S. cerevisiae

Mol Gen Genet, 1988 Apr, 212(1), 124 - 8
The Escherichia coli proB gene corrects the proline auxotrophy of Saccharomyces cerevisiae pro1 mutants; Orser CS et al.; We constructed plasmids carrying the Escherichia coli proB gene that encodes gamma-glutamyl kinase, under the control of the yeast GAL1 promoter . This construction was carried out with both the wild-type proB+ gene and a mutant allele, proB74, that specifies an enzyme resistant to feedback inhibition by proline . Yeast pro1 mutants harboring these plasmids are proline prototrophs . We conclude that the pro1 mutation results in a deficiency in the gamma-glutamyl kinase activity in Saccharomyces cerevisiae . Expression of the proB74 allele in yeast resulted in enhanced resistance to the proline analogue L-azetidine-2-carboxylate and in a 2.4-fold elevation of the intracellular free proline levels . This result suggests that gamma-glutamyl kinase is the rate limiting step in proline biosynthesis in yeast.

J Bacteriol, 1988 Apr, 170(4), 1878 - 86
Saccharomyces cerevisiae mutant with a partial defect in the synthesis of CDP-diacylglycerol and altered regulation of phospholipid biosynthesis; Klig LS et al.; A Saccharomyces cerevisiae mutant (cdg1 mutation) was isolated on the basis of an inositol excretion phenotype and exhibited pleiotropic deficiencies in phospholipid biosynthesis . Genetic analysis of the mutant confirmed that the cdg1 mutation represents a new genetic locus and that a defect in a single gene was responsible for the Cdg1 phenotype . CDP-diacylglycerol synthase activity in mutant haploid cells was 25% of the wild-type derepressed level . Biochemical and immunoblot analyses revealed that the defect in CDP-diacylglycerol synthase activity in the cdg1 mutant was due to a reduced level of the CDP-diacylglycerol synthase Mr-56,000 subunit rather than to an alteration in the enzymological properties of the enzyme . This defect resulted in a reduced rate of CDP-diacylglycerol synthesis, an elevated phosphatidate content, and alterations in overall phospholipid synthesis . Unlike wild-type cells, CDP-diacylglycerol synthase was not regulated in response to water-soluble phospholipid precursors . The cdg1 lesion also caused constitutive expression of inositol-1-phosphate synthase and elevated phosphatidylserine synthase . Phosphatidylinositol synthase was not affected in the cdg1 mutant.

Mol Cell Biol, 1988 Apr, 8(4), 1421 - 31
Functional organization of the retrotransposon Ty from Saccharomyces cerevisiae: Ty protease is required for transposition; Youngren SD et al.; We used several mutations generated in vitro to further characterize the functions of the products encoded by the TyB gene of the transpositionally active retrotransposon TyH3 from Saccharomyces cerevisiae . Mutations close to a core protein domain of TyB, which is homologous to retroviral proteases, have striking effects on Ty protein processing, the physiology of Ty viruslike particles, and transposition . The Ty protease is required for processing of both TyA and TyB proteins . Mutations in the protease resulted in the synthesis of morphologically and functionally aberrant Ty viruslike particles . The mutant particles displayed reverse transcriptase activity, but did not synthesize Ty DNA in vitro . Ty RNA was present in the mutant particles, but at very low levels . Transposition of a genetically tagged element ceased when the protease domain was mutated, demonstrating that Ty protease is essential for transposition . One of these mutations also defined a segment of TyB encoding an active reverse transcriptase . These results indicate that the Ty protease, like its retroviral counterpart, plays an important role in particle assembly, replication, and transposition of these elements.

