IV and ORF V) that lie in different translation reading frames . A comparison can be drawn between the synthesis of both CaMV proteins and the fusion protein in a yeast retrotransposon, Ty, resulting from a +1 frameshifting event which fuses two out-of-phase ORFs encoding the structural protein and the reverse transcriptase of Ty . For this reason, we constructed a yeast expression vector containing CaMV ORF VII fused to CaMV ORF III by a fragment of 452 bp including the overlapping region of ORF IV and ORF V, ORF VII and ORF III being used as reporter genes . We characterized two proteins (22 and 50 kDa) synthesized from this plasmid in the yeast expression system . We demonstrated that the 50-kDa polypeptide is not synthesized from a +1 frameshifting event but is probably a dimeric form of the 22-kDa protein . From this result we conclude that the CaMV reverse transcriptase is not produced by a mechanism of ribosomal frameshifting.

J Mol Evol, 1991 Feb, 32(2), 145 - 52
Evidence for cis- and trans-acting element coevolution of the 2-microns circle genome in Saccharomyces cerevisiae; Xiao W et al.; We compared the DNA sequence of the yeast 2-microns plasmid cis-acting STB and transacting REP1 partition loci of laboratory haploid and industrial amphiploid strains . Several industrial strains had a unique STB sequence (type 1) sharing only 70% homology with laboratory STB (type 2) . Type 1 plasmids had a REP1 protein with 6-10% amino acid substitutions when compared to REP1 of type 2 plasmids . All 2-microns variants that shared a similar STB consensus sequence exhibited a high degree of REP1 nucleotide and amino acid sequence conservation . These observations suggest molecular coevolution of trans-acting elements with cognate target DNA structure . Based on DNA sequencing and Southern hybridization analyses, we classified 2-microns variants into two main evolutionary lineages that differ at STB as well as REP1 loci . The role of molecular coevolution in yeast intra- and interspecies plasmid evolution was discussed.

J Biochem (Tokyo), 1991 Feb, 109(2), 276 - 87
Identification of the upstream activation sequences responsible for the expression and regulation of the PEM1 and PEM2 genes encoding the enzymes of the phosphatidylethanolamine methylation pathway in Saccharomyces cerevisiae; Kodaki T et al.; The yeast phosphatidylethanolamine methylation pathway is encoded by two structural genes, PEM1 and PEM2 . The abundance of their transcripts was coordinately repressed by myo-inositol and choline . The most upstream transcriptional start sites for PEM1 and PEM2 were mapped at positions -142 and -42 relative to their first ATG codons, respectively . Promoter deletion analysis defined the 5' boundary of the regulatory region of PEM1 between -336 and -332 and that of PEM2 between -177 and -158 . The 38-bp sequence between -336 and -299 from PEM1 and the 48-bp sequence between -177 and -130 from PEM2 conferred regulated transcription upon an upstream-activation-sequence-deficient test gene, CYC1-lacZ . Comparison of these two regions revealed the presence of a common octameric sequence, 5-CATRTGAA-3', which occurred twice in the 38-bp PEM1 regulatory region and once, followed by the 5'-AAACCCACACATG-3' GRFI site, in the 48-bp PEM2 regulatory region . When synthesized chemically and placed in front of CYC1-lacZ, a single copy of CATATGAA directed a rather low level of gene expression, but multiple copies produced high-level expression . In both cases, gene expression was sensitive to myo-inositol and choline . The synthesized GRFI site directed considerable but constitute lacZ expression . When used in conjunction with CATATGAA, synergistic, regulated gene expression was obtained . Hence CATRTGAA was concluded to play an important role in the myo-inositol-choline regulation of PEM1 and PEM2 . Binding proteins to these sequences were demonstrated by electrophoretic mobility shift assay . Protein binding to CATRTGAA was not competitive with binding to the GRFI sequence, and vice versa . CATRTGAA was also found in the upstream regions of other genes encoding phospholipid-synthesizing enzymes, such as choline kinase, phosphatidylserine synthase, and myo-inositol-1-phosphate synthase, known to be repressed by myo-inositol and choline.

