S. cerevisiae

Mol Cell Biol, 1991 Jan, 11(1), 202 - 12
SDC25, a CDC25-like gene which contains a RAS-activating domain and is a dispensable gene of Saccharomyces cerevisiae; Damak F et al.; In the yeast Saccharomyces cerevisiae, the CDC25 gene product activates adenylate cyclase through RAS1 and RAS2 gene products . We have recently described the cloning of a DNA fragment which suppresses the cdc25 mutation but not ras1, ras2, or cdc35 mutations . This fragment contains a 5'-truncated open reading frame which shares 47% identity with the C-terminal part of the CDC25 gene . We named the entire gene SDC25 . In this paper, we report the cloning, sequencing, and characterization of the complete SDC25 gene . The SDC25 gene is located on the chromosome XII close to the centromere . It is transcribed into a 4-kb-long mRNA that contains an open reading frame of 1,251 codons . Homology with the CDC25 gene extends in the N-terminal part, although the degree of similarity is lower than in the C-terminal part . In contrast with the C-terminal part, the complete SDC25 gene was found not to suppress the CDC25 gene defect . A deletion in the N-terminal part restored the suppressing activity, a result which suggests the existence of a regulatory domain . The SDC25 gene was found to be dispensable for cell growth under usual conditions . No noticeable phenotype was found in the deleted strain.

Methods Enzymol, 1991, 200, 605 - 27
Functional expression of mammalian adenosine cyclic monophosphate-dependent protein kinase in Saccharomyces cerevisiae; Zoller MJ et al.; The heterologous expression of protein kinases in E . coli has proved difficult and unpredictable . Although the v-abl protein kinase is successfully expressed in E . coli, our experiments on expression of yeast C subunits in E . coli produced large amounts of predominantly insoluble and inactive protein . Attempts to refold the protein proved unsuccessful . In contrast, a major fraction of mouse C alpha expressed in E . coli is soluble and the enzyme in the soluble fraction is active; however, certain mutant forms have proved to be unstable, difficult to purify, or insoluble . In addition, the E . coli system cannot be used to study the biological role of posttranslational modifications specific to eukaryotic systems . Several protein kinases have been expressed in soluble form in insect cells using baculovirus, suggesting that this system is generally more reliable than E . coli . However, the presence and nature of posttranslational modifications in insect cells may be different from that found in the natural source and may affect the biochemical function . In addition, baculovirus expression is not particularly useful for studying biological questions . Mouse C alpha and C beta have been overexpressed in NIH3T3 cells . This approach is useful in characterizing the biochemical properties of C alpha versus C beta, but it may not be an ideal system for studying mutant proteins since wild-type C subunits are still expressed from the chromosomal copies in this genetic background . This small level of wild type may make it difficult to analyze weakly functional mutants, which have activities less than 10% that of wild type . Several cell lines with altered subunits of cAMP-dependent protein kinase have been identified but a strain completely devoid of C subunit has not been adequately characterized for protein structure/function studies . Disruption of the genes encoding cAMP-dependent protein kinase in mammalian cells has not yet been accomplished . This chapter describes a method to express a C subunit of mammalian cAMP-dependent kinase in yeast . We have demonstrated that the mouse C alpha subunit can substitute for its yeast counterpart . Since at least one functional C subunit is required for viability, these results suggest that the yeast substrates important for viability are recognized by the mammalian C subunit . Although the sequence conservation between yeast and mouse C subunit is only about 50%, these results demonstrate that heterologous proteins with relatively low sequence conservation with their yeast counterparts can be functional in yeast.(ABSTRACT TRUNCATED AT 400 WORDS)

J Photochem Photobiol B, 1991 Jan, 8(2), 169 - 74
Luminescence spectra of exponential and stationary phase cultures of respiratory deficient Saccharomyces cerevisiae; Quickenden TI et al.; The spectral distributions of the luminescences emitted by the respiratory-deficient mutant of Saccharomyces cerevisiae and the normal yeast have been determined during the exponential phase of growth and during the stationary phase . The respiratory-deficient mutant gave a more intense emission in the visible region than did the normal yeast, but the UV intensities from the two yeasts did not differ greatly . These differences were explained in terms of higher O2- concentrations in the respiratory-deficient mutant which lead to enhanced visible region chemiluminescence from lipid peroxidation reactions.

J Bacteriol, 1991 Jan, 173(1), 255 - 61
DAL82, a second gene required for induction of allantoin system gene transcription in Saccharomyces cerevisiae; Olive MG et al.; Several highly inducible enzyme activities are required for the degradation of allantoin in Saccharomyces cerevisiae . Induction of these pathway enzymes has been shown to be regulated at transcription, and response to inducer is lost in dal81 and dal82/durM mutants . The similar phenotypes generated by dal81 and dal82 mutations prompted the question of whether they were allelic . We demonstrated that the DAL81 and DAL82 loci are distinct, unlinked genes situated on chromosomes IX and XIV . DAL82 gene expression did not respond to induction by the allantoin pathway inducer or to nitrogen catabolite repression . Expression was also not significantly affected by mutation of the dal80 locus . From the nucleotide sequence of the DAL82 gene, we deduced that it encodes a protein with a mass of 29,079 Da that may possess the structural motifs expected of a regulatory protein . This protein was shown to be required for the function mediated by the cis-acting upstream induction sequence situated in the 5'-flanking regions of the inducible allantoin pathway genes.

J Cell Biol, 1991 Jan, 112(1), 65 - 80
Sequence of the clathrin heavy chain from Saccharomyces cerevisiae and requirement of the COOH terminus for clathrin function; Lemmon SK et al.; The sequence of the clathrin heavy chain gene, CHC1, from Saccharomyces cerevisiae is reported . The gene encodes a protein of 1,653 amino acids that is 50% identical to the rat clathrin heavy chain (HC) (Kirchhausen, T., S . C . Harrison, E . P . Chow, R . J . Mattaliano, R . L . Ramachandran, J . Smart, and J . Brosius . 1987 . Proc . Natl . Acad . Sci . USA . 84:8805-8809) . The alignment extends over the complete length of the two proteins, except for a COOH-terminal extension of the rat HC and a few small gaps, primarily in the globular terminal domain . The yeast HC has four prolines in the region of the rat polypeptide that was proposed to form the binding site for clathrin light chains via an alpha-helical coiled-coil interaction . The yeast protein also lacks the COOH-terminal Pro-Gly rich segment present in the last 45 residues of the rat HC, which were proposed to be involved in the noncovalent association of HCs to form trimers at the triskelion vertex . To examine the importance of the COOH terminus of the HC for clathrin function, a HC containing a COOH-terminal deletion of 57 amino acids (HC delta 57) was expressed in clathrin-deficient yeast (chc1-delta) . HC delta 57 rescued some of the phenotypes (slow growth at 30 degrees, genetic instability, and defects in mating and sporulation) associated with the chc1-delta mutation to normal or near normal . Also, truncated HCs were assembled into triskelions . However, cells with HC delta 57 were temperature sensitive for growth and still displayed a major defect in processing of the mating pheromone alpha-factor . Fewer coated vesicles could be isolated from cells with HC delta 57 than cells with the wild-type HC . This suggests that the COOH-terminal region is not required for formation of trimers, but it may be important for normal clathrin-coated vesicle structure and function.

Genetics, 1991 Jan, 127(1), 75 - 85
The hyper-gene conversion hpr5-1 mutation of Saccharomyces cerevisiae is an allele of the SRS2/RADH gene; Rong L et al.; The HPR5 gene has been defined by the mutation hpr5-1 that results in an increased rate of gene conversion . This mutation suppresses the UV sensitive phenotype of rad18 mutations in hpr5-1 rad18 double mutants by channeling the aborted repair events into a recombination repair pathway . The HPR5 gene has been cloned and is shown to be allelic to the SRS2/RADH gene, a putative DNA helicase . The HPR5 gene, which is nonessential, is tightly linked to the ARG3 locus chromosome X . The hpr5-1 allele contains missense mutation in the putative ATP binding domain . A comparison of the recombination properties of the hpr5-1 allele and the null allele suggests that recombination events in hpr5 defective strains can be generated by several mechanisms . We propose that the HPR5 gene functions in the RAD6 repair pathway.

Trends Genet, 1991 Jan, 7(1), 28 - 33
RAS genes in Saccharomyces cerevisiae: signal transduction in search of a pathway; Broach JR; Ras proteins in budding yeasts initially appeared to regulate initiation of the cell cycle in response to nutrient availability . More recent work, while clarifying the mechanism of Ras-mediated signal transduction, has undermined our notion of the signal Ras transmits . We now suspect that Ras helps to coordinate cellular metabolism and mass accumulation, but what Ras responds to is not clear.

J Gen Microbiol, 1991 Jan, 137 ( Pt 1), 1 - 4
Inhibition of membrane Ca2(+)-ATPase of Saccharomyces cerevisiae by mating pheromone alpha-factor in vitro; Hiraga K et al.; Plasma membrane Ca2(+)-ATPase of Saccharomyces cerevisiae was solubilized and partially purified by calmodulin-affinity chromatography . The activity of Ca2(+)-ATPase isolated from MATa cells was inhibited by physiological concentrations of the mating pheromone alpha-factor in a dose-dependent manner . The enzyme prepared from a receptor-deficient sterile mutant cells (delta ste-2) was similarly inhibited by alpha-factor, but the enzyme from MAT alpha cells was resistant to the mating pheromone . We suggest that the inhibition may be involved in the alpha-factor-induced increase of Ca2+ uptake reaction of MATa cells.

Mol Cell Biol, 1991 Jan, 11(1), 27 - 37
Determinants for glycophospholipid anchoring of the Saccharomyces cerevisiae GAS1 protein to the plasma membrane; Nuoffer C et al.; A 125-kDa glycoprotein exposed on the surface of Saccharomyces cerevisiae cells belongs to a class of eucaryotic membrane proteins anchored to the lipid bilayer by covalent linkage to an inositol-containing glycophospholipid . We have cloned the gene (GAS1) encoding the 125-kDa protein (Gas1p) and found that the function of Gas1p is not essential for cell viability . The nucleotide sequence of GAS1 predicts a 60-kDa polypeptide with a cleavable N-terminal signal sequence, potential sites for N- and O-linked glycosylation, and a C-terminal hydrophobic domain . Determination of the anchor attachment site revealed that the C-terminal hydrophobic domain of Gas1p is removed during anchor addition . However, this domain is essential for addition of the glycophospholipid anchor, since a truncated form of the protein failed to become attached to the membrane . Anchor addition was also abolished by a point mutation affecting the hydrophobic character of the C-terminal sequence . We conclude that glycophospholipid anchoring of Gas1p depends on the integrity of the C-terminal hydrophobic domain that is removed during anchor attachment.

C R Seances Soc Biol Fil, 1991, 185(5), 312 - 8
{Isoprenoide pathway and cell proliferation in the yeast Saccharomyces cerevisiae}; Blanchard L et al.; Yeast mutants blocked in farnesyl diphosphate (FPP) synthetase have been isolated . Their specific phenotype is likely linked to a lowering in the FPP pool required for protein prenylation . The structural gene of FPP synthetase has been isolated . Complete inactivation of FPP synthetase by gene disruption is letal for the yeast cells.

Biomed Biochim Acta, 1991, 50(4-6), 711 - 6
Influence of yeast proteases on hirudin expression in Saccharomyces cerevisiae; Pohlig G et al.; Recombinant desulfatohirudin variant 1 is efficiently expressed and secreted from Saccharomyces cerevisiae . Chemical analysis of the secreted hirudin compounds revealed the presence of the full-length hirudin molecule as well as two degradation products that lack the C-terminal and in addition the penultimate amino acid, respectively . To eliminate the yeast proteases possibly involved in C-terminal hirudin proteolysis, we disrupted either the structural gene for endoprotease yscA (PRA1) or the gene encoding carboxypeptidase yscY (PRC1) . Both isogenic mutant strains secreted significantly higher amounts of full-length hirudin as compared to the parental strain . This suggests an involvement of carboxypeptidase yscY in hirudin proteolysis, since both protease disruptions lead to a lack in yscY activity; a yscA mutant accumulates the inactive yscY precursor . However, the strain devoid of protease yscA yielded significantly lower titers of total hirudin than the strain lacking yscY, but containing yscA.

Int J Biochem, 1991, 23(10), 1019 - 23
Different porphobilinogen-deaminase forms in wild and mutant strains of Saccharomyces cerevisiae . A possible correlation with its segregants behaviour; Correa Garcia SR et al.; 1 . Different porphobilinogen-deaminase (PBG-D) enzyme forms were found for D 27 and D 27/C6 (HEM R+) strains of Saccharomyces cerevisiae . 2 . PBG-D was partially purified and chromatographed on Sephadex G-100 in either the presence or absence of a protease inhibitor . For D 27 only one active peak was observed while for D 27/C6 strain two active peaks were found . 3 . A correlation between this differential behaviour and the presence of HEM R+ gene was looked for employing two segregants of one tetrad from D 27 and D 27/C6 mating.

Microbios, 1991, 67(272-273), 177 - 86
Co2+ and Ni2+ resistance in Saccharomyces cerevisiae associated with a reduction in the accumulation of Mg2+; Joho M et al.; A mutant strain of Saccharomyces cerevisiae (NR 6), which can be associated with a reduction in the accumulation of Mg2+ has been isolated . This mutant strain displays resistance to both Ni2+ and Co2+, but not, however, towards Cu2+, Cd2+, Mn2+, Zn2+ and Cr2O7(2-) . Both Co2+ and Ni2+ uptake by the mutant strain is less than by the wild type (CMR-50) . The inhibitory effects of Ni2+ and Co2+ on the growth of both strains NR-6 and CMR-50 can be cancelled by increasing the concentrations of Mg2+ in the medium and to a lower extent by the addition of Ca2+ which results in a decreased uptake of these metals . It therefore appears that the resistant mechanisms of this mutant strain NR-6 is due to a reduction in the uptake of Co2+ and Ni2+ via a Mg2+ transport system.

Acta Microbiol Pol, 1991, 40(1-2), 5 - 10
Decreased chemical mutagenesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae; Baranowska H et al.; In the DNA replication mutant of yeast cdc8 the frequency of chemically induced reversion of lys2-1 and hom2-1 was found to be reduced . Mutation induced by ethyl methanesulfonate (EMS) were greatly diminished in the strain homozygous for the cdc8-1 gene.

Chin J Biotechnol, 1991, 7(4), 253 - 61
Chemical synthesis, cloning and expression of human epidermal growth factor gene in Saccharomyces cerevisiae; Yuan H et al.; A gene coding for human epidermal growth factor (hEGF) has been chemically synthesized by solid-phase phosphoramidite method . The 173 base-pair synthetic DNA duplex consists of a structural gene encoding hEGF, a stop codon TGA at 3' end and some convenient restriction sites at both ends of the gene . The synthesis of the gene involved enzymatic joining of 8 oligonucleotides to form a DNA duplex which was cloned into vector M13mp18 . The recombinant colonies were identified by dot hybridization and restriction enzyme digestion . Its accuracy was confirmed by DNA sequence analysis . The hEGF DNA was inserted into yeast secretory vector YFD59 . The resulting expression plasmid YFD104 was introduced into yeast Saccharomyces cerevisiae . The binding assay showed that the yeast transformants could express and secrete hEGF.

J Basic Microbiol, 1991, 31(3), 195 - 205
{Purification and characterization of cAMP-dependent protein kinases of yeasts in a Saccharomyces cerevisiae wild strain and selected mutants of cAMP metabolism}; Sachse O et al.; Protein kinases represent a diverse family of enzymes that play a critical role in regulation . Among nearly 100 known protein kinases, the cAMP-dependent enzyme is best understood biochemically . Unlike other protein kinases, cAMP-dependent protein kinase consists of two different types of subunits that dissociate, a regulatory subunit (R), which is the receptor for cAMP, and a catalytic subunit (C) . In the absence of cAMP, the enzyme exists as an inactive tetramer, R2C2 . The binding of intracellular cAMP to the R subunit decreases the affinity of the R subunit for the C subunit by approximately four orders of magnitude and, under physiological conditions, leads to dissociation of the holoenzyme into R2(cAMP)4 dimer and two free C subunits that are catalytically active . Mutants of the cAMP metabolism, adenylate cyclase and cell cycle mutants, provided further information about protein synthesis and cellular growth in Saccharomyces cerevisiae . The purified protein kinases were divided into different types according to their elution profiles from the DEAE-cellulose matrix . Two types of cAMP-dependent and two types of cAMP-independent protein kinases were isolated from the wild strain . Differences in the activities of the kinases in the mutants showed a close relationship to the locus of the respective mutations in the cell-cycle . Some properties of the protein kinases are discussed with respect to individual mutations.

J Basic Microbiol, 1991, 31(3), 189 - 94
{Modification of intracellular cAMP and cGMP concentration in yeast wild strains and in selected mutants from Saccharomyces cerevisiae as a regulation model for higher eukaryotes}; Sachse O; The addition of D(+)-glucose (final concentration 50 mM) to a cell suspension of yeasts (wild type and several mutants of the cell cycle, the cAMP-dependent protein kinase system, and a mutant of the adenylate cyclase gene) triggers a rapid increase in the concentrations of cAMP and cGMP in the wild strain . In contrast to cAMP, an increase of cGMP was also found in the mutants . cAMP and cGMP have been characterized as second messengers in eucaryotic cells . Cyclic nucleotide activation of the protein kinases enables them to perform their only known function in eukaryotes, the phosphorylation of substrate proteins . The results, described here by using selected yeast mutants as a model for higher eukaryotes, indicate that there exist two different regulatory systems for the control of the cAMP and cGMP levels.

Curr Genet, 1991 Jan, 19(1), 9 - 14
Nucleotide sequence of the ERG12 gene of Saccharomyces cerevisiae encoding mevalonate kinase; Oulmouden A et al.; The nucleotide sequence of the ERG12 gene, encoding mevalonate kinase, from Saccharomyces cerevisiae is presented . The longest open reading frame may code for a protein containing 443 amino acids with a deduced relative molecular mass of 48,500 . The analysis of the nucleotide sequence reveals a complete identity with the yeast gene RAR1, isolated elsewhere by complementation of a rar1 mutation involved in the stability of plasmids with weak ARS . In addition, we show that mevalonate kinase is not a rate-limiting enzyme; however its sensitivity to FFP could be a key regulatory mechanism in the sterol pathway of yeast.

Biochem Biophys Res Commun, 1990 Dec 31, 173(3), 1205 - 11
Catalytically active monoamine oxidase type A from human liver expressed in Saccharomyces cerevisiae contains covalent FAD; Weyler W et al.; Monoamine oxidase type A from human liver cDNA was expressed in Saccharomyces cerevisiae . This enzyme's properties with respect to Km and Ki values for kynuramine and amphetamine, respectively, were similar to values for human placental enzyme . As expected, clorgyline inhibited the yeast enzyme at lower concentrations than deprenyl . Interestingly, the FAD cofactor was covalently attached and fluorescence properties of the enzyme bound prosthetic group indicate that it is attached to a cysteine residue, the same linkage observed in other monoamine oxidases . The yield of expressed enzyme is about 15 mg/l of culture with an A600 of 15 . It is suggested that covalent flavin attachment proceeds by an autoflavination mechanism.

Eur J Biochem, 1990 Dec 27, 194(3), 785 - 90
Molecular and biochemical characterization of the Dio-9-resistant pma1-1 mutation of the H(+)-ATPase from Saccharomyces cerevisiae; Van Dyck L et al.; The plasma-membrane H(+)-ATPase gene PMA1 was sequenced in four Dio-9-resistant strains of Saccharomyces cerevisiae, isolated independently . The same amino acid substitution Ala608----Thr was found in the four mutated strains . The mutant ATPase activity was decreased while the Km value for MgATP was increased . The ATPase efficiency (V/Km) of the mutant was reduced by a factor of 25 under acid conditions (pH 5.5), and by a factor of 10 at physiological pH (pH 6.6) . The mutation also strongly reduces the inhibition by vanadate of ATPase activity, suggesting that the altered amino acid is involved in phosphate binding and/or in the E1-E2 transition.

J Biol Chem, 1990 Dec 25, 265(36), 22418 - 25
A novel pathway of import of alpha-mannosidase, a marker enzyme of vacuolar membrane, in Saccharomyces cerevisiae; Yoshihisa T et al.; We have investigated the vacuolar delivery of alpha-mannosidase, a marker enzyme of the vacuolar membrane in the yeast Saccharomyces cerevisiae, and found that the enzyme has several unique characteristics in its biosynthesis and vacuolar delivery . alpha-Mannosidase has no typical signal sequence (Yoshihisa, T., and Anraku, Y . (1989) Biochem . Biophys . Res . Commun . 163, 908-915) but is located on the inner surface of the vacuolar membrane . The enzyme is synthesized as a 107-kDa polypeptide and converted to a 73-kDa polypeptide . Although the conversion depends on a vacuolar processing protease, proteinase A, it is much slower (t1/2 = 10 h) than the proteinase A-dependent processing of other vacuolar proteins . None of Asn-X-Thr/Ser sites on the 107-kDa alpha-mannosidase or on two alpha-mannosidase-invertase fusion proteins that are localized inside the vacuole receives N-linked oligosaccharide, whereas those sites on a carboxypeptidase Y-alpha-mannosidase fusion protein are N-glycosylated . The newly synthesized alpha-mannosidase is normally delivered to the vacuole and converted to the 73-kDa polypeptide even when the secretory pathway is blocked by a subset of sec mutations . These characteristics are different from those of other vacuolar proteins targeted to the vacuole via the secretory pathway . We conclude that alpha-mannosidase is delivered to the vacuole in a novel pathway separate from the secretory pathway.

J Biol Chem, 1990 Dec 25, 265(36), 22321 - 8
Spermidine biosynthesis in Saccharomyces cerevisiae . Biosynthesis and processing of a proenzyme form of S-adenosylmethionine decarboxylase; Kashiwagi K et al.; We have cloned and sequenced the Saccharomyces cerevisiae gene for S-adenosylmethionine decarboxylase . This enzyme contains covalently bound pyruvate which is essential for enzymatic activity . We have shown that this enzyme is synthesized as a Mr 46,000 proenzyme which is then cleaved post-translationally to form two polypeptide chains: a beta subunit (Mr 10,000) from the amino-terminal portion and an alpha subunit (Mr 36,000) from the carboxyl-terminal portion . The protein was overexpressed in Escherichia coli and purified to homogeneity . The purified enzyme contains both the alpha and beta subunits . About half of the alpha subunits have pyruvate blocking the amino-terminal end; the remaining alpha subunits have alanine in this position . From a comparison of the amino acid sequence deduced from the nucleotide sequence with the amino acid sequence of the amino-terminal portion of each subunit (determined by Edman degradation), we have identified the cleavage site of the proenzyme as the peptide bond between glutamic acid 87 and serine 88 . The pyruvate moiety, which is essential for activity, is generated from serine 88 during the cleavage . The amino acid sequence of the yeast enzyme has essentially no homology with S-adenosylmethionine decarboxylase of E . coli (Tabor, C . W., and Tabor, H . (1987) J . Biol . Chem . 262, 16037-16040) and only a moderate degree of homology with the human and rat enzymes (Pajunen, A., Crozat, A., Janne, O . A., Ihalainen, R., Laitinen, P . H., Stanley, B., Madhubala, R., and Pegg, A . E . (1988) J . Biol . Chem . 263, 17040-17049); all of these enzymes are pyruvoyl-containing proteins . Despite this limited overall homology the cleavage site of the yeast proenzyme is identical to the cleavage sites in the human and rat proenzymes, and seven of the eight amino acids adjacent to the cleavage site are identical in the three eukaryote enzymes.

Gene, 1990 Dec 15, 96(2), 181 - 8
Negative regulatory elements of the Saccharomyces cerevisiae PHO system: interaction between PHO80 and PHO85 proteins; Gilliquet V et al.; The negative regulatory genes, PHO80 and PHO85, involved in transcriptional regulation of the yeast repressible acid-phosphatase-encoding gene, PHO5, have been cloned . Expression of PHO80 and PHO85 has been studied by means of lacZ fusions . We show here that these expressions are inorganic phosphate (Pi) independent and that they are controlled by the PHO80 gene product; moreover, PHO80 expression is controlled by PHO85 . We also present genetic evidence for an interaction between the PHO80 and PHO85 proteins: increased PHO85 gene dosage partially compensates for the pho80-1 mutation and this effect is allele-specific . The pho80-1 allele has been cloned and sequenced . The mutation changes Gly229 to Asp . This region was shown to be essential for PHO80 function by C-terminal deletion analysis.

Gene, 1990 Dec 15, 96(2), 161 - 9
Isolation and characterization of the ZWF1 gene of Saccharomyces cerevisiae, encoding glucose-6-phosphate dehydrogenase; Nogae I et al.; Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first step of the pentose phosphate pathway, a reaction that generates NADPH . We have isolated ZWF1, the Saccharomyces cerevisiae gene that encodes G6PD, and identified its transcript and transcription start point . Expression of ZWF1 appears not to be regulated, consistent with its 'housekeeping' role . Null mutants lacking G6PD appear to grow normally, but are more sensitive than wild type to oxidizing agents that presumably reduce the level of NADPH . This suggests that G6PD has a major role in NADPH production in yeast . Regulation of GAL1 expression appears normal in zwf1 mutants, suggesting that the pentose phosphate pathway is not involved in glucose repression . The predicted amino acid sequence of yeast G6PD is highly similar to the sequence of the Drosophila, human, and rat enzymes, except near its N terminus, where the yeast and Drosophila sequences diverge from that of human and rat.

J Biol Chem, 1990 Dec 15, 265(35), 21997 - 2003
Expression of human vitamin D receptor in Saccharomyces cerevisiae . Purification, properties, and generation of polyclonal antibodies; Sone T et al.; We have cloned a cDNA encoding the human vitamin D receptor (VDR) into a high copy yeast plasmid controlled transcriptionally by the copper-inducible metallothionein (CUP-1) promoter to produce YEpV1 . Introduction of this plasmid in the protease deficient Saccharomyces cerevisiae strain BJ 3505 and subsequent growth in the presence of copper and 1,25-dihydroxyvitamin D3 leads to the synthesis of intact VDR comprising over 0.5% of total soluble protein . The VDR was purified to near homogeneity from similarly induced yeast cultures by ammonium sulfate precipitation, and sequential DNA-cellulose and DEAE-Sephadex chromatography, and then characterized for physical and functional properties . The purified VDR associated with a specific synthetic DNA sequence comprising the vitamin D response element as assayed through bandshift analysis . Binding, however, required the presence of a mammalian cell protein factor that also enhances vitamin D response element interaction by mammalian cell-derived VDR . Polyclonal antibodies raised in rabbits against the purified VDR further retarded the receptor/nuclear factor/DNA complex in these analyses . These studies, together with our previous experiments that demonstrate reconstitution of a vitamin D-dependent transcription system in yeast, show that the VDR can be produced and purified from yeast in a functional form.

Nucleic Acids Res, 1990 Dec 11, 18(23), 7099 - 107
Characterization of TPI gene expression in isogeneic wild-type and gcr1-deletion mutant strains of Saccharomyces cerevisiae; Scott EW et al.; In Saccharomyces cerevisiae the enzymes of glycolysis constitute 30-60 percent of the soluble protein . GCR1 gene function is required for high level glycolytic gene expression . We have undertaken a biochemical and genetic characterization of TPI, a gene affected by gcr1 lesions . Northern analysis showed that steady-state levels of TPI transcripts are severely reduced in gcr1 mutant strains . However, primer extension experiments revealed that TPI transcripts isolated from wild-type and gcr1 mutant strains have identical 5' ends . To map the 5' boundary of TPI controlling region, we employed a TPI::lacZ gene fusion carrying 3.5 kb 5' to the translational start of the TPI structural gene . Nuclease Bal31 deletion analysis demonstrated that sequences sufficient for high level expression of TPI reside within 392 nucleotides preceding the start of the structural gene . We have identified GRF1/RAPI/TUF-binding site positioned 339 to 349 bp 5' to the translation start of TPI . DNA band shift assays were carried out with wild-type and gcr1 deletion mutant strains, and similar patterns of band shifting were observed.

J Biol Chem, 1990 Dec 5, 265(34), 20813 - 21
Isolation and characterization of QCR9, a nuclear gene encoding the 7.3-kDa subunit 9 of the Saccharomyces cerevisiae ubiquinol-cytochrome c oxidoreductase complex . An intron-containing gene with a conserved sequence occurring in the intron of COX4; Phillips JD et al.; A nuclear gene (QCR9) encoding the 7.3-kDa subunit 9 of the mitochondrial cytochrome bc1 complex from Saccharomyces cerevisiae has been isolated from a yeast genomic library by hybridization with a degenerate oligonucleotide corresponding to nine amino acids proximal to the N terminus of purified subunit 9 . QCR9 includes a 195-base pair open reading frame capable of encoding a protein of 66 amino acids and having a predicted molecular weight of 7471 . The N-terminal methionine of subunit 9 is removed posttranslationally because the N-terminal sequence of the purified protein begins with serine 2 . The ATG triplet corresponding to the N-terminal methionine is separated from the open reading frame by an intron . The intron is 213 base pairs long and contains previously reported 5' donor, 3' acceptor, and TACTAAC sequences necessary for splicing . The splice junctions, as well as the 5' end of the message, were confirmed by isolation and sequencing of a cDNA copy of QCR9 . In addition, the intron contains a nucleotide sequence in which 15 out of 18 nucleotides are identical with a sequence in the intron of COX4, the nuclear gene encoding cytochrome c oxidase subunit 4 . The deduced amino acid sequence of the yeast subunit 9 is 39% identical with that of a protein of similar molecular weight from beef heart cytochrome bc1 complex . If conservative substitutions are allowed for, the two proteins are 56% similar . The predicted secondary structure of the 7.3-kDa protein revealed a single possible transmembrane helix, in which the amino acids conserved between beef heart and yeast are asymmetrically arranged along one face of the helix, implying that this domain of the protein is involved in a conserved interaction with another hydrophobic protein of the cytochrome bc1 complex . Two yeast strains, JDP1 and JDP2, were constructed in which QCR9 was deleted . Both strains grew very poorly, or not at all, on nonfermentable carbon sources and exhibited, at most, only 5% of wild-type ubiquinol-cytochrome c oxidoreductase activity . Optical spectra of mitochondrial membranes from the deletion strains revealed slightly reduced levels of cytochrome b . When JDP1 and JDP2 were complemented with a plasmid carrying QCR9, the resulting yeast grew normally on ethanol/glycerol and exhibited normal cytochrome c reductase activities and optical spectra . These results indicate that QCR9 encodes a 7.3-kDa subunit of the bc1 complex that is required for formation of a fully functional complex.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1990 Dec 5, 265(34), 21216 - 22
Cell cycle control by Ca2+ in Saccharomyces cerevisiae; Iida H et al.; We established an experimental system suitable for study of cell cycle regulation by Ca2+ in the yeast Saccharomyces cerevisiae . Systematic cell cycle analysis using media containing various concentrations of Ca2+, a Ca2(+)-ionophore (A23187), and a Ca2(+)-chelator {ethylenebis(oxyethylenenitrilo)}tetraacetic acid (EGTA) revealed that simultaneous addition of 10 microM A23187 and 10 mM EGTA to cells growing in a Ca2(+)-deficient medium at 22 degrees C caused rapid decrease in intracellular Ca content and resulted in transient G1 arrest followed by block mostly at G2/M, as revealed by flow cytometry . Recovery from G1 arrest was not due to coordinated initiation of DNA synthesis and bud emergence: unbudded cells with S or G2/M DNA were observed . Examination of terminal phenotype suggested that Ca2+ was required at all the stages of the cell cycle except for the initiation of DNA synthesis . The intracellular cAMP level decreased within 10 min of addition of A23187 and EGTA . No significant transient G1 arrest was observed in cells incubated with 8-Br-cAMP, or RAS2val19 and delta bcy1 mutants, which produce a high level of cAMP and have constitutively activated cAMP-dependent protein kinase, respectively . These results indicate that Ca2+ is essential for cell cycle progression and suggest that Ca2+ may regulate the cAMP level . This system will be useful for genetic and molecular studies on cell cycle events regulated by Ca2+.

J Cell Biol, 1990 Dec, 111(6 Pt 1), 2573 - 86
BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin; Berlin V et al.; BIK1 function is required for nuclear fusion, chromosome disjunction, and nuclear segregation during mitosis . The BIK1 protein colocalizes with tubulin to the spindle pole body and mitotic spindle . Synthetic lethality observed in double mutant strains containing a mutation in the BIK1 gene and in the gene for alpha- or beta-tubulin is consistent with a physical interaction between BIK1 and tubulin . Furthermore, over- or underexpression of BIK1 causes aberrant microtubule assembly and function, bik1 null mutants are viable but contain very short or undetectable cytoplasmic microtubules . Spindle formation often occurs strictly within the mother cell, probably accounting for the many multinucleate and anucleate bik1 cells . Elevated levels of chromosome loss in bik1 cells are indicative of defective spindle function . Nuclear fusion is blocked in bik1 x bik1 zygotes, which have truncated cytoplasmic microtubules . Cells overexpressing BIK1 initially have abnormally short or nonexistent spindle microtubules and long cytoplasmic microtubules . Subsequently, cells lose all microtubule structures, coincident with the arrest of division . Based on these results, we propose that BIK1 is required stoichiometrically for the formation or stabilization of microtubules during mitosis and for spindle pole body fusion during conjugation.

EMBO J, 1990 Dec, 9(13), 4569 - 75
Saccharomyces cerevisiae 3-methyladenine DNA glycosylase has homology to the AlkA glycosylase of E . coli and is induced in response to DNA alkylation damage; Chen J et al.; We previously cloned a DNA fragment from Saccharomyces cerevisiae that suppressed the alkylation sensitivity of Escherichia coli glycosylase deficient mutants and we showed that it apparently contained a gene for 3-methyl-adenine DNA glycosylase (MAG) . Here we establish the identity of the MAG gene by sequence analysis and describe its in vivo function and expression in yeast cells . The MAG DNA glycosylase specifically protects yeast cells against the killing effects of alkylating agents . It does not protect cells against mutation; indeed, it appears to generate mutations which presumably result from those apurinic sites produced by the glycosylase that escape further repair . The MAG gene, which we mapped to chromosome V, is not allelic with any of the RAD genes and appears to be allelic to the unmapped MMS-5 gene . From its sequence the MAG glycosylase is predicted to contain 296 amino acids and have a molecular weight of 34,293 daltons . A 137 amino acid stretch of the MAG glycosylase displays 27.0% identity and 63.5% similarity with the E . coli AlkA glycosylase . Transcription of the MAG gene, like that of the E . coli alkA gene, is greatly increased when yeast cells are exposed to relatively non-toxic levels of alkylating agents.

EMBO J, 1990 Dec, 9(13), 4563 - 8
Cloning and expression in Escherichia coli of a gene for an alkylbase DNA glycosylase from Saccharomyces cerevisiae; a homologue to the bacterial alkA gene; Berdal KG et al.; An alkylation repair deficient mutant of Escherichia coli (tag ada), lacking DNA glycosylase activity for removal of alkylated bases, was transformed by a genomic yeast DNA library and clones selected which survived plating on medium containing the alkylating agent methylmethane sulphonate . Three distinct yeast clones were identified which were able to suppress the alkylation sensitive phenotype of the bacterial mutant . Restriction enzyme analysis revealed common DNA fragments present in all three clones spanning 2 kb of yeast DNA . DNA from this region was sequenced and analysed for possible translation of polypeptides with any homology to either the Tag or the AlkA DNA glycosylases of E . coli . One open reading frame of 296 amino acids was identified encoding a putative protein with significant homology to AlkA . DNA containing the open reading frame was subcloned in E . coli expression vectors and cell extracts assayed for alkylbase DNA glycosylase activity . It appeared that such activity was expressed at levels sufficiently high for enzyme purification . The molecular weight of the purified protein was determined by SDS-PAGE to be 35,000 daltons, in good agreement with the 34,340 value calculated from the sequence . The yeast enzyme was able to excise 7-methylguanine as well as 3-methyladenine from dimethyl sulphate treated DNA, confirming the related nature of this enzyme to the AlkA DNA glycosylase from E . coli.

