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