Gene, 1988 Mar 31, 63(2), 175 - 85
Mapping and sequencing of the dihydrofolate reductase gene (DFR1) of Saccharomyces cerevisiae; Barclay BJ et al.; The dihydrofolate reductase gene (DFR1) from Saccharomyces cerevisiae has been mapped and sequenced . The gene was isolated on an 8.8-kb BamHI fragment from a yeast genomic library by screening of Escherichia coli transformants for resistance to trimethoprim . A 1.8-kb SalI-BamHI fragment which was able to confer methotrexate resistance in yeast also complemented an E . coli DHFR-deficient (folA) mutant . Nucleotide sequence analysis revealed that the yeast DFR1 gene encoded a polypeptide with a predicted Mr of 24230 . The deduced sequence of 211 amino acid residues showed considerable homology with DHFRs from both bacterial and animal sources . The codon bias index of the DFR1 coding region is 0.0083, which indicates a random pattern of codon usage . The upstream region contains two consensus sequences required for binding of the yeast's positive regulatory factor, GCN4, suggesting that the DFR1 gene might be subject to the amino acid general control . Several potential 'TATA' boxes are located in the sequence 5' to the gene . Located in the 3' flanking region are homologies with several canonical sequences thought to be required for efficient transcription termination in yeast . We also mapped the DFR1 gene to a position 1.4 cM proximal to the MET7 locus on chromosome XV.

Biochem Biophys Res Commun, 1988 Mar 30, 151(3), 1346 - 51
Accumulation of processing intermediates of the RAS2 protein in strain 112 of Saccharomyces cerevisiae; Breviario D et al.; Strain 112 (RAS1 RAS2) contains a naturally occurring mutation which significantly retards processing of the RAS2 gene product . This mutation, resulting in the accumulation of precursor forms of RAS2 protein, has been assigned by genetic analysis to a single chromosomal locus distinct from the RAS2 locus . In addition to the known precursor molecule of 41000 daltons (p41), 112 cells accumulate within the soluble fraction an intermediate form of RAS2 (p40-1), which migrates, in SDS-polyacrylamide gel, between p41 and the fully processed, membrane-bound 40,000 daltons (p40) product . We propose for RAS2 protein processing the following sequence of events: p41 greater than p40-1 greater than p40 where p40-1 represents a RAS2 intermediate required for the targeting of the protein to the plasma membrane.

Biochim Biophys Acta, 1988 Mar 30, 933(1), 212 - 22
The definition of mitochondrial H+ ATPase assembly defects in mit- mutants of Saccharomyces cerevisiae with a monoclonal antibody to the enzyme complex as an assembly probe; Hadikusumo RG et al.; mit- mutants with genetically defined mutations in the mitochondrial structural genes of the H+-ATPase membrane subunits 6, 8 and 9 were analysed to determine the H+-ATPase assembly defects that resulted as a consequence of the mutations . These include mutants which do not synthesize one of the membrane subunits and mutants which can synthesize these subunits, but in an altered form . Protein subunits which can still be assembled to the defective H+-ATPase in these mutants were determined by immunoprecipitation using a monoclonal antibody to the beta-subunit of the enzyme complex . The results suggest that the assembly pathway of the mitochondrially synthesized H+-ATPase subunits involves the sequential addition of subunits 9, 8 and 6 to a membrane-bound F1-sector . In addition to subunits of the F0- and F1-sectors, two other polypeptides (Mr = 18,000 and Mr = 25,000) are associated with the yeast H+-ATPase . These polypeptides were not observed in the immunoprecipitates obtained from mutants in which the F0-sector is not properly assembled.

FEBS Lett, 1988 Mar 14, 229(2), 383 - 7
Sequence of the gene encoding phosphoglycerate mutase from Saccharomyces cerevisiae; White MF et al.; The gene encoding yeast phosphoglycerate mutase was isolated, and its sequence was determined . The gene specifies a protein of 246 amino acids, and contains no introns . The sequence shows a strong codon bias . The upstream untranslated portion of the gene contains a CT-rich block such as is found in many highly expressed yeast genes, but does not have the associated CAAG sequence.