EMBO J, 1990 Dec, 9(13), 4503 - 9
Identification of essential elements in U14 RNA of Saccharomyces cerevisiae; Jarmolowski A et al.; The U14 RNA of Saccharomyces cerevisiae is a small nucleolar RNA (snoRNA) required for normal production of 18S rRNA . Depletion of U14 results in impaired processing of pre-rRNA, deficiency in 18S-containing intermediates and marked under-accumulation of mature 18S RNA . The present report describes results of functional mapping of U14, by a variety of mutagenic approaches . Special attention was directed at assessing the importance of sequence elements conserved between yeast and mouse U14 as well as other snoRNA species . Functionality was assessed in a test strain containing a galactose dependent U14 gene . The results show portions of three U14 conserved regions to be required for U14 accumulation or function . These regions include bases in: (i) the 5'-proximal box C region, (ii) the 3'-distal box D region, and (iii) a 13 base domain complementary to 18S rRNA . Point and multi-base substitution mutations in the snoRNA conserved box C and box D regions prevent U14 accumulation . Mutations in the essential 18S related domain do not effect U14 levels, but do disrupt synthesis of 18S RNA, indicating that this region is required for function . Taken together, the results suggest that the box C and box D regions influence U14 expression or stability and that U14 function might involve direct interaction with 18S RNA.

EMBO J, 1990 Dec, 9(13), 4347 - 58
The CHL 1 (CTF 1) gene product of Saccharomyces cerevisiae is important for chromosome transmission and normal cell cycle progression in G2/M; Gerring SL et al.; We have analyzed the CTF1 gene, identified in a screen for mutants with decreased chromosome transmission fidelity and shown to correspond to the previously identified chl1 mutation . Chl1 null mutants exhibited a 200-fold increase in the rate of chromosome III missegregation per cell division, and near wild-type rates of marker homozygosis on this chromosome by mitotic recombination . Analysis of the segregation of a marker chromosome indicated that sister chromatid loss (1:0 segregation) and sister chromatid non-disjunction (2:0 segregation) contributed equally to chromosome missegregation . A genomic clone of CHL1 was isolated and used to map its physical position on chromosome XVI . Nucleotide sequence analysis of CHL1 revealed a 2.6 kb open reading frame with a 99 kd predicted protein sequence that contained two PEST sequences and was 23% identical to the coding region of a nucleotide excision repair gene, RAD3 . Domains of homology between these two predicted protein sequences included a helix-turn-helix motif and an ATP binding site containing a helicase consensus . Mutants lacking the CHL1 gene product are viable and display two striking, and perhaps interrelated, phenotypes: extreme chromosome instability and a delay in cell cycle progression in G2/M . This delay is independent of the cell cycle checkpoint that requires the function of the RAD9 gene.

Proc Natl Acad Sci U S A, 1990 Dec, 87(23), 9338 - 42
Control of mitochondrial gene expression in the yeast Saccharomyces cerevisiae; Biswas TK; Mitochondrial promoters in Saccharomyces cerevisiae contain an identical octanucleotide {sequence: see text} sequence present just upstream of the initiation site (at the left end of the arrow) . Studies have shown that the transcription rates of mitochondrial genes vary from 7- to 15-fold . The nucleotide at position +2 regulates the efficiency of mitochondrial promoters but does not affect the specificity of initiation . The data presented herein demonstrate that the variable transcription rates of mitochondrial genes are due to different levels of transcriptional initiation . The rate of first phosphodiester bond formation between a purine and a pyrimidine on a weak promoter is much lower than that of purine-purine on a strong promoter . A dinucleotide corresponding to positions +1 and +2 acts in vitro as a primer, bypassing the first phosphodiester bond formation at the initiation site . When these dinucleotides were used to prime transcription, the activities of the strong and weak promoters were found to be identical . In heparin-challenge experiments, there is no significant effect of dinucleotide on heparin-resistant DNA-RNA polymerase complex formation . These results indicate that the low level of transcription from the weak mitochondrial promoter is due to the slow rate of formation of the first phosphodiester bond.

Mol Cell Biol, 1990 Dec, 10(12), 6742 - 54
Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae; Herman PK et al.; VPS34 gene function is required for the efficient localization of a variety of vacuolar proteins . We have cloned and sequenced the wild-type VPS34 gene in order to gain a better understanding of the role of its protein product in this intracellular sorting pathway . Interestingly, disruption of the VPS34 locus resulted in a temperature-sensitive growth defect, indicating that the VPS34 gene is essential for vegetative growth only at elevated growth temperatures . As with the original vps34 alleles, vps34 null mutants exhibited severe vacuolar protein sorting defects and possessed a morphologically normal vacuolar structure . The VPS34 gene DNA sequence identifies an open reading frame that could encode a hydrophilic protein of 875 amino acids . The predicted protein sequence lacks any apparent signal sequence or membrane-spanning domains, suggesting that Vps34p does not enter the secretory pathway . Results from immunoprecipitation experiments with antiserum prepared against a TrpE-Vps34 fusion protein were consistent with this prediction: a rare, unglycosylated protein of approximately 95,000 Da was detected in extracts of wild-type Saccharomyces cerevisiae cells . Cell fractionation studies indicated that a significant portion of the Vps34p is found associated with a particulate fraction of yeast cells . This particulate Vps34p was readily solubilized by treatment with 2 M urea but not with Triton X-100, suggesting that the presence of Vps34p in this pelletable structure is mediated by protein-protein interactions . vp34 mutant cells also exhibited a defect in the normal partitioning of the vacuolar compartment between mother and daughter cells during cell division . In more than 80% of the delta vps34 dividing cells examined, no vacuolar structures were observed in the newly emerging bud, whereas in wild-type dividing cells, more than 95% of the buds had a detectable vacuolar compartment . Our results suggest that the Vps34p may act as a component of a relatively large intracellular structure that functions to facilitate specific steps of the vacuolar protein delivery and inheritance pathways.

Mol Cell Biol, 1990 Dec, 10(12), 6554 - 64
Characterization of RAD9 of Saccharomyces cerevisiae and evidence that its function acts posttranslationally in cell cycle arrest after DNA damage; Weinert TA et al.; In eucaryotic cells, incompletely replicated or damaged chromosomes induce cell cycle arrest in G2 before mitosis, and in the yeast Saccharomyces cerevisiae the RAD9 gene is essential for the cell cycle arrest (T.A . Weinert and L . H . Hartwell, Science 241:317-322, 1988) . In this report, we extend the analysis of RAD9-dependent cell cycle control . We found that both induction of RAD9-dependent arrest in G2 and recovery from arrest could occur in the presence of the protein synthesis inhibitor cycloheximide, showing that the mechanism of RAD9-dependent control involves a posttranslational mechanism(s) . We have isolated and determined the DNA sequence of the RAD9 gene, confirming the DNA sequence reported previously (R . H . Schiestl, P . Reynolds, S . Prakash, and L . Prakash, Mol . Cell . Biol . 9:1882-1886, 1989) . The predicted protein sequence for the Rad9 protein bears no similarity to sequences of known proteins . We also found that synthesis of the RAD9 transcript in the cell cycle was constitutive and not induced by X-irradiation . We constructed yeast cells containing a complete deletion of the RAD9 gene; the rad9 null mutants were viable, sensitive to X- and UV irradiation, and defective for cell cycle arrest after DNA damage . Although Rad+ and rad9 delta cells had similar growth rates and cell cycle kinetics in unirradiated cells, the spontaneous rate of chromosome loss (in unirradiated cells) was elevated 7- to 21-fold in rad9 delta cells . These studies show that in the presence of induced or endogenous DNA damage, RAD9 is a negative regulator that inhibits progression from G2 in order to preserve cell viability and to maintain the fidelity of chromosome transmission.

Mol Cell Biol, 1990 Dec, 10(12), 6500 - 11
Characterization of TUP1, a mediator of glucose repression in Saccharomyces cerevisiae; Williams FE et al.; The TUP1 and CYC8 (= SSN6) genes of Saccharomyces cerevisiae play a major role in glucose repression . Mutations in either TUP1 or CYC8 eliminate or reduce glucose repression of many repressible genes and induce other phenotypes, including flocculence, failure to sporulate, and sterility of MAT alpha cells . The TUP1 gene was isolated in a screen for genes that regulate mating type (V.L . MacKay, Methods Enzymol . 101:325-343, 1983) . We found that a 3.5-kb restriction fragment was sufficient for complete complementation of tup1-100 . The gene was further localized by insertional mutagenesis and RNA mapping . Sequence analysis of 2.9 kb of DNA including TUP1 revealed only one long open reading frame which predicts a protein of molecular weight 78,221 . The predicted protein is rich in serine, threonine, and glutamine . In the carboxyl region there are six repeats of a pattern of about 43 amino acids . This same pattern of conserved residues is seen in the beta subunit of transducin and the yeast CDC4 gene product . Insertion and deletion mutants are viable, with the same range of phenotypes as for point mutants . Deletions of the 3' end of the coding region produced the same mutant phenotypes as did total deletions, suggesting that the C terminus is critical for TUP1 function . Strains with deletions in both the CYC8 and TUP1 genes are viable, with phenotypes similar to those of strains with a single deletion . A deletion mutation of TUP1 was able to suppress the snf1 mutation block on expression of the SUC2 gene encoding invertase.

Mol Cell Biol, 1990 Dec, 10(12), 6389 - 96
gcr2, a new mutation affecting glycolytic gene expression in Saccharomyces cerevisiae; Uemura H et al.; Screening of a mutagenized strain carrying a multicopy ENO1-'lacZ fusion plasmid revealed a new mutation affecting most glycolytic enzyme activities in a pattern resembling that caused by gcr1: levels in the range of 10% of wild-type levels on glycerol plus lactate but somewhat higher on glucose . The recessive single nuclear gene mutation, named gcr2-1, was unlinked to gcr1, and GCR1 in multiple copies did not restore enzyme levels . GCR2 was obtained by complementation from a YCp50 genomic library; the complemented strain had normal enzyme levels, as did a strain with GCR2 in multiple copies . GCR2 in multiple copies did not suppress gcr1 . A chromosomal gcr2 null mutant was constructed; its pattern of enzyme activities resembled that of the gcr2-1 mutant and, like the gcr2-1 mutant, its growth defect on glucose was only partial (in contrast to the glucose negativity of the gcr1 mutant) . Northern (RNA) analysis showed that gcr2 and gcr1 affect ENO1 mRNA levels.

Mol Cell Biol, 1990 Dec, 10(12), 6123 - 31
A suppressor of an RNA polymerase II mutation of Saccharomyces cerevisiae encodes a subunit common to RNA polymerases I, II, and III; Archambault J et al.; RNA polymerase II (RNAPII) is a complex multisubunit enzyme responsible for the synthesis of pre-mRNA in eucaryotes . The enzyme is made of two large subunits associated with at least eight smaller polypeptides, some of which are common to all three RNA polymerase species . We have initiated a genetic analysis of RNAPII by introducing mutations in RPO21, the gene encoding the largest subunit of RNAPII in Saccharomyces cerevisiae . We have used a yeast genomic library to isolate plasmids that can suppress a temperature-sensitive mutation in RPO21 (rpo21-4), with the goal of identifying gene products that interact with the largest subunit of RNAPII . We found that increased expression of wild-type RPO26, a single-copy, essential gene encoding a 155-amino-acid subunit common to RNAPI, RNAPII, and RNAPIII, suppressed the rpo21-4 temperature-sensitive mutation . Mutations were constructed in vitro that resulted in single amino acid changes in the carboxy-terminal portion of the RPO26 gene product . One temperature-sensitive mutation, as well as some mutations that did not by themselves generate a phenotype, were lethal in combination with rpo21-4 . These results support the idea that the RPO26 and RPO21 gene products interact.

Mol Cell Biol, 1990 Dec, 10(12), 6103 - 13
Role of IME1 expression in regulation of meiosis in Saccharomyces cerevisiae; Smith HE et al.; Two signals are required for meiosis and spore formation in the yeast Saccharomyces cerevisiae: starvation and the MAT products a1 and alpha 2, which determine the a/alpha cell type . These signals lead to increased expression of the IME1 (inducer of meiosis) gene, which is required for sporulation and sporulation-specific gene expression . We report here the sequence of the IME1 gene and the consequences of IME1 expression from the GAL1 promoter . The deduced IME1 product is a 360-amino-acid protein with a tyrosine-rich C-terminal region . Expression of PGAL1-IME1 in vegetative a/alpha cells led to moderate accumulation of four early sporulation-specific transcripts (IME2, SPO11, SPO13, and HOP1); the transcripts accumulated 3- to 10-fold more after starvation . Two sporulation-specific transcripts normally expressed later (SPS1 and SPS2) did not accumulate until PGAL1-IME1 strains were starved, and the intact IME1 gene was not activated by PGAL1-IME1 expression . In a or alpha cells, which lack alpha 2 or a1, expression of PGAL1-IME1 led to the same pattern of IME2 and SPO13 expression as in a/alpha cells, as measured with ime2::lacZ and spo13::lacZ fusions . Thus, in wild-type strains, the increased expression of IME1 in starved a/alpha cells can account entirely for cell type control, but only partially for nutritional control, of early sporulation-specific gene expression . PGAL1-IME1 expression did not cause growing cells to sporulate but permitted efficient sporulation of amino acid-limited cells, which otherwise sporulated poorly . We suggest that IME1 acts primarily as a positive regulator of early sporulation-specific genes and that growth arrest is an independent prerequisite for execution of the sporulation program.

EMBO J, 1990 Dec, 9(13), 4339 - 46
Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes; Sneddon AA et al.; Two genes (PPH21 and PPH22) encoding the yeast homologues of protein serine-threonine phosphatase 2A have been cloned from a Saccharomyces cerevisiae genomic library using a rabbit protein phosphatase 2A cDNA as a hybridization probe . The PPH genes are genetically linked on chromosome IV and are predicted to encode polypeptides each with 74% amino acid sequence identity to rabbit type 2A protein phosphatase, indicating once again the extraordinarily high degree of sequence conservation shown by protein-phosphatases from different species . The two PPH genes show less than 10% amino acid sequence divergence from each other and while disruption of either PPH gene alone is without any major effect, the double disruption is lethal . This indicates that protein phosphatase 2A activity is an essential cellular function in yeast . Measurement of type 2A protein phosphatase activity in yeast strains lacking one or other of the genes indicates that they account for most, if not all, protein phosphatase 2A activity in the cell.

J Bacteriol, 1990 Dec, 172(12), 6892 - 9
Catabolism of bis(5'-nucleosidyl) tetraphosphates in Saccharomyces cerevisiae; Plateau P et al.; Bis(5'-adenosyl) tetraphosphate (Ap4A) phosphorylase II (P . Plateau, M . Fromant, J . M . Schmitter, J . M . Buhler, and S . Blanquet, J . Bacteriol . 171:6437-6445, 1989) was obtained in a homogeneous form through a 40,000-fold purification, starting from a Saccharomyces cerevisiae strain devoid of Ap4A phosphorylase I activity . The former enzyme behaves as a 36.8K monomer . As with Ap4A phosphorylase I, the addition of divalent cations is required for the expression of activity . Mn2+, Mg2+, and Ca2+ sustain phosphorolysis by the two enzymes, whereas Co2+ and Cd2+ stimulate only phosphorylase II activity . All bis(5'-nucleosidyl) tetraphosphates assayed (Ap4A, Ap4C, Ap4G, Ap4U, Gp4G, and Gp4U) are substrates of the two enzymes . However, Ap4A phosphorylase II shows a marked preference for A-containing substrates . The two enzymes catalyze adenosine 5'-phosphosulfate phosphorolysis or an exchange reaction between Pi and the beta-phosphate of any nucleoside diphosphate . They can also produce Ap4A at the expense of ATP and ADP . The gene (APA2) encoding Ap4A phosphorylase II was isolated and sequenced . The deduced amino acid sequence shares 60% identity with that of Ap4A phosphorylase I . Disruption of APA2 and/or APA1 shows that none of these genes is essential for the viability of Saccharomyces cerevisiae . The concentrations of all bis(5'-nucleosidyl) tetraphosphates are increased in an apa1 apa2 double mutant, as compared with the parental wild-type strain . The factor of increase is 5 to 50 times, depending on the nucleotide . This observation supports the conclusion that, in vivo, Ap4A phosphorylase II, like Ap4A phosphorylase I, participates in the catabolism rather than the synthesis of the bis(5'-nucleosidyl) tetraphosphates.

Biochem Int, 1990 Dec, 22(5), 791 - 5
Decreased synthesis of alkali-soluble glucan in a cell-wall mutant of Saccharomyces cerevisiae; Blagoeva J et al.; In vivo studies and quantitative measurements of glucans provide evidence for a decreased rate of synthesis and a lower amount of alkali-soluble glucan in cells of the osmotically fragile VY1160 mutant of the yeast Saccharomyces cerevisiae . Combined genetic and biochemical analysis shows that the srb1 mutation is responsible for the reduction of alkali-soluble glucan . Data on beta(1----3) glucan synthase activity did not indicate the participation of the enzyme in the in vivo synthesis of alkali-soluble glucan and suggest the existence of other glucan synthases in Saccharomyces cerevisiae.

Mol Cell Biol, 1990 Dec, 10(12), 6482 - 90
Cell cycle arrest caused by CLN gene deficiency in Saccharomyces cerevisiae resembles START-I arrest and is independent of the mating-pheromone signalling pathway; Cross FR; Null mutations in three genes encoding cyclin-like proteins (CLN1, CLN2, and CLN3) in Saccharomyces cerevisiae cause cell cycle arrest in G1 (cln arrest) . In cln1 cln2 cln3 strains bearing plasmids containing the CLN3 (also called WHI1 or DAF1) coding sequence under the transcriptional control of a galactose-regulated promoter, shift from galactose to glucose medium (shutting off synthesis of CLN3 mRNA) allowed completion of cell cycles in progress but caused arrest in the ensuing unbudded G1 phase . Cell growth was not inhibited in arrested cells . Cell division occurred in glucose medium even if cells were arrested in S phase during the initial 2 h of glucose treatment, suggesting that CLN function may not be required in the cell cycle after S phase . However, when the coding sequence of the hyperactive C-terminal truncation allele CLN3-2 (formerly DAF1-1) was placed under GAL control, cells went through multiple cycles before arresting after a shift from galactose to glucose . These results suggest that the C terminus of the wild-type protein confers functional instability . cln-arrested cells are mating competent . However, cln arrest is distinct from constitutive activation of the mating-factor signalling pathway because cln-arrested cells were dependent on the addition of pheromone both for mating and for induction of an alpha-factor-induced transcript, FUS1, and because MATa/MAT alpha (pheromone-nonresponsive) strains were capable of cln arrest in G1 (although a residual capacity for cell division before arrest was observed in MATa/MAT alpha strains) . These results are consistent with a specific CLN requirement for START transit.

Proc Natl Acad Sci U S A, 1990 Dec, 87(24), 9665 - 9
Mutants of Saccharomyces cerevisiae defective in the farnesylation of Ras proteins; Goodman LE et al.; Ras proteins are post-translationally modified by farnesylation . In the present investigation, we identified an activity in crude soluble extracts of yeast cells that catalyzes the transfer of a farnesyl moiety from farnesyl pyrophosphate to yeast RAS2 protein . RAS2 proteins having a C-terminal Cys-Ali-Ali-Xaa sequence (where Ali is an aliphatic amino acid and Xaa is the unspecified C-terminal amino acid) served as substrates for this reaction, whereas RAS2 proteins with an altered or deleted Cys-Ali-Ali-Xaa sequence did not . A yeast mutant, dpr1/ram1, originally isolated as a Ras-processing mutant was shown to be defective in farnesyltransferase activity . In addition, another mutant, ram2, also was defective in the transferase activity . These results demonstrate that at least two genes, DPR1/RAM1 and RAM2, are required for the farnesyltransferase activity in yeast.

Proc Natl Acad Sci U S A, 1990 Dec, 87(23), 9406 - 10
Nucleotide sequence and promoter analysis of SPO13, a meiosis-specific gene of Saccharomyces cerevisiae; Buckingham LE et al.; The SPO13 gene, required for meiosis I segregation in Saccharomyces cerevisiae, produces two developmentally regulated transcripts (1.0 and 1.4 kilobases) that differ in length at their 5' ends . The shorter transcript is sufficient to complement the spo13-1 mutation and contains a major open reading frame encoding a highly basic protein of 33.4 kilodaltons . A fragment upstream (-170 to -8) of the open reading frame confers meiosis-specific transcription on a spo13-HIS3 fusion . Deletions at the 5' end of spo13-lacZ fusions define a region between -140 and -80 that is essential for meiosis-specific expression . This region acts in an orientation-independent manner and is responsive to the MAT-RME regulatory cascade . It contains a 10-base-pair sequence, TAGCCGCCGA, found in a number of meiosis-specific genes, that appears to be required for SPO13 expression . This sequence is identical to URS1, a ubiquitous mitotic repressor element.

Mol Cell Biol, 1990 Dec, 10(12), 6362 - 73
Transcriptional derepression of the Saccharomyces cerevisiae HSP26 gene during heat shock; Susek RE et al.; hsp26, the small heat shock protein of Saccharomyces cerevisiae, accumulates in response to heat and other types of stress . It also accumulates during the normal course of development, as cells enter stationary phase growth or begin to sporulate (S . Kurtz, J . Rossi, L . Petko, and S . Lindquist, Science 231:1154-1157, 1986) . Analysis of deletion and insertion mutations demonstrated that transcriptional control plays a critical role in regulating HSP26 expression . The HSP26 promoter was found to be complex and appears to contain repressing elements as well as activating elements . Several upstream deletion mutations resulted in strong constitutive expression of HSP26 . Furthermore, upstream sequences from the HSP26 gene repressed the constitutive expression of a heterologous heat shock gene . We propose that basal repression and heat-induced depression of transcription play major roles in regulating the expression of HSP26 . None of the recombinant constructs that we analyzed separated cis-regulatory sequences responsible for heat shock regulation from those responsible for developmental regulation of HSP26 . Depression of HSP26 transcription may be the general mechanism of HSP26 induction in yeast cells . This regulatory scheme is very different from that described for the regulation of most other heat shock genes.

Mycopathologia, 1990 Dec, 112(3), 165 - 72
Effect of nystatin, amphotericin B and amphotericin B methyl ester on Saccharomyces cerevisiae with different lipid composition; de Resende MA et al.; Saccharomyces cerevisiae was cultured under anaerobiosis in semi-complete medium to which either palmitoleic or oleic acid was added . Cells were grown at 20 degrees C or 30 degrees C . The levels of total lipids, total sterols, and phospholipids were higher in cells grown at 20 degrees C than at 30 degrees C . The effects of nystatin (NYS), amphotericin B (AMB), and amphotericin B methyl ester (AME) were evaluated by determining cell viability and liberation of intracellular compounds . The loss of cell viability is higher in the first 30 minutes of incubation with the drugs and is the same regardless of the type of cells obtained . Low molecular weight compounds and ions such as K+ are liberated a few minutes after incubation with the drugs whereas proteins and substances absorbing at 260 nm are liberated later . Phosphate liberation comes after K+ and before compounds of higher molecular weights.

Genetics, 1990 Dec, 126(4), 823 - 35
An RME1-independent pathway for sporulation control in Saccharomyces cerevisiae acts through IME1 transcript accumulation; Kao G et al.; The RES1-1 mutation was isolated on the basis of its ability to allow MATa/MAT alpha diploid Saccharomyces cerevisiae cells to express a late sporulation-regulated gene, SPR3, in the presence of excess copies of RME1 . RME1 is a repressor of meiosis that is normally expressed in cells that lack the a1/alpha 2 repressor encoded by MAT . The RES1-1 mutation also supports sporulation in mat-insufficient diploids . This phenotype does not result from a failure to express RME1 and is not due to activation of the silent copies of mating type information . RES1-1 activates sporulation by allowing IME1 accumulation in all cell types, irrespective of the presence of the MAT products . IME1 is still responsive to RME1 in RES1-1 cells, since double mutants (rme1 RES1-1) that are deficient at MAT can sporulate better than either single mutant . RES1-1 is not an allele of IME1.

Genetics, 1990 Dec, 126(4), 799 - 812
kem mutations affect nuclear fusion in Saccharomyces cerevisiae; Kim J et al.; We have identified mutations in three genes of Saccharomyces cerevisiae, KEM1, KEM2 and KEM3, that enhance the nuclear fusion defect of kar1-1 yeast during conjugation . The KEM1 and KEM3 genes are located on the left arm of chromosome VII . Kem mutations reduce nuclear fusion whether the kem and the kar1-1 mutations are in the same or in different parents (i.e., in both kem kar1-1 X wild-type and kem X kar 1-1 crosses) . kem 1 X kem 1 crosses show a defect in nuclear fusion, but kem 1 X wild-type crosses do not . Mutant kem 1 strains are hypersensitive to benomyl, lose chromosomes at a rate 10-20-fold higher than KEM+ strains, and lose viability upon nitrogen starvation . In addition, kem 1/kem 1 diploids are unable to sporulate . Cells containing a kem 1 null allele grow very poorly, have an elongated rod-shape and are defective in spindle pole body duplication and/or separation . The KEM 1 gene, which is expressed as a 5.5-kb mRNA transcript, contains a 4.6-kb open reading frame encoding a 175-kD protein.

Curr Genet, 1990 Dec, 18(6), 531 - 6
Expression of the Aspergillus niger glucose oxidase gene in A . niger, A . nidulans and Saccharomyces cerevisiae; Whittington H et al.; We report the cloning of the Aspergillus niger glucose oxidase gene and its use to elevate glucose oxidase productivity in A . niger by increasing the gene dosage . In addition, the gene has been introduced into A . nidulans where it provides the novel capacity to produce glucose oxidase . A plasmid, in which DNA encoding the mature form of glucose oxidase was preceded by a Saccharomyces cerevisiae secretion signal, effected high-level production of extracellular glucose oxidase in this yeast.

J Gen Microbiol, 1990 Dec, 136 ( Pt 12), 2537 - 43
Transient increase in Ca2+ influx in Saccharomyces cerevisiae in response to glucose: effects of intracellular acidification and cAMP levels; Eilam Y et al.; Influx of 45Ca2+ into Saccharomyces cerevisiae was measured under experimental conditions which enabled measurements of initial rate of transport across the plasma membrane, without interference by the vacuolar Ca2+ transport system . Addition of glucose or glycerol to the cells, after pre-incubation in glucose-free medium for 5 min, caused a rapid, transient increase in 45Ca2+ influx, reaching a peak at 3-5 min after addition of substrate . Ethanol, or glycerol added with antimycin A, had no effect on 45Ca2+ influx . We have shown previously that this increase is not mediated by an effect of the substrates on intracellular ATP levels . Changes in membrane potential accounted for only a part of the glucose-stimulated 45Ca2+ influx . The roles of intracellular acidification and changes in cellular cAMP in mediating the effects of glucose on 45Ca2+ influx were examined . After a short preincubation in glucose-free medium addition of glucose caused a decrease in the intracellular pH, {pH}i, which reached a minimum value after 3 min . A transient increase in the cellular cAMP level was also observed . Addition of glycerol also caused intracellular acidification, but ethanol or glycerol added with antimycin A had no effect on {pH}i . Artificial intracellular acidification induced by exposure to isobutyric acid or to CCCP caused a transient rise in Ca2+ influx but the extent of the increase was smaller than that caused by glucose, and the time-course was different . We conclude that intracellular acidification may be responsible for part of the glucose stimulation of Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1990 Nov 26, 194(1), 293 - 9
Photoaffinity labelling of the purine-cytosine permease of Saccharomyces cerevisiae; Chirio MC et al.; 8-Azidoadenine was used as a photoaffinity reagent to characterize the purine-cytosine permease of Saccharomyces cerevisiae . It is a potent competitive inhibitor of cytosine uptake and irradiation of the cells incubated with the label induced the irreversible inactivation of cytosine uptake . Addition of excess cytosine prevented this labelling which was restricted to the outer face of the plasma membrane since it was not accumulated by the cells . In the strain with the amplified purine-cytosine permease gene the maximum cytosine uptake rate was increased 4-5-fold relative to wild type without a modification of the Michaelis constant of uptake (Kt); no uptake could be measured in the deleted strain . The relative amounts of specific labelling determined for the cells and for membrane preparations were 0, 1 and 4 for the null, the wild-type and the amplified strains, respectively . One major band specifically labelled by {3H}azidoadenine, corresponding to a polypeptide with an apparent molecular mass of 45 kDa, was observed in the wild type, amplified in the strain carrying the multicopy plasmid and not detected in the deleted strain . Therefore this polypeptide corresponds to the purine-cytosine permease.

Nucleic Acids Res, 1990 Nov 25, 18(22), 6559 - 64
Interactions of PRP2 protein with pre-mRNA splicing complexes in Saccharomyces cerevisiae; King DS et al.; PRP2 protein of Saccharomyces cerevisiae is required for the pre-mRNA splicing reaction but not for the early stages of spliceosome assembly . Using anti-PRP2 antibodies we demonstrate that PRP2 protein is associated with spliceosomes prior to, and throughout step 1 of the splicing reaction . Heat-inactivated prp2 protein, by contrast, does not seem to associate with spliceosomes . By elution of electrophoretically distinct spliceosomal complexes from non-denaturing gels we identify the specific complex with which PRP2 initially interacts in the pathway of spliceosome assembly.

J Biol Chem, 1990 Nov 25, 265(33), 20057 - 60
Saccharomyces cerevisiae STE14 gene is required for COOH-terminal methylation of a-factor mating pheromone; Marr RS et al.; Saccharomyces cerevisiae a-factor is a dodecapeptide pheromone in which the carboxyl group of the COOH-terminal cysteine residue is methyl-esterified and the sulfhydryl side chain is conjugated in thioether linkage to a farnesyl moiety . We found that MAT a ste14 mutant cells secreted a biologically inactive form of a-factor which had more hydrophilic character than the wild-type pheromone . The authentic pheromone could be metabolically labeled with {methyl-3H}methionine, and the resulting COOH-terminal methyl ester could be removed by mild alkaline hydrolysis . In contrast, a-factor secreted by ste14 mutants did not incorporate a base-labile 3H-methyl moiety . Base treatment converted the normal pheromone into a form which was biologically inactive and which comigrated with the ste14 form of the peptide upon thin-layer chromatography . These results indicate that STE14 gene function is required for COOH-terminal methylation of a-factor.

J Biol Chem, 1990 Nov 25, 265(33), 20144 - 9
The OLE1 gene of Saccharomyces cerevisiae encodes the delta 9 fatty acid desaturase and can be functionally replaced by the rat stearoyl-CoA desaturase gene; Stukey JE et al.; Strains of Saccharomyces cerevisiae bearing the ole1 mutation are defective in unsaturated fatty acid (UFA) synthesis and require UFAs for growth . A previously isolated yeast genomic fragment complementing the ole1 mutation has been sequenced and determined to encode the delta 9 fatty acid desaturase enzyme by comparison of primary amino acid sequence to the rat liver stearoyl-CoA desaturase . The OLE1 structural gene encodes a protein of 510 amino acids (251 hydrophobic) having an approximate molecular mass of 57.4 kDa . A 257-amino acid internal region of the yeast open reading frame aligns with and shows 36% identity and 60% similarity to the rat liver stearoyl-CoA desaturase protein . This comparison disclosed three short regions of high consecutive amino acid identity (greater than 70%) including one 11 of 12 perfect residue match . The predicted yeast enzyme contains at least four potential membrane-spanning regions and several shorter hydrophobic regions that align exactly with similar sequences in the rat liver protein . An ole1 gene-disrupted yeast strain was transformed with a yeast-rat chimeric gene consisting of the promoter region and N-terminal 27 codons of OLE1 fused to the rat desaturase coding sequence . Fusion gene transformants displayed near equivalent growth rates and modest lipid composition changes relative to wild type yeast control implying a significant conservation of delta 9 desaturase tertiary structure and efficient interaction between the rat desaturase and yeast cytochrome b5.

Biochim Biophys Acta, 1990 Nov 16, 1029(2), 211 - 7
Regulation of potassium fluxes in Saccharomyces cerevisiae; Ramos J et al.; To investigate the regulation of K+ fluxes in Saccharomyces cerevisiae the dependence of K+ efflux and Rb+ influx on {K+}i, pHi, {Na+}i, membrane potential, cell volume, and turgor pressure were studied in cells with different K+ contents . By decreasing the cell volume with osmotic shocks and the cellular pH with butyric acid the following was found . (1) The K+ efflux induced by uncouplers decreases simultaneously with the decrease of the K+ content of the cell, but the process was insensitive to {K+}i, pHi, cell volume and turgor pressure . The internal presence of Na+ inhibited this K+ efflux . (2) The increase of the Vmax of Rb+ influx observed in low-K+ cells is due to the decrease of the pHi and probably mediated by the increase of the activity of the plasma membrane ATPase . The Vmax is independent of {K+}i, {Na+}i, cell volume and turgor pressure . (3) The decrease in the Km of Bt+ influx observed in low-K+ cells does not depend directly on {K+}i, pHi, cell volume or turgor pressure . If Na+ is present, {Na+}i might be directly involved in the regulation of the Km.

Biochem Biophys Res Commun, 1990 Nov 15, 172(3), 1310 - 6
Total in vitro maturation of the Saccharomyces cerevisiae a-factor lipopeptide mating pheromone; Marcus S et al.; The a-factor mating pheromone, produced by Saccharomyces cerevisiae a haploid cells, is post-translationally modified in a manner analogous to that of the ras proto-oncogene product . A consensus C-terminal amino acid sequence, -CAAX (C is cysteine, A is aliphatic amino acid, and X is any amino acid), is the target of these modifications, which include isoprenylation (essential for Ras function), proteolysis of the -AAX sequence, and carboxy methyl esterification . Recently, the RAM/DPR1 gene product was shown to be a component of the activity responsible for isoprenylation of both Ras and a-factor . In this report, we present an in vitro assay which not only detects a-factor isoprenylation, but also proteolysis and carboxy methyl esterification, and directly demonstrates, biochemically, the order of these processing events . This a-factor maturation assay may prove useful for screening agents which block any of the steps involved in the post-translational modification of the a-factor and Ras -CAAX sequences . Such agents would be potential anti-Ras-related cancer therapeutic drugs.

Eur J Biochem, 1990 Nov 13, 193(3), 675 - 80
The C-terminal part of the CDC25 gene product plays a key role in signal transduction in the glucose-induced modulation of cAMP level in Saccharomyces cerevisiae; Van Aelst L et al.; In the yeast Saccharomyces cerevisiae, addition of glucose to cells grown under glucose-derepressed conditions induces a transient rise in the intracellular level of cAMP . This modulation requires functional elements of the cAMP-producing pathway, adenylate cyclase, ras proteins and the product of CDC25 gene . To determine whether or not the CDC25 gene product is a transducing element in the signal-transmission pathway leading from glucose to ras adenylate cyclase we have made use of the mutated allele RAS2Ile152 whose gene product uncouples the product of CDC25 from adenylate cyclase, but does not promotes other secondary phenotypes . The transient increase in cAMP is lost in cells lacking a functional CDC25 gene product, although they produce a normal amount of cAMP with the RAS2Ile152 gene . This result demonstrates the requirement of CDC25 for mediation of glucose signal transmission . The fact that cells grow normally on glucose in the absence of glucose-induced cAMP signaling confirms that this signaling pathway is not essential for growth on glucose . To further analyze the role of the CDC25 gene product we have made use of truncated versions of the gene . The results show that the C-terminal part of the gene alone is able to mediate glucose-induced activation of the RAS adenylate cyclase pathway.

FEBS Lett, 1990 Nov 12, 274(1-2), 19 - 22
The glucose-induced polyphosphoinositides turnover in Saccharomyces cerevisiae is not dependent on the CDC25-RAS mediated signal transduction pathway; Frascotti G et al.; Recently the polyphosphoinositides (PI) turnover has been related to the control of growth and cell cycle also in Saccharomyces cerevisiae, and the RAS2 and RAS1 gene products have been shown to be involved in the stimulation of PI turnover in G0/G1 arrested yeast cells . Here we show that addition of glucose to previously glucose-starved cells, stimulates, the PI turnover with fast kinetics also in yeast cells that were not arrested in the G0/G1 phase of the cell cycle . In addition PI turnover is equally stimulated in temperature sensitive cdc25-1 and cdc25-5 strains at restrictive temperature, as well as in ras1, ras2-ts strain, suggesting that PI turnover stimulation is not dependent on the CDC25-RAS mediated signal transduction pathway.