Biotechnology (N Y), 1991 Feb, 9(2), 183 - 7
Saccharomyces cerevisiae strains that overexpress heterologous proteins; Sleep D et al.; We describe a system that facilitates the selection of host mutants that overproduce a range of secreted and internally produced heterologous proteins in Saccharomyces cerevisiae . These mutants were initially selected for their ability to oversecrete recombinant human albumin (rHA), as detected by a direct visual assay that relies upon antibody precipitation in solid media . Yeast strains that were able to synthesize and secrete increased levels of rHA also produced elevated levels of internally expressed proteins including alpha 1-antitrypsin Pittsburgh variant and plasminogen activator inhibitor type 2.

Eur J Biochem, 1991 Jan 30, 195(2), 439 - 48
Purification, biosynthesis and cellular localization of a major 125-kDa glycophosphatidylinositol-anchored membrane glycoprotein of Saccharomyces cerevisiae; Fankhauser C et al.; The yeast Saccharomyces cerevisiae has been shown to contain a major 125-kDa membrane glycoprotein which is anchored in the lipid bilayer by a glycophosphatidylinositol anchor . This protein was purified to near homogeneity and was used to raise a rabbit antibody . Biosynthesis of the 125-kDa protein was studied by immunoprecipitation of 35SO4-labeled material from wild-type cells or a secretion mutant (sec18) in which the vesicular traffic from the endoplasmic reticulum (ER) to the Golgi is blocked . The 125-kDa protein is first made in the ER as a 105-kDa precursor which already contains a glycophosphatidylinositol anchor and which is slowly transformed into the 125-kDa form upon chase (t1/2 approximately 10-15 min) . The 105-kDa precursor can be reduced to an 83-kDa form by the enzymatic removal of N-glycans . The removal of N-glycans from the mature 125-kDa protein yields a 95-kDa species . Thus, removal of the N-glycans does not reduce the ER and mature forms to the same molecular mass, indicating that not only elongation of N-glycans but also another post-translational modification takes place during maturation . Selective tagging of surface proteins by treatment of 35SO4-labeled cells with trinitrobenzene sulfonic acid at 0 C followed by immunoprecipitation of the tagged proteins shows that the 125-kDa protein, but not the 105-kDa precursor, becomes transported to the cell surface . This tagging of cells after various lengths of chase also shows that the surface appearance of the protein is biphasic with about one half of the mature 125-kDa protein remaining intracellular for over 2 h . Glycosylation and/or glycophosphatidylinositol anchor addition is important for the stability of the 125-kDa protein since the protein remains undetectable in sec53, a temperature-sensitive mutant which does not make GDP-mannose at 37 C and does not add glycophosphatidylinositol anchors at 37 degrees C.

FEBS Lett, 1991 Jan 28, 278(2), 234 - 8
ATP13, a nuclear gene of Saccharomyces cerevisiae essential for the expression of subunit 9 of the mitochondrial ATPase; Ackerman SH et al.; The respiratory deficient nuclear mutant of Saccharomyces cerevisiae, N9-168, assigned to complementation group G95 was previously shown to lack subunit 9, one of the three mitochondrially encoded subunits of the Fo component of the mitochondrial ATPase . As a consequence of the structural defect in Fo, the ATPase activity of G95 mutants is not inhibited by rutamycin . The absence of subunit 9 in N9-168 has been correlated with a lower steady-state level of its mRNA and an increase in higher molecular weight precursor transcripts . These results suggest that the mutation is most likely to affect either translation of the oli1 mRNA or processing of the primary transcript . We have isolated a nuclear gene, designated ATP13, which complements the respiratory defect and restores rutamycin-sensitive ATPase in G95 mutants . Disruption of ATP13 induces a respiratory deficiency which is not complemented by G95 mutants . The nucleotide sequence of ATP13 indicates a primary translation product with an Mapp of 42,897 . The protein has a basic amino terminal signal sequence that is cleaved upon import into mitochondria . No significant primary structure homology is detected with any protein in the most recent libraries.