Biochim Biophys Acta, 1988 Mar 11, 968(3), 408 - 17
Guanine nucleotide regulation of adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae; Becker JM et al.; Adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae was examined . Among various permeabilization procedures, including organic solvents, detergents and other reagents, dimethylsulfoxide (DMSO) and digitonin treatments resulted in the highest recovery of adenylate cyclase activity . Incubation of cells at 30 degrees C with digitonin at 0.01% to 0.1%, or DMSO at 20% to 40% for 15 to 30 min gave optimal adenylate cyclase activity . The enzyme activity in digitonin-permeabilized cells could be supported only by Mn2+, whereas Mg2+ with or without guanine nucleotides did not support cyclase activity . DMSO-permeabilized cells exhibit efficient Mn2+- and Mg2+/Gpp{NH}p-dependent stimulation . Furthermore, digitonin added to yeast membranes at a 1:50 detergent to protein ratio (w/w) abolishes guanyl nucleotide regulation without significantly affecting the Mn2+-supported cyclase activity . The superiority of DMSO is further supported by the fact that recovery of adenylate cyclase activity is better in the DMSO-treated cells than in the digitonin-treated cells . DMSO most probably causes less disturbance of the fabric of the native cell . We conclude that digitonin, but not DMSO, uncouples the catalytic unit of adenylate cyclase from the regulatory GTP binding (ras) proteins.

Biochim Biophys Acta, 1988 Mar 4, 959(1), 9 - 19
Inhibition of sterol biosynthesis in Saccharomyces cerevisiae by N,N-diethylazasqualene and derivatives; Balliano G et al.; The ability of some azasqualene derivatives to inhibit yeast cell growth was compared with their inhibition activity on squalene-2,3-oxide cyclase (EC 5.4.99.7) both in living cells and in microsome preparations . Among the compounds tested, N,N-diethylazasqualene showed the best correlation between the activity on squalene-2,3-oxide cyclase and its inhibition of yeast growth . The N-oxide derivative, N,N-diethylazasqualene N-oxide, which was as active as the amine in microsomes, was much less active in living cells, probably because it could not easily penetrate the cell wall . Kinetic analysis of the inhibitory activity of compounds on squalene-2,3-oxide cyclase revealed a sharp difference between N,N-diethylazasqualene and its N-oxide; the former showed a non-competitive-type inhibition, whereas the latter behaved as a competitive inhibitor.

Curr Genet, 1988 Mar, 13(3), 207 - 17
Regulation of isoleucine-valine biosynthesis in Saccharomyces cerevisiae; Holmberg S et al.; The threonine deaminase gene (ILV1) of Saccharomyces cerevisiae has been designated "multifunctional" since Bollon (1974) indicated its involvement both in the catalysis of the first step in isoleucine biosynthesis and in the regulation of the isoleucine-valine pathway . Its role in regulation is characterized by a decrease in the activity of the five isoleucine-valine enzymes when cells are grown in the presence of the three branched-chain amino acids, isoleucine, valine and leucine (multivalent repression) . We have demonstrated that the regulation of AHA reductoisomerase (encoded by ILV5) and branched-chain amino acid transaminase is unaffected by the deletion of ILV1, subsequently revealing that the two enzymes can be regulated in the absence of threonine deaminase . Both threonine deaminase activity and ILV1 mRNA levels increase in mutants (gcd2 and gcd3) having constitutively depressed levels of enzymes under the general control of amino acid biosynthesis, as well as in response to starvation for tryptophan and branched-chain amino acid imbalance . Thus, the ILV1 gene is under general amino acid control, as is the case for both the ILV5 and the transaminase gene . Multivalent repression of reductoisomerase and transaminase can be observed in mutants defective in general control (gcn and gcd), whereas this is not the case for threonine deaminase . Our analysis suggests that repression effected by general control is not complete in minimal medium . Amino acid dependent regulation of threonine deaminase is only through general control, while the branched-chain amino acid repression of AHA reducto isomerase and the transaminase is caused both by general control and an amino acid-specific regulation.