J Biol Chem, 1990 Nov 5, 265(31), 19122 - 7
Purification and characterization of Saccharomyces cerevisiae uridine monophosphate kinase; Ma JJ et al.; The SOC8 gene was isolated as an extragenic suppressor of cdc8 mutant cells . It has been suggested that SOC8 is allelic with the URA6 gene which was originally identified as a uridine monophosphate kinase . In this article, we describe the purification of the uridine monophosphate kinase from a yeast Saccharomyces cerevisae strain that overproduces the activity 8-fold . The protein was purified through Fast-Flow Q-Separose, phosphocellulose, blue-agarose, and fast protein liquid chromatography Superose 12 columns, and appears homogeneous by sodium dodecyl sulfate-polyacrylamide gel analysis . The uridine monophosphate kinase contains a single polypeptide with a molecular weight of 25,000, as evidence by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analysis . The amino acid composition has also been determined . Substrate specificity studies show that the relative activity of nucleoside monophosphates is in order of UMP greater than dUMP, and to a lesser extent, dTMP, GMP, and dGMP . The Km and Vm of UMP, dUMP, and dTMP have been determined.

J Biol Chem, 1990 Nov 5, 265(31), 18976 - 82
Purification of a site-specific endonuclease, I-Sce II, encoded by intron 4 alpha of the mitochondrial coxI gene of Saccharomyces cerevisiae; Wernette CM et al.; We have purified to near homogeneity a site-specific, double-stranded DNA endonuclease (I-Sce II) encoded by intron 4 alpha (aI4 alpha) of the yeast mitochondrial coxI gene . Our purification starts with a high salt extract of mitochondria isolated from a yeast strain that overproduces the enzyme because of a block in splicing of aI4 alpha . The final step of purification is an affinity column consisting of covalently bound double-stranded DNA multimers of a synthetic sequence, 5'-TTGGTCATCCAGAAGTAT-3', which contains the I-Sce II cleavage/recognition site . Typical yields of enzyme are 3-5% with a specific activity of approximately 500,000 units/mg, where 1 unit of activity cleaves 50 ng of DNA substrate/h at 30 degrees C . I-Sce II has a monomer molecular mass of 31 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Active enzyme purifies as a 55-kDa species, which we presume to be a homodimer . I-Sce II monomer comigrates with an in vivo synthesized mitochondrial translation product made in the strain that overproduces the enzyme . We conclude that I-Sce II is derived by proteolytic processing of a precursor polypeptide, p62, encoded by an in-frame fusion of coxI exons 1-4 with the downstream aI4 alpha reading frame . I-Sce II is most active at pH 7.5 and at 20-30 degrees C . Endonuclease activity is sensitive to salt and is dependent upon Mg2+ or Mn2+, but is unaffected by inclusion of ATP or GTP . I-Sce II is the first intron-encoded protein to be purified and characterized from yeast mitochondria.

Mol Gen Genet, 1990 Nov, 224(2), 257 - 63
Induction of "General Control" and thermotolerance in cdc mutants of Saccharomyces cerevisiae; Messenguy F et al.; In Saccharomyces cerevisiae starvation for a single amino acid activates the transcription of a set of genes belonging to different amino acid biosynthetic pathways (General Control, GC) . We show that mutants affected in GC regulation are also affected in their response to thermal stress . Moreover, growth conditions that are known to induce heat shock proteins induce the GC response . However, unlike heat shock proteins, the transcriptional activator of GC, GCN4, is not induced after a short exposure to heat, and in gcn mutant strains induction of heat resistance is normal.

Mutat Res, 1990 Nov, 242(3), 231 - 58
The detection of mitotic and meiotic chromosome gain in the yeast Saccharomyces cerevisiae: effects of methyl benzimidazol-2-yl carbamate, methyl methanesulfonate, ethyl methanesulfonate, dimethyl sulfoxide, propionitrile and cyclophosphamide monohydrate; Whittaker SG et al.; The diploid yeast strain BR1669 was used to study induction of mitotic and meiotic chromosome gain by selected chemical agents . The test relies on a gene dosage selection system in which hyperploidy is detected by the simultaneous increase in copy number of two alleles residing on the right arm of chromosome VIII: arg4-8 and cup1S (Rockmill and Fogel . 1988; Whittaker et al., 1988) . Methyl methanesulfonate (MMS) induced mitotic, but not meiotic, chromosome gain . Methyl benzimidazol-2-yl carbamate (MBC) and ethyl methanesulfonate (EMS) induced both mitotic and meiotic chromosome gain . Propionitrile, a polar aprotic solvent, induced only mitotic chromosome gain; a reliable response was only achieved by overnight incubation of treated cultures at 0 degrees C . MBC is postulated to act by binding directly to tubulin . The requirement for low-temperature incubation suggests that propionitrile also induces aneuploidy by perturbation of microtubular dynamics . The alkylating agents MMS and EMS probably induce recombination which might in turn perturb chromosome segregation . Cyclophosphamide monohydrate and dimethyl sulfoxide (DMSO) failed to induce mitotic or meiotic chromosome gain.

Curr Genet, 1990 Nov, 18(4), 293 - 301
Functional analysis of the sporulation-specific SPR6 gene of Saccharomyces cerevisiae; Kallal LA et al.; The SPR6 gene of Saccharomyces cerevisiae encodes a moderately abundant RNA that is present at high levels only during sporulation . The gene contains a long open reading frame that could encode a hydrophilic protein approximately 21 kDa in size . This protein is probably produced by the yeast, because the lacZ gene of Escherichia coli is expressed during sporulation when fused to SPR6 in the expected reading frame . SPR6 is inessential for sporulation; mutants that lack SPR6 activity sporulate normally and produce viable ascospores . Nonetheless, the SPR6 gene encodes a function that is relevant to sporulating cells; the wild-type allele can enhance sporulation in strains that are defective for several SPR functions . SPR6 is located on chromosome V, 14.4 centimorgans centromere-distal to MET6.

Genetics, 1990 Nov, 126(3), 535 - 47
A defect in mismatch repair in Saccharomyces cerevisiae stimulates ectopic recombination between homeologous genes by an excision repair dependent process; Bailis AM et al.; Null mutations in three recombination and DNA repair genes were studied to determine their effects on mitotic recombination between the duplicate AdoMet (S-adenosylmethionine) synthetase genes (SAM1 and SAM2) in Saccharomyces cerevisiae . SAM1 and SAM2, located on chromosomes XII and IV, respectively, encode functionally equivalent although differentially regulated AdoMet synthetases . These similar but not identical (homeologous) genes are 83% homologous at the nucleotide level and this identity is limited solely to the coding regions of the genes . Single frameshift mutations were introduced into the 5' end of SAM1 and the 3' end of SAM2 by restriction site ablation . The sequences surrounding these mutations differ significantly in their degree of homology to the corresponding area of the other gene . Mitotic ectopic recombination between the mutant sam genes occurs at a rate of 8.4 x 10(-9) in a wild-type genetic background . Gene conversion of the marker within the region of greater sequence homology occurs 20-fold more frequently than conversion of the marker within the region of relative sequence diversity . The relative orientation of the two genes prevents the recovery of translocations . Mitotic recombination between the sam genes is completely dependent on the DNA repair and recombination gene RAD52 . A mutation in PMS1, a mismatch repair gene, causes a 4.5-fold increase in the rate of ectopic recombination . RAD1, an excision repair gene, is required to observe this increased rate of ectopic conversion . In addition, RAD1 is involved in modulating the pattern of coconversion during recombination between the homeologous sam genes . These results suggest that interactions between mismatch repair, excision repair and recombinational repair functions are involved in determining the ectopic gene conversion frequency between the sam genes.

Mol Cell Biol, 1990 Nov, 10(11), 6097 - 100
Functional analysis of a duplicated linked pair of ribosomal protein genes in Saccharomyces cerevisiae; Donovan DM et al.; Ribosomal protein genes RP28 and S16A (RP55) are closely linked . Another set of this pair of genes exists in the genome (copy 2), genetically unlinked to copy 1 . By using gene replacement techniques, we have shown that RP28 from copy 1 is required for vegetative growth and that the cells need S16A from copy 2 to achieve maximum growth rate.

Mol Cell Biol, 1990 Nov, 10(11), 5927 - 36
The Saccharomyces cerevisiae SIN3 gene, a negative regulator of HO, contains four paired amphipathic helix motifs; Wang H et al.; The SIN3 gene (also known as SDI1) is a negative regulator of the yeast HO gene . Mutations in SIN3 suppress the requirement for the SWI5 activator for expression of the yeast HO gene and change the normal asymmetric pattern of HO expression in mother and daughter cells . Furthermore, the in vitro DNA-binding activity of several DNA-binding proteins is reduced in extracts prepared from sin3 mutants . We have cloned the SIN3 gene and determined that a haploid strain with a SIN3 gene disruption is viable . We determined the sequence of the SIN3 gene, which is predicted to encode a 175-kDa polypeptide with four paired amphipathic helix motifs . These motifs have been identified in the myc family of helix-loop-helix DNA-binding proteins and in the TPR family of regulatory proteins . The SIN3 transcript was mapped, and it was determined that the SIN3 transcript was absent in stationary-phase cells . Immunofluorescence microscopy with anti-SIN3 antibody demonstrated that SIN3 protein was present in nuclei . A comparison of restriction map and sequence data revealed that SIN3 is the same as regulatory genes UME4 and RPD1.

Mol Cell Biol, 1990 Nov, 10(11), 5921 - 6
A hypoxic consensus operator and a constitutive activation region regulate the ANB1 gene of Saccharomyces cerevisiae; Lowry CV et al.; We have identified a consensus operator sequence, YYYATTGTTCTC, which mediates the repression imposed by the ROX1 factor upon the members of the hypoxic gene regulon, which includes ANB1, HEM13, COX5b, and CYC7 . The members of the regulon were repressed with widely varying stringency, and the variation was correlated with the number and fidelity of operator sequences observed . ANB1 had two operators operating with unequal efficiency, each containing two copies of the operator sequence . Synthetic operator sequences introduced into an operator deletion were effective as monomers but much more so as dimers, consistent with cooperativity . The native operators both imposed ROX1 repression on the GAL1 gene, in either orientation, but the synthetic operators did not, indicating that the sequence context may be important . The repression and activation of ANB1 are independent spatially and functionally, since deletion of the operators did not reduce expression and since both the operator and activation regions functioned separately in the GAL1 UAS . The ANB1 UAS was constitutive, containing several elements distributed over a 300-bp region . There were two dT-rich segments, one of 51 bp and one of 165 bp, the latter capable of activating transcription by itself . Flanking segments containing GRF2 (REB1) and ABF1 (GF1) sites may contribute to activation but were not essential . The UAS showed a strongly preferred orientation.

Mol Cell Biol, 1990 Nov, 10(11), 5721 - 7
Nucleosome depletion alters the chromatin structure of Saccharomyces cerevisiae centromeres; Saunders MJ et al.; Saccharomyces cerevisiae centromeric DNA is packaged into a highly nuclease-resistant chromatin core of approximately 200 base pairs of DNA . The structure of the centromere in chromosome III is somewhat larger than a 160-base-pair nucleosomal core and encompasses the conserved centromere DNA elements (CDE I, II, and III) . Extensive mutational analysis has revealed the sequence requirements for centromere function . Mutations affecting the segregation properties of centromeres also exhibit altered chromatin structures in vivo . Thus the structure, as delineated by nuclease digestion, correlated with functional centromeres . We have determined the contribution of histone proteins to this unique structural organization . Nucleosome depletion by repression of either histone H2B or H4 rendered the cell incapable of chromosome segregation . Histone repression resulted in increased nuclease sensitivity of centromere DNA, with up to 40% of CEN3 DNA molecules becoming accessible to nucleolytic attack . Nucleosome depletion also resulted in an alteration in the distribution of nuclease cutting sites in the DNA surrounding CEN3 . These data provide the first indication that authentic nucleosomal subunits flank the centromere and suggest that nucleosomes may be the central core of the centromere itself.

Mol Cell Biol, 1990 Nov, 10(11), 5616 - 25
The SNF5 protein of Saccharomyces cerevisiae is a glutamine- and proline-rich transcriptional activator that affects expression of a broad spectrum of genes; Laurent BC et al.; The Saccharomyces cerevisiae SNF5 gene affects expression of both glucose- and phosphate-regulated genes and appears to function in transcription . We report the nucleotide sequence, which predicts that SNF5 encodes a 102,536-dalton protein . The N-terminal third of the protein is extremely rich in glutamine and proline . Mutants carrying a deletion of the coding sequence were viable but grew slowly, indicating that the SNF5 gene is important but not essential . Evidence that SNF5 affects expression of the cell type-specific genes MF alpha 1 and BAR1 at the RNA level extends the known range of SNF5 function . SNF5 is apparently required for expression of a wide variety of differently regulated genes . A bifunctional SNF5-beta-galactosidase fusion protein was localized in the nucleus by immunofluorescence . No DNA-binding activity was detected for SNF5 . A LexA-SNF5 fusion protein, when bound to a lexA operator, functioned as a transcriptional activator.

EMBO J, 1990 Nov, 9(11), 3691 - 7
Signal sequence for generation of mRNA 3' end in the Saccharomyces cerevisiae GAL7 gene; Abe A et al.; We have identified a signal sequence (designated core signal) necessary to specify formation of mRNA 3' end of the GAL7 gene in Saccharomyces cerevisiae within a DNA segment 26 bp long . The sequence was located 4-5 nucleotides upstream from the 3' end, i.e . the polyadenylation site, of the GAL7 mRNA . Replacement of a DNA segment encompassing the polyadenylation site with a pBR322 DNA, leaving the core signal intact, resulted in alteration of the mRNA 3' end by several nucleotides, suggesting the existence of an additional signal (designated end signal) at or near the polyadenylation site . The normal end formation was abolished when the core signal was placed in the reverse orientation . A considerable fraction of pre-mRNA synthesized in vitro with SP6 RNA polymerase on the template of a DNA fragment containing these signals was cleaved and polyadenylated presumably at the in vitro 3' end during incubation in a cell-free system of yeast . By contrast pre-mRNA synthesized on the template with the core signal alone was processed but much less efficiently . No such processing was seen when the pre-mRNA either lacked the core signal or contained it in the reverse orientation.

Genetics, 1990 Nov, 126(3), 625 - 37
Drosophila nonsense suppressors: functional analysis in Saccharomyces cerevisiae, Drosophila tissue culture cells and Drosophila melanogaster; Garza D et al.; Amber (UAG) and opal (UGA) nonsense suppressors were constructed by oligonucleotide site-directed mutagenesis of two Drosophila melanogaster leucine-tRNA genes and tested in yeast, Drosophila tissue culture cells and transformed flies . Suppression of a variety of amber and opal alleles occurs in yeast . In Drosophila tissue culture cells, the mutant tRNAs suppress hsp70:Adh (alcohol dehydrogenase) amber and opal alleles as well as an hsp70:beta-gal (beta-galactosidase) amber allele . The mutant tRNAs were also introduced into the Drosophila genome by P element-mediated transformation . No measurable suppression was seen in histochemical assays for Adhn4 (amber), AdhnB (opal), or an amber allele of beta-galactosidase . Low levels of suppression (approximately 0.1-0.5% of wild type) were detected using an hsp70:cat (chloramphenicol acetyltransferase) amber mutation . Dominant male sterility was consistently associated with the presence of the amber suppressors.

J Virol, 1990 Nov, 64(11), 5628 - 32
Rous sarcoma virus expression in Saccharomyces cerevisiae: processing and membrane targeting of the gag gene product; Bonnet D et al.; In avian cells, the product of the gag gene of Rous sarcoma virus, Pr76gag, has been shown to be targeted to the plasma membrane, to form virus particles, and then to be processed into mature viral gag proteins . To explore how these phenomena may be dependent upon cellular (host) factors, we expressed the Rous sarcoma virus gag gene in a lower eucaryote, Saccharomyces cerevisiae, and studied the behavior of the gag gene product . We show here that Pr76gag is processed in yeast cells and that this processing is specific, since it is abolished in a mutant in which the active site of the gag protease has been destroyed . In this mutant, the uncleaved precursor is found associated with the yeast plasma membrane, yet no virus particles were detected in cells or in the culture medium . From our results, we can speculate either that in yeast cells, a host protease initiates Pr76gag processing in the cytosol or that in avian cells, an inhibitor prevents the processing until the viral particle is formed.

Mol Cell Biol, 1990 Nov, 10(11), 5796 - 805
Dolichol phosphate mannose synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O mannosylation, and N glycosylation of protein in Saccharomyces cerevisiae; Orlean P; Glycosyl phosphatidylinositol (GPI) anchoring, N glycosylation, and O mannosylation of protein occur in the rough endoplasmic reticulum and involve transfer of precursor structures that contain mannose . Direct genetic evidence is presented that dolichol phosphate mannose (Dol-P-Man) synthase, which transfers mannose from GDPMan to the polyisoprenoid dolichol phosphate, is required in vivo for all three biosynthetic pathways leading to these covalent modifications of protein in yeast cells . Temperature-sensitive yeast mutants were isolated after in vitro mutagenesis of the yeast DPM1 gene . At the nonpermissive temperature of 37 degrees C, the dpm1 mutants were blocked in {2-3H}myo-inositol incorporation into protein and accumulated a lipid that could be radiolabeled with both {2-3H}myo-inositol and {2-3H}glucosamine and met existing criteria for an intermediate in GPI anchor biosynthesis . The likeliest explanation for these results is that Dol-P-Man donates the mannose residues needed for completion of the GPI anchor precursor lipid before it can be transferred to protein . Dol-P-Man synthase is also required in vivo for N glycosylation of protein, because (i) dpm1 cells were unable to make the full-length precursor Dol-PP-GlcNAc2Man9Glc3 and instead accumulated the intermediate Dol-PP-GlcNAc2Man5 in their pool of lipid-linked precursor oligosaccharides and (ii) truncated, endoglycosidase H-resistant oligosaccharides were transferred to the N-glycosylated protein invertase after a shift to 37 degrees C . Dol-P-Man synthase is also required in vivo for O mannosylation of protein, because chitinase, normally a 150-kDa O-mannosylated protein, showed a molecular size of 60 kDa, the size predicted for the unglycosylated protein, after shift of the dpm1 mutant to the nonpermissive temperature.

J Photochem Photobiol B, 1990 Nov, 7(2-4), 209 - 29
Genotoxicity of bergapten and bergamot oil in Saccharomyces cerevisiae; Averbeck D et al.; In order to determine the genotoxic potential of bergapten (5-methoxypsoralen (5-MOP} and bergamot oil (BO), the genetic effects of 5-MOP and BO (containing equivalent amounts of 5-MOP) were studied in haploid and diploid yeast (Saccharomyces cerevisiae) using solar simulated radiation (SSR) . At equal doses of SSR, equal concentrations of 5-MOP alone or 5-MOP in BO have a similar influence on survival and on the induction of cytoplasmic "petite" mutations, reverse and forward mutations, mitotic gene conversion and genetically aberrant colonies including mitotic crossing over . No reciprocity is found between SSR dose and 5-MOP concentration for cytotoxic, mutagenic and recombinogenic effects . In the presence of chemical filters (Parsol 1789, a UVA filter, and Parsol MCX, a cinnamate derivative acting as a UVB filter) considerable protection is observed against the induction of genetic effects by 5-MOP and BO containing 5-MOP in haploid and diploid cells . As indicated by the lower induction kinetics, the protection is higher than expected from the light-absorbing properties, suggesting photochemical interaction . The protection is slightly higher for BO than for 5-MOP . The induction of genetic effects by 5-MOP alone or BO containing 5-MOP is independent of oxygen . Experiments on suction blister fluids taken from patients after topical treatment with BO containing 5-MOP indicate that in comparison with water the bioavailability and thus the genotoxic effects of the compounds are decreased . Moreover, in addition to the filtering effect against the photoinduced genotoxic effects of BO, the presence of chemical filters apparently reduces the penetration of BO containing 5-MOP and provides a reduction in biological effectiveness.

J Biochem (Tokyo), 1990 Nov, 108(5), 859 - 65
Expression of cloned yeast NADPH-cytochrome P450 reductase gene in Saccharomyces cerevisiae; Murakami H et al.; The NADPH-cytochrome P450 reductase gene isolated from the yeast Saccharomyces cerevisiae {Yabusaki et al., J . Biochem . 103, 1004-1010 (1988)} was expressed on a multi-copy plasmid in the yeast . The transformed yeast cells with the recombinant plasmid carrying the reductase gene with a length of 3 kb produced the corresponding mRNA read from the original transcription initiation site under the control of its own promoter with a maximum length of 300 bp . The reductase content in the transformed cells was 25 times higher than that of the endogenous reductase . When the coding region for the reductase was placed between the alcohol dehydrogenase I gene promoter and the terminator of the expression vector pAAH5, the expression level was 32 times higher than at the endogenous level . These recombinant yeast strains showed enhanced cytochrome c reductase activity with increased cellular reductase levels . A simultaneous expression of yeast P450 reductase with rat P450c or bovine P450(17 alpha) resulted in 25 times or a 5 times increase in the corresponding P450-dependent monooxygenase activity of the recombinant yeast strains, respectively, as compared with that of the yeast cells expressing the corresponding P450 species . These results suggested that the overproduction of yeast P450 reductase with a simultaneous expression of the mammalian P450 species enhanced the P450c- and P450(17 alpha)-dependent monooxygenase activities in the recombinant yeast strains, probably due to the increased frequency of the interaction between yeast P450 reductase and P450c or P450(17 alpha) in the yeast microsomes.

Biochem Int, 1990 Nov, 22(4), 781 - 90
Molasses induced changes in Saccharomyces cerevisiae: alterations in plasma membrane structure and function and metallothionein level; Bhatnagar NB; Growth of yeast cells in molasses caused changes in plasma membrane properties . The polypeptide profile of plasma membrane prepared by sucrose density gradient centrifugation showed a range of new polypeptides above 55 kD which were not present in malt extract-glucose-yeast extract-peptone medium grown cells . Isolated new bands appeared in the range of 25-50 kD . Transport of glucose was markedly enhanced in molasses trained cells . Among the amino acids, L-lysine uptake was decreased while L-aspartate uptake was enhanced considerably . Finally the metallothionein levels in the cells grown in molasses was found to be increased significantly, possibly to protect the cells from the harmful effect of heavy metal ions.

Mol Cell Biol, 1990 Nov, 10(11), 5950 - 7
Molecular and expression analysis of the negative regulators involved in the transcriptional regulation of acid phosphatase production in Saccharomyces cerevisiae; Madden SL et al.; The PHO80 and PHO85 gene products encode proteins necessary for the repression of transcription from the major acid phosphatase gene (PHO5) of Saccharomyces cerevisiae . The deduced amino acid sequences of these genes have revealed that PHO85 is likely to encode a protein kinase, whereas no potential function has been revealed for PHO80 . We undertook several approaches to aid in the elucidation of the PHO80 function, including deletion analysis, chemical mutagenesis, and expression analysis . DNA deletion analysis revealed that residues from both the carboxy- and amino-terminal regions of the protein, amounting to a total of 21% of the PHO80 protein, were not required for function with respect to repressor activity . Also, 10 independent single-amino-acid changes within PHO80 which resulted in the failure to repress PHO5 transcription were isolated . Nine of the 10 missense mutations resided in two subregions of the PHO80 molecule . In addition, expression analysis of the PHO80 and PHO85 genes suggested that the PHO85 gene product was not necessary for PHO80 expression and that the PHO85 gene was expressed at much higher levels in the cell than was the PHO80 gene . Furthermore, high levels of PHO80 were shown to suppress the effect of a PHO85 deletion at a level close to full repression . Implications for the function of the negative regulators in this system are discussed.

J Cell Biol, 1990 Nov, 111(5 Pt 1), 1741 - 51
SSB-1 of the yeast Saccharomyces cerevisiae is a nucleolar-specific, silver-binding protein that is associated with the snR10 and snR11 small nuclear RNAs; Clark MW et al.; SSB-1, the yeast single-strand RNA-binding protein, is demonstrated to be a yeast nucleolar-specific, silver-binding protein . In double-label immunofluorescence microscopy experiments antibodies to two other nucleolar proteins, RNA Pol I 190-kD and fibrillarin, were used to reveal the site of rRNA transcription; i.e., the fibrillar region of the nucleolus . SSB-1 colocalized with fibrillarin in a double-label immunofluorescence mapping experiment to the yeast nucleolus . SSB-1 is located, though, over a wider region of the nucleolus than the transcription site marker . Immunoprecipitations of yeast cell extracts with the SSB-1 antibody reveal that in 150 mM NaCl SSB-1 is bound to two small nuclear RNAs (snRNAs) . These yeast snRNAs are snR10 and snR11, with snR10 being predominant . Since snR10 has been implicated in pre-rRNA processing, the association of SSB-1 and snR10 into a nucleolar snRNP particle indicates SSB-1 involvement in rRNA processing as well . Also, another yeast protein, SSB-36-kD, isolated by single-strand DNA chromatography, is shown to bind silver under the conditions used for nucleolar-specific staining . It is, most likely, another yeast nucleolar protein.

Yeast, 1990 Nov-Dec, 6(6), 491 - 9
The glucanase-soluble mannoproteins limit cell wall porosity in Saccharomyces cerevisiae; de Nobel JG et al.; The cell wall porosity of batch-grown Saccharomyces cerevisiae was maximal in the early exponential phase and fell off rapidly to lower levels in later growth phases . Treatment of stationary-phase cells with alpha-mannosidase restored wall porosity to the level of cells in early exponential phase . When cells in the early exponential phase were treated with alpha-mannosidase, or tunicamycin, an inhibitor of N-glycosylation, even higher porosities were obtained . Mutants with truncated mannan side-chains in their wall proteins also had very porous walls . The importance of the mannan side-chains for wall porosity was also seen during sexual induction . Treatment with alpha pheromone, which leads to the formation of wall proteins with shorter mannan side-chains, enhanced wall porosity . Disulphide bridges also affect cell wall porosity . They were predominantly found in the glucanase-soluble wall proteins . Because the main part of the mannan side-chains is also found in this family of wall proteins, our results demonstrate that the glucanase-soluble mannoproteins limit cell wall porosity in yeast.

Yeast, 1990 Nov-Dec, 6(6), 455 - 60
Ornithine decarboxylase in Saccharomyces cerevisiae: chromosomal assignment and genetic mapping of the SPE1 gene; Xie QW et al.; The gene for ornithine decarboxylase in Saccharomyces cerevisiae, SPE1, has been assigned to chromosome XI by the technique of transverse alternating pulsed field electrophoresis and DNA-DNA hybridization . Genetic mapping by tetrad analysis shows that the SPE1 gene is located on the left arm of chromosome XI, 6 cM from the LAP1 gene and 43 cM from the TRP3 gene . The spe10 mutation previously isolated in this laboratory is mapped to the N-terminal region of the SPE1 gene, and therefore should be designated as a spe1 allele.

J Cell Sci, 1990 Nov, 97 ( Pt 3), 517 - 25
Saccharomyces cerevisiae mutants lacking a functional vacuole are defective for aspects of the pheromone response; Dulic V et al.; The end1 mutant belongs to a group of four vacuolar protein sorting mutants (class C vps) that lack a morphologically distinguishable and functional vacuole . These mutants share several other phenotypes, such as the inability to grow at 37 degrees C or on nonfermentable carbon sources . We show that, as in the case of the end1 mutant, vps16, vps18 and vps33 mutants all internalize but do not degrade alpha-factor . In addition, all four mutants are defective for alpha-factor-induced projection formation to the same extent . A more detailed investigation of pheromone response in the end1 mutant reveals that one aspect of the early response (induction of FUS1) is as defective as late responses (cell cycle arrest and projection formation) . In contrast, another measure of the early response (induction of STE2) is normal . These data suggest that the biogenesis of a functional vacuole is necessary for optimal response to pheromone.

Biotechnol Prog, 1990 Nov-Dec, 6(6), 504 - 12
Slit scanning of Saccharomyces cerevisiae cells: quantification of asymmetric cell division and cell cycle progression in asynchronous culture; Block DE et al.; Slit scanning flow cytometry has been applied to the analysis of the cell cycle and cell-cycle-dependent events in Saccharomyces cerevisiae, yielding information on the low-resolution spatial distribution of cellular components in single cells of unperturbed cell populations . Because this process is rapid, large numbers of cells can be analyzed to give distributions of parameters in a given population . To study asymmetric cell division and cell cycle progression, forward-angle light scattering (FALS) signals together with fluorescence signals from acriflavine-stained nuclei have been measured in cells from exponentially growing yeast populations . An algorithm has been developed that assigns the position of the bud neck in the FALS signals so that both FALS and DNA signals can be analyzed in terms of the contributions from the mother cell and the cell bud . The data indicate that mother cell FALS, on average, remains constant while FALS due to the cell bud increases as a cell progresses through the cell cycle . By identifying mitotic cells and measuring their properties, we have found that the coefficient of variation for the distribution of FALS is smallest within the dividing cell population and largest within the newborn cell population, in accordance with the critical size control mechanism of yeast cell growth . The use of this experimental approach to provide data for statistical population models is discussed.

Gene, 1990 Oct 30, 95(1), 65 - 72
Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation, characterization and regulation of the SPO7 sporulation gene; Whyte W et al.; SPO7 is one of several previously identified genes from the yeast Saccharomyces cerevisiae that is required for sporulation but not for vegetative growth . The SPO7 gene has been cloned by functional complementation and physically mapped 15-16 kb to the left of CEN1 . Gene-disruption experiments confirmed that the cloned gene was the bona fide SPO7 gene . SPO7 codes for a 0.95-kb transcript that is expressed at approximately the same level in both vegetative and sporulating cells . The gene was sequenced and has the capacity to encode a 259-amino acid protein that does not appear to be related to other known proteins.

Biochem Biophys Res Commun, 1990 Oct 30, 172(2), 737 - 44
Constitutive and inducible expression of human cytochrome P450IA1 in yeast Saccharomyces cerevisiae: an alternative enzyme source for in vitro studies; Eugster HP et al.; A cDNA of human cytochrome P450IA1 was expressed in yeast Saccharomyces cerevisiae on a multicopy plasmid under the control of the constitutive GAPFL or the inducible PHO5 promoter . Microsomes of transformed yeast contained substantial amounts of the heterologous enzyme as determined by reduced CO-difference spectra (156-68 pmol/mg) . Enzyme kinetics with 7-ethoxyresorufin as substrate resulted in a Km value of 92 nM and a Vmax value of 223 pmol/mg/min, which is comparable to data obtained with human liver microsomes . The antimycotic drug ketoconazole (Ki = 22nM) as well as the isozyme specific P450 inhibitor alpha-naphthoflavone (Ki = 1.2 nM) were shown to be strong inhibitors of human P450IA1 . Taken together, these data show that heterologous P450 gene expression in yeast is a potent instrument for the study of enzyme specific parameters and might be used to answer further questions with regard to substrate specificity as well as drug interaction in a background with no interfering activities.

Gene, 1990 Oct 30, 95(1), 79 - 84
Sequencing and enhanced expression of the gene encoding diadenosine 5',5'''-P1, P4-tetraphosphate (Ap4A) phosphorylase in Saccharomyces cerevisiae; Kaushal V et al.; The gene, DTP, coding for diadenosine 5',5'''-P1, P4-tetraphosphate (Ap4A) phosphorylase was isolated from a Saccharomyces cerevisiae genomic DNA library in lambda gt11 . In yeast and Escherichia coli transformed with the multicopy vector, YEp352, containing the cloned DTP gene, the Ap4A phosphorylase was produced at levels nine- to 17-fold higher than in untransformed hosts . The nucleotide (nt) sequence was determined . The gene codes for a polypeptide chain of 321 amino acids (aa) . Two-aa sequence motifs of possible significance were identified: a potential adenine nt binding site and a potential phosphorylation site . The DTP gene is located on yeast chromosome III and is present as a single copy . Although multicopy vector expression increased the Ap4A phosphorylase activity ninefold above the endogenous activity in transformed yeast, the intracellular concentration of Ap4A did not decrease and the growth rate of the yeast was unchanged.

Gene, 1990 Oct 30, 95(1), 99 - 104
Sequences of the genes encoding argininosuccinate synthetase in Escherichia coli and Saccharomyces cerevisiae: comparison with methanogenic archaebacteria and mammals; Van Vliet F et al.; The nucleotide (nt) sequences of the genes encoding argininosuccinate synthetase from Escherichia coli K-12 (argG) and Saccharomyces cerevisiae (ARG1) were determined . The deduced amino-acid sequences were compared to each other and to their counterparts in two methanogens and in mammals . Three regions are highly conserved . Two of them appear to contain possible Walker-type nt-binding sites {Walker et al., EMBO J . 1 (1982) 945-951} and are therefore candidates for ATP-binding sites . The third region shows some similarity to a short portion of the N-proximal part of the PurA enzyme which catalyses an analogous reaction.

Nucleic Acids Res, 1990 Oct 25, 18(20), 6075 - 81
Synthesis of a gene for human serum albumin and its expression in Saccharomyces cerevisiae; Kalman M et al.; A 1761 base pairs long artificial gene coding for human serum albumin (HSA) has been prepared by a newly developed synthetic approach, resulting in the largest synthetic gene so far described . Oligonucleotides corresponding to only one strand of the HSA gene were prepared by chemical synthesis, while the complementary strand was obtained by a combination of enzymatic and cloning steps . 24 synthetic, 69-85 nucleotides long oligonucleotides covering the major part of the HSA gene (41-1761 nucleotides) were used as building blocks . Generally, four groups of 6-6 such oligonucleotides were successively cloned in pUC19 Escherichia coli vector to obtain about quarters of the gene as large fragments . Joining of these four fragments resulted in a cloned DNA coding for the 13-585 amino acid region of HSA, which was further supplemented with a double-stranded linker sequence coding for the amino terminal 12 amino acids . The completed structural gene composed of frequently used codons in the highly expressed yeast genes was then supplied with yeast regulatory sequences and the HSA expression cassette so obtained was inserted into an Escherichia coli-Saccharomyces cerevisiae shuttle vector . This vector was shown to direct the expression in Saccharomyces cerevisiae of correctly processed, mature HSA which was recognized by antiserum to HSA, and possessed the correct N-terminal amino acid sequence.

J Biol Chem, 1990 Oct 25, 265(30), 18554 - 60
The 31-kDa polypeptide is an essential subunit of the vacuolar ATPase in Saccharomyces cerevisiae; Foury F; The VMA4 gene encodes a 26.6-kDa hydrophilic polypeptide which exhibits 34% sequence identity with the E subunit of the vacuolar ATPase from bovine kidney microsomes . The chromosomal VMA4 gene was inactivated by a 171-base pair deletion followed by insertion of the URA3 gene within the coding sequence . Null vma4 haploid mutants are viable . However, their growth is considerably slowed down specially in non-acidic conditions; they are cold sensitive and thermo-sensitive, exhibit poor growth on glycerol medium, and do not accumulate in their vacuole the red pigment of ade2 strains . No bafilomycin-sensitive ATPase is detected in a vacuolar fraction . These properties shared by null mutants in the A, B, and C subunits of the vacuolar ATPase show that the VMA4 polypeptide is also an essential component of the vacuolar ATPase which has been conserved from yeast to mammals . The tightly linked VMA4 and MIP1 (encoding the mitochondrial DNA polymerase) genes are divergently transcribed from face-to-face promoters . About 250 base pairs upstream of the VMA4 gene, Homoll and RPG consensus for the binding of TUF (RAP/GRF1) protein are present, suggesting that the VMA4 gene belongs to this large family of genes involved in cellular growth and division whose transcription is regulated by the TUF protein.

J Biol Chem, 1990 Oct 25, 265(30), 18447 - 53
Roles of the VMA3 gene product, subunit c of the vacuolar membrane H(+)-ATPase on vacuolar acidification and protein transport . A study with VMA3-disrupted mutants of Saccharomyces cerevisiae; Umemoto N et al.; VMA3, a structure gene of the vacuolar membrane H(+)-ATPase subunit c of Saccharomyces cerevisiae, has been cloned and characterized . The VMA3 gene encodes a hydrophobic polypeptide with 160 amino acids as reported previously by Nelson and Nelson (Nelson, H., and Nelson, N . (1989) FEBS Lett . 247, 147-153) . Peptide sequence analysis indicated that the VMA3 gene product lacks N-terminal methionine and does not have a cleavable signal sequence . To investigate functional and structural roles of the subunit c for vacuolar acidification and protein transport to the vacuole, haploid mutants with the disrupted VMA3 gene were constructed . The vma3 mutants can grow in nutrient-enriched medium, but they have completely lost the vacuolar membrane H(+)-ATPase activity and the ability of vacuolar acidification in vivo . The subunit c was found to be indispensable for the assembly of subunits a and b of the H(+)-ATPase complex . The disruption of the VMA3 gene causes yeast cells with considerable lesions in vacuolar biogenesis and protein transport to the vacuole and inhibits endocytosis of lucifer yellow CH completely.