J Biol Chem, 1991 Jan 25, 266(3), 1383 - 9
Effect of membrane voltage on the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae; Seto-Young D et al.; A novel system for generating large interior positive membrane potentials in proteoliposomes was used to examine the effects of membrane voltage on reconstituted plasma membrane H(+)-ATPase from Saccharomyces cerevisiae . The membrane potential-generating system was dependent upon the lipophilic electron carrier tetracyanoquinodimethane, located within the bilayer, to mediate electron flow from vesicle entrapped ascorbate to external K3Fe(CN)6 . Membrane potential formation was followed by the potential-dependent probe oxonol V and was found to rapidly reach a steady-state which lasted at least 90 s . A membrane potential of approximately 254 mV was determined under optimal conditions and ATP hydrolysis by wild-type H(+)-ATPase was inhibited from 34 to 46% under these conditions . In contrast, membrane potential had little effect on pma1-105 mutant enzyme suggesting that it is defective in electrogenic proton translocation . Applied membrane voltage was also found to alter the sensitivity of wild-type enzyme to vanadate at concentrations less than 50 microM . These data suggest a coupling between the charge-transfer and ATP hydrolysis domains and establish a solid basis for future probing of the electrogenic properties of the yeast H(+)-ATPase.

FEMS Microbiol Lett, 1991 Jan 15, 61(2-3), 233 - 6
Isolation and characterization of maltose non utilizing (mnu) mutants mapping outside the MAL1 locus in Saccharomyces cerevisiae; Vanoni M et al.; The MAL1 locus of Saccharomyces cerevisiae comprises three genes necessary for maltose utilization . They include regulatory, maltose transport and maltase genes designated MAL1R, MAL1T and MAL1S respectively . Using a MAL1 strain transformed with an episomal, multicopy plasmid carrying the MAL2 locus, five recessive and one dominant mutant unable to grow on maltose, but still retaining a functional MAL1 locus were isolated . All the mutants could use glycerol, ethanol, raffinose and sucrose as a sole carbon source; expression of the maltase and maltose permease genes was severely and coordinately reduced . Only the dominant mutant failed to accumulate the MAL1R mRNA.

Gene, 1991 Jan 15, 97(2), 163 - 71
The pleiotropic UGA35(DURL) regulatory gene of Saccharomyces cerevisiae: cloning, sequence and identity with the DAL81 gene; Coornaert D et al.; The UGA35 gene of Saccharomyces cerevisiae (also called DURL) encodes a positive regulator of the expression of structural genes involved in 4-aminobutyric acid (GABA) and urea catabolisms . The UGA35 gene has been cloned by complementation of function and identified by chromosomal gene replacement . The sequence of this regulatory gene and its flanking regions has been established . Our data reveal an open reading frame of 2892 nt, corresponding to 964 amino acids (aa) . The deduced UGA35 aa sequence shares several similarities with that of other regulatory proteins, suggesting that the UGA35 gene encodes a DNA-binding transcriptional activator . We also show that UGA35 and the DAL81 regulatory gene controlling allantoin and urea catabolisms are one and the same gene . This means that the same factor is required for specific induction of three distinct catabolic pathways, namely those involved in GABA, urea and allantoin utilization as nitrogen sources.

Biochem Biophys Res Commun, 1991 Jan 15, 174(1), 204 - 10
Proteinase B is, indeed, not required for chitin synthetase 1 function in Saccharomyces cerevisiae; Silverman SJ et al.; Previous genetic evidence led to the conclusion that proteinase B of yeast was not involved in the function of chitin synthetase 1 (Chs1), based on the demonstration of normal septum formation, cell division and chitin deposition in mutants devoid of the proteinase (Zubenko, G.S., Mitchell, A.P., and Jones, E.W . (1979) Proc . Natl . Acad . Sci . USA 76, 2395-2399) . Later, however, it was found that the essential enzyme for septum formation is chitin synthetase 2, whereas Chs1 acts as an auxiliary enzyme, whose absence results in daughter cell lysis under acidic conditions (Cabib, E., Sburlati, A., Bowers, B . and Silverman, S.J . (1989) J . Cell Biol . 108, 1665-1672) . By using the lytic behavior as a criterion, we have now found that prb1 strains are not defective in Chs1 function . Certain strains contain a recessive suppressor of lysis which could mask the Chs1 defect . However, appropriate crosses and transformation experiments showed that the prb1 mutants do not harbor the suppressor . It may now be concluded with confidence that proteinase B is not required for chitin synthetase 1 function.