Genetika, 1988 Mar, 24(3), 568 - 71
{Ploidy of a mutant Saccharomyces cerevisiae defective in homologous recombination}; Semenova VD et al.; The ploidy of a mutant of Saccharomyces cerevisiae defective in recombination (Rec-) has been determined using tetrad analysis and flowing fluorometry . Evidence is obtained that the effect of the Rec- phenotype, i.e . the increase of the stability of plasmids with 2 mkm DNA ori replication in the yeast cirO cells is not the result of the diploidy in cells of the Rec- mutant developed in the process of transformation.

Mol Cell Biol, 1988 Mar, 8(3), 1309 - 18
The a-factor pheromone of Saccharomyces cerevisiae is essential for mating; Michaelis S et al.; The Saccharomyces cerevisiae pheromone a-factor is produced by a cells and interacts with alpha cells to cause cell cycle arrest and other physiological responses associated with mating . Two a-factor structural genes, MFA1 and MFA2, have been previously cloned with synthetic probes based on the a-factor amino acid sequence (A . Brake, C . Brenner, R . Najarian, P . Laybourn, and J . Merryweather, cited in M.-J . Gething {ed.}, Protein transport and secretion, 1985) . We have examined the function of these genes in a-factor production and mating by construction and analysis of chromosomal null mutations . mfa1 and mfa2 single mutants each exhibited approximately half the wild-type level of a-factor activity and were proficient in mating, whereas the mfa1 mfa2 double mutant produced no a-factor and was unable to mate . These results demonstrate that both genes are functional, that each gene makes an equivalent contribution to the a-factor activity and mating capacity of a cells, and that a-factor plays an essential role in mating . Strikingly, exogenous a-factor did not alleviate the mating defect of the double mutant, suggesting that an a cell must be producing a-factor to be an effective mating partner.

Mol Cell Biol, 1988 Mar, 8(3), 1253 - 8
A DNA sequence conferring high postmeiotic segregation frequency to heterozygous deletions in Saccharomyces cerevisiae is related to sequences associated with eucaryotic recombination hotspots; White JH et al.; The meiotic behavior of two graded series of deletion mutations in the ADE8 gene in Saccharomyces cerevisiae was analyzed to investigate the molecular basis of meiotic recombination . Postmeiotic segregation (PMS) was observed for a subset of the deletion heterozygosities, including deletions of 38 to 93 base pairs . There was no clear relationship between deletion length and PMS frequency . A common sequence characterized the novel joint region in the alleles which displayed PMS . This sequence is related to repeated sequences recently identified in association with recombination hotspots in the human and mouse genomes . We propose that these particular deletion heterozygosities escape heteroduplex DNA repair because of fortuitous homology to a binding site for a protein.

Mol Cell Biol, 1988 Mar, 8(3), 1179 - 85
Domain structure and functional analysis of the carboxyl-terminal polyacidic sequence of the RAD6 protein of Saccharomyces cerevisiae; Morrison A et al.; The RAD6 gene of Saccharomyces cerevisiae, which is required for normal tolerance of DNA damage and for sporulation, encodes a 172-residue protein whose 23 carboxyl-terminal residues are almost all acidic . We show that this polyacidic sequence appends to RAD6 protein as a polyanionic tail and that its function in vivo does not require stoichiometry of length . RAD6 protein was purified to near homogeneity from a yeast strain carrying a RAD6 overproducing plasmid . Approximately the first 150 residues of RAD6 protein composed a structural domain that was resistant to proteinase K and had a Stokes radius typical of a globular protein of its calculated mass . The carboxyl-terminal polyacidic sequence was sensitive to proteinase K, and it endowed RAD6 protein with an aberrantly large Stokes radius that indicates an asymmetric shape . We deduce that RAD6 protein is monomeric and comprises a globular domain with a freely extending polyacidic tail . We tested the phenotypic effects of partial or complete deletion of the polyacidic sequence, demonstrating the presence of the shortened proteins in the cell by using antibody to RAD6 protein . Removal of the entire polyacidic sequence severely reduced sporulation but only slightly affected survival after UV irradiation or UV-induced mutagenesis . Strains with deletions of all but the first 4 or 15 residues of the polyacidic sequence were phenotypically almost wild type or wild type, respectively . We conclude that the intrinsic activity of RAD6 protein resides in the globular domain, that the polyacidic sequence has a stimulatory or modifying role evident primarily in sporulation, and that only a short section apparently functions as effectively as the entire polyacidic sequence.