Nucleic Acids Res, 1990 Oct 25, 18(20), 5975 - 9
Cytidines in tRNAs that are required intact by ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and Saccharomyces cerevisiae; Hegg LA et al.; Individual species of tRNA from Escherichia coli were treated with hydrazine/3 M NaCl to modify cytidine residues . The chemically modified tRNAs were used as substrate for ATP/CTP: tRNA nucleotidyltransferases from E . coli and yeast, with {alpha-32P}ATP as cosubstrate . tRNAs that were labeled were analyzed for their content of modified cytidines . Cytidines at positions 74 and 75 were found to be required chemically intact for interaction with both enzymes . C56 was also required intact by the E . coli enzyme in all tRNAs, and by the yeast enzyme in several instances . C61 was found to be important in seven of 14 tRNAs with the E . coli enzyme but only in four of 13 tRNAs with that from yeast . Our results support a model in which nucleotidyltransferase extends from the 3' end of its tRNA substrate across the top of the stacked array of bases in the accepter- and psi-stems to the corner of the molecule where the D- and psi-loops are juxtaposed.

Biochemistry, 1990 Oct 16, 29(41), 9651 - 9
Evidence for an S-farnesylcysteine methyl ester at the carboxyl terminus of the Saccharomyces cerevisiae RAS2 protein; Stimmel JB et al.; The protein products of yeast and mammalian ras genes are posttranslationally modified to give mature forms that are localized to the inner surface of the plasma membrane . We have previously demonstrated that the mature form of the Saccharomyces cerevisiae RAS2 gene product is methyl esterified at a modified C-terminal cysteine residue . Here we provide evidence that this residue is an S-farnesylcysteine alpha-carboxyl methyl ester . This result establishes common posttranslational modifications for RAS proteins and fungal sex factors . These polypeptides exhibit sequence similarities at their C-termini that appear to be the critical recognition elements for a common set of modification enzymes . In mammalian cells, proteins with analogous C-terminal sequences appear to be prenylated and carboxyl methylated by a similar mechanism.

Gene, 1990 Oct 15, 94(2), 209 - 16
Expression and secretion of rice alpha-amylase by Saccharomyces cerevisiae; Kumagai MH et al.; We report the high level expression and secretion of rice alpha-amylase isozyme by Saccharomyces cerevisiae . Transcription of this gene was under control of the yeast enolase promoter . The synthesized protein had an approximate molecular size of 45 kDa and a pI of approx 4.7 to 5.0 . The rice alpha-amylase signal peptide was recognized and efficiently processed by yeast and the active, glycosylated enzyme was secreted into the culture media . This enzyme was purified to homogeneity by affinity chromatography and its enzymatic properties were characterized . The Km and Vmax were found to be similar to those of alpha-amylases from other organisms . The high level of secretion observed in these studies may be due to the unique features of the rice signal peptide and/or to the glycosylation of the recombinant enzyme.

FEBS Lett, 1990 Oct 15, 272(1-2), 85 - 8
Isolation and characterization of human reg protein produced in Saccharomyces cerevisiae; Itoh T et al.; reg was originally identified as a gene expressed during the regeneration of insulin-producing pancreatic beta-cells of the rat . We built an expression vector containing human reg cDNA to drive Saccharomyces cerevisiae to synthesize the reg protein, and purified it from the culture medium . The 144-amino acid sequence of the recombinant protein was consistent with that deduced from the cDNA and genomic DNA sequence except that the signal sequence of 22 amino acids was eliminated, and the amino-terminal residue of the protein was pyroglutamic acid . The secondary structure of the reg protein was predicted by determination of the intramolecular cystine linkage and of alpha-helix and beta-sheet contents.

J Biol Chem, 1990 Oct 15, 265(29), 17441 - 5
Recognition of the tRNA-like structure in tobacco mosaic viral RNA by ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and Saccharomyces cerevisiae; Hegg LA et al.; The 3'-terminal tRNA-like structure of the tobacco mosaic virus RNA interacts with ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli or yeast in much the same manner as do tRNAs . Primary sites of interaction cluster near the 3' end and in the loop proposed to be analogous to the psi-loop of a tRNA . Some modified bases in the tRNA-like structure inhibit interaction with nucleotidyltransferase, yet the analogous bases in a tRNA do not . The location of some of these nucleotides within the analog to the psi-loop suggests that this structure differs slightly from its counterpart in a tRNA . The location of other such bases in the helical stem near the 3' end can be explained if the pseudoknot is disrupted by these modified bases or if the tertiary structure of the RNA is altered in the enzyme-RNA complex . A partially denatured secondary structure that persists on denaturing gels is proposed.

Biochem Biophys Res Commun, 1990 Oct 15, 172(1), 61 - 9
Overexpression of the CDC25 gene, an upstream element of the RAS/adenylyl cyclase pathway in Saccharomyces cerevisiae, allows immunological identification and characterization of its gene product; Vanoni M et al.; The product of the START gene CDC25, an upstream element of the RAS/adenylyl cyclase pathway in Saccharomyces cerevisiae, was identified using specific antibodies raised against a chimeric beta-galactosidase/CDC25 protein . The CDC25 protein is poorly expressed and can be detected only when the CDC25 gene is overexpressed under the control of the galactose-inducible GAL1-10 strong promoter elements . It has a molecular weight of 180,000, is not glycosylated and is strongly associated with the particulate fraction . After deletion of residues 1255-1550 the protein is found in the soluble fraction.

Nucleic Acids Res, 1990 Oct 11, 18(19), 5823 - 8
Translation of the Saccharomyces cerevisiae tcm1 gene in the absence of a 5'-untranslated leader; Maicas E et al.; The role of eukaryotic 5'-untranslated messenger RNA leaders is not entirely clear, since they share little sequence similarity among each other . The importance of the leader in determining the efficiency of translation initiation was addressed here by examining the polyribosome distribution of several leader-deletion alleles of the yeast tcm1 gene (coding for ribosomal protein L3) . Shortening of this 22-nucleotide leader, or complete removal of it (the first nucleotide of the mRNA becoming the A of the translation initiation codon AUG) permitted translation, albeit reduced . Further deletion of as few as the first two nucleotides of the initiation codon leads to a substantial reduction in ribosome loading, which is compatible with inefficient initiation at the next downstream, out-of-frame, AUG triplet . A second measure of translation initiation was obtained by assaying qualitatively for the production of biologically active L3 protein using growth-resistance to trichodermin . This experiment indicates that ribosomes can recognize the correct initiation codon even in the complete absence of a leader . We conclude that the 5'-untranslated leader of the yeast tcm1 gene is not essential for accurate translation initiation, but enhances its efficiency.

J Biol Chem, 1990 Oct 5, 265(28), 17005 - 11
Subunit 6 regulates half-of-the-sites reactivity of the dimeric cytochrome bc1 complex in Saccharomyces cerevisiae; Schmitt ME et al.; We have characterized the activities of the cytochrome bc1 complex in mitochondrial membranes from a yeast strain in which we deleted the nuclear gene (QCR6, COR3) which codes for the highly acidic subunit 6 of the bc1 complex . The chromosomal copy of QCR6 was replaced with a plasmid derived copy of QCR6, in which the entire coding region of QCR6 was replaced with the yeast LEU2 gene . The resulting deletion strain, MES8, contained no detectable mRNA for QCR6, and the cytochrome bc1 complex purified from the deletion strain lacked subunit 6 . The deletion strain respired and grew on nonfermentable carbon sources such as ethanol and glycerol . Ubiquinol-cytochrome c reductase activity of mitochondria from the deletion strain was decreased 50% under conditions where the activity is zero order with respect to cytochrome c, and there was a similar decrease in the first-order rate constant for cytochrome c reduction . The loss of bc1 complex activities, observed at physiological ionic strengths, was reversible . Both the zero order rate and the first-order rate constant for cytochrome c reduction could be recovered to those of the parental strain by measuring these activities in mitochondrial membranes under conditions of low ionic strength . The zero order rate and first-order rate constant for cytochrome c reduction in membranes from the parent, wild-type yeast showed essentially no change coincident with this change in ionic strength . The 50% drop in both turnover number and first-order rate constant of ubiquinol-cytochrome c reductase activity indicates that half of the cytochrome bc1 complexes are inactive in the deletion strain at physiological ionic strengths . Inhibition by myxothiazol of cytochrome c reductase activity of mitochondrial membranes from the deletion strain showed an ionic strength-dependent lag in the titration curve that extended to the point where half of the inhibitor sites are filled . This lag was not observed with membranes from the wild-type, parent strain . This response to the inhibitor is consistent with half of the cytochrome bc1 complexes being inactive in mitochondria from the deletion strain at physiological ionic strength, but with both active and inactive complexes still able to bind inhibitor . The reversible, half-of-the-sites reactivity indicates that the bc1 complex must be dimeric in situ, in agreement with previous findings that the complexes isolated from fungal (Leonard, K., Wingfield, P., Arad, T., and Weiss, H . (1981) J . Mol . Biol . 149, 259-274) and mammalian (Nalecz, M . J., Bolli, R., and Azzi, A . (1985) Arch . Biochem . Biophys . 236, 619-628) mitochondria are structural dimers.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1990 Oct 2, 29(39), 9159 - 68
Steady-state fluorescence and time-resolved fluorescence monitor changes in tryptophan environment in arginase from Saccharomyces cerevisiae upon removal of catalytic and structural metal ions; Green SM et al.; Yeast arginase is a trimeric protein of identical subunits, each containing three tryptophans . Time-resolved fluorescence and steady-state fluorescence were employed to monitor the effects of removing the weakly bound catalytic Mn2+ as well as the tightly bound structural Zn2+/Mn2+ . Resolution of the total native emission spectrum into decay-associated spectra (DAS) yielded components with lifetimes of 0.1, 1.2, and 4.0 ns . Upon removal of the catalytic metal, the intensities increased approximately 20% while the lifetimes increased less than 10%, and the DAS were unchanged except in intensity . The two major components are well resolved, but the 0.1-ns term is small and dominated by scattered excitation . In contrast, removal of the structural metal increased decay times to 0.2, 1.8, and 5.3 ns . More important, both native DAS red-shifted and became indistinguishable . These data suggest that removal of the catalytic metal does little to change the microenvironments of the individual tryptophans while removal of the structural metal causes partial unfolding of the protein . The excitation spectra for the active and inactive trimers were resolved into their excitation DAS (IEDAS), suggesting ground-state heterogeneity of the fluorescent species . In contrast, the excitation spectra of arginase without the structural metal could not be resolved due to the indistinguishable DAS . The tryptophans are quenched by acrylamide but not by cesium or iodide . Global analysis of the acrylamide quenching data resulted in two quenching decay-associated spectra (QDAS) which correlated well with the DAS . Since the apoenzyme does not exhibit tryptophan accessibility to either positive or negative ionic quenchers, one must assume that the "unfolded" monomeric protein retains considerable tertiary structure.

Biochemistry, 1990 Oct 2, 29(39), 9106 - 10
Channeling of TCA cycle intermediates in cultured Saccharomyces cerevisiae; Sumegi B et al.; Oxidation of {3-13C}propionate was studied in cultured yeast cells, and the distribution of label in the 2- and 3-positions of alanine was detected by 13C NMR . {3-13C}Propionate forms {2-13C}succinyl-CoA in the mitochondria which then enters the citric acid cycle and forms malate through two symmetrical intermediates, succinate and fumarate . If these symmetrical intermediates randomly diffuse from one enzyme to the next in mitochondria as is normally assumed, then 13C labeling in malate C2 and C3 must be equal . However, any direct transfer of metabolites from site to site between succinate thiokinase, succinate dehydrogenase, and fumarase would result in an uneven distribution of 13C in malate C2 and C3 and any molecules derived from malate . Since pyruvate may be derived from malate via the malic enzyme and subsequently converted into alanine by transamination, any 13C asymmetry in alanine C2 and C3 must directly reflect the 13C distribution in the malate pool . During oxidation of {3-13C}propionate, we detect a significant quantity of labeled alanine, where 13C enrichment in C3 is significantly higher than that in C2 . Inhibition of succinate dehydrogenase with malonate or creating conditions that increase the chances of a back-reaction (from malate to fumarate) result in a significant decrease in the asymmetric labeling of alanine . Ubiquinone-deficient yeast cells (having only 10% of the oxidative capacity of wild-type cells) could slowly oxidize propionate, but in this case the 13C labeling was equal in the C2 and C3 of alanine, showing that isotope randomization had occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1990 Oct, 224(1), 57 - 64
A GCN4 protein recognition element is not sufficient for GCN4-dependent regulation of transcription in the ARO7 promoter of Saccharomyces cerevisiae; Schmidheini T et al.; The gene ARO7 encodes the monofunctional enzyme chorismate mutase, a branch point enzyme in the aromatic amino acid biosynthetic pathway in Saccharomyces cerevisiae . We investigated the transcription of the ARO7 gene . Three 5' ends at positions -36, -56 and -73 and the 3' end of the transcripts 146 bp downstream of the translational stop codon were mapped . As in the promoters of other aromatic amino acid biosynthetic genes, a recognition element for the GCN4 transcriptional activator of amino acid biosynthesis is located 425 base pairs (bp) upstream of the first transcriptional start point . This element binds GCN4 specifically in vitro . Northern analysis and determination of the specific enzyme activity reveals however, that the element is not sufficient to mediate transcriptional regulation by GCN4 in vivo . We thus suggest that in addition to a consensus sequence capable of binding the GCN4 protein other factors like, for example, chromatin structure, determine whether a recognition site for a transcription factor functions as an upstream activation sequence.

Genes Dev, 1990 Oct, 4(10), 1775 - 89
Isolation of two developmentally regulated genes involved in spore wall maturation in Saccharomyces cerevisiae; Briza P et al.; During sporulation of Saccharomyces cerevisiae, the four haploid nuclei generated by meiosis are encapsulated within multilayered spore walls . Taking advantage of the natural fluorescence imparted to yeast spores by the presence of a dityrosine-containing macromolecule in the spore wall, we identified and cloned two genes, termed DIT1 and DIT2, which are required for spore wall maturation . Mutation of these genes has no effect on the efficiency of spore formation or spore viability . The mutant spores, however, fail to accumulate the spore wall-specific dityrosine and lack the outermost layer of the spore wall . The absence of this cross-linked surface layer reduces the resistance of the spores to lytic enzymes, to ether, and to elevated temperature . Expression of the DIT and DIT2 genes is restricted to sporulating cells, with the DIT1 transcripts accumulating at the time of prospore enclosure and just prior to the time of dityrosine biosynthesis . Both genes act in a spore-autonomous manner implying that at least some of the activities responsible for forming the outermost layer of the spore wall reside within the developing spore rather than in the surrounding ascal cytoplasm . As the DIT2 gene product has significant homology with cytochrome P-450s, DIT2 may be responsible for catalyzing the oxidation of tyrosine residues in the formation of dityrosine.

Curr Genet, 1990 Oct, 18(3), 187 - 92
A recessive mutant allele of the HNM1 gene of Saccharomyces cerevisiae is responsible for hyper-resistance to nitrogen mustard; Haase E et al.; A screening of haploid yeast strains for enhanced resistance to nitrogen mustard (HN2) yielded a recessive mutant allele, hnm1, that conferred hyper-resistance (HYR) to HN2 . Diploids, homo- or heterozygous for the HNM1 locus, exhibit normal wild-type like resistance while homozygosity for hnm1 leads to the phenotype HYR to HN2 . The hnm1 mutation could be found in yeast strains proficient or deficient in different DNA repair systems . In these mostly HN2-sensitive haploid repair-deficient mutants, hnm1 acted as a partial suppressor of HN2 sensitivity . All isolated recessive mutations conferring hyper-resistance belonged to a single complementation group . The HYR to HN2 phenotype was maximally expressed in growing cells and was associated with reduced mutability by HN2 . HNM1 most probably controls uptake of HN2 which would be impaired in the hnm1 mutants.

Curr Genet, 1990 Oct, 18(3), 181 - 5
Resistance to cadmium is under control of the CAD2 gene in the yeast Saccharomyces cerevisiae; Tohoyama H et al.; A cadmium-resistant strain, X3382-3A, which is able to grow in a medium containing 0.2 mM cadmium sulfate, was picked out from our laboratory stock strains of Saccharomyces cerevisiae . The cadmium resistance of this strain is controlled by a single dominant nuclear gene, denoted as CAD2 . The locus of CAD2 was mapped by gene linkage to a site 15.5 centimorgans to the right of the his7 locus on the right arm of chromosome II . The cadmium resistance of the strain carrying CAD2 was evaluated for its properties of cadmium uptake, cadmium distribution and cadmium-metallothionein formation, in comparison with those of some other strains . The results suggest that the novel type of cadmium resistance controlled by CAD2 does ot involve production of a cadmium-metallothionein.

Curr Genet, 1990 Oct, 18(3), 175 - 9
Role of the CDC8 gene in the repair of single strand breaks in DNA of the yeast Saccharomyces cerevisiae; Baranowska H et al.; It has been found that the repair of single strand breaks is defective in the DNA replication mutants cdc8-1 and cdc8-3 of Saccharomyces cerevisiae both in permissive (23 degrees C) and restrictive conditions (36 degrees C) . In permissive conditions we observed a significant delay in single strand break repair in a diploid strain HB7 (cdc8-1/cdc8-1), as compared with the wild-type strain . Under restrictive conditions no repair was observed, but rather degradation of MMS-damaged DNA occurred . It has been also found that the repair of single strand breaks in yeast is inhibited by cycloheximide but not by hydroxyurea.

Genetics, 1990 Oct, 126(2), 301 - 8
The DAF2-2 mutation, a dominant inhibitor of the STE4 step in the alpha-factor signaling pathway of Saccharomyces cerevisiae MAT alpha cells; Cross FR; A dominant mutation (DAF2-2) resulting in resistance to the mating pheromone alpha-factor in Saccharomyces cerevisiae MATa cells was identified and characterized genetically . Whereas wild-type cells induce a high level of the FUS1 mRNA from a low baseline on exposure to alpha-factor, DAF2-2 cells were constitutive producers of an intermediate level of FUS1 RNA; the level was increased only modestly by alpha-factor . FUS1 constitutivity required STE4, STE5 and STE18, but did not require STE2, the alpha-factor receptor gene . DAF2-2 suppressed the alpha-factor supersensitivity of a STE2 C-terminal truncation, and suppressed lethality due to scg1 mutations . Thus DAF2-2 may act by uncoupling the signaling pathway from alpha-factor binding at some point in the pathway between Scg1 inactivation and the action of Ste4, Ste5 and Ste18; this uncoupling might occur at the expense of partial constitutive activation of the pathway . DAF2-2 suppressed the unconditional cell-cycle arrest phenotype of a dominant "constitutive signaling" allele of STE4 (STE4Hpl), although the constitutive FUS1 phenotype of DAF2-2 was suppressed by ste4 null mutations; therefore DAF2-2 may directly affect the performance of the STE4 step.

Proc Natl Acad Sci U S A, 1990 Oct, 87(19), 7653 - 7
Detection of heteroduplex DNA molecules among the products of Saccharomyces cerevisiae meiosis; Lichten M et al.; We have used denaturant-gel electrophoresis to provide a physical demonstration of heteroduplex DNA in the products of yeast meiosis . We examined heteroduplex formation at arg4-nsp, a G.C----C.G transversion that displays a moderately high level of postmeiotic segregation . Of the two possible arg4-nsp/ARG4 mismatches (G.G and C.C), only C.C was detected in spores from mismatch repair-competent (Pms1+) diploids . In contrast, C.C and G.G were present at nearly equal levels in spores from Pms1- diploids . These results confirm previous suggestions that postmeiotic segregation spores contain heteroduplex DNA at the site of the marker in question, that C.C is repaired less frequently than is G.G, and that the PMS1 gene product plays a role in mismatch correction . Combined with the observation that Pms1+ ARG4/arg4-nsp diploids produce 3 times more 3+:5m (wildtype:mutant) tetrads (+, +, +/m, m) than 5+:3m tetrads (+, +/m, m, m), these results indicate that, during meiosis, formation of heteroduplex DNA at ARG4 involves preferential transfer of the sense (nontranscribed) strand of the DNA duplex.

Proc Natl Acad Sci U S A, 1990 Oct, 87(19), 7424 - 8
Chitin synthase I and chitin synthase II are not required for chitin synthesis in vivo in Saccharomyces cerevisiae; Bulawa CE et al.; In Saccharomyces cerevisiae, the polysaccharide chitin forms the primary division septum between mother cell and bud . Two related enzymes, chitin synthase I and chitin synthase II (UDP-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxyglucosyltransferase, EC 2.4.1.16), have been identified and their structural genes, CHS1 and CHS2, respectively, have been cloned and sequenced . Gene disruption experiments led to the conclusion that CHS2 is essential for cell division {Silverman, S.J., Sburlati, A., Slater, M.L . & Cabib, E . (1988) Proc . Natl . Acad . Sci . USA 85, 4735-4739}, whereas CHS1 is not . We repeated the disruption of CHS2 and determined that it is not essential for vegetative growth . The viability of chs1::HIS3 chs2::TRP1 spores is influenced by strain background and germination conditions . The double disruption mutant has no detectable chitin deficiency in vivo, as judged by quantitative assay and by staining cells with Calcofluor . Assay of membrane preparations from the double disruption mutant indicates the presence of chitin synthetic activity . Unlike the CHS gene products, this third activity is not stimulated by trypsin . Characterization of the double disruption mutant revealed abnormalities in morphology and nuclear migration.

Mutat Res, 1990 Oct, 232(2), 313 - 26
Analysis of interactions between mutagens, I . Heat and ultraviolet light in Saccharomyces cerevisiae; Ager DD et al.; A new mathematical approach to the description of interaction data (Ager and Haynes, 1987) is applied here to the interaction between heat and ultraviolet light (UV) in Saccharomyces cerevisiae . A strong synergism for cell killing is found to be associated with large increases in gene conversion (of up to 8-fold), and mutation (of up to 14-fold) . Analysis of the interaction data for both wild-type and repair-deficient strains indicates that the heat-UV synergism arises via the inhibition of two different repair pathways . Unambiguous conclusions regarding the molecular mechanisms by which these repair processes are inhibited cannot be drawn on the basis of dose-response data alone . However, this approach does enable one to make well defined, empirical comparisons of the nature and kinetics of such interactions.

J Cell Biol, 1990 Oct, 111(4), 1451 - 64
The SPA2 gene of Saccharomyces cerevisiae is important for pheromone-induced morphogenesis and efficient mating; Gehrung S et al.; Upon exposure to mating pheromone, Saccharomyces cerevisiae undergoes cellular differentiation to form a morphologically distinct cell called a "shmoo" . Double staining experiments revealed that both the SPA2 protein and actin localize to the shmoo tip which is the site of polarized cell growth . Actin concentrates as spots throughout the shmoo projection, while SPA2 localizes as a sharp patch at the shmoo tip . DNA sequence analysis of the SPA2 gene revealed an open reading frame 1,466 codons in length; the predicted protein sequence contains many internal repeats including a nine amino acid sequence that is imperfectly repeated 25 times . Portions of the SPA2 sequence exhibit a low-level similarity to proteins containing coiled-coil structures . Yeast cells containing a large deletion of the SPA2 gene are similar in growth rate to wild-type cells . However, spa2 mutant cells are impaired in their ability to form shmoos upon exposure to mating pheromone, and they do not mate efficiently with other spa2 mutant cells . Thus, we suggest that the SPA2 protein plays a critical role in cellular morphogenesis during mating, perhaps as a cytoskeletal protein.

J Bacteriol, 1990 Oct, 172(10), 5816 - 27
Association of glyoxylate and beta-oxidation enzymes with peroxisomes of Saccharomyces cerevisiae; McCammon MT et al.; Although peroxisomes are difficult to identify in Saccharomyces cerevisiae under ordinary growth conditions, they proliferate when cells are cultured on oleic acid . We used this finding to study the protein composition of these organelles in detail . Peroxisomes from oleic acid-grown cells were purified on a discontinuous sucrose gradient; they migrated to the 46 to 50% (wt/wt) sucrose interface . The peroxisomal fraction was identified morphologically and by the presence of all of the enzymes of the peroxisomal beta-oxidation pathway . These organelles also contained a significant but minor fraction of two enzymes of the glyoxylate pathway, malate synthase and malate dehydrogenase-2 . The localization of malate synthase in peroxisomes was confirmed by immunoelectron microscopy . It is postulated that glyoxylate pathway enzymes are readily and preferentially released from peroxisomes upon cell lysis, accounting for their incomplete recovery from isolated organelles . Small uninduced peroxisomes from glycerol-grown cultures were detected on sucrose gradients by marker enzymes . Under these conditions, catalase, acyl-coenzyme A oxidase, and malate synthase cofractionated at equilibrium close to the mitochondrial peak, indicating smaller, less dense organelles than those from cells grown on oleic acid . Peroxisomal membranes from oleate cultures were purified by buoyant density centrifugation . Three abundant proteins of 24, 31, and 32 kilodaltons were observed.

EMBO J, 1990 Oct, 9(10), 3225 - 32
The adenylate cyclase/protein kinase cascade regulates entry into meiosis in Saccharomyces cerevisiae through the gene IME1; Matsuura A et al.; Entry into meiosis in Saccharomyces cerevisiae cells is regulated by starvation through the adenylate cyclase/cAMP-dependent protein kinase (AC/PK) pathway . The gene IME1 is also involved in starvation control of meiosis . Multicopy IME1 plasmids overcome the meiotic deficiency of bcy1 and of RASval19 diploids . Double mutants ime1 cdc25 and ime1 ras2 are sporulation deficient . These results suggest that IME1 comes after the AC/PK cascade . Furthermore, the level of IME1 transcripts is affected by mutations in the AC/PK genes CDC25, CYR1 and BCY1 . Moreover, the addition of cAMP to a cyr1-2 diploid suppresses IME1 transcription . The presence in a bcy1 diploid of IME1 multicopy plasmids does not cure the failure of bcy1 cells to arrest as unbudded cells following starvation and to enter the G0 state (thermotolerance, synthesis of unique G0 proteins) . This indicates that the pathway downstream of the AC/PK cascade branches to control meiosis through IME1, and to control entry into G0 and cell cycle initiation, independently of IME1.

Mol Cell Biol, 1990 Oct, 10(10), 5553 - 7
The DNA damage-inducible gene DIN1 of Saccharomyces cerevisiae encodes a regulatory subunit of ribonucleotide reductase and is identical to RNR3; Yagle K et al.; The sequence of the DIN1 gene of Saccharomyces cerevisiae is identical to RNR3, a gene encoding a DNA damage-inducible regulatory subunit of ribonucleotide reductase . Two sequence elements located upstream of DIN1 (RNR3) are homologous to putative DNA damage regulatory elements in the promoter of the reductase catalytic subunit gene, RNR2 . The transcript start sites for DIN1(RNR3) have been localized, and induction by different agents has been compared with other DNA damage-regulated genes.

Mol Cell Biol, 1990 Oct, 10(10), 5295 - 304
Phenotypic consequences of tubulin overproduction in Saccharomyces cerevisiae: differences between alpha-tubulin and beta-tubulin; Weinstein B et al.; Overexpression of alpha- and beta-tubulin genes in Saccharomyces cerevisiae, separately or together, leads to accumulation of large excesses of each of the polypeptides and arrest of cell division . However, other consequences of overexpression of these genes differ in several ways . As shown previously (D . Burke, P . Gasdaska, and L . Hartwell, Mol . Cell . Biol . 9:1049-1059, 1989), overexpression of beta-tubulin leads, at early times, to loss of microtubule structures and loss of viability . Eventually, the excess beta-tubulin forms abnormal structures . We show here that, in contrast, overexpression of alpha-tubulin led to none of these phenotypes and in fact could suppress each of the phenotypes associated with beta-tubulin accumulation . Truncated forms of beta-tubulin that were not competent to carry out microtubule functions also failed to elicit the beta-tubulin-specific phenotypes when overexpressed . The data support the hypothesis that beta-tubulin in excess over alpha-tubulin is uniquely toxic, perhaps because it interferes with normal microtubule assembly.

Mol Cell Biol, 1990 Oct, 10(10), 5286 - 94
Regulation of tubulin levels and microtubule assembly in Saccharomyces cerevisiae: consequences of altered tubulin gene copy number; Katz W et al.; Microtubule organization in the cytoplasm is in part a function of the number and length of the assembled polymers . The intracellular concentration of tubulin could specify those parameters . Saccharomyces cerevisiae strains constructed with moderately decreased or increased copy numbers of tubulin genes provide an opportunity to study the cellular response to a steady-state change in tubulin concentration . We found no evidence of a mechanism for adjusting tubulin concentrations upward from a deficit, nor did we find a need for such a mechanism: cells with no more than 50% of the wild-type tubulin level were normal with respect to a series of microtubule-dependent properties . Strains with increased copies of both alpha- and beta-tubulin genes, or of alpha-tubulin genes alone, apparently did down regulate their tubulin levels . As a result, they contained greater than normal concentrations of tubulin but much less than predicted from the increase in gene number . Some of this down regulation occurred at the level of protein . These strains were also phenotypically normal . Cells could contain excess alpha-tubulin protein without detectable consequences, but perturbations resulting in excess beta-tubulin genes may have affected microtubule-dependent functions . All of the observed regulation of levels of tubulin can be explained as a response to toxicity associated with excess tubulin proteins, especially if beta-tubulin is much more toxic than alpha-tubulin.

Mol Cell Biol, 1990 Oct, 10(10), 5235 - 43
Ribosomal protein L30 is dispensable in the yeast Saccharomyces cerevisiae; Baronas-Lowell DM et al.; In the yeast Saccharomyces cerevisiae, L30 is one of many ribosomal proteins that is encoded by two functional genes . We have cloned and sequenced RPL30B, which shows strong homology to RPL30A . Use of mRNA as a template for a polymerase chain reaction demonstrated that RPL30B contains an intron in its 5' untranslated region . This intron has an unusual 5' splice site, C/GUAUGU . The genomic copies of RPL30A and RPL30B were disrupted by homologous recombination . Growth rates, primer extension, and two-dimensional ribosomal protein analyses of these disruption mutants suggested that RPL30A is responsible for the majority of L30 production . Surprisingly, meiosis of a diploid strain carrying one disrupted RPL30A and one disrupted RPL30B yielded four viable spores . Ribosomes from haploid cells carrying both disrupted genes had no detectable L30, yet such cells grew with a doubling time only 30% longer than that of wild-type cells . Furthermore, depletion of L30 did not alter the ratio of 60S to 40S ribosomal subunits, suggesting that there is no serious effect on the assembly of 60S subunits . Polysome profiles, however, suggest that the absence of L30 leads to the formation of stalled translation initiation complexes.

Mol Cell Biol, 1990 Oct, 10(10), 5087 - 97
Multiple positive and negative cis-acting elements mediate induced arginase (CAR1) gene expression in Saccharomyces cerevisiae; Kovari L et al.; Expression of the arginase (CAR1) gene in Saccharomyces cerevisiae is induced by arginine or its analog homoarginine . Induction has been previously shown to require a negatively acting upstream repression sequence, which maintains expression of the gene at a low level in the absence of inducer . The objective of this work was to identify the cis-acting elements responsible for CAR1 transcriptional activation and response to inducer . We identified three upstream activation sequences (UASs) that support transcriptional activation in a heterologous expression vector . Two of these UAS elements function in the absence of inducer, whereas the third functions only when inducer is present . One of the inducer-independent UAS elements exhibits significant homology to the Sp1 factor-binding sites identified in simian virus 40 and various mammalian genes.

Mol Cell Biol, 1990 Oct, 10(10), 5071 - 6
Farnesyl cysteine C-terminal methyltransferase activity is dependent upon the STE14 gene product in Saccharomyces cerevisiae; Hrycyna CA et al.; Membrane extracts of sterile Saccharomyces cerevisiae strains containing the a-specific ste14 mutation lack a farnesyl cysteine C-terminal carboxyl methyltransferase activity that is present in wild-type a and alpha cells . Other a-specific sterile strains with ste6 and ste16 mutations also have wild-type levels of the farnesyl cysteine carboxyl methyltransferase activity . This enzyme activity, detected by using a synthetic peptide sequence based on the C-terminus of a ras protein, may be responsible not only for the essential methylation of the farnesyl cysteine residue of a mating factor, but also for the methylation of yeast RAS1 and RAS2 proteins and possibly other polypeptides with similar C-terminal structures . We demonstrate that the farnesylation of the cysteine residue in the peptide is required for the methyltransferase activity, suggesting that methyl esterification follows the lipidation reaction in the cell . To show that the loss of methyltransferase activity is a direct result of the ste14 mutation, we transformed ste14 mutant cells with a plasmid complementing the mating defect of this strain and found that active enzyme was produced . Finally, we demonstrated that a similar transformation of cells possessing the wild-type STE14 gene resulted in sixfold overproduction of the enzyme . Although more complicated possibilities cannot be ruled out, these results suggest that STE14 is a candidate for the structural gene for a methyltransferase involved in the formation of isoprenylated cysteine alpha-methyl ester C-terminal structures.

DNA Cell Biol, 1990 Oct, 9(8), 603 - 14
Expression of bovine cytochrome P450c21 and its fused enzymes with yeast NADPH-cytochrome P450 reductase in Saccharomyces cerevisiae; Sakaki T et al.; Recombinant plasmids for expression of bovine cytochrome P450c21 (pA gamma 2), both P450c21 and yeast NADPH-cytochrome P450 reductase (pAR gamma 1), P450c21/yeast reductase fused enzymes (pAF gamma R1, pAF gamma R2, and pAF gamma R20), and yeast reductase/P450c21 fused enzymes (pAFR gamma 1 and pAFR gamma 2) were constructed by using expression vector pAAH5 . The plasmids were each introduced into the yeast Saccharomyces cerevisiae AH22 cells . The recombinant yeast strains AH22/pA gamma 2 (Y21) and AH22/pAR gamma 1 (Y21R) produced 2-3 X 10(3) molecules of P450c21 per cell . The cultures of both strains converted progesterone and 17 alpha-hydroxyprogesterone into 11-deoxycorticosterone and 11-deoxycortisol, respectively . The 21-hydroxylase activity per cell of the strain Y21R was about three times higher than that of the strain Y21, probably due to overproduction of yeast reductase . The recombinant yeast strains AH22/pAF gamma R1 (Y21RF1), AH22/pAF gamma R2 (Y21RF2), and AH22/pAF gamma R20 (Y21RF20) produced about 1.1-2.0 X 10(4) molecules per cell of the corresponding P450c21/yeast reductase fused enzymes . The specific 21-hydroxylase activity toward 17 alpha-hydroxyprogesterone per cell of the strains Y21RF1, Y21RF2, and Y21RF20 was about 21, 28, and 49 times higher than that of the strain Y21, respectively . Thus, the fused enzymes were superior to P450c21 in the specific activity and in the expression level in the yeast . The Km values for 17 alpha-hydroxyprogesterone of P450c21 in the strains Y21 and Y21R, and of the fused enzymes in the strains Y21RF1 and Y21RF2 were 0.29, 0.30, 0.67, and 0.65 microM, respectively . The Vmax values of P450c21 in the strains Y21 and Y21R, and of the fused enzymes in the strains Y21RF1 and Y21RF2 were 28, 124, 151, and 222 moles/min.mole P450c21 or fused enzyme, respectively . These results indicated that the fused enzymes showed lower affinity for the substrate, probably due to structural modification and higher reaction rates through efficient intramolecular electron transfer as compared with those of P450c21 . While the strain AH22/pAFR gamma 2 (YR21F2) produced about 3 X 10(4) molecules per cell of the reductase/P450c21 fused enzyme, the specific 21-hydroxylase activity of the fused enzyme toward 17 alpha-hydroxyprogesterone was extremely low, suggesting that the structure of the fused enzyme might not be suited for electron transfer in yeast microsomes.