J Biol Chem, 1991 Jan 15, 266(2), 863 - 72
The OPI1 gene of Saccharomyces cerevisiae, a negative regulator of phospholipid biosynthesis, encodes a protein containing polyglutamine tracts and a leucine zipper; White MJ et al.; In Saccharomyces cerevisiae, recessive mutations at the OPI1 locus result in constitutively derepressed expression of inositol 1-phosphate synthase, the product of the INO1 gene . Many of the other enzymes involved in phospholipid biosynthesis are also expressed at high derepressed levels in opi1 mutants . Thus, the OPI1 gene is believed to encode a negative regulator that is required to repress a whole subset of structural genes encoding for phospholipid biosynthetic enzymes . In this study, the OPI1 gene was mapped to chromosome VIII and cloned . When transformed into an opi1 mutant, the cloned DNA was capable of complementing the mutant phenotype and restoring correct regulation to the INO1 structural gene . Construction of two opi1 disruption alleles and subsequent genetic analysis of strains bearing these alleles confirmed that the cloned DNA was homologous to the genomic OPI1 locus . Furthermore, the OPI1 gene was found to be nonessential to the organism since mutants bearing the null allele were viable and exhibited a phenotype similar to that of previously isolated opi1 mutants . Similar to other opi1 mutants, the opi1 disruption mutants accumulated INO1 mRNA constitutively to a level 2-3-fold higher than that observed in wild-type cells . The cloned OPI1 gene was sequenced, and translation of the open reading frame predicted a protein composed of 404 amino acid residues with a molecular weight of 40,036 . The predicted Opi1 protein contained a well defined heptad repeat of leucine residues that has been observed in other regulatory proteins . In addition, the predicted protein contained polyglutamine residue stretches which have also been reported in yeast genes having regulatory functions . Sequencing of opi1 mutant alleles, isolated after chemical mutagenesis, revealed that several were the result of a chain termination mutation located within the largest polyglutamine residue stretch.

J Biol Chem, 1991 Jan 15, 266(2), 723 - 7
Isolation and complete amino acid sequence of the mitochondrial ATP synthase epsilon-subunit of the yeast Saccharomyces cerevisiae; Arselin G et al.; All five subunits of yeast mitochondrial F1-ATPase have been isolated by reverse-phase high performance liquid chromatography . This procedure allows micro-preparative purification of all the subunits with 60% recoveries . The complete amino acid sequence of the epsilon-subunit has been established . This has been achieved by the sequence analysis of subnanomole amounts of the intact molecule and that of peptides derived by enzymatic digestion with endoproteinase Arg-C and by chemical cleavage with hydroxylamine . Yeast ATP synthase epsilon-subunit is composed of 61 residues with a calculated molecular mass of 6612 Da . This polypeptide is rather basic since it contains 7 basic residues and 3 acidic residues . This study shows a slight similarity with the bovine epsilon-subunit ATP synthase since there are 16 identical residues.

Gene, 1991 Jan 15, 97(2), 173 - 82
Nucleotide sequence of the exo-1,3-beta-glucanase-encoding gene, EXG1, of the yeast Saccharomyces cerevisiae; Vazquez de Aldana CR et al.; The nucleotide (nt) sequence of the Saccharomyces cerevisiae gene (EXG1) encoding extracellular exo-1,3-beta-glucanases (EXG) I and II was determined . An open reading frame of 1344 bp codes for a 448-amino acid (aa) polypeptide, with a calculated Mr of 51,307, which contains two potential N-glycosylation sites . The EXG1 DNA hybridizes to a 1.7-kb transcript whose 5' end maps to a position 98 bp upstream from the site of initiation of protein synthesis . Comparison of the N-terminal aa sequence deduced from the nt sequence with that of the purified EXGII revealed the existence of an extra 40-aa peptide in the precursor protein containing a Lys-Arg peptidase-processing site at the junction with the mature, extracellular form . The N-terminal region of the putative precursor is a very hydrophobic segment with structural features resembling those of signal peptides of secreted proteins . The Mr of the mature EXG polypeptide deduced from the nt sequence is 46,385 . The 5'- and 3'-flanking regions of the EXG1 gene have structural features in common with other yeast genes.

FEBS Lett, 1991 Jan 14, 278(1), 26 - 30
Molecular analysis of revertants from a respiratory-deficient mutant affecting the center o domain of cytochrome b in Saccharomyces cerevisiae; Tron T et al.; In bc complexes, cytochrome b plays a major role in electron transfer and in proton translocation across the membrane . Several inhibitor-resistant and respiratory-deficient mutants have already been used to study the structure-function relationships of this integral membrane protein . We describe here the selection and the molecular analysis of revertants from a thermo-sensitive mit-mutant of known nucleotide changes . Among 80 independent pseudo-wild type revertants screened by DNA-labelled oligonucleotide hybridization, 33 have been sequenced . Eight suppressor mutations, affecting a region critical for both the function and the binding of center o inhibitors (end of helix C) were identified . Two of them were found to be more resistant to myxothiazol.