Mol Gen Genet, 1988 Mar, 211(3), 455 - 8
Mitotic gene conversion of large DNA heterologies in Saccharomyces cerevisiae; Aguilera A; Gene conversion of large DNA heterologous fragments has been shown to take place efficiently in Saccharomyces cerevisiae . It has been found that a 2.6 kb LEU2 DNA fragment in a multicopy plasmid was replaced by a 3.1 kb PGI1 chromosomal DNA fragment, when both fragments were flanked by homologous DNA regions . Gene conversion was asymmetric in a total of 481 recombinants analyzed . In contrast, truncated PGI1 or LEU2 genes in multicopy plasmids, gave no recombinants that restored a complete plasmid copy of these genes in a total of 242 recombinants studied, confirming that a conversion tract is disrupted by a heterologous region . The asymmetry of the events detected suggest that gene conversion of large DNA heterologies involves a process whereby a gap first covers one heterologous fragment and then this is followed by new DNA synthesis using the other heterologous fragment as a template . Therefore, it is likely that large DNA heterologies are converted by a double-strand gap repair mechanism.

Mol Gen Genet, 1988 Mar, 211(3), 430 - 4
The sporulation capable (sca) mutation of Saccharomyces cerevisiae is an allele of the SIR2 gene; Margolskee JP; We have used the special properties of the spo13-1 mutation in order to study the regulation of yeast meiosis by the mating type loci . We have found that both the rme1-1 mutation and the sca mutation allow haploid meiosis in spo13-1 strains . Therefore, haploid meiosis is regulated in the same manner as diploid meiosis . Unlike rme1-1, the sca mutation allows meiosis through derepression of the silent mating type cassettes; sca strains can sporulate only because they express both MATa and MAT alpha information . We have found further that sca is an allele of SIR2, one of the genes involved in repression of the silent cassettes . Therefore, the RME1 gene is the only known candidate for a master negative regulator through which the MAT locus controls meiosis.

Genetics, 1988 Mar, 118(3), 411 - 5
Maintenance of the 2 micron circle plasmid of Saccharomyces cerevisiae by sexual transmission: an example of a selfish DNA; Futcher B et al.; Many eukaryotic mobile elements have been identified, but few have any obvious function . This has led to the proposal that many such elements may be parasitic DNA . We have used the 2 micron circle plasmid of Saccharomyces cerevisiae as a model system to investigate the maintenance of a cryptic genetic element . We find that under certain conditions this plasmid can spread through experimental populations despite demonstrable selection against it . This spread is dependent upon outbreeding, suggesting that cell to cell transmission of the plasmid during the yeast sexual cycle can counterbalance selection, and maintain the plasmid in populations . This result provides experimental support for the idea that some mobile elements may be parasitic DNA.

DNA, 1988 Mar, 7(2), 117 - 26
Regulated secretion of MuGM-CSF in Saccharomyces cerevisiae via GAL1:MF alpha 1 prepro sequences; Shaw KJ et al.; Murine granulocyte-macrophage colony-stimulating factor (GM-CSF) was expressed in Saccharomyces cerevisiae using a novel regulated secretion system . This system involves the fusion of the GAL1 upstream regulatory region to the signal sequence of the alpha mating pheromone, and the integration of this GAL1:MF alpha 1 prepro:MuGM-CSF construct into the yeast chromosome . These constructs were very stable under both selective and nonselective conditions: after 30 generations of growth no plasmid loss was observed . The expression and secretion of MuGM-CSF were analyzed by biological assays and Western blots of yeast culture medium and yeast cell extracts . Expression of MuGM-CSF was regulated by galactose induction . In addition, expression levels were proportional to the number of tandem copies of the gene inserted into the yeast chromosome.