Mol Cell Biol, 1990 Oct, 10(10), 5455 - 63
Studies of an 800-kilobase DNA stretch of the Drosophila X chromosome: comapping of a subclass of scaffold-attached regions with sequences able to replicate autonomously in Saccharomyces cerevisiae; Brun C et al.; We have previously mapped scaffold-attached regions (SARs) on an 800-kilobase DNA walk from the Drosophila X chromosome . We have also previously shown that the strength of binding, i.e., the ability of SARs to bind to all nuclear scaffolds or only to a fraction of them varied from one SAR to another one . In the present study, 71 of the 85 subfragments that bind scaffolds and 38 fragments that do not bind scaffolds were tested for their ability to promote autonomous replicating sequence (ARS) activity in Saccharomyces cerevisiae . Sixteen SAR-containing fragments from the chromosome walk were also examined for association to yeast nuclear scaffolds in vitro . All identified ARSs (a total of 27) were present on SAR-containing fragments, except two, which were adjacent to SARs . There is thus a correlation between ARS and SAR activities, and this correlation defines a SAR subclass . Moreover, the presence of an ARS on a DNA fragment appeared to be highly correlated with the strength of binding . The binding activity was highly conserved from Drosophila melanogaster to yeast . These data suggest that Drosophila DNA sequences responsible for binding to components of the nuclear scaffold from either D . melanogaster or yeast may be involved in the process of heterologous extrachromosomal replication in yeasts.

Int J Pept Protein Res, 1990 Oct, 36(4), 362 - 73
Solution phase synthesis of Saccharomyces cerevisiae a-mating factor and its analogs; Xue CB et al.; The solution phase synthesis of the Saccharomyces cerevisiae a-mating factor and nonfarnesylated and nonmethylated a-factor analogs are reported . The a-factor, a lipopeptide with the sequence Tyr-Ile-Ile-Lys-Gly-Val-Phe-Trp-Asp-Pro-Ala-Cys(S-Farnesyl)OCH3 was synthesized by the condensation of the amine terminal protected decapeptide with the carboxyl terminal farnesylated dipeptide using benzotriazol-l-yloxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP reagent) as the coupling agent . The synthesis of the decapeptide involved 5 + 5 fragment coupling with the BOP reagent and the successful application of 9-fluorenylmethyl ester(OFm) and 9-fluorenylmethoxycarbonyl(Fmoc) groups for the protection of Asp and Lys side chains and Tyr alpha-amine and of phenacyl esters (OPa) for alpha-carboxyl protection . The OFm and Fmoc groups tolerated repeated couplings and were completely stable to zinc powder in acetic acid, a condition under which the OPa group was removed . The synthesis of the nonfarnesylated alpha-factor was accomplished by the coupling of the decapeptide with tetrapeptide (Ala-CysOCH3)2 followed by the deprotection of the OFm and Fmoc groups with piperidine and the cleavage of the disulfide bond with zinc powder in acetic acid . The nonmethylated a-factor was prepared by 10 + 2 fragment coupling using OFm protection of the dipeptide carboxyl group followed by removal of all protecting groups with piperidine . Attempts to saponify a-factor were not successful . The synthetic nonfarnesylated and nonmethylated a-mating pheromones were 100-1000 times less active than the a-factor, indicating that although the methyl ester and the farnesyl group are not essential for biological activity, they are necessary for high potency.

Proc Natl Acad Sci U S A, 1990 Oct, 87(19), 7628 - 32
Circular single-stranded RNA replicon in Saccharomyces cerevisiae; Matsumoto Y et al.; Circular RNA replicons have been reported in plants and, in one case, in animal cells . We describe such an element in yeast . In certain yeast strains, a 20S RNA species appears on transfer of cells to acetate medium . This phenotype shows cytoplasmic (non-Mendelian) inheritance and the 20S RNA is associated with 23-kDa protein subunits as a 32S particle . We demonstrate that yeast 20S RNA is an independent replicon with no homology to host genomic, mitochondrial, or 2-microns plasmid DNA or to the L-A, L-BC, or M1 double-stranded RNA viruses of yeast . The circularity of the 20S RNA is shown by the apparent absence of 3' and 5' ends, by two-dimensional gel electrophoresis, and by electron microscopy . Replication of yeast 20S RNA proceeds through an RNA-RNA pathway, and a 10,000-fold amplification occurs on shift to acetate medium . The copy number of 20S RNA is also reduced severalfold by the SKI gene products, a host antiviral system that also lowers the copy numbers of yeast double-stranded RNA viruses . Yeast 20S RNA and the hepatitis delta virus show some similarities.

J Biol Chem, 1990 Sep 25, 265(27), 16216 - 20
Isolation of a temperature-sensitive mutant with an altered tRNA nucleotidyltransferase and cloning of the gene encoding tRNA nucleotidyltransferase in the yeast Saccharomyces cerevisiae; Aebi M et al.; We have isolated a yeast mutant, ts352, that is temperature-sensitive for growth . The mutation has a general effect on mRNA metabolism and a specific effect on tRNA biosynthesis . Cells shifted to the nonpermissive temperature accumulate tRNAs that are shorter than mature tRNAs . The increased ability of these tRNAs to accept ATP demonstrates that growth of the ts352 mutant at the nonpermissive temperature results in accumulation of tRNA with defective 3' ends . The activity of ATP (CTP):tRNA-specific tRNA nucleotidyltransferase can readily be measured in extracts from wild type but not mutant cells . We have cloned and sequenced the wild type allele of the ts352 gene and find significant similarity between the yeast protein sequence predicted from the DNA sequence and the protein predicted from the sequence of the Escherichi coli tRNA nucleotidyltransferase gene . Expression of the yeast gene on a multicopy plasmid increases the activity of the tRNA nucleotidyltransferase in extracts . We conclude that the defect in the ts352 mutant is in the gene coding for yeast tRNA nucleotidyltransferase and that we have isolated the yeast gene that codes for this enzyme.

Eur J Biochem, 1990 Sep 24, 192(3), 761 - 5
Membrane topography of the subunits of ubiquinol-cytochrome-c oxidoreductase of Saccharomyces cerevisiae . The 14-kDa and the 11-kDa subunits face opposite sides of the mitochondrial inner membrane; Hemrika W et al.; The topology of the subunits of ubiquinol-cytochrome-c oxidoreductase of the yeast Saccharomyces cerevisiae has been determined using a digitonin/proteinase K assay . With this assay we were able selectively to disrupt the mitochondrial membranes and to identify the subunits which became proteinase-K sensitive after disruption of either the outer or both outer and inner membranes . This approach confirmed previous indications for the localization of the core I protein, cytochrome c1, cytochrome b, the FeS protein and the 17-kDa subunit, while it also provided direct evidence for the site of accessibility to proteinase K of the 14-kDa and 11-kDa subunits . The 14-kDa subunit faces the mitochondrial matrix and the 11-kDa subunit faces the intermembrane space.

J Mol Biol, 1990 Sep 20, 215(2), 211 - 3
Crystallographic studies of 3-ketoacylCoA thiolase from yeast Saccharomyces cerevisiae; Zeelen JP et al.; Good diffracting crystals of 3-ketoacylCoA thiolase (EC 2.3.1.16) from yeast Saccharomyces cerevisiae have been obtained . The crystals diffract to at least 2.4 A . The space group of these crystals is P2(1)2(1)2(1), with cell dimensions a = 71.8 A, b = 93.8 A and c = 119.9 A . There is one dimer per asymmetric unit.

Biochemistry, 1990 Sep 18, 29(37), 8827 - 34
Protein chemical and kinetic characterization of recombinant porcine ribonuclease inhibitor expressed in Saccharomyces cerevisiae; Vicentini AM et al.; A cDNA encoding porcine ribonuclease inhibitor was used to express this protein in yeast under control of the PHO5 promoter . The recombinant protein was purified to homogeneity with a yield of 0.2 mg/g of yeast cells (wet weight) and was found to be indistinguishable from the inhibitor isolated from porcine liver on the basis of the following criteria: the amino acid composition, the number of free sulfhydryl groups, the molecular weight of the native and the denatured protein, peptide mapping, and amino acid sequence analysis of the N- and C-terminal regions of the protein . A simple method was developed for measuring accurately the slow, tight-biding kinetics of the inhibition of ribonuclease by ribonuclease inhibitor . From the dependence of the observed inhibition constant on the substrate concentration, it could be concluded that RI was competitive with the substrate UpA . The dependence of the observed association rate constant on the substrate concentration was consistent with a two-step mechanism in which the substrate only competed in the second (isomerization) step . The values for the inhibition constant for the inhibition of RNase by the recombinant inhibitor, 67 fM, the association rate constant, 1.5 x 10(8) M-1.s-1, and the dissociation rate constant, 8.3 x 10(-6) s-1, were in good agreement with those obtained for the porcine liver RNase inhibitor.

Biochim Biophys Acta, 1990 Sep 18, 1046(2), 214 - 22
Biochemical characterization and regulation of cardiolipin synthase in Saccharomyces cerevisiae; Tamai KT et al.; Cardiolipin (CL) synthase activity was characterized in mitochondrial extracts of the yeast Saccharomyces cerevisiae and was shown for the first time to utilize CDP-diacylglycerol as a substrate . CL synthase exhibited a pH optimum of 9.0 . Maximal activity was obtained in the presence of 20 mM magnesium with a Triton X-100: phospholipid ratio of 1:1 . The apparent Km values for phosphatidylglycerol and CDP-diacylglycerol were 1 mM and 36 microM, respectively . CL synthase activity was maximal at 45 degrees C and heat inactivation studies showed that the enzyme retained greater than 75% of its activity at temperatures up to 55 degrees C . To study the regulation of CL synthase, the enzyme was assayed in cells grown under conditions known to affect general phospholipid synthesis . Unlike many phospholipid biosynthetic enzymes including PGP synthase, which catalyzes the initial step in CL biosynthesis, CL synthase was not repressed in cells grown in the presence of the phospholipid precursor inositol . Detailed procedures for the enzymatic synthesis of 32P-labelled substrates are described.

Antimicrob Agents Chemother, 1990 Sep, 34(9), 1660 - 5
Effects of lovastatin (mevinolin) on sterol levels and on activity of azoles in Saccharomyces cerevisiae; Lorenz RT et al.; The hypocholesterolemic drug lovastatin (mevinolin) was found to be very effective in lowering the sterol levels of the wild-type yeast Saccharomyces cerevisiae . Lovastatin dramatically decreased the steryl ester content from 2.62 to 0.8 micrograms/mg (dry weight), whereas the free sterol content decreased only from 2.79 to 2.24 micrograms/mg (dry weight) when lovastatin was present in the medium at 10 micrograms/ml . At higher concentrations (100 micrograms/ml), lovastatin nearly abolished the accumulation of steryl esters and decreased the free sterol concentration to less than 1.3 micrograms/mg (dry weight) . As a result of the lowered sterol levels, proportional amounts of exogenous sterol were taken up from the medium during aerobic, respiratory conditions . Nearly all of the exogenous sterol taken up was partitioned into the free sterol fraction . The inhibition of sterol esterification in the presence of lovastatin was dependent on heme synthesis . The result of these combined effects caused the MICs of three azole antifungal drugs (ketoconazole, clotrimazole, and miconazole) to be lowered from 6- to 32-fold when lovastatin was present in the medium at 10 micrograms/ml.

Appl Environ Microbiol, 1990 Sep, 56(9), 2853 - 7
Saccharomyces cerevisiae membrane sterol modifications in response to growth in the presence of ethanol; Walker-Caprioglio HM et al.; Membranes isolated from yeasts grown in the presence of ethanol do not display the thermally induced transition in diphenylhexatriene anisotropy that is seen in control cells when they are exposed to ethanol in vitro . The total sterol content of the cells that were exposed to ethanol during growth is reduced, with no steryl esters being detected . A greater proportion of the total sterol pool is ergosterol in cells grown in the presence of alcohol . The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase is reduced by ethanol in vitro . Ethanol-exposed cells take up more exogenous sterol under aerobic conditions than do control cells . The presence of ethanol during growth reduces the activity of the plasma membrane enzyme, chitin synthase, as well as increasing the thermosensitivity of this enzyme.

Appl Environ Microbiol, 1990 Sep, 56(9), 2777 - 83
Inducible overexpression of the FUM1 gene in Saccharomyces cerevisiae: localization of fumarase and efficient fumaric acid bioconversion to L-malic acid; Peleg Y et al.; Cloning of the Saccharomyces cerevisiae FUM1 gene downstream of the strong GAL10 promoter resulted in inducible overexpression of fumarase in the yeast . The overproducing strain exhibited efficient bioconversion of fumaric acid to L-malic acid with an apparent conversion value of 88% and a conversion rate of 80.4 mmol of fumaric acid/h per g of cell wet weight, both of which are much higher than parameters known for industrial bacterial strains . The only product of the conversion reaction was L-malic acid, which was essentially free of the unwanted by-product succinic acid . The GAL10 promoter situated upstream of a promoterless FUM1 gene led to production and correct distribution of the two fumarase isoenzyme activities between cytosolic and mitochondrial subcellular fractions . The amino-terminal sequence of fumarase contains the mitochondrial signal sequence since (i) 92 of 463 amino acid residues from the amino terminus of fumarase are sufficient to localize fumarase-lacZ fusions to mitochondria and (ii) fumarase and fumarase-lacZ fusions lacking the amino-terminal sequence are localized exclusively in the cytosol . The possibility that both mitochondrial and cytosolic fumarases are derived from the same initial translation product is discussed.

Mol Gen Genet, 1990 Sep, 223(3), 474 - 80
Deletion analysis of the ARG4 promoter of Saccharomyces cerevisiae: a poly(dAdT) stretch involved in gene transcription; Thiry-Blaise LM et al.; Transcription of the ARG4 gene of Saccharomyces cerevisiae is regulated by general control of amino acid biosynthesis but not by a specific regulatory mechanism . Three deletion mutants (delta I, delta II, delta III) successively removing DNA sequences upstream from the coding sequence have been phenotypically analyzed after insertion into a single copy plasmid . As expected, delta I, which lacks the sequences upstream to -155, including the two putative upstream activation sequences (UAS), was unable to derepress argininosuccinate lyase biosynthesis under conditions of amino acid starvation . In delta II (deleted up to -126) the enzyme activity was very low and cells harbouring this allele were arginine dependent . These drastic phenotypic changes can be attributed to the loss of 12 out of 14 dA residues from positions -124 to -137 . This poly (dAdT) sequence most likely serves as an upstream promoter element for constitutive expression of ARG4 . The delta III deletion removes all 5' sequences including the putative TATA box . This inactive allele has been successfully used for selecting yeast promoters of unknown origin.

Mol Gen Genet, 1990 Sep, 223(3), 449 - 56
TUF factor binds to the upstream region of the pyruvate decarboxylase structural gene (PDC1) of Saccharomyces cerevisiae; Butler G et al.; The upstream activation site of the pyruvate decarboxylase gene, PDC1, of Saccharomyces cerevisiae contains an RPG box, and mediates the increase in expression of a PDC1-lacZ fusion gene during growth on glucose . Oligonucleotide replacement experiments indicate that the RPG box functions as an absolute activator of expression, but other elements (possibly CTTCC repeats) are required for carbon source regulation, and maximal expression . Gel retardation and oligonucleotide competition experiments suggest that the DNA binding factor TUF interacts with the RPG box in the upstream region of PDC1 . Binding of TUF factor is not carbon source dependent in in vitro experiments, and is probably not responsible for glucose induction of PDC1 expression.

Mol Gen Genet, 1990 Sep, 223(3), 394 - 400
Identification of CBS2 as a mitochondrial protein in Saccharomyces cerevisiae; Michaelis U et al.; The nuclear genome encoded yeast protein CBS2 is required for translational activation of mitochondrial cytochrome b RNA . Genetic studies have shown that the target sequence of the CBS2 protein is the 5' untranslated leader sequence of cytochrome b RNA . Here we report on the intracellular localization of CBS2 . CBS2 protein, expressed in Escherichia coli and prepared from inclusion bodies, was used as an antigen to raise a polyclonal rabbit antiserum . Affinity-purified CBS2 antibodies detect a 45 kDa protein in mitochondrial lysates of wild-type cells, which is absent in a strain in which the CBS2 gene has been deleted . The protein is overexpressed in mitochondrial extracts of a transformant carrying the CBS2 gene on a high copy number plasmid, but undetectable in the post-mitochondrial supernatant . Intramitochondrial localization of CBS2 was verified by in vitro import of CBS2 protein that had been synthesized in a reticulocyte lysate programmed with CBS2 mRNA transcribed in vitro . Mitochondrial import of CBS2 is not accompanied by any detectable proteolytic processing.

Mutagenesis, 1990 Sep, 5(5), 453 - 9
Analysis of nine known or suspected spindle poisons for mitotic chromosome malsegregation using Saccharomyces cerevisiae D61.M; Albertini S; We tested nine (cadmium chloride, chloral hydrate, colchicine, diazepam, econazole nitrate, hydroquinone, pyrimethamine, thiabendazole, thimerosal) of the 10 known or suspected spindle poisons of the coordinated programme to study aneuploidy induction sponsored by the Commission of the European Communities using Saccharomyces cerevisiae D61.M (mitotic chromosomal malsegregation system) . Mitotic malsegregation of chromosome VII was induced by chloral hydrate, thiabendazole and thimerosal . Diazepam, colchicine, cadmium chloride, econazole nitrate, hydroquinone and pyrimethamine revealed no induction of chromosomal malsegregation.

Mol Gen Genet, 1990 Sep, 223(2), 241 - 8
Transformation and recombination in rad mutants of Saccharomyces cerevisiae; Simon JR et al.; Disruption/deletion mutations in genes of the RAD52 epistasis group of Saccharomyces cerevisiae were examined for their effects on recombination between single- and double-stranded circular DNA substrates and chromosomal genes in a transformation assay . In rad50 mutants there was a small reduction in recombination with single-stranded DNA at the leu2-3, 112 allele; in addition there was an almost complete elimination of recombination at trp1-1 for both single- and double-stranded DNA . Reintroduction of a wild-type RAD50 gene on a replicating plasmid carrying CEN4 restored recombinational competence at trp1-1, indicating that rad50 is defective in gene replacement of this allele . In rad52 mutants a reduction of 30%-50% in recombination involving either single- or double-stranded circular DNA was observed in each experiment when compared to the wild type . This reduction of recombination in rad52 mutants was similar for recombination at the ura3-52 mutant locus where only integration events have been observed, and at the trp1-1 mutant locus, where recombination occurs predominantly by gene replacement . Neither the rad54 nor the rad57 mutations had a significant effect on recombination with single- or double-stranded DNA substrates.

Yeast, 1990 Sep-Oct, 6(5), 411 - 5
Mapping of the trifunctional fatty acid synthetase gene FAS2 on chromosome XVI of Saccharomyces cerevisiae; Siebenlist U et al.; The trifunctional FAS2 gene encoding subunit alpha of the Saccharomyces cerevisiae fatty acid synthetase complex was mapped on the left arm of chromosome XVI 24 centimorgans proximal to GAL4 and 39 centimorgans distal to PEP4 relative to the centromere . Mapping was achieved by three independent methods: meiotic co-segregation of FAS2 and ARO7 in recombination-deficient spo11-mutants: tetrad analysis of crosses between FAS2, GAL4 and PEP4; and Southern hybridization of purified FAS2 DNA with individual yeast chromosomes separated by pulsed-field gel electrophoresis.

Yeast, 1990 Sep-Oct, 6(5), 383 - 401
Chromosome III of Saccharomyces cerevisiae: an ordered clone bank, a detailed restriction map and analysis of transcripts suggest the presence of 160 genes; Yoshikawa A et al.; Using lambda phage vector EMBL4, we isolated 344 clones containing segments of chromosome III of Saccharomyces cerevisiae, analysed their physical structure with eight restriction enzymes and sorted the data in contiguous groups with computer programmes . Furthermore, we performed Southern hybridizations between the sorted contiguous clone groups and interrelated them into larger groups . In this way, we constructed an ordered clone bank that covers almost the whole of chromosome III with a single gap of several kilobases in length . The consensus physical map thus obtained totals 334.6 kb, which is in good agreement with the size of this chromosome estimated by pulsed-field gel electrophoresis . Southern hybridization analysis with the DNA probes containing telomere-specific sequences showed that the bank contained a telomere at a position corresponding to the right arm terminus of chromosome III . Also, five Ty elements were found to be present . To estimate the number of genes on this chromosome and to analyse their levels of expression, we performed a series of Northern hybridization experiments using total poly(A)+ RNA from vegetatively growing cells and appropriate restriction enzyme fragments from the bank . Thus, we identified a total of 156 transcripts on chromosome III, indicating, on an average, one gene in every 2 kb on this chromosome . The transcripts were visually categorized into five groups according to their apparent levels of expression . It was found that the genes located near both termini are expressed only at low levels and that highly expressed genes are rather scattered over the chromosome.

EMBO J, 1990 Sep, 9(9), 2899 - 904
Differential repair of UV damage in Saccharomyces cerevisiae is cell cycle dependent; Terleth C et al.; In the yeast Saccharomyces cerevisiae the transcriptionally active MAT alpha locus is repaired preferentially to the inactive HML alpha locus after UV irradiation . Here we analysed the repair of both loci after irradiating yeast cells at different stages of the mitotic cell cycle . In all stages repair of the active MAT alpha locus occurs at a rate of 30% removal of dimers per hour after a UV dose of 60 J/m2 . The inactive HML alpha is repaired as efficiently as MAT alpha following irradiation in G2 whereas repair of HML alpha is less efficient in the other stages . Thus differential repair is observed in G1 and S but not in G2 . Apparently, in G2 a chromatin structure exists in which repair does not discriminate between transcriptionally active and inactive DNA or, alternatively, an additional repair mechanism might exist which is only operational during G2.

Mol Cell Biol, 1990 Sep, 10(9), 4935 - 41
SPT6, an essential gene that affects transcription in Saccharomyces cerevisiae, encodes a nuclear protein with an extremely acidic amino terminus; Swanson MS et al.; SPT6 is an essential gene of Saccharomyces cerevisiae that appears to play a role in transcription . Mutations in the SPT6 (SSN20, CRE2) gene suppress delta insertion mutations in the 5' regions of HIS4 and LYS2 and mutations in cis- and/or trans-acting elements that are required for expression of SUC2 and ADH2 . We report here that SPT6 encodes a 170-kilodalton highly charged protein with an extremely acidic amino terminus . By use of an epitope-tagged SPT6 protein, we have determined by indirect immunofluorescence that the SPT6 protein is located in the nucleus.

Mol Cell Biol, 1990 Sep, 10(9), 4757 - 69
Two systems of glucose repression of the GAL1 promoter in Saccharomyces cerevisiae; Flick JS et al.; Expression of the GAL1 gene in Saccharomyces cerevisiae is strongly repressed by growth on glucose . We show that two sites within the GAL1 promoter mediate glucose repression . First, glucose inhibits transcription activation by GAL4 protein through UASG . Second, a promoter element, termed URSG, confers glucose repression independently of GAL4 . We have localized the URSG sequences responsible for glucose repression to an 87-base-pair fragment located between UASG and the TATA box . Promoters deleted for small (20-base-pair) segments that span this sequence are still subject to glucose repression, suggesting that there are multiple sequences within this region that confer repression . Extended deletions across this region confirm that it contains at least two and possibly three URSG elements . To identify the gene products that confer repression upon UASG and URSG, we have analyzed glucose repression mutants and found that the GAL83, REG1, GRR1, and SSN6 genes are required for repression mediated by both UASG and URSG . In contrast, GAL82 and HXK2 are required only for UASG repression . A mutation designated urr1-1 (URSG repression resistant) was identified that specifically relieves URSG repression without affecting UASG repression . In addition, we observed that the SNF1-encoded protein kinase is essential for derepression of both UASG and URSG . We propose that repression of UASG and URSG is mediated by two independent pathways that respond to a common signal generated by growth on glucose.

Mol Cell Biol, 1990 Sep, 10(9), 4744 - 56
The N-terminal TPR region is the functional domain of SSN6, a nuclear phosphoprotein of Saccharomyces cerevisiae; Schultz J et al.; The SSN6 protein functions as a negative regulator of a variety of genes in Saccharomyces cerevisiae and is required for normal growth, mating, and sporulation . It is a member of a family defined by a repeated amino acid sequence, the TPR (tetratricopeptide repeat) motif . Here, we have used specific antibody to identify and characterize the SSN6 protein . Both SSN6 and a bifunctional SSN6-beta-galactosidase fusion protein were localized in the nucleus by immunofluorescence staining . The N-terminal one-third of the protein containing the TPR units was identified as the region that is important for SSN6 function . Analysis of four nonsense alleles, isolated as intragenic suppressors of an ssn6::URA3 insertion, revealed that polypeptides truncated after TPR unit 7 provide SSN6 function . Deletion analysis suggested that TPR units are required but that 4 of the 10 TPR units are sufficient . In addition, deletion studies indicated that three very long, homogeneous tracts of polyglutamine and poly(glutamine-alanine) are dispensable . Previous genetic evidence suggested the SSN6 protein as a possible target of the SNF1 protein kinase . Here, we show that the C terminus of SSN6 is phosphorylated in vivo and that the SNF1 kinase is not responsible for most of the phosphorylation . Finally, SSN6 has a modest effect on the maintenance of minichromosomes.

Mol Cell Biol, 1990 Sep, 10(9), 4737 - 43
The RPC31 gene of Saccharomyces cerevisiae encodes a subunit of RNA polymerase C (III) with an acidic tail; Mosrin C et al.; The RPC31 gene encoding the C31 subunit of Saccharomyces cerevisiae RNA polymerase C (III) has been isolated, starting from a C-terminal fragment cloned on a lambda gt11 library . It is unique on the yeast genome and lies on the left arm of chromosome XIV, very close to a NotI site . Its coding sequence perfectly matches the amino acid sequence of two oligopeptides prepared from purified C31 . It is also identical to the ACP2 gene previously described as encoding an HMG1-like protein (W . Haggren and D . Kolodrubetz, Mol . Cell . Biol . 8:1282-1289, 1988) . Thus, ACP2 and RPC31 are allelic and encode a subunit of RNA polymerase C . The c31 protein has a highly acidic C-terminal tail also found in several other chromatin-interacting proteins, including animal HMG1 . Outside this domain, however, there is no appreciable homology to any known protein . The growth phenotypes of a gene deletion, of insertions, and of nonsense mutations indicate that the C31 protein is strictly required for cell growth and that most of the acidic domain is essential for its function . Random mutagenesis failed to yield temperature-sensitive mutants, but a slowly growing mutant was constructed by partial suppression of a UAA nonsense allele of RPC31 . Its reduced rate of tRNA synthesis in vivo relative to 5.8S rRNA supports the hypothesis that the C31 protein is a functional subunit of RNA polymerase C.

Mol Cell Biol, 1990 Sep, 10(9), 4678 - 84
Differential repair of UV damage in rad mutants of Saccharomyces cerevisiae: a possible function of G2 arrest upon UV irradiation; Terleth C et al.; After UV irradiation, the transcriptionally active MAT alpha locus in Saccharomyces cerevisiae is preferentially repaired compared with the inactive HML alpha locus . The effect of rad mutations from three different epistasis groups on differential repair was investigated . Three mutants, rad9, rad16, and rad24, were impaired in the removal of UV dimers from the inactive HML alpha locus, whereas they had generally normal repair of the active MAT alpha locus . Since RAD9 is necessary for G2 arrest after UV irradiation, we propose that the G2 stage plays a role in making the dimers accessible for repair, at least in the repressed HML alpha locus.

Mol Cell Biol, 1990 Sep, 10(9), 4486 - 94
Expression and function of a human initiator tRNA gene in the yeast Saccharomyces cerevisiae; Francis MA et al.; We showed previously that the human initiator tRNA gene, in the context of its own 5'- and 3'-flanking sequences, was not expressed in Saccharomyces cerevisiae . Here we show that switching its 5'-flanking sequence with that of a yeast arginine tRNA gene allows its functional expression in yeast cells . The human initiator tRNA coding sequence was either cloned downstream of the yeast arginine tRNA gene, with various lengths of intergenic spacer separating them, or linked directly to the 5'-flanking sequence of the yeast arginine tRNA coding sequence . The human initiator tRNA made in yeast cells can be aminoacylated with methionine, and it was clearly separated from the yeast initiator and elongator methionine tRNAs by RPC-5 column chromatography . It was also functional in yeast cells . Expression of the human initiator tRNA in transformants of a slow-growing mutant yeast strain, in which three of the four endogenous initiator tRNA genes had been inactivated by gene disruption, resulted in enhancement of the growth rate . The degree of growth rate enhancement correlated with the steady-state levels of human tRNA in the transformants . Besides providing a possible assay for in vivo function of mutant human initiator tRNAs, this work represents the only example of the functional expression of a vertebrate RNA polymerase III-transcribed gene in yeast cells.

Mol Gen Genet, 1990 Sep, 223(3), 426 - 32
Site-directed mutagenesis of the Saccharomyces cerevisiae CDC25 gene: effects on mitotic growth and cAMP signalling; Schomerus C et al.; A potential membrane-interacting site within the essential growth-controlling carboxy-terminal region of the CDC25 protein was interrupted by a lethal mutation (1461 Tyr----Asp and 1462 Leu----Arg) . The elimination of two potential phosphorylation sites found in the same region (1489 Thr----Pro and 1584 Ser----Pro) does not affect growth but completely prevents glucose-induced cAMP signalling in the double mutant, whereas the single mutants produce normal or slightly retarded cAMP signals . A cluster of five potential targets for cAMP-dependent phosphorylation at the amino-terminal region could be deleted without affecting phenotypic properties . It is concluded that the carboxy-terminal 137 residues of the CDC25 protein are involved in three different functions: control of mitotic growth, glucose-induced hyperactivation of adenylate cyclase, and feed-back inhibition of cAMP synthesis.

FEMS Microbiol Lett, 1990 Sep 1, 59(1-2), 43 - 8
Two glycosylation patterns for a single protein (exoglucanase) in Saccharomyces cerevisiae; Ramirez M et al.; Exoglucanases (beta-glucosidases) I and II secreted into the culture medium by Saccharomyces cerevisiae were purified from cell cultures harvested at the early exponential phase of growth in order to avoid contamination of the second by a new immunologically-related material . The amino acid composition of the purified enzymes was roughly the same . In addition, both exoglucanases exhibited an identical NH2-terminal sequence (50 residues) . These results confirm our previous results about the identity of the protein moieties of both enzymes . Exoglucanase I appears to arise by elongation of one or both short oligosaccharides present in enzyme II.

Proc Natl Acad Sci U S A, 1990 Sep, 87(17), 6550 - 4
Evidence for a heat shock transcription factor-independent mechanism for heat shock induction of transcription in Saccharomyces cerevisiae; Kobayashi N et al.; Transcription of the DNA damage-responsive gene, DDRA2, of Saccharomyces cerevisiae is activated by heat shock treatment as well as by mutagen/carcinogen exposure . Deletion analysis of upstream noncoding sequence indicated that sequences between approximately -190 and -140 base pairs were necessary for heat shock and DNA damage regulation of transcription . Fusion of this region to a CYC1-lacZ reporter gene demonstrated that the sequence between -202 and -165 base pairs was sufficient for basal level and heat shock-induced expression . This DNA sequence was unable to bind heat shock transcription factor as judged by binding competition experiments in vitro . These results indicate that yeast possesses a second, heat shock transcription factor-independent mechanism for activating transcription in response to thermal stress.

Mol Cell Biol, 1990 Sep, 10(9), 4439 - 46
G protein mutations that alter the pheromone response in Saccharomyces cerevisiae; Stone DE et al.; The GPA1 gene of Saccharomyces cerevisiae encodes a G alpha protein that couples the membrane-bound pheromone receptors to downstream elements in the mating response pathway . We have isolated seven mutant alleles of GPA1 that confer pheromone resistance: G50D (a glycine-to-aspartate change at position 50), G322E, G322R, E355K, E364K, G470D, and an E364K-G470D double mutant . All of the mutations lie within large regions that are highly conserved between Gpa1 and four other G alpha proteins; four of the changes are located in domains with proposed functions . On the basis of a gentic analysis, the pheromone-unresponsive GPA1 alleles can be divided into two classes: those that encode constitutively activated proteins and those that encode proteins unable to respond to the upstream signal . Our results support the hypothesis that the activated form of Gpa1 stimulates adaptation to pheromone.

Genetika, 1990 Sep, 26(9), 1667 - 70
{Mapping the xrs2 and him1 genes of Saccharomyces cerevisiae by a method based on the chromosome destabilization effect}; Koval'tsova SV et al.; The methods for chimeric chromosomes' destabilization and the standard tetrad analysis have been used for mapping of the yeast genes XRS2 and HIM1 . The genes are localized in the right arm of chromosome IV in the following order: XRS2-31cM-ADE8-28.5cM-HIM1.

J Bacteriol, 1990 Sep, 172(9), 4927 - 35
Role of NAD-linked glutamate dehydrogenase in nitrogen metabolism in Saccharomyces cerevisiae; Miller SM et al.; We cloned GDH2, the gene that encodes the NAD-linked glutamate dehydrogenase in the yeast Saccharomyces cerevisiae, by purifying the enzyme, making polyclonal antibodies to it, and using the antibodies to screen a lambda gt11 yeast genomic library . A yeast strain with a deletion-disruption allele of GDH2 which replaced the wild-type gene grew very poorly with glutamate as a nitrogen source, but growth improved significantly when the strain was also provided with adenine or other nitrogenous compounds whose biosynthesis requires glutamine . Our results indicate that the NAD-linked glutamate dehydrogenase catalyzes the major, but not sole, pathway for generation of ammonia from glutamate . We also isolated yeast mutants that lacked glutamate synthase activity and present evidence which shows that normally NAD-linked glutamate dehydrogenase is not involved in glutamate biosynthesis, but that if the enzyme is overexpressed, it may function reversibly in intact cells.

Proc Natl Acad Sci U S A, 1990 Sep, 87(17), 6689 - 92
Molecular cloning of a gene encoding an ARS binding factor from the yeast Saccharomyces cerevisiae; Biswas EE et al.; We report the isolation of the gene for origin binding factor 1 (OBF1) from the yeast Saccharomyces cerevisiae by screening a yeast genomic DNA library in lambda gt11 with an ARS-specific oligonucleotide probe . One recombinant encoded a fusion protein of approximately 180 kDa that bound ARS-specific oligonucleotide probes in vitro . The restriction map of this gene was determined after isolation of the complete gene by screening a yeast genomic DNA library in YEp24 . Characterization of the gene for OBF1 by pulsed-field gel electrophoresis and Northern and Southern blot analyses demonstrated that (i) the gene is located in chromosome IV, (ii) the gene is a single-copy gene, (iii) the mRNA is approximately 3.8 kilobases, which could code for an approximately 130-kDa polypeptide, consistent with the reported size of OBF1 . An antibody, affinity-purified using the lysogen-encoded fusion protein, specifically detected an approximately 130-kDa polypeptide in yeast extract . The isolation of the gene for OBF1 should allow further analysis of the mechanism of action of this protein in vitro and in vivo.

Biochem Biophys Res Commun, 1990 Aug 31, 171(1), 138 - 42
In vivo expression of rat liver c-erbA beta thyroid hormone receptor in yeast (Saccharomyces cerevisiae); Lu C et al.; To study thyroid hormone receptor (TR), we developed an in vivo expression system in yeast by using a copper-responsive yeast metallothionein promoter and ubiquitin-fusion protein technology . The cDNA encoding full-length rat liver TR beta was expressed under the control of copper . The {125I}T3 binding activities to yeast extracts were significantly correlated with the added copper sulfate into the medium . Partially purified TR from the transformed yeast had a high hormone binding affinity (Kd = 0.34) for T3 and could bind thyroid hormone response element in gel retardation analysis.

Biochemistry, 1990 Aug 28, 29(34), 7911 - 7
Interaction of a Saccharomyces cerevisiae strand exchange stimulatory factor with DNA; Norris D et al.; In the preceding paper (Norris & Kolodner, 1990), we described the purification of a Mr 33,000 polypeptide which dramatically stimulated the activity of SEP1, the yeast mitotic strand exchange protein . In this paper, we characterized this new protein, which was designated SF1, in the absence of SEP1 . SF1 had a sedimentation coefficient of 1.7 S and a Stokes radius of 30 A, which was consistent with a calculated native molecular weight of 31,000, indicating that SF1 existed in solution as a monomer . Filter binding assays showed that SF1 bound preferentially to single-stranded rather than double-stranded DNA . Fluorescence spectroscopy analysis indicated that SF1 occluded approximately eight nucleotides when bound to single-stranded DNA and exhibited a dissociation constant, KD, of 2.83 x 10(-6) M . The binding of SF1 to single-stranded DNA was noncooperative and appeared to involve at least one tyrosine residue . SF1, in the absence of SEP1, stimulated the renaturation of homologous single-stranded DNA, suggesting that it might act directly in some phase of the strand exchange reaction.