FEBS Lett, 1991 Jan 14, 278(1), 123 - 6
Inhibition by different amino acids of the aspartate kinase and the homoserine kinase of the yeast Saccharomyces cerevisiae; Ramos C et al.; In this paper, we describe a simple method to measure the yeast homoserine kinase and aspartate kinase activities, independently but in the same extract . With this method, we have determined some kinetic parameters for the physiological substrates of both enzymes, and investigated the inhibition exerted by different amino acids on these activities . Of all natural amino acids tested, only threonine inhibits effectively both enzymatic activities, although to a different degree . We did not find the reported inhibition by L-homoserine over the aspartate kinase . Altogether the data point to the aspartate kinase and to the threonine as the key factors in the regulation of this route.

Biochemistry, 1991 Jan 8, 30(1), 248 - 52
Cloning and characterization of the mitochondrial phosphate transport protein gene from the yeast Saccharomyces cerevisiae; Phelps A et al.; We have cloned the gene of the Saccharomyces cerevisiae phosphate transport protein (PTP), a member of the mitochondrial anion transport protein gene family . As PTP has a blocked N-terminus, we prepared three peptides . Oligonucleotides, based on their sequences, were used to screen a Yep24-housed genomic library . A total of 2073 bases of clone Y22 code for a 311 amino acid protein (Mr 32,814), which has similarities to the anion transport proteins: a triplicate gene structure and 6 hydrophobic segments . Typical for PTP, the triplicate gene structure possesses the X-Pro-X-(Asp/Glu)-X-X-(Lys/Arg)-X-(Arg/Lys)-X (X is an unspecified amino acid) motif and the very high homology only between the first and second repeat . The 6 hydrophobic segments harbor most of the 116 amino acids that are conserved between the yeast and the beef proteins . An N-terminal-extended signal sequence, as found in the beef protein, is absent . The yeast protein has about 33% fewer basic and acidic amino acids and five fewer Cys residues than the beef protein . The protein is insensitive to N-ethylmaleimide since Cys-42 (beef) has been replaced with a Thr . Mersalyl sensitivity has been retained and must be due to one of its three cysteines . Among these three cysteines, only Cys-28, located in the first hydrophobic segment, is conserved between the yeast and the beef protein.

Arch Microbiol, 1991, 155(4), 320 - 4
Effect of L-azetidine 2-carboxilic acid on the activity of the general amino-acid permease from Saccharomyces cerevisiae var . ellipsoideus; Iglesias R et al.; Addition of the L-proline analogue L-azetidine 2-carboxylic acid to growing cultures of Saccharomyces cerevisiae var . ellipsoideus promoted fast deactivation of the general aminoacid permease, measured as L-valine uptake, without an immediate decrease in the growth rate . Cells preincubated with the analogue for 3 h were unable to restore either growth ability or general aminoacid permease activity in analogue-free medium . Eadie-Hofstee plots of L-valine uptake in the presence of the analogue are consistent with a strong reduction in the number of active molecules of the general amino-acid permease located in the plasma membrane . Inhibitory effects on protein synthesis were seen after preincubations of the yeast with the analogue for 3 h although a 30 min preincubation had no effect.

Yeast, 1991 Jan, 7(1), 1 - 14
RCS1, a gene involved in controlling cell size in Saccharomyces cerevisiae; Gil R et al.; Cloning and sequencing of RCS1, Saccharomyces cerevisiae gene whose product seems to be involved in timing the budding event of the cell cycle, is described . A haploid strain in which the 3'-terminal region of the chromosomal copy of the gene has been disrupted produces cells that are, on average, twice the size of cells of the parental strain . The critical size for budding in the mutant is similarly increased, and the disruption mutation is dominant in a diploid heterozygous for the RCS1 gene . Spores from this diploid have a reduced ability to germinate, the effect being more pronounced in the spores carrying the disrupted copy of RCS1 . However, disrupted cells recover from alpha-factor treatment equally as well as wild-type cells.