J Cell Biol, 1988 Mar, 106(3), 557 - 66
Histone H3 and H4 gene deletions in Saccharomyces cerevisiae; Smith MM et al.; The genome of haploid Saccharomyces cerevisiae contains two nonallelic sets of histone H3 and H4 genes . Strains with deletions of each of these loci were constructed by gene replacement techniques . Mutants containing deletions of either gene set were viable, however meiotic segregants lacking both histone H3 and H4 gene loci were inviable . In haploid cells no phenotypic expression of the histone gene deletions was observed; deletion mutants had wild-type growth rates, were not temperature sensitive for growth, and mated normally . However, diploids homozygous for the H3-H4 gene deletions were slightly defective in their growth and cell cycle progression . The generation times of the diploid mutants were longer than wild-type cells, the size distributions of cells from exponentially growing cultures were skewed towards larger cell volumes, and the G1 period of the mutant cells was longer than that of the wild-type diploid . The homozygous deletion of the copy-II set of H3-H4 genes in diploids also increased the frequency of mitotic chromosome loss as measured using a circular plasmid minichromosome assay.

Yeast, 1988 Mar, 4(1), 41 - 6
Purification of isocitrate lyase from Saccharomyces cerevisiae; Lopez-Boado YS et al.; Isocitrate lyase purified to homogeneity from Saccharomyces cerevisiae was composed of four identical subunits with a molecular mass of 75 kDa . The enzyme was most active at pH 7.0 in the presence of 5 mM-Mg2+ . The Km value for threo-Ds-isocitrate was 1.4 mM . Isocitrate lyase was shown to be thermostable at 50 degrees C for 60 min at a high salt concentration, but rapidly lost activity at -20 degrees C or by dialysis.

J Gen Microbiol, 1988 Mar, 134 ( Pt 3), 785 - 90
Regulation of trehalase activity during the cell cycle of Saccharomyces cerevisiae; Van Doorn J et al.; Synchronous cultures of Saccharomyces cerevisiae prepared by selection of small unbudded cells from an elutriating rotor were used to measure trehalase activity during the cell cycle . After the small cells had been removed from the rotor, the remainder was used to prepare asynchronous control cultures . Both synchronous and control cultures were studied for two cell cycles . In asynchronous cultures the trehalase activity of crude cell lysates rose continuously . In synchronized populations trehalase activity increased from the beginning of budding onwards . However, around the period of cell division the enzyme activity dropped rapidly but transiently by more than 5-fold . The same changes were found during the second budding cycle . Measurements of invertase and glucose-6-phosphate dehydrogenase activities in the same synchronous and asynchronous cultures revealed a continuous increase for both enzymes . Incubation of cell lysates with cAMP-dependent protein kinase before assaying for trehalase resulted in a 2-fold enhancement of enzyme activity in asynchronous control cultures . In synchronized cells this treatment also led to a significant stimulation of trehalase activity, and largely abolished the cell-cycle-dependent oscillatory pattern of enzyme activity . These results suggest that the activity of trehalase during the cell cycle is regulated, presumably at the post-translational level, by a phosphorylation-dephosphorylation mechanism.

Yeast, 1988 Mar, 4(1), 47 - 59
Expression of env sequences of the bovine leukemia virus (BLV) in the yeast Saccharomyces cerevisiae; Brantl S et al.; DNA sequences of the envelope (env) gene of the bovine leukemia virus (BLV) were expressed in the yeast Saccharomyces cerevisiae . Two yeast promoters, the repressible PHO5 promoter and the constitutive PGK promoter, were used to construct four expression plasmids comprising either a sequence of the surface antigen gp51 or a (gp51 + gp30) sequence . The expressed heterologous gene products were characterized by Western blot analysis and competitive radioimmunoassay . By means of Northern blot analysis the steady-state level of env-specific mRNA was analysed . The highest expression rate was obtained from recombinant plasmid YEpSG 94 comprising a gp51 sequence--a 630 base pair fragment containing 70% of the gp51 but lacking the N terminus--as well as the PHO5 promoter including PHO5 signal sequence and the PHO5 terminator . The recombinant gp51 was partially glycosylated but the PHO5 signal peptide did not seem to be cleaved off . No immunoreactive material could be found in the periplasm or in the culture medium . By means of monoclonal antibodies directed against eight different epitopes of viral gp51, all four sequential antigenic determinants were detected in the AH 216 (YEpSG 94) expression product.