Biochemistry, 1990 Aug 28, 29(34), 7903 - 11
Purification of a strand exchange stimulatory factor from Saccharomyces cerevisiae; Norris D et al.; The SEP1 strand exchange protein of Saccharomyces cerevisiae catalyzes the formation of heteroduplex DNA joints between single-strand circles and homologous linear duplexes in vitro . Previous work {Kolodner, R., Evans, D . H., & Morrison, P . T . (1987) Proc . Natl . Acad . Sci . U.S.A . 84, 5560-5564} showed that the optimal stoichiometry of SEP1 in this reaction was 1 SEP1 monomer per 12-14 nucleotides of single-stranded DNA . The work presented here describes the purification and characterization of a 33,000-dalton yeast protein that permits SEP1 to catalyze joint molecule formation at much lower stoichiometries . In the presence of this second factor, which has been designated SF1 for stimulatory factor 1, the optimal amount of SEP1 dropped to 1 SEP1 monomer per 725 nucleotides of single-stranded DNA . At this concentration of SEP1, the rate of joint molecule formation increased approximately 3-fold over that seen in the unstimulated reaction (no SF1) . Titration experiments indicated that when the concentration of SEP1 was reduced over 300-fold to 1 SEP1 molecule per 5800 nucleotides of single-stranded DNA, the stimulated reaction had the same rate and extent of joint molecule formation as the unstimulated reaction . The optimal amount of SF1 was 1 molecule of SF1 per 20 nucleotides of single-stranded DNA . Electron microscopic analysis showed that a bona fide strand exchange reaction produced the joint molecules in the stimulated reaction . The stimulated reaction had requirements that were essentially identical with those seen in the unstimulated reaction, including a lack of dependence on ATP . SF1 aggregated single-stranded and double-stranded DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 74 - 9
In vivo and in vitro analysis of structure-function relationships in ribosomal protein L25 from Saccharomyces cerevisiae; Rutgers CA et al.; We have developed a combination of in vivo and in vitro methods which allows us to determine the effect of practically every structural change, deletions as well as point mutations, on various biological functions of a ribosomal protein (r-protein) . We have used this approach to delineate the functional domains of r-protein L25 from Saccharomyces cerevisiae . By analysis of the intracellular distribution of fusion proteins carrying various portions of L25 linked to Escherichia coli beta-galactosidase we traced the nuclear localization signal(s) of L25 to the region encompassing the N-terminal 61 amino acids of the protein . On the other hand, using in vitro prepared fragments of L25 we located the domain responsible for its specific binding to 26S rRNA to the region between amino acids 61 and 135 . In order to be able to analyze the effect of mutations in L25 on ribosome biogenesis and function in vivo we constructed a mutant yeast strain in which the chromosomal L25 gene is placed under control of the inducible yeast GAL promoter . Since this strain is unable to grow on glucose as a carbon source the L25 gene must be essential for cell viability . Growth on glucose can be restored by introduction of a wild-type L25 gene on a plasmid, demonstrating that under these conditions the cells are dependent upon an extrachromosomally supplied copy of the gene.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 155 - 9
Translation initiation factor-dependent extracts from Saccharomyces cerevisiae; Altmann M et al.; Translation initiation factor 4A- and 4E-dependent extracts were developed from Saccharomyces cerevisiae and used to study factor requirements for translation of individual mRNAs in vitro . Whereas all mRNAs tested required eIF-4A, mRNAs devoid of secondary structure in their 5' untranslated region did not require exogenous eIF-4E for translation . The latter included alfalfa mosaic virus RNA4, mRNA containing the untranslated region of tobacco mosaic virus RNA and mRNA containing part of the untranslated region of poliovirus RNA . Furthermore, initiation of translation on mRNAs containing part of the untranslated region of poliovirus RNA is most likely internal.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 140 - 5
Expression of translation initiation factor 4A from yeast and mouse in Saccharomyces cerevisiae; Prat A et al.; The eukaryotic translation initiation factor 4A (eIF-4A) plays an important role in regulating initiation . To analyze its function in yeast, we carried out a mutational analysis of the TIF1 and TIF2 genes, which encode eIF-4A . Expression of these two yeast genes has also been investigated at the transcriptional level and it has been found that both are expressed in wild-type yeast cells . Analysis of the expression of eIF-4A-beta-galactosidase fusion proteins reveals that the TIF2 gene is more highly expressed than the TIF1 gene . Interestingly, the yeast eIF-4A protein shows a high degree of amino acid sequence similarity to the mouse homologue . However, we find that the mammalian factor does not support protein synthesis in yeast either in vivo or in vitro.

FEBS Lett, 1990 Aug 20, 269(1), 53 - 9
The Saccharomyces cerevisiae CDC25 gene product is a 180 kDa polypeptide and is associated with a membrane fraction; Garreau H et al.; In the yeast Saccharomyces cerevisiae, the CDC25 gene product is supposed to interact with ras proteins and adenylate cyclase for progression through the cell division cycle . To identify the CDC25 gene product, we raised antibodies against two hybrid proteins, encoded by in-frame fusions between the E . coli lacZ gene and two different parts of the CDC25 gene . By protein immuno-blotting, we were able to identify the CDC25 gene product as a 180 kDa polypeptide, which we named p180CDC25 . It was detected only when the CDC25 gene was overexpressed in a proteases-deficient yeast strain . Subcellular fractionation experiments showed that p180CDC25, as well as ras proteins, is attached to the membrane, even after treatments which release peripheral membrane proteins.

Eur J Biochem, 1990 Aug 17, 191(3), 769 - 74
Molecular cloning of the gene for the E1 alpha subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae; Steensma HY et al.; The E1 alpha and E1 beta subunits of the pyruvate dehydrogenase complex from the yeast Saccharomyces cerevisiae were purified . Antibodies raised against these subunits were used to clone the corresponding genes from a genomic yeast DNA library in the expression vector lambda gt11 . The gene encoding the E1 alpha subunit was unique and localized on a 1.7-kb HindIII fragment from chromosome V . The identify of the gene was confirmed in two ways . (a) Expression of the gene in Escherichia coli produced a protein that reacted with the anti-E1 alpha serum . (b) Gene replacement at the 1.7-kb HindIII fragment abolished both pyruvate dehydrogenase activity and the production of proteins reacting with anti-E1 alpha serum in haploid cells . In addition, the 1.7-kb HindIII fragment hybridized to a set of oligonucleotides derived from amino acid sequences from the N-terminal and central regions of the human E1 alpha peptide . We propose to call the gene encoding the E1 alpha subunit of the yeast pyruvate dehydrogenase complex PDA1 . Screening of the lambda gt11 library using the anti-E1 beta serum resulted in the reisolation of the RAP1 gene, which was located on chromosome XIV.

J Biol Chem, 1990 Aug 15, 265(23), 13888 - 9
Human recombinant factor XIII from Saccharomyces cerevisiae . Crystallization and preliminary x-ray data; Bishop PD et al.; Crystals of human recombinant factor XIII from the yeast Saccharomyces cerevisiae have been grown from solutions of ammonium sulfate at pH 5.8 . The crystals are orthorhombic, with space group P2(1)2(1)2 and unit cell dimensions gamma a = 101.2, b = 182.7, and c = 93.4 A . The asymmetric unit consists of one a2 dimer of molecular mass 166 kDa . A 3.5-A resolution data set for the native protein has been collected . Practical resolution limits for these crystals have not been determined, but reflections have been observed to a Bragg spacing of 2.8-A resolution.

J Biol Chem, 1990 Aug 15, 265(23), 13871 - 7
Purification and characterization of glycogen synthase from a glycogen-deficient strain of Saccharomyces cerevisiae; Peng ZY et al.; Chromatography of wild-type yeast extracts on DEAE-cellulose columns resolves two populations of glycogen synthase I (glucose-6-P-independent) and D (glucose-6-P-dependent) (Huang, K . P., Cabib, E . (1974) J . Biol . Chem . 249, 3851-3857) . Extracts from a glycogen-deficient mutant strain, 22R1 (glc7), yielded only the D form of glycogen synthase . Glycogen synthase D purified from either wild-type yeast or from this glycogen-deficient mutant displayed two polypeptides with molecular masses of 76 and 83 kDa on sodium dodecyl sulfate-gel electrophoresis in a protein ratio of about 4:1 . Phosphate analysis showed that glycogen synthase D from either strain of yeast contained approximately 3 phosphates/subunit . The 76- and 83-kDa bands of the mutant strain copurified through a variety of procedures including nondenaturing gel electrophoresis . These two polypeptides showed immunological cross-reactivity and similar peptide maps indicating that they are structurally related . The relative amounts of these two forms remained constant during purification and storage of the enzyme and after treatment with cAMP-dependent protein kinase or with protein phosphatases . The two polypeptides were phosphorylated to similar extent in vitro by the catalytic subunit of mammalian cyclic AMP-dependent protein kinase . Phosphorylation of the enzyme in the presence of labeled ATP followed by tryptic digestion and reversed phase high performance liquid chromatography yielded two labeled peptides from each of the 76- and 83-kDa subunits . Treatment of wild-type yeast with Li+ increased the glycogen synthase activity, measured in the absence of glucose-6-P, by approximately 2-fold, whereas similar treatment of the glc7 mutant had no effect . The results of this study indicate that the GLC7 gene is involved in a pathway that regulates the phosphorylation state of glycogen synthase.

Biochemistry, 1990 Aug 14, 29(32), 7531 - 8
Isopentenyldiphosphate:dimethylallyldiphosphate isomerase: construction of a high-level heterologous expression system for the gene from Saccharomyces cerevisiae and identification of an active-site nucleophile; Street IP et al.; Isopentenyldiphosphate:dimethylallyldiphosphate isomerase (IPP isomerase) is an enzyme in isoprene metabolism which catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks for the pathway . The gene encoding IPP isomerase has recently been isolated from Saccharomyces cerevisiae {Anderson, M . S., Muehlbacher, M., Street, I.P., Proffitt, J., & Poulter, C . D . (1989) J . Biol . Chem . 264, 19169-19175} . A heterologous expression system was constructed for the gene and used to overexpress IPP isomerase in Escherichia coli . In transformants carrying the expression vector, IPP isomerase activity was increased by over 100,000-fold relative to that of the untransformed host strain . The overexpressed enzyme constitutes 30-35% of the total soluble cell protein and can be purified to homogeneity in two steps . Recombinant IPP isomerase was indistinguishable from that purified from yeast . 3-(Fluoromethyl)-3-butenyl diphosphate (FIPP) is a specific active-site-directed inhibitor of IPP isomerase from Claviceps purpurea {Muehlbacher, M., & Poulter, C . D . (1988) Biochemistry 27, 7315-7328} . Inactivation of yeast IPP isomerase by FIPP was active-site-directed, and inhibition resulted in formation of a stoichiometric enzyme-inhibitor complex . The site of covalent attachment in the enzyme-inhibitor complex was determined by inactivating IPP isomerase with {4-3H}FIPP, followed by digestion of the labeled enzyme with trypsin and purification of the resulting radioactive peptides by reversed-phase high-performance liquid chromatography . The primary site of attachment was Cys-139.

Nucleic Acids Res, 1990 Aug 11, 18(15), 4409 - 16
The 26S rRNA binding ribosomal protein equivalent to bacterial protein L11 is encoded by unspliced duplicated genes in Saccharomyces cerevisiae; Pucciarelli MG et al.; Transformant phages expressing L15, a yeast ribosomal protein which binds to 26S rRNA and interacts with the acidic ribosomal proteins, were isolated by screening a yeast cDNA expression library in lambda gt11 with specific monoclonal antibodies . Using yeast DNA HindIII fragments that hybridize with the cDNA insert from the L15-expressing clones, minilibraries were prepared in pUC18, which were afterward screened with the same cDNA probe . In this way, plasmids carrying two different types of genomic DNA inserts were obtained . The inserts were subcloned and sequenced and we found a similar coding sequence in both cases flanked by 5' and 3' regions with very low homology . Sequences homologous to the consensus TUF-binding UAS boxes are present in the 5' flanking regions of both genes . Southern analysis revealed the presence of two copies of the L15 gene in the Saccharomyces cerevisiae genome, which are located in different chromosomes . The encoded amino acid sequence corresponds, as expected, to protein L15 and shows a high similarity to bacterial ribosomal protein L11.

J Biol Chem, 1990 Aug 5, 265(22), 13391 - 9
Essential role for induced Ca2+ influx followed by {Ca2+}i rise in maintaining viability of yeast cells late in the mating pheromone response pathway . A study of {Ca2+}i in single Saccharomyces cerevisiae cells with imaging of fura-2; Iida H et al.; We established an experimental system for measuring the cytosolic-free Ca2+ concentration ({Ca2+}i) in individual Saccharomyces cerevisiae cells using fura-2 as a Ca2(+)-specific probe in conjunction with digital image processing and examined changes in {Ca2+}i in response to alpha-factor in single cells of a mating type . The addition of alpha-factor to a cells raised {Ca2+}i to several hundred nanomolar in the cells from a basal level of approximately 100 nM, simultaneous with the induction of Ca2+ influx . When the cells were incubated with alpha-factor in a Ca2(+)-deficient medium, Ca2+ influx was greatly reduced, and the rise in {Ca2+}i was not detected . This indicates that the alpha-factor-induced rise in {Ca2+}i is generated by Ca2+ influx through the plasma membrane and not by release from internal stores . In the Ca2(+)-deficient medium, a cells died specifically after they had changed into cells with one projection on the cell surface . This indicates that the rise in {Ca2+}i is essential for the late response to alpha-factor . The duration of Ca2+ requirement for maintaining viability was limited to this stage, and the earlier and later stages were not affected by Ca2+ deprivation . Mating between a and alpha mating type cells was impaired in this medium due to cell death at and before the stage of conjugation . These findings are the first evidence for an essential role for mobilized Ca2+ in the yeast life cycle.

J Biol Chem, 1990 Aug 5, 265(22), 13370 - 8
Structural and functional studies of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase produced in Escherichia coli . Evidence for an acyl-enzyme intermediate; Rudnick DA et al.; Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase has been efficiently expressed in Escherichia coli and subsequently purified to homogeneity using phosphocellulose chromatography . The interactions between apoenzyme and its acyl-CoA and peptide ligands were examined by an isoelectric focusing gel shift assay, circular dichroism, and fluorescence spectroscopy, and a continuous assay of enzyme activity which measures the release of CoA from acyl-CoA using the thiol-specific reagent 5-5'-dithiobis-2-nitrobenzoate . Addition of myristoyl-CoA (without a substrate peptide) results in the formation of a high affinity reaction intermediate which can be operationally defined by the appearance of a more acidic enzyme isoform and by quenching of the tryptophan emission with a maximal difference at 340 nm . Circular dichroism spectroscopy indicates that these changes are accompanied by minimal changes in the enzyme's secondary structure . Incubation of purified NMT with {1-14C} myristoyl-CoA, followed by chymotryptic digestion, denaturing polyacrylamide gel electrophoresis, and treatment with hydroxylamine yielded results that are highly suggestive of a covalent ester-linked acyl-enzyme complex . Edman degradation of chymotryptic peptides has narrowed the site of interaction to a domain spanning Arg42 to Thr220 of the 455 amino acid acyltransferase . An octapeptide containing Gly but not Ala at position 1 is able to reverse the change in pI and reduce the quenching almost entirely . These data suggest a preferred order or ping-pong reaction mechanism with the acyl-CoA substrate binding event occurring first . They also indicate that Gly1 is absolutely necessary for the reaction to proceed forward from the acyl-enzyme reaction intermediate.

Mol Cell Biol, 1990 Aug, 10(8), 4379 - 83
Construction of an opal suppressor by oligonucleotide-directed mutagenesis of a Saccharomyces cerevisiae tRNA(Trp) gene; Atkin AL et al.; In vitro mutagenesis was used to create putative opal suppressor alleles of a tRNA(Trp) gene of Saccharomyces cerevisiae . The construct with the requisite anticodon change did not result in an active suppressor, whereas when a second change was introduced into the portion of the gene encoding the intron, an active and specific opal suppressor was produced . We propose that the secondary structure of transcripts from the first mutant may prevent efficient pre-tRNA processing, whereas normal processing occurs with the double mutant.

Genome, 1990 Aug, 33(4), 596 - 603
Branched chain amino acid regulation of the ILV2 locus in Saccharomyces cerevisiae; Xiao W et al.; Mutant regulatory loci of the branched pathway for the biosynthesis of isoleucine-valine and leucine were identified with the unusual phenotype of an amino acid dependent auxotrophy . Two mutant loci, bcs1 and bcs2, conferred branched chain amino acid sensitivity and showed independent segregation . Linkage studies defined bcs1 as a cis-acting regulatory site of ILV2 (SMR1) . ILV2 upstream deletion analyses and high-copy transformation of the positive regulatory locus LEU3 ruled out the possibility of LEU3 protein binding palindromes mediating the branched chain amino acid dependent auxotrophy . In the presence of leucine and valine, the general amino acid control system (GCN4) was epistatic to bcs1 and bcs2, and under nonstarvation conditions GCN4 strains showed an increased acetolactate synthase activity over gcn4 strains . Thus in addition to general regulation of ILV2, GCN4 functions in basal level expression when the locus is subject to specific repression by pathway end product.

Curr Genet, 1990 Aug, 18(2), 97 - 103
The influence of GAP promoter variants on hirudin production, average plasmid copy number and cell growth in Saccharomyces cerevisiae; Janes M et al.; The yeast Saccharomyces cerevisiae has been engineered to synthesize and secrete desulfato-hirudin (hirudin), a thrombin inhibitor from the leech Hirudo medicinalis . The synthetic gene coding for hirudin was expressed constitutively under the control of four size-variants of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) and cloned into a 2 mu based multicopy yeast vector . The constitutive action of the four promoter variants was confirmed by demonstrating that the expression and secretion of hirudin is growth-related . The different efficiencies of the promoter variants not only affected hirudin expression but also led to changes in several cellular parameters, such as cell growth, average plasmid copy number and plasmid stability . The observed changes show that yeast cells establish a specific equilibrium for each promoter variant . We conclude, that the adjustment of cellular parameters in response to the expression levels of a heterologous protein is regulated by two counteracting selective forces: (1) the need for complementation of the auxotrophic host marker by the plasmid-encoded selection gene which, in the case of dLEU2, requires several plasmid copies; and (2) a selective advantage of cells with a lower copy number enabling them to escape the burden of heterologous protein production.

Photochem Photobiol, 1990 Aug, 52(2), 395 - 400
Characterization of RAD4 gene required for ultraviolet-induced excision repair of Saccharomyces cerevisiae propagated in Escherichia coli without inactivation; Choi IS et al.; The previously isolated RAD4 gene designated as pPC1 from the genomic library of Saccharomyces cerevisiae (Yoon et al., 1985, Korean J . Genetics 7, 97-104) appeared to propagate in Escherichia coli and yet retained its complementing activity to rad4 mutants without inactivation . The subcloned RAD4 gene was found to be localized within a 2.5 kb DNA fragment flanking Bg1II and BamHI sites in the insert DNA, and was shown to have the same restriction map as a yeast chromosomal DNA, as determined by Southern hybridization . Tetrad analysis and pulse-field chromosome mapping have revealed that the cloned RAD4 gene can be mapped and integrated into the yeast chromosome V, the actual site of this gene . DNA-tRNA hybridization has shown that the isolated RAD4 gene did not contain a suppressor tRNA gene . These results have indicated that the pPC1 is a functional RAD4 gene playing a unique role involved in the nucleotide excision repair of yeast without any genetic change during amplification in E . coli.

Chromosoma, 1990 Aug, 99(4), 281 - 8
Heterogeneity and maintenance of centromere plasmid copy number in Saccharomyces cerevisiae; Resnick MA et al.; We developed a novel approach to quantitate the heterogeneity of centromere number in yeast, and the cellular capacity for excess centromeres . Small circular plasmids were constructed to contain the CUP1 metallothionein gene . ARS1 (autonomously replicating sequence) and a conditionally functional centromere (GAL1-GAL10 promoter controlled centromere) . The CUP1 gene provided a gene dosage marker, and therefore a genetic determinant of plasmid copy number . Growth of cells on glucose is permissive for centromere function, while growth on galactose renders the centromere nonfunctional and the plasmids are segregated in an asymmetric fashion . We identified "lines" of cells containing increased numbers of plasmids after transformation . Cell lines containing as many as five to ten active centromeres are stably maintained in the absence of genetic selection . Thus haploid yeast cells can tolerate a 50% increase in their centromere number without affecting progression through the cell cycle . This system provides the opportunity to address issues of specific cellular controls on centromere copy number.

Genetics, 1990 Aug, 125(4), 763 - 74
Mitotic transmission of artificial chromosomes in cdc mutants of the yeast, Saccharomyces cerevisiae; Palmer RE et al.; In the yeast, Saccharomyces cerevisiae, cell division cycle (CDC) genes have been identified whose products are required for the execution of different steps in the cell cycle . In this study, the fidelity of transmission of a 14-kb circular minichromosome and a 155-kb linear chromosome fragment was examined in cell divisions where specific CDC products were temporarily inactivated with either inhibitors, or temperature sensitive mutations in the appropriate CDC gene . All of the cdc mutants previously shown to induce loss of endogenous linear chromosomes also induced loss of a circular minichromosome and a large linear chromosome fragment in our study (either 1:0 or 2:0 loss events) . Therefore, the efficient transmission of these artificial chromosomes depends upon the same trans factors that are required for the efficient transmission of endogenous chromosomes . In a subset of cdc mutants (cdc6, cdc7 and cdc16), the rate of minichromosome loss was significantly greater than the rate of loss of the linear chromosome fragment, suggesting that a structural feature of the minichromosome (nucleotide content, length or topology) makes the minichromosome hypersensitive to the level of function of these CDC gene products . In another subset of cdc mutants (cdc7 and cdc17), the relative rate of 1:0 events to 2:0 events differed for the minichromosome and chromosome fragment, suggesting that the type of chromosome loss event observed in these mutants was dependent upon chromosome structure . Finally, we show that 2:0 events for the minichromosome can occur by both a RAD52 dependent and RAD52 independent mechanism . These results are discussed in the context of the molecular functions of the CDC products.

Genetics, 1990 Aug, 125(4), 739 - 52
Isolation and characterization of PEP5, a gene essential for vacuolar biogenesis in Saccharomyces cerevisiae; Woolford CA et al.; pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases . The PEP5 gene was isolated from a genomic DNA library by complementation of the pep5-8 mutation . Deletion analysis localized the complementing activity to a 3.3-kb DNA fragment . DNA sequence analysis of the PEP5 gene revealed an open reading frame of 1029 codons with a calculated molecular mass for the encoded protein of 117,403 D . Deletion/disruption of the PEP5 gene did not kill the cells . The resulting strains grow very slowly at 37 degrees . The disruption mutant showed greatly decreased activities of all vacuolar hydrolases examined, including PrA, PrB, CpY, and the repressible alkaline phosphatase . Apparently normal precursors forms of the proteases accumulated in pep5 mutants, as did novel forms of PrB antigen . Antibodies raised to a fusion protein that contained almost half of the PEP5 open reading frame allowed detection by immunoblot of a protein of relative molecular mass 107 kD in extracts prepared from wild-type cells . Cell fractionation showed the PEP5 gene product is enriched in the vacuolar fraction and appears to be a peripheral vacuolar membrane protein.

Genetics, 1990 Aug, 125(4), 729 - 38
"Alternative self-diploidization" or "ASD" homothallism in Saccharomyces cerevisiae: isolation of a mutant, nuclear-cytoplasmic interaction and endomitotic diploidization; Ono B et al.; A mutant of Saccharomyces cerevisiae representing a novel life cycle, named "alternative self-diploidization" or "ASD" homothallism, was obtained fortuitously . In this life cycle, MAT alpha (or MATa) haplophase and MAT alpha/MAT alpha (or MATa/MATa) diplophase alternate . Germinated cells are haploid and mating . They soon become nonmating and sporogenous as they vegetatively grow . They sooner or later diploidize presumably via endomitosis . The diploid cells haploidize via normal meiosis . A single recessive nuclear mutation, named asd 1-1, is responsible for "ASD" homothallism . In the rho 0 cytoplasm, asd 1-1 cells mate even if at a low efficiency and fail to diploidize . Since pet mutations do not have such effects, we conclude that a certain mitochondrial function other than respiration is required for manifestation of "ASD" homothallism . That is, "ASD" homothallism is the result of some sort of nuclear-cytoplasmic interaction.

Proc Natl Acad Sci U S A, 1990 Aug, 87(16), 6286 - 90
Genetic evidence for an interaction between SIR3 and histone H4 in the repression of the silent mating loci in Saccharomyces cerevisiae; Johnson LM et al.; Repression of transcription from the silent mating loci (HML alpha and HMRa) is essential for mating ability in Saccharomyces cerevisiae . This silencing is known to require at least five proteins (SIR1, SIR2, SIR3, SIR4, and histone H4) and is accompanied by a change in chromatin structure . We show here that four positions of histone H4 (N-terminal residues 16, 17, 18, and 19) are crucial to silencing . HML alpha and HMRa are efficiently repressed when these positions are occupied by basic amino acids but are derepressed when substituted with glycine . These results suggest that acetylation of Lys-16 would lead to derepression of the silent mating loci . Three strong extragenic suppressors of the latter H4 mutations were isolated and determined to be located in SIR3 . These suppressors allow high mating efficiencies in cells expressing either wild-type H4 or H4 containing single amino acid substitutions . They did not allow efficient mating in a strain that contained an H4 N-terminal deletion . These results indicate that the SIR3 mutations do not bypass the requirement for the H4 N terminus but, rather, allow repression in the presence of a less than optimal H4 N terminus . This provides a link between one of the SIR proteins and a component of chromatin.

Mutat Res, 1990 Aug, 231(2), 177 - 86
High levels of chromosome instability in polyploids of Saccharomyces cerevisiae; Mayer VW et al.; The yeast Saccharomyces cerevisiae was used to study the genetic consequences of polyploidy in a unicellular organism . Isogenic diploid (2N), triploid (3N) and tetraploid (4N) strains with a genetically marked chromosome VII (cyh2-leu1-CEN7-ade6) were constructed and were used to follow the loss of one, two or three chromosome VII's during mitosis . We found that as ploidy increased, the frequency of loss of a single chromosome VII increased: Loss of one copy of chromosome VII occurred at a rate nearly 30-fold higher in triploids and approximately 1000-fold higher in tetraploids than in the diploid . Loss of two or three copies occurred at an even greater frequency . These findings suggest either that aneuploidy (3N-1, 3N-2, 4N-1, 4N-2, 4N-3) increases genome instability or that multiple chromosome loss events occur at high frequency . Polyploidy appears to dramatically increase chromosome loss, presumably due to the inability of the cell to undergo proper chromosome segregation . The biological significance and possible causes for the instability of polyploidy in unicellular organisms such as yeast are discussed.

Biochim Biophys Acta, 1990 Aug 1, 1040(1), 95 - 101
Non-sterol structural probes of the lanosterol 14 alpha-demethylase from Saccharomyces cerevisiae; Wright GD et al.; A number of non-sterol iron-liganding molecules were used to probe the active site of the lanosterol 14 alpha-demethylase from Saccharomyces cerevisiae . Simple bi- and tricyclic aromatic amines were found to exhibit Type II binding spectra with the demethylase . Stereochemical and positional effects appear to play critical roles in the binding of these compounds to the demethylase . These compounds have been used to generate additional active-site structural information on this enzyme, currently a target for the development of new antifungal agents.

Biochim Biophys Acta, 1990 Aug 1, 1040(1), 71 - 6
Reactive sulfhydryl groups in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase; Cardemil E et al.; Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) is inactivated by several thiol- and vicinal dithiol-specific reagents . Titration experiments of the enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) show the presence of reactive monothiol and vicinal dithiol groups, whose modifications lead to enzyme inactivation . The enzyme is also inactivated by N-(1-pyrenyl)iodoacetamide (PyrIAM), with a binding stoichiometry of approx . 2 mol per mol of enzyme subunit . A high level of pyrene excimer fluorescence is detected on the labeled enzyme, thus implying the reaction of the reagent with two spatially close sulfhydryl groups in the protein . The carboxykinase is not completely inactivated by different vicinal dithiol-specific reagents, thus implying a catalytically non-essential character for these groups . From substrate protection experiments of the enzyme inactivation by DTNB, PyrIAM and vicinal dithiol-specific reagents, it is concluded that the loss of enzyme activity is caused by the modification of both thiol and vicinal dithiol groups in the substrate binding region.

Mutat Res, 1990 Aug, 241(4), 369 - 77
Mutagenic and recombinogenic consequences of DNA-repair inhibition during treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea in Saccharomyces cerevisiae; Ferguson LR; The yeast Saccharomyces cerevisiae has been used as a model system to explore whether the clinical combination of the antitumour agent BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) with DNA-repair inhibitors would affect the drug's mutagenic or recombinogenic potential . Preliminary experiments suggested that mitotic crossing-over and other mutagenic events are controlled in a separate fashion . BCNU was more toxic in yeast derivatives with specific defects in any of the three recognised major DNA repair pathways than in the DNA-repair-proficient parent strain . However, in a diploid homozygous for rad18, BCNU showed enhanced mutagenic and recombinogenic potential . Both of these effects were reduced in a comparable rad3 strain, and mitotic crossing-over but not other types of mutagenic event eliminated in the rad52 derivative . Experiments were performed in the presence of three DNA-repair inhibitors which are currently in clinical use and which might be available for combination chemotherapy . Hydroxyurea and amsacrine themselves caused mitotic crossing-over and other events, and did not reduce mutagenic or recombinogenic potential of the BCNU . Hydroxyurea actually decreased toxicity of the BCNU . Caffeine, however, showed some effect in enhancing toxicity and decreasing both mutagenic and recombinogenic potential of the drug . Development of more specific repair inhibitors related to amsacrine or to caffeine, using these repair-deficient strains as model systems, might lead to an enhanced clinical potential of this bisalkylating drug and related compounds.

J Bacteriol, 1990 Aug, 172(8), 4352 - 8
Thermotolerance is independent of induction of the full spectrum of heat shock proteins and of cell cycle blockage in the yeast Saccharomyces cerevisiae; Barnes CA et al.; Cells of the yeast Saccharomyces cerevisiae are known to acquire thermotolerance in response to the stresses of starvation or heat shock . We show here through the use of cell cycle inhibitors that blockage of yeast cells in the G1, S, or G2 phases of the mitotic cell cycle is not a stress that induces thermotolerance; arrested cells remained as sensitive to thermal killing as proliferating cells . These G1- or S-phase-arrested cells were unimpaired in the acquisition of thermotolerance when subjected to a mild heat shock by incubation at 37 degrees C . One cell cycle inhibitor, o-phenanthroline, did in fact cause cells to become thermotolerant but without induction of the characteristic pattern of heat shock proteins . Thermal induction of heat shock protein synthesis was unaffected; the o-phenanthroline-treated cells could still synthesize heat shock proteins upon transfer to 37 degrees C . Use of a novel mutant conditionally defective only for the resumption of proliferation from stationary phase (M . A . Drebot, G . C . Johnston, and R . A . Singer, Proc . Natl . Acad . Sci . USA 84:7948-7952, 1987) indicated that o-phenanthroline inhibition produces a stationary-phase arrest, a finding which is consistent with the increased thermotolerance and regulated cessation of proliferation exhibited by the inhibited cells . These findings show that the acquired thermotolerance of cells is unrelated to blockage of the mitotic cell cycle or to the rapid synthesis of the characteristic spectrum of heat shock proteins.

Mol Cell Biol, 1990 Aug, 10(8), 4130 - 8
Structure and molecular analysis of RGR1, a gene required for glucose repression of Saccharomyces cerevisiae; Sakai A et al.; An RGR1 gene product is required to repress expression of glucose-regulated genes in Saccharomyces cerevisiae . The abnormal morphology of rgr1 cells was studied . Scanning and transmission electron microscopic observations revealed that the cell wall of the daughter cell remained attached to that of mother cell . We cloned the RGR1 gene by complementation and showed that the cloned DNA was tightly linked to the chromosomal RGR1 locus . The cloned RGR1 gene suppressed all of the phenotypes caused by the mutation and encoded a 3.6-kilobase poly(A)+ RNA . The RGR1 gene is located on chromosome XII, as determined by pulsed-field gel electrophoresis, and we mapped rgr1 between gal2 and pep3 by genetic analysis . rgr1 was shown to be a new locus . We also determined the nucleotide sequence of RGR1, which was predicted to encode a 123-kilodalton protein . The null mutation resulted in lethality, indicating that the RGR1 gene is essential for growth . On the other hand, a carboxy-terminal deletion of the gene caused phenotypes similar to but more severe than those caused by the original mutation . The amount of reserve carbohydrates was reduced in rgr1 cells . Possible functions of the RGR1 product are discussed.

Mol Cell Biol, 1990 Aug, 10(8), 4089 - 99
Isolation, sequencing, and disruption of the yeast CKA2 gene: casein kinase II is essential for viability in Saccharomyces cerevisiae; Padmanabha R et al.; Casein kinase II of Saccharomyces cerevisiae contains two distinct catalytic subunits, alpha and alpha', which are encoded by the CKA1 and CKA2 genes, respectively . Null mutations in the CKA1 gene do not confer a detectable phenotype (J . L.-P . Chen-Wu, R . Padmanabha, and C . V . C . Glover, Mol . Cell . Biol . 8:4981-4990, 1988), presumably because of the presence of the CKA2 gene . We report here the cloning, sequencing, and disruption of the CKA2 gene . The alpha' subunit encoded by the CKA2 gene is 60% identical to the CKA1-encoded alpha subunit and 55% identical to the Drosophila alpha subunit (A . Saxena, R . Padmanabha, and C . V . C . Glover, Mol . Cell . Biol . 7:3409-3417, 1987) . Deletions of the CKA2 gene were constructed by gene replacement techniques . Haploid cells in which the CKA2 gene alone is disrupted show no detectable phenotype, but haploid cells carrying disruptions in both the CKA1 and CKA2 genes are inviable . Cells in which casein kinase II activity is depleted increase substantially in size prior to growth arrest, and a significant fraction of the arrested cells exhibit a pseudomycelial morphology . Disruption of the activity also results in flocculation . Yeast strains lacking both endogenous catalytic subunit genes can be rescued by expression of the alpha and beta subunits of Drosophila casein kinase II or by expression of the Drosophila alpha subunit alone, suggesting that casein kinase II function has been conserved through evolution.

Mol Cell Biol, 1990 Aug, 10(8), 3873 - 83
Adenylate cyclase in Saccharomyces cerevisiae is a peripheral membrane protein; Mitts MR et al.; The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cyclic AMP (cAMP) from ATP, and two RAS polypeptides, which mediate stimulation of cAMP synthesis of guanine nucleotides . By analogy to the mammalian enzyme, models of yeast adenylate cyclase have depicted the enzyme as a membrane protein . We have concluded that adenylate cyclase is only peripherally bound to the yeast membrane, based on the following criteria: (i) substantial activity was found in cytoplasmic fractions; (ii) activity was released from membranes by the addition of 0.5 M NaCl; (iii) in the presence of 0.5 M NaCl, activity in detergent extracts had hydrodynamic properties identical to those of cytosolic or NaCl-extracted enzyme; (iv) antibodies to yeast adenylate cyclase identified a full-length adenylate cyclase in both membrane and cytosol fractions; and (v) activity from both cytosolic fractions and NaCl extracts could be functionally reconstituted into membranes lacking adenylate cyclase activity . The binding of adenylate cyclase to the membrane may have regulatory significance; the fraction of activity associated with the membrane increased as cultures approached stationary phase . In addition, binding of adenylate cyclase to membranes appeared to be inhibited by cAMP . These results indicate the existence of a protein anchoring adenylate cyclase to the membrane . The identity of this protein remains unknown.

