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 expo