Genetics, 1988 Mar, 118(3), 401 - 10
Physical lengths of meiotic and mitotic gene conversion tracts in Saccharomyces cerevisiae; Judd SR et al.; Physical lengths of gene conversion tracts for meiotic and mitotic conversions were examined, using the same diploid yeast strain in all experiments . This strain is heterozygous for a mutation in the URA3 gene as well as closely linked restriction site markers . In cells that had a gene conversion event at the URA3 locus, it was determined by Southern analysis which of the flanking heterozygous restriction sites had co-converted . It was found that mitotic conversion tracts were longer on the average than meiotic tracts . About half of the tracts generated by spontaneous mitotic gene conversion included heterozygous markers 4.2 kb apart; none of the meiotic conversions included these markers . Stimulation of mitotic gene conversion by ultraviolet light or methylmethanesulfonate had no obvious effect on the size or distribution of the tracts . Almost all conversion tracts were continuous.

J Bacteriol, 1988 Mar, 170(3), 1399 - 402
Identification of a third nuclear protein-coding gene required specifically for posttranscriptional expression of the mitochondrial COX3 gene is Saccharomyces cerevisiae; Kloeckener-Gruissem B et al.; A third nuclear protein-coding gene termed PET122 has been shown to be required for a post-transcriptional step in expression of the mitochondrial COX3 gene is Saccharomyces cerevisiae . pet122 mutants fail to produce cytochrome c oxidase subunit III, which is the polypeptide product of the COX3 gene, but produce normal amounts of mature COX3 mRNA . A strain bearing the pet122-1 allele is amber suppressible and correctly processes the 5' end of COX3 mRNA . Therefore, the PET122 gene product is a protein required for the expression of COX3 at some step after transcription and 5'-end processing of its transcript.

J Theor Biol, 1988 Feb 21, 130(4), 481 - 92
A mathematical model of recombinational amplification of the 2 mu plasmid in the yeast Saccharomyces cerevisiae; Wittrup KD et al.; A mathematical model of 2 mu plasmid recombinational amplification in Saccharomyces cerevisiae has been developed, based on mechanisms of 2 mu recombination and replication presented in the literature . A probabilistic description reveals the limits inherent in the recombinational mode of plasmid amplification . These limits correspond well with values calculated from reported results . In the model, copy number control is effected by the constitutive expression of a repressor of recombinase expression . Estimation of the model parameters is accomplished via a set of heuristic rules which restrict the feasible parameter space considerably . It is demonstrated that many parameter sets arbitrarily chosen from the feasible parameter space reproduce the observed characteristics of 2 mu plasmid amplification: rapid correction of downward copy number deviations, with a lack of strict control of steady-state copy number.