Antimicrob Agents Chemother, 1991 Jan, 35(1), 170 - 3
Differential inhibition of chitin synthetases 1 and 2 from Saccharomyces cerevisiae by polyoxin D and nikkomycins; Cabib E; Polyoxin D, nikkomycin X, and nikkomycin Z are all competitive inhibitors of chitin synthetase 2 (Chs2), the essential enzyme for primary septum formation in Saccharomyces cerevisiae, and of Chs1, a repair enzyme . However, Chs2 is more resistant to these antibiotics than Chs1 . When Co2+, the best stimulator of Chs2, was used in the assay for this enzyme, the differences in the Ki values for nikkomycins between the two isozymes reached 3 orders of magnitude . These results point to differences in the active sites of the two isozymes . Polyoxin D was much more effective than nikkomycin Z in inhibiting cell growth . This underlines the importance of the choice of enzyme and of assay conditions when cell wall-synthesizing enzymes are used in screens for possible antifungal agents.

J Bacteriol, 1991 Jan, 173(2), 834 - 41
Characterization of vanadate-dependent NADH oxidation stimulated by Saccharomyces cerevisiae plasma membranes; Minasi LA et al.; Plasma membrane-stimulated vanadate-dependent NADH oxidation has been characterized in Saccharomyces cerevisiae . This activity is specific for vanadate, because molybdate, a similar metal oxide, did not substitute for vanadate in the reaction . Vanadate-dependent plasma membrane-stimulated NADH oxidation activity was dependent on the concentrations of vanadate, NADH, and NADPH and required functional plasma membranes; no stimulation occurred in the presence of boiled membranes or bovine serum albumin . The dependence of membrane-stimulated vanadate-dependent NADH oxidation was not linearly dependent on added membrane protein . The activity was abolished by the superoxide anion scavenger superoxide dismutase and was stimulated by paraquat and NADPH . These data are consistent with the previously proposed chain reaction for vanadate-dependent NADH oxidation . The role of the plasma membrane appears to be to stimulate superoxide radical formation, which is coupled to NADH oxidation by vanadate . 51V-nuclear magnetic resonance studies are consistent with the hypothesis that a phosphovanadate anhydride is the stimulatory oxyvanadium species in the phosphate buffers used at pHs 5.0 and 7.0 . In phosphate buffers, compared with acetate buffers, the single vanadate resonance was shifted upfield at both pH 5.0 and pH 7.0, which is characteristic of the phosphovanadate anhydride . Since the cell contains an excess of phosphate to vanadate, the phosphovanadate anhydride may be involved in membrane-mediated vanadate-dependent NADH oxidation in vivo.

Mol Cell Biol, 1991 Jan, 11(1), 425 - 39
In vivo pre-tRNA processing in Saccharomyces cerevisiae; O'Connor JP et al.; We have surveyed intron-containing RNAs of the yeast Saccharomyces cerevisiae by filter hybridization with pre-tRNA intron-specific oligonucleotide probes . We have classified various RNAs as pre-tRNAs, splicing intermediates, or excised intron products according to apparent size and structure . Linear, excised intron products were detected, and one example was isolated and sequenced directly . Additional probes designed to detect other precursor sequences were used to verify the identification of several intermediates . Pre-tRNA species with both 5' leader and 3' extension, with 3' extension only, and with mature ends were distinguished . From these results, we conclude that the processing reactions used to remove the 5' leader and 3' extension from the transcript are ordered 5' end trimming before 3' end trimming . Splicing intermediates containing the 5' exon plus the intron were detected . The splice site cleavage reactions are probably ordered 3' splice site cleavage before 5' splice site cleavage . Surprisingly, we also detected a splicing intermediate with the 5' leader and a spliced product with both 5' leader and 3' extension . Evidently, splicing and end trimming are not ordered relative to each other, splicing occurring either before or after end trimming.