Mol Gen Genet, 1990 Aug, 223(1), 97 - 106
HSP12, a new small heat shock gene of Saccharomyces cerevisiae: analysis of structure, regulation and function; Praekelt UM et al.; We have isolated a new small heat shock gene, HSP12, from Saccharomyces cerevisiae . It encodes a polypeptide of predicted Mr 12 kDa, with structural similarity to other small heat shock proteins . HSP12 gene expression is induced several hundred-fold by heat shock and on entry into stationary phase . HSP12 mRNA is undetectable during exponential growth in rich medium, but low levels are present when cells are grown in minimal medium . Analysis of HSP12 expression in mutants affected in cAMP-dependent protein phosphorylation suggests that the gene is regulated by cAMP as well as heat shock . A disruption of the HSP12 coding region results in the loss of an abundant 14.4 kDa protein present in heat shocked and stationary phase cells . It also leads to the induction of the heat shock response under conditions normally associated with low-level HSP12 expression . The HSP12 disruption has no observable effect on growth at various temperatures, nor on the ability to acquire thermotolerance.

Mol Cell Biol, 1990 Aug, 10(8), 4303 - 13
IRA2, a second gene of Saccharomyces cerevisiae that encodes a protein with a domain homologous to mammalian ras GTPase-activating protein; Tanaka K et al.; The IRA1 gene is a negative regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae . To identify other genes involved in this pathway, we screened yeast genomic DNA libraries for genes that can suppress the heat shock sensitivity of the ira1 mutation on a multicopy vector . We identified IRA2, encoding a protein of 3,079 amino acids, that is 45% identical to the IRA1 protein . The region homologous between the IRA1 protein and ras GTPase-activating protein is also conserved in IRA2 . IRA2 maps 11 centimorgans distal to the arg1 locus on the left arm of chromosome XV and was found to be allelic to glc4 . Disruption of the IRA2 gene resulted in (i) increased sensitivity to heat shock and nitrogen starvation, (ii) sporulation defects, and (iii) suppression of the lethality of the cdc25 mutant . Analysis of disruption mutants of IRA1 and IRA2 indicated that IRA1 and IRA2 proteins additively regulate the RAS-cyclic AMP pathway in a negative fashion . Expression of the IRA2 domain homologous with GAP is sufficient for complementation of the heat shock sensitivity of ira2, suggesting that IRA down regulates RAS activity by stimulating the GTPase activity of RAS proteins.

Protein Eng, 1990 Aug, 3(8), 739 - 44
Expression of human asparagine synthetase in Saccharomyces cerevisiae; Van Heeke G et al.; Human asparagine synthetase was expressed in the yeast Saccharomyces cerevisiae . The identity of the expressed protein was confirmed by immunoblotting and in vitro enzymatic activity . The recombinant enzyme was shown to have both the ammonia- and glutamine-dependent asparagine synthetase activity in vitro . In contrast to overproduction in Escherichia coli, the expressed protein was found to be soluble in the yeast cell . Furthermore, expression in yeast made it possible to isolate non-degraded human asparagine synthetase which had also the N-terminal methionine correctly processed . The yeast expression plasmid was constructed for optimal production of the recombinant enzyme . In addition, unique restriction enzyme sites that bracket the first five codons of the human asparagine synthetase gene were introduced . This will allow the use of oligonucleotide cassette mutagenesis to investigate the role of the N-terminal amino acids in asparagine synthetase enzymatic activity.

Agric Biol Chem, 1990 Aug, 54(8), 2023 - 8
Quantification of physical and cyto-physiological conditions for the electrofusion of Saccharomyces cerevisiae; Noda K et al.; Various conditions for obtaining hybrids of the auxotrophic mutants SH1509 and SH1512 of Saccharomyces cerevisiae by electrofusion were investigated . An AC field of 400 Vp/cm and a DC field of 2 square pulses (7 kV/cm; 60 microsec each) at an interval of 0.5 sec were effective . Treatment with 0.2 (SH1509) or 1.0 mg/ml (SH1512) Zymolyase for 1 or 1.5 hr was essential . As to the molarity of the osmotic stabilizer (sorbitol), the hybrid yield peaked at 0.6 M . The presence of CaCl2 (up to 0.4 mM) or 0.1 mM CaCl2 with 0.1 mM MgCl2 enhanced the yield . The temperature of the spheroplast suspension during pulsations also affected the yield, the most suitable temperature being 28 degrees C.

Agric Biol Chem, 1990 Aug, 54(8), 2009 - 15
The enzymatic and molecular characteristics of Saccharomycopsis alpha-amylase secreted from Saccharomyces cerevisiae; Matsui I et al.; The Saccharomycopsis fibuligera alpha-amylase (Sfamy) gene was expressed in Saccharomyces cerevisiae . The highest productivity of Sfamy was 70 mg per liter of culture broth . We purified Sfamy from the culture broth and identified the NH2 terminal primary sequence . This sequence suggests that the Sfamy gene product is synthesized as a pre-pro-precursor, and the pro-sequence is cleaved after a Lys-Arg sequence with the calpain-like endopeptidase encode by the KEX2 gene, resulting in mature Sfamy protein composed of 468 amino acids . Furthermore, the enzyme Sfamy is a glycoprotein in which one N-linked sugar chain containing mannose residues is attached to the Asn residue at the 198 position . The Km and kcat values were 1.1 x 10(-4) M and 1.4 x 10(2) sec-1, respectively, using amylose (the degree of polymerization n = 18) as a substrate . Moreover, the secondary structure, the location of the secondary elements including alpha-helix, beta-sheet, and loop, and tertiary structure were predicted theoretically on the basis of the molecular structure of Aspergillus oryzae alpha-amylase . Taka-amylase A (TAA) . These results indicate that Sfamy protein is composed of main (M) and C-terminal (C) domains . The molecular structure of M domain closely resembles that of TAA, but the C domain appears to be more compact than that of TAA because of deletions at three regions forming turns and one region forming alpha-helix.

Biochem Biophys Res Commun, 1990 Jul 31, 170(2), 470 - 6
Strain-specific lethal effect of the adenovirus E1a protein on Saccharomyces cerevisiae; Wada T et al.; Various adenovirus E1a proteins, including 13S protein, 12S protein and three other derivatives of 13S protein with deletions were expressed in Saccharomyces cerevisiae . Both the C-terminal 67 residues and the 13S unique domain are required for the nuclear targeting in yeast . The N-terminus containing multiple functional domains appears to be involved in the G1 arrest of diploid yeast and two other regions, the region containing amino acid residues between 122 and 139, and the 67 residues of the C-terminus are required for the lethal effect on haploid yeast . The latter effect, however, is dependent on strains . Thus, the yeast system may be utilized for functional dissection of E1a protein by further analyzing metabolic consequences.

J Biol Chem, 1990 Jul 15, 265(20), 11576 - 80
Model peptides reveal specificity of N alpha-acetyltransferase from Saccharomyces cerevisiae; Lee FJ et al.; N alpha-Acetylation is a major co-translational modification occurring at the alpha-NH2 group of eukaryotic cytosolic proteins . In order to understand better the specificity of N alpha-acetyltransferase, we used the purified enzyme from yeast (Lee, F.-J . S., Lin, L.-W., and Smith J . A . (1988) J . Biol . Chem . 263, 14948-14955) and synthetic peptides mimicking the NH2 terminus of yeast and human proteins . Alcohol dehydrogenase I-(1-24) and 8 of the 19 synthetic analogues with substitutions at the NH2-terminal residue were N alpha-acetylated with varying efficiency . Penultimate amino acid substitutions, except for proline, had little influence on N alpha-acetylation . Substitution of sequences from N alpha-acetylated proteins into the yeast sequences which cannot be N alpha-acetylated demonstrated that not only the first 3 NH2-terminal residues but also more carboxyl-terminal residues were important for determining the specificity of N alpha-acetyltransferase . Two other peptides mimicking yeast mitochondrial cytochrome c oxidase (subunit VI) and ATPase inhibitor, which are naturally non-acetylated, were efficiently acetylated . In addition, recombinant human alcohol dehydrogenase I and basic fibroblast growth factor, which are naturally N alpha-acetylated, were not acetylated post-translationally.

Nucleic Acids Res, 1990 Jul 11, 18(13), 3923 - 31
The upstream activating sequence for L-leucine gene regulation in Saccharomyces cerevisiae; Tu H et al.; The upstream activating sequence (UAS) conferring leucine-specific regulation of transcription in Saccharomyces cerevisiae was identified by analysis of the LEU2 promoter and by comparison to other genes regulated by leucine . The UAS was localized with deletions and cloned synthetic DNA . Point mutations and sequence rearrangements were used to identify important basepairs and to construct an improved UAS with increased regulation and expression . The improved UAS contains a core ten basepair, GC-rich, palindromic sequence, which is sufficient to confer minimal levels of activation and regulation, within a 36 basepair palindromic sequence which confers maximal activation and regulation . Deletions downstream of the UAS indicated that the UAS must act in conjunction with at least one other site, perhaps a TATAA region, in order to confer high levels of activation . Tandem copies of the UAS in front of LEU2 increased expression and regulation . Tandem UAS elements in trans on a multi-copy 2 mu-based plasmid decreased expression and regulation . These results are consistent with a model that the UAS serves as the DNA-binding site for diffusible activation factor(s), possibly the LEU3 gene product.

Gene, 1990 Jul 2, 91(1), 123 - 6
Cloning and nucleotide sequence of the CDC23 gene of Saccharomyces cerevisiae; Doi A et al.; As a preliminary step for studying the function and intracellular behavior of the product of the CDC23 gene, a cell cycle gene of Saccharomyces cerevisiae, we cloned the CDC23 gene and determined its nucleotide (nt) sequence . The nt sequence contains an open reading frame (ORF) of 1878 nt coding for a protein of 626 amino acids (aa) . The CDC23 gene is expressed as a 2.4- to 2.5-kb major transcript . The putative protein product of the CDC23 gene has a potential Ca2(+)-binding site near its N terminus, and has four contiguous repeats of a consensus sequence consisting of 34 aa near its C terminus . Regions that can be regarded as consisting of repeats of the same consensus sequence were found in the published aa sequences of the products of three other yeast genes.

Mol Gen Genet, 1990 Jul, 222(2-3), 192 - 200
Functional analysis of ARGRI and ARGRIII regulatory proteins involved in the regulation of arginine metabolism in Saccharomyces cerevisiae; Qiu HF et al.; We present here a functional analysis of ARGRI and ARGRIII regulatory proteins which are involved together with ARGRII in specific regulation of arginine anabolic and catabolic pathways . Unlike ARGRII, ARGRI and ARGRIII have no transcriptional activation capacity . The first 60 amino acids of ARGRI (out of 177) are dispensable for its activity . The functional domain of the protein is located in the region of homology with MCM1 and SRF proteins . ARGRIII contains in its C-terminal portion a stretch of 17 aspartate residues which are indispensable for arginine regulation . Gene disruption of the ARGRIII gene impairs the growth of the mutant on rich medium, showing that ARGRIII has a pleiotropic role in the cell.

Mol Biol (Mosk), 1990 Jul-Aug, 24(4), 1126 - 33
{The nature of N-terminal signal sequence determines the type of intracellular distribution and effectiveness of export of human growth hormone in Saccharomyces cerevisiae}; Tsiomenko AB et al.; Various N-terminal signal peptides (SP) were tested to investigate a human growth hormone (hGH) synthesis, processing and intracellular sorting in yeast . Maximal level of hGH was observed in the case when the mature hGH gene was placed under the control of PHO5 promoter . In this case about 90% of hGH was localized in the cytosol, but some portion was trustworthly detected in microsomes and periplasma in spite of the absence of SP . Addition of own or PHO5 SP resulted in lowering of the synthesis and a difficulty in the prehGH processing . In this case the immunoreactive products were localized mainly in periplasma and vacuoles and to a lesser degree in the cytosol . When hGH gene was placed under the control of the yeast MF alpha 1 promoter and alpha-factor preprosegment was used as SP more then a half (67%) of hGH processed correctly was exported in a medium, the rest was detected in vacuole (17%) and periplasma (8%).

Mol Biol (Mosk), 1990 Jul-Aug, 24(4), 1037 - 41
{Deletion analysis of the SUP2 gene in Saccharomyces cerevisiae}; Kushnirov VV et al.; The sup2 mutations of the yeast Saccharomyces cerevisiae or plasmid-mediated amplification of the wild type SUP2 gene lead to suppression of different types of nonsense mutations . The Sup2 protein includes a C-terminal region homologous to elongation factor EF-1 alpha and an unique N-terminal region . The SUP2 is an essential gene . The functional role of different regions of the SUP2 gene was investigated, by deleting them without disruption of the reading frame . Such constructs were maintained in yeast on episomal or centromeric plasmids . It was shown that the region, homologous to EF-1 alpha is necessary for viability, while the remaining N-terminal part is nonessential . The region of the first 154 amino acids is necessary and sufficient for the suppressor effect, caused by plasmid-mediated amplification of the SUP2 gene.

Curr Genet, 1990 Jul, 18(1), 7 - 12
Expression of the avian gag-myc oncogene in Saccharomyces cerevisiae; Durrens P et al.; The gag-myc oncogenic sequence of the avian retrovirus MC29 was first inserted in a multicopy expression vector allowing its expression in Saccharomyces cerevisiae . The oncogene transcripts were detected in yeast by Northern blot hybridization and gag-myc proteins were revealed by immunoprecipitation . On solid medium, the average size of gag-myc transformant colonies was smaller than control . In liquid cultures, the gag-myc strains had a doubling time of 4.7 h compared with 3.1 h in the controls . In one of the recipient strains, and after an initial transient period of 5 days, the gag-myc transformants became physiologically indistinguishable from control . In another recipient strain, the slow-growth phenotype is permanent . Plasmid instability is increased in gag-myc transformants . When a single copy of the gag-myc gene was inserted in a yeast chromosome, no phenotype was observed, showing that slow growth is the consequence of plasmid loss.

Curr Genet, 1990 Jul, 18(1), 23 - 7
Fate of highly expressed proteins destined to peroxisomes in Saccharomyces cerevisiae; Hartig A et al.; Import of proteins into organelles usually requires a cis-acting targeting signal . Analysis of various hybrid proteins, consisting of mouse DHFR and parts of catalase A from Saccharomyces cerevisiae, revealed that fusion proteins containing the N-terminal 126 amino acids, or less, of catalase A remain in the cytosol whereas fusion proteins containing 140, or more, N-terminal amino acids of catalase A form large aggregates inside the cell . These protein bodies, which lack a surrounding membrane, copurified with peroxisomes on cell fractionation . The peroxisomal targeting signal of catalase A does not reside at the C-terminus or at the N-terminus.

Curr Genet, 1990 Jul, 18(1), 1 - 5
Genetic control of plasmid DNA double-strand gap repair in yeast, Saccharomyces cerevisiae; Glaser VM et al.; The repair of double-strand gaps (DSGs) in the plasmid DNA of radiosensitive mutants of Saccharomyces cerevisiae has been analyzed . The proportion of repair events that resulted in complete plasmid DNA DSG recovery was close to 100% in Rad+ cells . Mutation rad55 does not influence the efficiency and preciseness of DSG repair . The mutant rad57, which is capable of recombinational DNA DSB repair, resulted in no DSG recovery . Mutation rad53 substantially inhibits the efficiency of DSG repair but does not influence the precision of repair . Plasmid DNA DSG repair is completely blocked by mutations rad50 and rad54.

J Biochem (Tokyo), 1990 Jul, 108(1), 4 - 6
Sexual response of Saccharomyces cerevisiae: phosphorylation of yeast glyoxalase I by a cell extract of mating factor-treated cells; Inoue Y et al.; The phosphorylation of glyoxalase I was observed when the phosphatase-treated enzyme was incubated in the presence of {gamma-32P}ATP and a cell extract prepared from alpha-type yeast cells which had been treated with the culture supernatant of a-type yeast cells . The phosphorylated protein was identified as glyoxalase I by using anti-glyoxalase I rabbit immunoglobulin G.

Yeast, 1990 Jul-Aug, 6(4), 353 - 61
Analysis of the THR4 region on chromosome III of the yeast Saccharomyces cerevisiae; Mannhaupt G et al.; The gene encoding threonine synthase (THR4) from the yeast Saccharomyces cerevisiae was cloned by complementation of a thr4 mutant . This gene was also found on a lambda clone (5239) consisting of a fragment of chromosome III inserted in the vector lambdaMG3 . The THR4 gene encodes a protein of 514 amino acids (M.W . 58 kDa), which has extensive homologies with E . coli threonine synthase (thrC) and B subtilis threonine synthase . The 5' flanking region of the gene contains three regulatory sequences {TGACT(C)} for the general amino acid control (GCN) . About 130 bp downstream of the THR4 gene another large open reading frame (563 amino acids) is found in the opposite orientation . This may imply that this open reading frame, called CTR86, shares a terminator region with THR4 . The function of the protein encoded by CTR86 is not yet clear, but the fact that the upstream region contains a GCN4 responsive site suggests that the gene product may also be involved in amino acid biosynthesis.

Yeast, 1990 Jul-Aug, 6(4), 345 - 52
An essential gene in Saccharomyces cerevisiae shares an upstream regulatory element with PRP4; Petersen-Bjorn S et al.; ORF2 is an essential gene immediately upstream of PRP4 (formerly RNA4), a gene involved in nuclear mRNA processing in Saccharomyces cerevisiae . The two genes are arranged head-to-head . An 8 base-pair conserved sequence element is found upstream of both genes, as well as upstream of certain other genes that are known to be involved in pre-mRNA processing . Through deletion analysis we have found that both of the conserved sequence elements are important for transcription of both genes . We have cloned ORF2 and have isolated temperature-sensitive orf2 mutants . The phenotype of these mutants does not suggest a role for ORF2 in mRNA processing . The deduced amino acid sequence of ORF2 indicates significant similarity to DPR1, a gene encoding a protein that is involved in the carboxy-terminal processing of G-protein.

Genetics, 1990 Jul, 125(3), 487 - 94
Centromeric regions control autonomous segregation tendencies in single-division meiosis of Saccharomyces cerevisiae; Sharon G et al.; We have previously shown that yeast cdc5 or cdc14 homozygotes can be led through a single-division meiosis in which some of the chromosomes segregate reductionally whereas others, within the same cell, segregate equationally . Chromosomes XI tend to segregate reductionally, whereas chromosomes IV tend to segregate equationally . In this report we present experiments with cdc5 homozygous strains, in which the centromeres of one or both chromosomes XI was replaced by the centromeric region from chromosome IV . Analysis of the products of single-division meioses in these strains demonstrates that the choice between reductional or equational segregation is directed by sequences in the vicinity of the centromeres . Although the choice is made separately for each individual chromosome, the analysis also reveals the existence of a system responsible for coordinated segregation of the two chromosomes of a given pair.

Genetics, 1990 Jul, 125(3), 475 - 85
Mixed segregation of chromosomes during single-division meiosis of Saccharomyces cerevisiae; Sharon G et al.; Normal meiosis consists of two consecutive cell divisions in which all the chromosomes behave in a concerted manner . Yeast cells homozygous for the mutation cdc5, however, may be directed through a single meiotic division of a novel type . Dyad analysis of a cdc5/cdc5 strain with centromere-linked markers on four different chromosomes has shown that, in these meioses, some chromosomes within a given cell segregate reductionally whereas others segregate equationally . The choice between the two types of segregation in these meioses is made individually by each chromosome pair . Different chromosome pairs exhibit different segregation tendencies . Similar results were obtained for cells homozygous for cdc14.

Proc Natl Acad Sci U S A, 1990 Jul, 87(13), 5109 - 13
Transfer of a yeast artificial chromosome carrying human DNA from Saccharomyces cerevisiae into mammalian cells; Pachnis V et al.; To test the feasibility of transferring yeast artificial chromosomes (YACs) into mammalian cells, we modified a YAC that carries approximately 450 kilobases (kb) of human DNA, by inserting a neomycin-resistance gene . Saccharomyces cerevisiae cells carrying this YAC were fused by polyethylene glycol to mouse L cells and G418-resistant colonies were obtained . A high percentage of these clones contained virtually intact YAC sequences as revealed by "Alu fingerprint" analysis and restriction enzyme analysis using pulsed-field gel electrophoresis . Furthermore, the YAC sequences were stably integrated into the mouse chromosomes, as shown by in situ hybridization and by the stability of the G418 resistance . These results establish that large segments of the mammalian genome, cloned in yeast, can be efficiently transferred into cultured mammalian cells.

J Cell Biol, 1990 Jul, 111(1), 131 - 42
CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae; Adams AE et al.; Budding in the yeast Saccharomyces cerevisiae involves a polarized deposition of new cell surface material that is associated with a highly asymmetric disposition of the actin cytoskeleton . Mutants defective in gene CDC24, which are unable to bud or establish cell polarity, have been of great interest with regard to both the mechanisms of cellular morphogenesis and the mechanisms that coordinate cell-cycle events . To gain further insights into these problems, we sought additional mutants with defects in budding . We report here that temperature-sensitive mutants defective in genes CDC42 and CDC43, like cdc24 mutants, fail to bud but continue growth at restrictive temperature, and thus arrest as large unbudded cells . Nearly all of the arrested cells appear to begin nuclear cycles (as judged by the occurrence of DNA replication and the formation and elongation of mitotic spindles), and many go on to complete nuclear division, supporting the hypothesis that the events associated with budding and those of the nuclear cycle represent two independent pathways within the cell cycle . The arrested mutant cells display delocalized cell-surface deposition associated with a loss of asymmetry of the actin cytoskeleton . CDC42 maps distal to the rDNA on chromosome XII and CDC43 maps near lys5 on chromosome VII.

Mol Cell Biol, 1990 Jul, 10(7), 3541 - 50
TEC1, a gene involved in the activation of Ty1 and Ty1-mediated gene expression in Saccharomyces cerevisiae: cloning and molecular analysis; Laloux I et al.; Ty and Ty-mediated gene expression observed in haploid cells of Saccharomyces cerevisiae depends on several determinants, some of which are required for the expression of haploid-specific genes . We report here the cloning and molecular analysis of TEC1 . TEC1 encodes a 486-amino-acid protein that is a trans-acting factor required for full Ty1 expression and Ty1-mediated gene activation . However, mutation or deletion of the TEC1 gene had little effect on total Ty2 transcript levels . Our analysis provides clear evidence that TEC1 is not involved in mating or sporulation processes . Unlike most of the proteins involved in Ty and adjacent gene expression, the product of TEC1 has no known cellular function . Although there was no mating-type effect on TEC1 expression, our results indicate that the TEC1 and the a/alpha diploid controls on Ty1 expression are probably not cumulative.

Curr Genet, 1990 Jul, 18(1), 17 - 22
Blockage to exonuclease III digestion in the chromatin of Saccharomyces cerevisiae maps to the in vitro--determined binding site of a trans-acting regulatory factor; Feaver WJ et al.; A series of transposable element-induced mutations at the HIS4 locus in Saccharomyces cerevisiae have been attributed to the transposition of a Ty element into the 5' regulatory region of this gene . Various Ty-containing His+ revertants have been isolated and the HIS4/Ty junction region sequenced . The only difference found in this region between a His- and a weak His+ strain was a single point mutation, an A----G transition . The position of Ty remained unaltered . Examination of lacZ fusion plasmids further implicated this A----G transition as being responsible for the altered phenotype, the bp transition representing an allele of a cis-acting regulatory element . Subsequent gel retardation and methylation interference experiments revealed that this A----G mutation enabled the binding of a trans-acting factor (TyBf) in vitro . In this paper we show that the TyBf binding site is in a region of chromatin hypersensitive to digestion by DNase I . The binding site is protected in vivo from digestion with exonuclease III, suggesting the presence of a bound protein in His+ ("on") but not His- ("off") Ty-containing strains . We propose that a trans-acting factor binding in vivo, presumably TyBf, is responsible for the activation of HIS4 expression in these insertion mutants.

Proc Natl Acad Sci U S A, 1990 Jul, 87(13), 5011 - 5
Protein kinase C in Saccharomyces cerevisiae: comparison with the mammalian enzyme; Ogita K et al.; Protein kinase C (PKC) was detected in the yeast Saccharomyces cerevisiae with bovine myelin basic protein as the phosphate acceptor . The enzyme was purified at least 500-fold by a four-step column chromatographic procedure (phenyl-Sepharose CL-4B, Mono Q, Heparin-5PW, and hydroxyapatite) . The molecular mass was approximately 90 kDa, as estimated by gel-filtration analysis . Yeast PKC was activated by the simultaneous addition of Ca2+, diacylglycerol, and phosphatidylserine . Free arachidonic acid alone could activate the enzyme to some extent . However, yeast PKC did not respond significantly to tumor-promoting phorbol esters . GTP did not serve as phosphate donor . The yeast enzyme showed substrate specificity distinctly different from that of mammalian PKCs . H1 histone and protamine were poor substrates . With myelin basic protein as a model substrate, yeast PKC phosphorylated threonyl residues preferentially, whereas rat brain PKCs phosphorylated seryl residues preferentially . Further studies should elucidate the role of yeast PKC in cellular regulation and cell cycle control.

J Cell Biol, 1990 Jul, 111(1), 143 - 52
Molecular characterization of CDC42, a Saccharomyces cerevisiae gene involved in the development of cell polarity; Johnson DI et al.; The Saccharomyces cerevisiae CDC42 gene product is involved in the morphogenetic events of the cell division cycle; temperature-sensitive cdc42 mutants are unable to form buds and display delocalized cell-surface deposition at the restrictive temperature (Adams, A . E . M., D . I . Johnson, R . M . Longnecker, B . F . Sloat, and J . R . Pringle . 1990 . J . Cell Biol . 111:131-142) . To begin a molecular analysis of CDC42 function, we have isolated the CDC42 gene from a yeast genomic DNA library . The use of the cloned DNA to create a deletion of CDC42 confirmed that the gene is essential . Overexpression of CDC42 under control of the GAL10 promoter was not grossly deleterious to cell growth but did perturb the normal pattern of selection of budding sites . Determination of the DNA and predicted amino acid sequences of CDC42 revealed a high degree of similarity in amino acid sequence to the ras and rho (Madaule, P., R . Axel, and A . M . Myers . 1987 . Proc . Natl . Acad . Sci . 84:779-783) families of gene products . The similarities to ras proteins (approximately 40% identical or related amino acids overall) were most pronounced in the regions that have been implicated in GTP binding and hydrolysis and in the COOH-terminal modifications leading to membrane association, suggesting that CDC42 function also involves these biochemical properties . The similarities to the rho proteins (approximately 60% identical or related amino acids overall) were more widely distributed through the coding region, suggesting more extensive similarities in as yet undefined biochemical properties and functions.

J Bacteriol, 1990 Jul, 172(7), 4115 - 7
Enzymes of phosphoinositide synthesis in secretory vesicles destined for the plasma membrane in Saccharomyces cerevisiae; Kinney AJ et al.; CDP-diacylglycerol synthase, phosphatidylinositol synthase, and phosphatidylinositol kinase activities were associated with post-Golgi apparatus secretory vesicles destined for the plasma membrane of Saccharomyces cerevisiae . These results suggest that the plasma membrane is capable of synthesizing both CDP-diacylglycerol and phosphatidylinositol as well as phosphorylating phosphatidylinositol.

J Bacteriol, 1990 Jul, 172(7), 3584 - 9
Genetic assessment of stationary phase for cells of the yeast Saccharomyces cerevisiae; Drebot MA et al.; Starvation of cells of the yeast Saccharomyces cerevisiae causes cessation of proliferation and acquisition of characteristic physiological properties . The stationary-phase state that results represents a unique developmental state, as shown by a novel conditional phenotype (M . A . Drebot, G . C . Johnston, and R . A . Singer, Proc . Natl . Acad . Sci . USA 84:7948-7952, 1987): mutant cells cannot proliferate at the restrictive temperature when stimulated to reenter the mitotic cell cycle from stationary phase but are unaffected and continue proliferation indefinitely if transferred to the restrictive temperature during exponential growth . We have exploited this reentry mutant phenotype to demonstrate that the same stationary-phase state is generated by nitrogen, sulfur, or carbon starvation and by the cdc25-1 mutation, which conditionally impairs the cyclic AMP-mediated signal transduction pathway . We also show that heat shock, a treatment that elicits physiological perturbations associated with stationary phase, does not cause cells to enter stationary phase . The physiological properties associated with stationary phase therefore do not result from residence in stationary phase but from the stress conditions that bring about stationary phase.

Mol Gen Genet, 1990 Jul, 222(2-3), 393 - 9
The Saccharomyces cerevisiae NPR1 gene required for the activity of ammonia-sensitive amino acid permeases encodes a protein kinase homologue; Vandenbol M et al.; The NPR1 gene of Saccharomyces cerevisiae plays a central role in controlling permease activity; its product is required to promote the activity of at least six distinct transport systems for nitrogenous nutrients under conditions of nitrogen catabolite derepression . We report here the nucleotide sequence of the cloned NPR1 gene . The predicted amino acid sequence indicates that NPR1 encodes a protein of 86 kDa which appears to be organized into two distinct structural domains . The amino-terminal domain of NPR1 (residues 1 to 440) contains 26% serine residues and several regions strongly enriched for PEST residues suggesting a short half-life for the NPR1 protein . The carboxy-terminal region of NPR1 contains consensus sequences characteristic of the catalytic domains of protein kinases . Therefore, NPR1-dependent positive control of nitrogen transport systems most likely involves protein phosphorylation . Northern analysis indicates that the absence of general amino acid permease (GAP1) activity in npr1 mutants is not due to reduction in transcription or messenger stability . Hence, the NPR1 protein probably acts at the post-transcriptional level . Proteins that may serve as substrates for phosphorylation are discussed.

Appl Environ Microbiol, 1990 Jul, 56(7), 2125 - 32
The prepro-peptide of Mucor rennin directs the secretion of human growth hormone by Saccharomyces cerevisiae; Hiramatsu R et al.; An aspartic proteinase, Mucor pusillus rennin (MPR), of filamentous fungus Mucor pusillus, is efficiently secreted from a transformant of Saccharomyces cerevisiae containing the intact MPR gene . To test the usefulness of the MPR leader peptide in secretion of heterologous proteins from yeast cells, several plasmids encoding the fusion proteins composed of different parts of the NH2-terminal region of prepro-MPR and human growth hormone (hGH) were constructed . The parts of the leader peptide upstream of hGH were the whole prepro-peptide following the NH2-terminal region of mature MPR in JGH1, the intact pre-sequence and a part of the pro-sequence in JGH2, and the putative signal sequences of the NH2-terminal 18 and 22 amino acids in JGH3 and JGH7, respectively . When the hGH genes fused to these leader sequences were expressed in yeast cells under the control of the yeast GAL7 promoter, proteins of various sizes immunoreactive with the anti-hGH antibody were secreted into the medium . Among the plasmids mentioned above, JGH2 directed the greatest secretion of the protein of 23 kilodaltons in size, which contained the expected NH2-terminal amino acid sequence of an additional eight amino acids derived from the pro-peptide of MPR . The addition of the GAL10 terminator downstream of the hGH gene in JGH2 resulted in a greater than three- to fivefold increase in the secretion, whereas the insertion of the GAL4 gene, which is a positive regulator for the GAL system, had no significant effect . The improved yield of the total protein of hGH secreted into the medium reached approximately 10 mg/liter.

J Biol Chem, 1990 Jun 25, 265(18), 10419 - 23
Cloning and characterization of the iron-sulfur subunit gene of succinate dehydrogenase from Saccharomyces cerevisiae; Lombardo A et al.; We describe the cloning and characterization of the complete gene for the iron-sulfur protein subunit of succinate dehydrogenase (EC 1.3.99.1) from Saccharomyces cerevisiae . The promoter and coding sequence have been cloned into an Escherichia coli-yeast shuttle vector . The cloned gene complements the defect in a succinate dehydrogenase-deficient yeast mutant isolated by us, and gene expression is fully responsive to induction by glucose deprivation, indicating that the promoter is intact.

Nucleic Acids Res, 1990 Jun 11, 18(11), 3281 - 5
Regulated expression of the Saccharomyces cerevisiae DNA repair gene RAD7 in response to DNA damage and during sporulation; Jones JS et al.; The RAD7 gene of Saccharomyces cerevisiae affects the proficiency of excision repair of DNA damaged by UV light . Here, we report our studies on the regulation of the RAD7 gene in response to UV irradiation and during sporulation . RAD7 transcript levels increased 6-fold within 40 min of exposure of cells to 37 J/m2 of UV light . Higher UV doses also elicited rapid increases in the level of RAD7 mRNA . RAD7 mRNA levels increased in sporulating MATa/MAT alpha diploid cells, but not in the asporogenous MATa/MATa strain exposed to sporulation conditions . The increase in RAD7 mRNA level in MATa/MAT alpha cells was 15-fold after 6 h and 9-fold after 7 h in sporulation medium; thereafter, RAD7 mRNA levels declined . Periodic transcription of RAD7 during sporulation suggests a role for RAD7 in this process.

Nucleic Acids Res, 1990 Jun 11, 18(11), 3219 - 25
Characterisation of sequences required for RNA initiation from the PGK promoter of Saccharomyces cerevisiae; Rathjen J et al.; In the phosphoglycerate kinase (PGK) gene of yeast, as in other highly expressed yeast genes, the sequences surrounding the site of RNA initiation have a loosely conserved structure of a CT rich stretch followed by the tetranucleotide CAAG . Using internal deletions and insertions we have identified the elements in the PGK promoter which are required for correct RNA initiation at the CAAG sequence at -39 . The results indicate that two different components of the PGK promoter contribute to correct RNA initiation, the TATA homologies, located at -152 and -113, and the sequences at the site of initiation . Both TATA elements can function in RNA initiation . Deletion of the upstream TATA element, TATAI, results in slightly heterogeneous RNA initiation, but the majority of the RNA initiates correctly . Deletion of both the PGK TATA elements results in the majority of the RNA initiating at sites downstream from the wild-type I site, within the structural gene between +40 to +80 . The CT rich box is not essential for correct mRNA initiation as shown by deletion analysis . The site of RNA initiation in the PGK promoter appears to be determined by sequences located immediately 5' of the CAAG sequence motif . This short sequence, ACAGATC, when located the correct distance from the TATA elements may be sufficient to determine a discrete initiation site.

J Biol Chem, 1990 Jun 5, 265(16), 9194 - 200
Copper uptake in wild type and copper metallothionein-deficient Saccharomyces cerevisiae . Kinetics and mechanism; Lin CM et al.; The mechanism of copper uptake in Saccharomyces cerevisiae has been investigated using a combination of 64Cu2+ and atomic absorption spectrophotometry . A wild type copper-resistant CUP 1R-containing strain and a strain carrying a deletion of the CUP1 locus (yeast copper metallothionein) exhibited quantitatively similar saturable energy-dependent 64Cu2+ uptake when cultures were pregrown in copper-free media (medium {Cu} approximately 15 nM) . The kinetic constants for uptake by the wild type strain were Vmax = 0.21 nmol of copper/min/mg of protein and Km = 4.4 microM . This accumulation of 64Cu2+ represented net uptake as confirmed by atomic absorption spectrophotometry . This uptake was not seen in glucose-starved cells, but was supported in glycerol- and ethanol-grown ones . Uptake was inhibited by both N3- and dinitrophenol and was barely detectable in cultures at 4 degrees C . When present at 50 microM, Zn2+ and Ni2+ inhibited by 50% indicating that this uptake process was relatively selective for Cu2+ . 64Cu2+ accumulation was qualitatively and quantitatively different in cultures either grown in or preincubated with cold Cu2+ . Either treatment resulted in the appearance of a fast phase (t 1/2 approximately 1 min) of 64Cu2+ accumulation which represented isotopic exchange since it did not lead to an increase in the mass of cell-associated copper; also, it was not energy-dependent . Exchange of 64Cu2+ into this pool was not inhibited by Zn2+ . Pretreatment with Cu2+ caused a change in the rate of net accumulation as well; a 3-h incubation of cells in 5 microM medium Cu2+ caused a 1.6-fold increase in the velocity of energy-dependent uptake . Prior addition of cycloheximide abolished this Cu2(+)-dependent increase and, in fact, inhibited the 64Cu2+ uptake velocity by greater than 85% . The exchangeable pool was also absent in cycloheximide, Cu2(+)-treated cells suggesting that exchangeable Cu2+ derived from the copper taken up initially by the energy-dependent process . The thionein deletion mutant was similar to wild type in response to medium Cu2+ and cycloheximide indicating that copper metallothionein is not directly involved in Cu2+ uptake (as distinct from retention) in yeast.