Mol Cell Biol, 1991 Jan, 11(1), 38 - 46
Intramitochondrial functions regulate nonmitochondrial citrate synthase (CIT2) expression in Saccharomyces cerevisiae; Liao XS et al.; We have examined the effects of perturbation of mitochondrial function on expression of two nuclear genes encoding the mitochondrial and peroxisomal forms of citrate synthase in Saccharomyces cerevisiae, CIT1 and CIT2 . CIT2 expression was as much as 30-fold higher in {rho0} petites, than in isochromosomal {rho+} cells, whereas CIT1 expression was slightly down regulated in {rho0} cells . CIT2 expression was also increased in {rho+} cells by inhibition of respiration with antimycin A or in {rho+} cells containing a disruption of the CIT1 gene . These effects were additive, and together they approached the level of CIT2 expression seen in {rho0} cells . Experiments using heterologous gene fusions showed that all of the effects leading to increased expression of CIT2 were transcriptionally controlled through 5'-flanking CIT2 DNA sequences . Analysis of {rho+} and {rho0} cells containing disruptions of CIT1 and CIT2, singly and in combination, showed that the peroxisomal citrate synthase could partially spare the mitochondrial isoform for growth yield in {rho+} but not in {rho0} cells . These studies suggest a physiological role for increased expression of CIT2 in cells with altered mitochondrial function . They also provide additional evidence for a retrograde path of communication from mitochondria to the nucleus in yeast cells.

Mol Cell Biol, 1991 Jan, 11(1), 370 - 80
Isolation, nucleotide sequence analysis, and disruption of the MDH2 gene from Saccharomyces cerevisiae: evidence for three isozymes of yeast malate dehydrogenase; Minard KI et al.; The major nonmitochondrial isozyme of malate dehydrogenase (MDH2) in Saccharomyces cerevisiae cells grown with acetate as a carbon source was purified and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a subunit molecular weight of approximately 42,000 . Enzyme assays and an antiserum prepared against the purified protein were used to screen a collection of acetate-nonutilizing (acetate-) yeast mutants, resulting in identification of mutants in one complementation group that lack active or immunoreactive MDH2 . Transformation and complementation of the acetate- growth phenotype was used to isolate a plasmid carrying the MDH2 gene from a yeast genomic DNA library . The amino acid sequence derived from complete nucleotide sequence analysis of the isolated gene was found to be extremely similar (49% residue identity) to that of yeast mitochondrial malate dehydrogenase (molecular weight, 33,500) despite the difference in sizes of the two proteins . Disruption of the MDH2 gene in a haploid yeast strain produced a mutant unable to grow on minimal medium with acetate or ethanol as a carbon source . Disruption of the MDH2 gene in a haploid strain also containing a disruption in the chromosomal MDH1 gene encoding the mitochondrial isozyme produced a strain unable to grow with acetate but capable of growth on rich medium with glycerol as a carbon source . The detection of residual malate dehydrogenase activity in the latter strain confirmed the existence of at least three isozymes in yeast cells.

Mol Cell Biol, 1991 Jan, 11(1), 322 - 8
A poly(dA.dT) tract is a component of the recombination initiation site at the ARG4 locus in Saccharomyces cerevisiae; Schultes NP et al.; An initiation site for meiotic gene conversion is located in the promoter region of the ARG4 locus in Saccharomyces cerevisiae . We have tested the hypothesis that the initiation site is identical with the promoter by making a series of small deletions that remove specific promoter elements . Disruption of most promoter elements does not lower the level of gene conversion in ARG4, and analysis of RNA levels at the time of recombination in meiosis reveals no direct correlation between the level of ARG4 transcript and the level of gene conversion in ARG4 . However, deletion of a tract of 14 A residues located at the peak of the gene conversion gradient decreases the number of gene conversion events stimulated by the initiation site to 25 to 35% of the normal level . We conclude that the poly(dA.dT) tract is responsible for most but not all of the high levels of meiotic gene conversion observed in ARG4.

Mol Cell Biol, 1991 Jan, 11(1), 213 - 7
A chicken beta-actin gene can complement a disruption of the Saccharomyces cerevisiae ACT1 gene; Karlsson R et al.; Recently it was demonstrated that beta-actin can be produced in Saccharomyces cerevisiae by using the expression plasmid pY beta actin (R . Karlsson, Gene 68:249-258, 1988), and several site-specific mutants are now being produced in a protein engineering study . To establish a system with which recombinant actin mutants can be tested in vivo and thus enable a correlation to be made with functional effects observed in vitro, a yeast strain lacking endogenous yeast actin and expressing exclusively beta-actin was constructed . This strain is viable but has an altered morphology and a slow-growth phenotype and is temperature sensitive to the point of lethality at 37 degrees C.

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