J Biol Chem, 1990 Jun 5, 265(16), 8979 - 82
Accurate initiation by RNA polymerase II in a whole cell extract from Saccharomyces cerevisiae; Woontner M et al.; We have developed a simple procedure for isolating a transcriptional extract from whole yeast cells which obviates the requirement for nuclear isolation . Detection of accurate mRNA initiation by RNA polymerase II in the extract requires the use of a sensitive assay, recently described by Kornberg and co-workers (Lue, N . F., Flanagan, P . M., Sugimoto, K., and Kornberg, R . D . (1989) Science 246, 661-664) that involves activation by a GAL4-VP16 fusion protein and a template lacking guanosine residues in the coding strand . The extract is prepared from fresh or frozen yeast cells by disruption with glass beads and fractionation of proteins by ammonium sulfate precipitation . The alpha-amanitin-sensitive transcripts synthesized in the assay were identical to those produced in a parallel assay using a yeast nuclear extract . The activity of the whole cell extract is lower per mg of protein than a nuclear extract but proportional to the volume of the nucleus relative to the whole cell . The optimal ranges for several reaction components including template, mono- and divalent cations, and nucleotide substrate concentration were determined . Under optimal conditions the whole cell extract produced a maximum of approximately 1 X 10(-2) transcripts/template molecule in 30 min.

Mol Gen Genet, 1990 Jun, 222(1), 25 - 32
Interchromosomal and intrachromosomal recombination in rad 18 mutants of Saccharomyces cerevisiae; Schiestl RH et al.; The frequency of intra- and interchromosomal recombination was determined in RAD18 and rad18 deletion and rad18-3 mutant strains . It was found that spontaneous interchromosomal recombination at trp5, his1, ade2, and MAT was elevated 10- to 70-fold in the rad18-3 and rad18 delta mutants as compared to the RAD+ strains . On the other hand the frequencies of spontaneous intrachromosomal recombination for the his3 delta 3', his3 delta 5' and the his4C-, his4A- duplications and for heterothallic mating type switching were only marginally elevated in the rad18 deletion mutant, and recombination between ribosomal DNA repeats was only 2-fold elevated in the rad18-3 mutant . These differences may be due to a haploid versus diploid specific difference . However interchromosomal recombination was elevated 40-fold and intrachromosomal recombination was only marginally (1.5-fold) elevated in a diploid homozygous for rad18 delta, arguing against a haploid versus diploid specific difference . Possible explanations for the difference in the elevated levels of intra- versus interchromosomal spontaneous recombination are discussed.

Antimicrob Agents Chemother, 1990 Jun, 34(6), 989 - 93
In vivo effects of fenpropimorph on the yeast Saccharomyces cerevisiae and determination of the molecular basis of the antifungal property; Marcireau C et al.; The effects of fenpropimorph on sterol biosynthesis and growth of Saccharomyces cerevisiae were examined to pinpoint the mode of action of fungicides that inhibit ergosterol biosynthesis . Taking advantage of sterol auxotrophy and sterol permeability in mutant strains, we show that growth inhibition is strongly correlated with inhibition of sterol biosynthesis . We confirm that in vivo and at low concentrations, fenpropimorph inhibits delta 8----delta 7-sterol isomerase, and in addition, when it is used at higher concentrations, it inhibits delta 14-sterol reductase . We show also that the fungistatic effect of fenpropimorph is not due to the accumulation of abnormal sterols in treated cells but is linked to the specific inhibition of ergosterol biosynthesis, leading to the arrest of cell proliferation in the unbudded G1 phase of the cell cycle.

Curr Genet, 1990 Jun, 17(6), 535 - 6
Chromosome location of a family of genes encoding different acidic ribosomal proteins in Saccharomyces cerevisiae; Remacha M et al.; DNA probes from the genes encoding the acidic ribosomal proteins L44, L44' and L45, as well as from reporter genes for chromosomes IV, VII, XII and XV, have been hybridised to Southern blots of Saccharomyces cerevisiae DNA resolved by pulsed field gel electrophoresis . The protein L44' and protein L45 genes have been found to hybridise to chromosome IV, identified by the CAT1 gene probe, while the protein L44 probe hybridises with a band containing chromosomes VII and XV, identified by the ATPase 1 and HIS3 genes respectively.

Genetics, 1990 Jun, 125(2), 333 - 40
Chromosomal assignment of mutations by specific chromosome loss in the yeast Saccharomyces cerevisiae; Wakem LP et al.; Yeast 2-microns plasmids were integrated near the centromere of a different chromosome in each of 16 cir0 mapping strains of Saccharomyces cerevisiae . The specific chromosomes containing the integrated 2-microns plasmid DNA were lost at a high frequency after crossing the cir0 strains to cir+ strains . A recessive mutation in a cir+ strain can then be easily assigned to its chromosome using this set of mapping strains, since the phenotype of the recessive mutation will be manifested only in diploids having the integrated 2-microns plasmid and the unmapped mutation on homologous chromosomes.

Genetics, 1990 Jun, 125(2), 321 - 31
Extragenic suppressors of mar2(sir3) mutations in Saccharomyces cerevisiae; Lin CI et al.; The silent mating-type genes (HML and HMR) of Saccharomyces cerevisiae are kept under negative transcriptional control by four trans-acting MAR (or SIR) loci . We have isolated extragenic suppressors of the mar2-1 mutation which, based on genetic complementation tests, define two additional loci involved in regulating the expression of HML and HMR . A strain with the genotype HMLa MAT alpha HMRa mar2-1 is sterile due to the simultaneous expression of a and alpha information . Two mutants exhibiting an alpha phenotype (which may result from the restoration of MAR/SIR repression) were isolated and genetically characterized . The mutations in these strains: (1) are recessive, (2) are capable of suppressing a mar2-deletion mutation, (3) are unlinked to MAT, (4) complement one another as well as the previously identified sum1-1 mutation, and (5) are not new alleles of the known MAR/SIR loci . We designate these new regulatory loci SUM2 and SUM3 (suppressor of mar) . Unlike the sum1-1 mutation, suppression by sum2-1 and sum3-1 is mar2-locus specific . Both sum2-1 and sum3-1 affect the expression of a information at the HM loci . Transcript analysis shows a significant reduction in HMLa and HMRa gene transcription in mar2-1 sum2-1 and mar2-1 sum3-1 cells . Furthermore, we have found genetic evidence to suggest that mar2-1 sum2-1 cells exhibit only partial expression of silent alpha information . We conclude that the SUM2 and SUM3 gene products are required for expression of the HM loci and act downstream of the MAR2 (SIR3) gene function . Possible mechanisms for the action of the SUM gene products are discussed.

Genetics, 1990 Jun, 125(2), 313 - 20
Direct selection for mutants with increased K+ transport in Saccharomyces cerevisiae; Vidal M et al.; Saccharomyces cerevisiae cells containing a deletion of TRK1, the gene encoding the high affinity potassium transporter, retain only low affinity uptake of this ion and consequently lose the ability to grow in media containing low levels (0.2 mM) of potassium . Using a trk1 delta strain, we selected spontaneous Trk+ pseudorevertants that regained the ability to grow on low concentrations of potassium . The revertants define three unlinked extragenic suppressors of trk1 delta . Dominant RPD2 mutations and recessive rpd1 and rpd3 mutations confer increased potassium uptake in trk1 delta cells . Genetic evidence suggests that RPD2 mutations are alleles of TRK2, the putative low affinity transporter gene, whereas rpd1 and rpd3 mutations increase TRK2 activity: (1) RPD2 mutations are closely linked to trk2, and (2) trk2 mutations are epistatic to both rpd1 and rpd3 . rpd1 maps near pho80 on chromosome XV and rpd3 maps on the left arm of chromosome XIV, closely linked to kre1.

Genetics, 1990 Jun, 125(2), 305 - 12
TRK2 is required for low affinity K+ transport in Saccharomyces cerevisiae; Ko CH et al.; TRK1, the gene encoding the high affinity K+ transporter in Saccharomyces cerevisiae, is nonessential due to the existence of a functionally independent low affinity transporter . To identify the gene(s) encoding the low affinity K+ transporter, we screened trk1 delta cells for mutants (Kla-) that require higher concentrations of K+ in the medium to support growth . trk1 delta trk2 mutants require up to tenfold higher concentrations of K+ to exhibit normal growth compared to trk1 delta TRK2 cells . K+ and 86Rb+ transport assays demonstrate that the mutant phenotype is due to defective K+ transport (uptake) . Each of 38 independent mutants contains a mutation in the same gene, TRK2 . Cells deficient for both high and low affinity K+ transport (trk1 delta trk2) exhibit hypersensitivity to low extracellular pH that can be suppressed by high concentrations of K+ but not Na+ . TRK1 completely suppresses both the K+ transport defect and low pH hypersensitivity of trk2 cells, suggesting that TRK1 and TRK2 are functionally independent.

Genetics, 1990 Jun, 125(2), 281 - 91
Analysis of the GAL3 signal transduction pathway activating GAL4 protein-dependent transcription in Saccharomyces cerevisiae; Bhat PJ et al.; The Saccharomyces cerevisiae GAL/MEL regulon genes are normally induced within minutes of galactose addition, but gal3 mutants exhibit a 3-5-day induction lag . We have discovered that this long-term adaptation (LTA) phenotype conferred by gal3 is complemented by multiple copies of the GAL1 gene . Based on this result and the striking similarity between the GAL3 and GAL1 protein sequences we attempted to detect galactokinase activity that might be associated with the GAL3 protein . By both in vivo and in vitro tests the GAL3 gene product does not appear to catalyze a galactokinase-like reaction . In complementary experiments, Escherichia coli galactokinase expressed in yeast was shown to complement the gal1 but not the gal3 mutation . Thus, the complementation activity provided by GAL1 is not likely due to galactokinase activity, but rather due to a distinct GAL3-like activity . Overall, the results indicate that GAL1 encodes a bifunctional protein . In related experiments we tested for function of the LTA induction pathway in gal3 cells deficient for other gene functions . It has been known for some time that gal3gal1, gal3gal7, gal3gal10, and gal3 rho- are incapable of induction . We constructed isogenic haploid strains bearing the gal3 mutation in combination with either gal15 or pgi1 mutations: the gal15 and pgi1 blocks are not specific for the galactose pathway in contrast to the gal1, gal7 and gal10 blocks . The gal3gal5 and gal3pgi1 double mutants were not inducible, whereas both the gal5 and pgi1 single mutants were inducible . We conclude that, in addition to the GAL3-like activity of GAL1, functions beyond the galactose-specific GAL1, GAL7 and GAL10 enzymes are required for the LTA induction pathway.

Biochem J, 1990 Jun 1, 268(2), 309 - 15
Expression of human glutathione S-transferases in Saccharomyces cerevisiae confers resistance to the anticancer drugs adriamycin and chlorambucil; Black SM et al.; Adaptation and resistance to chemicals in the environment is a critical part of the evolutionary process . As a result, a wide variety of defence systems that protect cells against chemical insult have evolved . Such chemical resistance mechanisms appear to play a central role in determining the sensitivity of human tumours to treatment with chemotherapeutic drugs . The glutathione S-transferases (GST) are important detoxification enzymes whose over-expression has been associated with drug-resistance . In order to evaluate this possibility we have expressed the human Alpha-class and Pi-class GST cDNAs that encode GST B1B1 and GST pi in the yeast Saccharomyces cerevisiae . The expression of GST B1B1 or GST pi resulted in a marked reduction in the cytotoxic effects of chlorambucil, a bifunctional alkylating agent, and an anthracycline, adriamycin . These data provide direct evidence that the over-expression of GST in cells can confer resistance to anticancer drugs.

Biotechnol Appl Biochem, 1990 Jun, 12(3), 341 - 5
Nucleotide sequence of a gene which enhances the activity of glyoxalase I in Saccharomyces cerevisiae; Inoue Y et al.; A gene whose product enhances glyoxalase I activity in yeast Saccharomyces cerevisiae cells was cloned as a 6-kbp DNA fragment in the vector YEp13 . The DNA fragment was subcloned in approximately 2 kbp and its base sequence determined . An open reading frame with 318 nucleotide pairs (encoding 106 amino acids) was found . According to Northern blotting analysis, the yeast transformed with the hybrid plasmid harboring the 2-kbp fragment yielded two different transcripts . From the DNA sequence of the fragment, it is deduced that the gene product would contain seven cysteine residues, five of which would be located near the carboxy terminus of the peptide.

Proc Natl Acad Sci U S A, 1990 Jun, 87(12), 4665 - 9
A DNA replication enhancer in Saccharomyces cerevisiae; Walker SS et al.; We have dissected the autonomously replicating sequence ARS121 using site-directed in vitro mutagenesis . Three domains important for origin function were identified; one of these is essential and contains an 11-base-pair sequence resembling the canonical ARS core consensus; the second region, deletion of which affects the efficiency of the origin, is located 3' to the T-rich strand of the essential sequence and encompasses several elements with near matches to the ARS core consensus; the third region, containing two OBF1 DNA-binding sites and located 5' to the essential sequence, also affects the efficiency of the ARS . Here we demonstrate that a synthetic OBF1 DNA-binding site can substitute for the entire third domain in origin function . A dimer of the synthetic binding site, fused to a truncated origin containing only domains one and two, restored the origin activity to the levels of the wild-type ARS . The stimulation of origin function by the synthetic binding site was relatively orientation independent and could occur at distances as far as 1 kilobase upstream to the essential domain . Based on these results we conclude that the OBF1 DNA-binding site is an enhancer of DNA replication . We suggest that the DNA-binding site and the OBF1 protein are involved in the regulation of the activation of nuclear origins of replication in Saccharomyces cerevisiae.

J Bacteriol, 1990 Jun, 172(6), 2855 - 61
Isolation and characterization of a new mutant of Saccharomyces cerevisiae with altered synthesis of 5-aminolevulinic acid; Carvajal E et al.; A new gene, RHM1, required for normal production of 5-aminolevulinic acid by Saccharomyces cerevisiae, was identified by a novel screening method . Ethyl methanesulfonate treatment of a fluorescent porphyric strain bearing the pop3-1 mutation produced nonfluorescent or weakly fluorescent mutants with defects in early stages of tetrapyrrole biosynthesis . Class I mutants defective in synthesis of 5-aminolevulinate regained fluorescence when grown on medium supplemented with 5-aminolevulinate, whereas class II mutants altered in later biosynthetic steps did not . Among six recessive class I mutants, at least three complementation groups were found . One mutant contained an allele of HEM1, the structural gene for 5-aminolevulinate synthase, and two mutants contained alleles of the regulatory gene CYC4 . The remaining mutants contained genes complementary to both hem1 and cyc4 . Mutant strain DA3-RS3/68 contained mutant gene rhm1, which segregated independently of hem1 and cyc4 during meiosis . 5-Aminolevulinate synthase activity of the rhm1 mutant was 35 to 40% of that of the parental pop3-1 strain, whereas intracellular 5-aminolevulinate concentration was only 3 to 4% of the parental value . Transformation of an rhm1 strain with a multicopy plasmid containing the cloned HEM1 gene restored normal levels of 5-aminolevulinate synthase activity, but intracellular 5-aminolevulinate was increased to only 9 to 10% of normal . We concluded that RHM1 could control either targeting of 5-aminolevulinate synthase to the mitochondrial matrix or the activity of the enzyme in vivo.

Mol Cell Biol, 1990 Jun, 10(6), 3262 - 7
Transcriptional regulation of SSA3, an HSP70 gene from Saccharomyces cerevisiae; Boorstein WR et al.; The SSA3 gene of Saccharomyces cerevisiae, a member of the HSP70 multigene family, is expressed at low levels under optimal growth conditions and is dramatically induced in response to heat shock . Sequences coinciding with two overlapping heat shock elements, located 156 base pairs upstream of the transcribed region, were necessary and sufficient for regulation of heat induction . The SSA3 promoter was also activated in an ssa1ssa2 double-mutant strain . This increase in the expression of SSA3 was mediated via the same upstream activating sequences that activated transcription in response to heat shock.

Mol Cell Biol, 1990 Jun, 10(6), 3256 - 7
The Saccharomyces cerevisiae DNA repair gene RAD2 is regulated in meiosis but not during the mitotic cell cycle; Madura K et al.; The expression of the RAD2 gene of Saccharomyces cerevisiae is elevated upon DNA damage . Here, we show that RAD2 transcript levels also rise approximately eightfold during meiosis but remain constant during the mitotic cell cycle . The period of maximal RAD2 mRNA accumulation during meiosis is consistent with a possible role of RAD2 in a late stage of recombination, in mismatch repair of heteroduplexes, or both.

Mol Cell Biol, 1990 Jun, 10(6), 2941 - 9
Mutations in Saccharomyces cerevisiae which confer resistance to several amino acid analogs; McCusker JH et al.; Four new complementation groups of mutations which confer resistance to several amino acid analogs in Saccharomyces cerevisiae are described . These mutants were isolated on medium containing urea as the nitrogen source, in contrast to previous studies that had used medium containing proline . All four resistance to amino acid analog (raa) complementation groups appear to confer resistance by reducing amino acid analog and amino acid uptake . In some genetic backgrounds, raa leu2 and raa thr4 double mutants are inviable, even on rich medium . The raa4 mutation may affect multiple amino acid transport systems, since raa4 mutants are unable to use proline as a nitrogen source . raa4 is, however, unlinked to a previously described amino acid analog resistance and proline uptake mutant, aap1, or to the general amino acid permease mutant gap1 . Both raa4 and gap1 prevent uptake of {3H}leucine in liquid cultures . The raa1, raa2, and raa3 mutants affect only a subset of the amino acid analogs and amino acids affected by raa4 . The phenotypes of raa1, -2, and -3 mutants are readily observed on agar plates but are not seen in uptake and incorporation of amino acids measured in liquid media.

Mol Cell Biol, 1990 Jun, 10(6), 2809 - 19
Complementary transcripts from two genes necessary for normal meiosis in the yeast Saccharomyces cerevisiae; Malavasic MJ et al.; The SPO12 gene, which is required for meiosis I chromosome division during sporulation of the yeast Saccharomyces cerevisiae, has been isolated . DNA sequencing has identified an open reading frame of 173 codons that encodes the putative SPO12 protein and has no significant sequence similarities to known genes . The last 15 amino acids of this putative protein have a high negative charge, which appears to be required for function . A second sporulation-specific gene, designated SPO16, was found adjacent to SPO12 and shown to be necessary for efficient spore formation . The two genes are encoded on opposite DNA strands with only 103 nucleotides between the termination codons . Up to 700 nucleotides of the SPO12 and SPO16 transcripts are complementary, and the 3' untranslated region of the longest SPO16 transcript is complementary to all or nearly all of the SPO12 mRNA . A strain homozygous for an insertion which removes the complementarity between the SPO12 and SPO16 mRNAs has an efficiency of sporulation, number of spores per ascus, and spore viability identical to those of a wild-type strain . The complementarity therefore has either no function or only a subtle function in meiosis and sporulation.

Mol Cell Biol, 1990 Jun, 10(6), 2544 - 53
SNF6 encodes a nuclear protein that is required for expression of many genes in Saccharomyces cerevisiae; Estruch F et al.; The SNF6 gene appears to affect transcription from a variety of promoters in Saccharomyces cerevisiae . The gene was cloned, and sequence analysis revealed two completely overlapping open reading frames of 996 and 1092 nucleotides on opposite strands . The SNF6 coding sequence was identified by selective mutagenesis . The predicted 37,604-dalton SNF6 protein is highly charged but overall neutral . A bifunctional SNF6-beta-galactosidase fusion protein was localized in the nucleus, as judged by immunofluorescence microscopy . The N terminus of SNF6 contains a sequence homologous to nuclear localization signals and was sufficient to direct beta-galactosidase to the nucleus . The 5' ends of the SNF6 RNA were heterogeneous and included ends mapping downstream from the first ATG codon . Construction of a frameshift mutation provided evidence that translational initiation at the second ATG yields a partially functional SNF6 product . Null mutations in SNF6 caused a wider range of pleiotropic defects than the previously isolated point mutation, including slow growth . Genetic and molecular evidence suggested that SNF6 is functionally related to the SNF2 and SNF5 genes . These genes may function together to affect transcription.

Mol Cell Biol, 1990 Jun, 10(6), 2485 - 91
RAD10, an excision repair gene of Saccharomyces cerevisiae, is involved in the RAD1 pathway of mitotic recombination; Schiestl RH et al.; The RAD10 gene of Saccharomyces cerevisiae is required for the incision step of excision repair of UV-damaged DNA . We show that the RAD10 gene is also required for mitotic recombination . The rad10 delta mutation lowered the rate of intrachromosomal recombination of a his3 duplication in which one his3 allele has a deletion at the 3' end and the other his3 allele has a deletion at the 5' end (his3 delta 3' his3 delta 5') . The rate of formation of HIS3+ recombinants in the rad10 delta mutant was not affected by the rad1 delta mutation but decreased synergistically in the presence of the rad10 delta mutation in combination with the rad52 delta mutation . These observations indicate that the RAD1 and RAD10 genes function together in a mitotic recombination pathway that is distinct from the RAD52 recombination pathway . The rad10 delta mutation also lowered the efficiency of integration of linear DNA molecules and circular plasmids into homologous genomic sequences . We suggest that the RAD1 and RAD10 gene products act in recombination after the formation of the recombinogenic substrate . The rad1 delta and rad10 delta mutations did not affect meiotic intrachromosomal recombination of the his3 delta 3' his3 delta 5' duplication or mitotic and meiotic recombination of ade2 heteroalleles located on homologous chromosomes.

Mol Cell Biol, 1990 Jun, 10(6), 2458 - 67
Isolation of the gene encoding the Saccharomyces cerevisiae centromere-binding protein CP1; Baker RE et al.; CP1 is a sequence-specific DNA-binding protein of the yeast Saccharomyces cerevisiae which recognizes the highly conserved DNA element I (CDEI) of yeast centromeres . We cloned and sequenced the gene encoding CP1 . The gene codes for a protein of molecular weight 39,400 . When expressed in Escherichia coli, the CP1 gene directed the synthesis of a CDEI-binding protein having the same gel mobility as purified yeast CP1 . We have given the CP1 gene the genetic designation CEP1 (centromere protein 1) . CEP1 was mapped and found to reside on chromosome X, 2.0 centimorgans from SUP4 . Strains were constructed in which most of CEP1 was deleted . Such strains lacked detectable CP1 activity and were viable; however, CEP1 gene disruption resulted in a 35% increase in cell doubling time and a ninefold increase in the rate of mitotic chromosome loss . An unexpected consequence of CP1 gene disruption was methionine auxotrophy genetically linked to cep1 . This result and the recent finding that CDEI sites in the MET25 promoter are required to activate transcription (D . Thomas, H . Cherest, and Y . Surdin-Kerjan, J . Mol . Biol . 9:3292-3298, 1989) suggest that CP1 is both a kinetochore protein and a transcription factor.

J Histochem Cytochem, 1990 Jun, 38(6), 823 - 7
Cytochemical localization of mercury in Saccharomyces cerevisiae treated with mercuric chloride; Whittaker SG et al.; We describe a cytochemical method for localizing mercury at the electron microscopic level in the yeast Saccharomyces cerevisiae . After addition of a lethal concentration of mercuric chloride to growing yeast cells, mercury was associated with the cell wall and cytoplasmic membrane . Little or no mercury was present in the cytoplasm . Electron probe X-ray microanalysis (EPMA) confirmed that the cytochemical reaction, visualized as mercury-silver complexes, was localized in dense bodies consisting of a core of mercury sulfide polymers surrounded by a shell of silver atoms.

Biotechnol Appl Biochem, 1990 Jun, 12(3), 276 - 83
Immunoaffinity chromatography of diadenosine 5',5'''-P1,P4-tetraphosphate phosphorylase from Saccharomyces cerevisiae; Avila DM et al.; Diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) phosphorylase has been isolated previously using classical protein isolation techniques {A . Guranowski and S . Blanquet (1985) J . Biol . Chem . 260, 3542-3547} . A protein A-Sepharose immunoaffinity column was prepared to simplify the purification procedure . The immunoaffinity column was prepared using specific polyclonal antibodies to Ap4A phosphorylase covalently coupled to protein A-Sepharose with dimethyl pimelimidate by a modification of the procedure of C . Schneider et al . {(1982) J . Biol . Chem . 257, 10,766-10,769} . The specific activity of the immunoaffinity-purified enzyme showed an increase equivalent to the specific activity obtained by chromatography on DEAE-cellulose and hydroxyapatite columns.

EMBO J, 1990 Jun, 9(6), 1727 - 35
The plant G box promoter sequence activates transcription in Saccharomyces cerevisiae and is bound in vitro by a yeast activity similar to GBF, the plant G box binding factor; Donald RG et al.; G box and I box sequences of the Arabidopsis thaliana ribulose-bisphosphate-1,5-carboxylase small subunit (RBCS) promoter are required for expression mediated by the Arabidopsis rbcS-1A promoter in transgenic tobacco plants and are bound in vitro by factors from plant nuclear extracts termed GBF and GA-1, respectively . We show here that a -390 to -60 rbcS-1A promoter fragment containing the G box and two I boxes activates transcription from a truncated iso-1-cytochrome c (CYC1) gene promoter in Saccharomyces cerevisiae . Mutagenesis of either the rbcS-1A G box or both I box sequences eliminated the expression mediated by this fragment . When polymerized, I box oligonucleotides were also capable of enhancing expression from the truncated CYC1 promoter . Single-copy G box sequences from the Arabidopsis rbcS-1A, Arabidopsis Adh and tomato rbcS-3A promoters were more potent activators and were used in mobility shift assays to identify a DNA binding activity in yeast functionally similar to GBF . In methylation interference experiments, the binding specificity of the yeast protein was indistinguishable from that obtained with plant nuclear extracts.

J Bacteriol, 1990 Jun, 172(6), 3009 - 14
Specificity of the mutator effect caused by disruption of the RAD1 excision repair gene of Saccharomyces cerevisiae; Kunz BA et al.; Disruption of RAD1, a gene controlling excision repair in the yeast Saccharomyces cerevisiae, increased the frequency of spontaneous forward mutation in a plasmid-borne copy of the SUP4-o gene . To characterize this effect in detail, a collection of 249 SUP4-o mutations arising spontaneously in the rad1 strain was analyzed by DNA sequencing . The resulting mutational spectrum was compared with that derived from an examination of 322 spontaneous SUP4-o mutations selected in an isogenic wild-type (RAD1) strain . This comparison revealed that the rad1 mutator phenotype was associated with increases in the frequencies of single-base-pair substitution, single-base-pair deletion, and insertion of the yeast retrotransposon Ty . In the rad1 strain, the relative fractions of these events and their distributions within SUP4-o exhibited features similar to those for spontaneous mutagenesis in the isogenic RAD1 background . The increase in the frequency of Ty insertion argues that Ty transposition can be activated by unrepaired spontaneous DNA damage, which normally would be removed by excision repair . We discuss the possibilities that either translesion synthesis, a reduced fidelity of DNA replication, or a deficiency in mismatch correction might be responsible for the majority of single-base-pair events in the rad1 strain.

Mol Cell Biol, 1990 Jun, 10(6), 3027 - 35
Divergent overlapping transcripts at the PET122 locus in Saccharomyces cerevisiae; Ohmen JD et al.; PET122 is one of three nuclear genes specifically required for translation of the mitochondrial mRNA for cytochrome c oxidase subunit III in Saccharomyces cerevisiae . The nucleotide sequence of 2,862 base pairs (bp) of yeast genomic DNA encompassing the PET122 locus shows very close spacing between the PET122 gene (254 codons) and two unidentified open reading frames, termed ORF2 and ORF3 . ORF2 is encoded by the same strand of DNA as PET122 and is located 53 bp downstream of PET122, while ORF3 is encoded on the opposite strand and is located 215 bp upstream of PET122 . Five transcripts, with sizes of 2.9, 2.3, 2.1, 1.5, and 1.4 kilobases (kb), are produced from this locus . The 2.1- and 1.4-kb transcripts encode ORF3, the 1.5-kb transcript encodes ORF2, and the 2.9- and 2.3-kb transcripts encode PET122 . A particularly interesting feature of the ORF3-PET122-ORF2 transcription unit is a 535-base overlap between the 2.3-kb PET122 transcript produced from one strand and a 2.1-kb ORF3 transcript produced from the opposite strand . Similarly, the 2.9-kb PET122 transcript overlaps the 2.1-kb ORF3 transcript by more than 900 bases and the 1.5-kb ORF3 transcript by at least 200 bases . Hence, these pairs of transcripts are antisense to one another and have the potential to regulate, in an interdependent fashion, the posttranscriptional expression of ORF3 and PET122.

Antonie Van Leeuwenhoek, 1990 Jun, 58(1), 49 - 55
Localization of glucoamylase genes of Saccharomyces cerevisiae by pulsed field gel electrophoresis; Bignell GR et al.; The chromosomal locations of four glucoamylase-specifying genes in the yeast Saccharomyces cerevisiae have been determined . Chromosomes were separated by pulsed field gel electrophoresis and blots were probed with radiolabelled STA2 and marker DNA from specific yeast chromosomes . The three genes encoding extracellular glucoamylases, STA1 (DEX2), STA2 (DEX1) and STA3 (DEX3) are located on chromosomes IV, II and XIV, respectively . SGA, specifying the sporulation-specific glucoamylase, was positioned on chromosome IX.

Biochem J, 1990 Jun 1, 268(2), 401 - 7
Purification, characterization and partial amino acid sequence of glycogen synthase from Saccharomyces cerevisiae; Carabaza A et al.; Glycogen synthase from Saccharomyces cerevisiae was purified to homogeneity . The enzyme showed a subunit molecular mass of 80 kDa . The holoenzyme appears to be a tetramer . Antibodies developed against purified yeast glycogen synthase inactivated the enzyme in yeast extracts and allowed the detection of the protein in Western blots . Amino acid analysis showed that the enzyme is very rich in glutamate and/or glutamine residues . The N-terminal sequence (11 amino acid residues) was determined . In addition, selected tryptic-digest peptides were purified by reverse-phase h.p.l.c . and submitted to gas-phase sequencing . Up to eight sequences (79 amino acid residues) could be aligned with the human muscle enzyme sequence . Levels of identity range between 37 and 100%, indicating that, although human and yeast glycogen synthases probably share some conserved regions, significant differences in their primary structure should be expected.

Mol Cell Biol, 1990 Jun, 10(6), 2966 - 72
Mutations in cell division cycle genes CDC36 and CDC39 activate the Saccharomyces cerevisiae mating pheromone response pathway; de Barros Lopes M et al.; Conditional mutations in the genes CDC36 and CDC39 cause arrest in the G1 phase of the Saccharomyces cerevisiae cell cycle at the restrictive temperature . We present evidence that this arrest is a consequence of a mutational activation of the mating pheromone response . cdc36 and cdc39 mutants expressed pheromone-inducible genes in the absence of pheromone and conjugated in the absence of a mating pheromone receptor . On the other hand, cells lacking the G beta subunit or overproducing the G alpha subunit of the transducing G protein that couples the receptor to the pheromone response pathway prevented constitutive activation of the pathway in cdc36 and cdc39 mutants . These epistasis relationships imply that the CDC36 and CDC39 gene products act at the level of the transducing G protein . The CDC36 and CDC39 gene products have a role in cellular processes other than the mating pheromone response . A mating-type heterozygous diploid cell, homozygous for either the cdc36 or cdc39 mutation, does not exhibit the G1 arrest phenotype but arrests asynchronously with respect to the cell cycle . A similar asynchronous arrest was observed in cdc36 and cdc39 cells where the pheromone response pathway had been inactivated by mutations in the transducing G protein . Furthermore, cdc36 and cdc39 mutants, when grown on carbon catabolite-derepressing medium, did not arrest in G1 and did not induce pheromone-specific genes at the restrictive temperature.

Mol Cell Biol, 1990 Jun, 10(6), 2539 - 43
Mutational mapping of RAS-responsive domains of the Saccharomyces cerevisiae adenylyl cyclase; Colicelli J et al.; Large deletion and small insertion mutations in the adenylyl cyclase gene of Saccharomyces cerevisiae were used to map regions required for activation by RAS protein in vitro . The amino-terminal 605 amino acids were found to be dispensable for responsiveness to RAS protein . All other deletions in adenylyl cyclase destroyed its ability to respond to RAS . Small insertion mutations within the leucine-rich repeat region also prevented RAS responsiveness, while other insertions did not.

Biotechnology (N Y), 1990 Jun, 8(6), 543 - 6
Characterization of recombinant factor XIIIa produced in Saccharomyces cerevisiae; Rinas U et al.; Recombinant factor XIIIa (FXIIIa), produced in Saccharomyces cerevisiae, was recovered as a fully active cytosolic component and rigorously compared to natural F XIIIa from human placenta with respect to physicochemical and functional properties . Identical parameters were found in SDS polyacrylamide gel electrophoresis, analytical ultracentrifugation and HPLC gel filtration, and all spectral characteristics including derivative UV absorbance, fluorescence and circular dichroism were identical . Similarly, the interaction of both proteins with polyclonal antibodies directed against the entire FXIIIa or its N-terminal 4 kD activation peptide were identical . Furthermore, thrombin cleavage and fibrin cross-linking showed indistinguishable patterns . The only difference we observed was with respect to endgroup analysis . The recombinant protein is homogeneous, whereas placental FXIIIa shows multiple electrophoretic bands caused by microheterogeneity in the C-terminal part of the protein.

Appl Microbiol Biotechnol, 1990 Jun, 33(3), 302 - 6
Integration of heterologous genes into the chromosome of Saccharomyces cerevisiae using a delta sequence of yeast retrotransposon Ty; Sakai A et al.; Distribution of a delta (delta) sequence of the Ty element on a chromosome of the yeast Saccharomyces cerevisiae was analysed by pulsed-field gel electrophoresis . More than 100 copies of the delta sequence were nonrandomly distributed on the chromosome . Using the delta sequence as a recombination site, mouse alpha-amylase and human beta-endorphin genes were introduced into the chromosomal DNA . The integration occurred on a particular chromosome in each case and the copy number was estimated as three to five . It was also found that single- or multi-copy integration occurred at a single or multiple sites on the particular chromosome . The integrants secreted alpha-amylase and beta-endorphin by three-to fivefold compared with single-copy integrants . This type of integration was mitotically stable over a period of 50 generations under non-selective conditions.

Agric Biol Chem, 1990 Jun, 54(6), 1499 - 504
Chromosomal transformation in Saccharomyces cerevisiae with DNA isolated by pulse field gel electrophoresis; Goto K et al.; We isolated and purified yeast chromosome DNA molecules using pulse field gel electrophoresis (PFG) . The isolated DNA had nearly the same size as the native chromosomal DNA on PFG . We could directly transform Saccharomyces cerevisiae yeasts with it, and obtain transformants that were selected by complementation of several markers . They had new chromosome DNA bands observed on PFG . The new chromosome was very stable during mitosis and mating processes, and each of the three homologous chromosomes in the derivative zygotes of transformants was separated equally in daughter cells.

Gene, 1990 May 31, 90(1), 99 - 104
Nucleotide sequence and characterization of temperature-sensitive pol1 mutants of Saccharomyces cerevisiae; Lucchini G et al.; We have analyzed the effects of temperature-sensitivity (ts)-conferring mutations in the Saccharomyces cerevisiae DNA polymerase I-encoding gene on cell growth, in vivo DNA synthesis, intrachromosomal gene conversion and pop-out recombination . Also, we have identified the molecular defect responsible for the ts phenotype . Two mutant alleles (cdc17-1, cdc17-2) were originally identified as cell-cycle mutations, while a third mutation (hpr3) was found during a genetic screening for mutants with a hyper-recombination phenotype . Both cdc17-2 and hpr3 cells complete one round of cell division and DNA replication after shift to nonpermissive temperature, before being arrested as dumbbell-shaped cells . Conversely, the cdc17-1 mutation immediately blocks growth and DNA synthesis at 37 degrees C . No substantial difference was observed in the frequency of intrachromosomal gene conversion and pop-out recombination events, when hpr3 and cdc17-1 were compared to the previously characterized pol1-1 mutant . These two frequencies were ten- to 30-fold above wild-type level at semipermissive temperature . In each mutant, a single bp substitution, causing the replacement of Gly residues by either Asp (cdc17-1, cdc17-2) or Glu (hpr3) in yeast DNA polymerase I is responsible for the ts phenotype.

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