Eur J Biochem, 1988 Feb 15, 172(1), 205 - 11
Modifications of oxidative phosphorylations in mitochondria isolated from a mutant of Saccharomyces cerevisiae . Possible alterations of the phosphate transport; Manon S et al.; Mutants of Saccharomyces cerevisiae were isolated which supported two unlinked nuclear mutations conferring thermosensitivity and cold sensitivity respectively, and a mitochondrial one conferring paromomycin sensitivity . Mitochondria isolated from such a mutant exhibited modifications of several phosphate-requiring functions: (a) kinetic parameters of the phosphate dependence of ATP synthesis were modified; (b) in the absence of phosphate the inner mitochondrial membrane exhibited a high proton leakage; (c) mutant mitochondria always exhibited a poor respiratory control and required tenfold more phosphate to reach a maximal state 3 of respiration; (d) phosphate transport, as measured by swelling experiments, was mersalyl-insensitive and, consequently, state 3 of the respiration and ATP synthesis remained less mersalyl-sensitive than in wild-type mitochondria . Analysis of the mitochondrial metabolism of diploid and segregant strains indicates that these modifications are related to the cryosensitive phenotype; however, at present, a cooperative effect of the mitochondrial mutation cannot be eliminated . It is proposed that the phosphate carrier itself or a regulatory element was modified.

Eur J Biochem, 1988 Feb 15, 172(1), 179 - 84
Inactivation of the gene encoding the 11-kDa subunit VIII of the ubiquinol-cytochrome-c oxidoreductase in Saccharomyces cerevisiae; Maarse AC et al.; The single nuclear gene encoding the 11-kDa subunit VIII of the ubiquinol-cytochrome-c oxidoreductase (complex III) in Saccharomyces cerevisiae has been inactivated by a one-step gene disruption procedure . Inactivation results in a loss of ubiquinol-cytochrome-c oxidoreductase activity (less than 1% wild type) and respiratory deficiency . Cells lacking the 11-kDa protein also display lowered steady-state levels of other complex-III subunits encoded by nuclear genes including the 14-kDa subunit VII and the Rieske Fe-S protein and of the mitochondrially encoded cytochrome b . The steady-state levels of the transcripts from the genes encoding these proteins are however not reduced . The results strongly imply that the 11-kDa protein plays an important role in regulating the synthesis of complex III at the post-transcriptional level, most likely assembly . Separation of chromosomes by pulsed-field gel electrophoresis of DNA of wild-type and of the mutant lacking the 11-kDa-protein gene followed by Southern blot analysis reveals that the latter gene is located on chromosome X rather than on XII as reported by Van Loon et al . {Mol . Gen . Genet . 197 (1984) 219-224}.

Biochem Biophys Res Commun, 1988 Feb 15, 150(3), 1144 - 8
Cyclic AMP control of GTP pools in Saccharomyces cerevisiae; Pall ML; Previous studies have shown that GTP and cyclic AMP have similar effects on the regulation of sporulation in the yeast Saccharomyces cerevisiae . Declines in either nucleotide can trigger sporulation . These results raise the question whether either nucleotide influences the pool of the other . The current study shows that a cyclic AMP deficiency produces a decline in GTP pools and cyclic AMP readdition quickly increases GTP pools . UTP but not CTP shows a similar pattern of control to that shown by GTP . These results suggest that cyclic AMP effects on sporulation and possibly other cell properties may be mediated in part or in whole by GTP . They provide support for the hypothesis that GTP has a general role in stimulating cellular growth and proliferation.

J Biochem (Tokyo), 1988 Feb, 103(2), 321 - 6
Secretion of an active nonglycosylated form of the repressible acid phosphatase of Saccharomyces cerevisiae in the presence of tunicamycin at low temperatures; Mizunaga T et al.; The role of mannan chains in the formation and secretion of active acid phosphatase of yeast (Saccharomyces cerevisiae), a repressible cell surface mannoprotein, was studied in yeast protoplast systems by using tunicamycin at various temperatures . At 30 degrees C, tunicamycin-treated protoplasts did not produce active acid phosphatase; however, at 25 or 20 degrees C they formed and secreted active enzyme . This form of acid phosphatase gave 59-, 57-, and 55-kDa bands on SDS-PAGE which neither bound to concanavalin A Sepharose, nor changed in molecular weight upon treatment with endoglycosidase H, indicating that the peptides are nonglycosylated . The nonglycosylated form, like its glycosylated counterpart, is a dimer on the basis of gel permeation chromatography . The Km for para-nitrophenyl-phosphate and Ki for inorganic phosphate of both glycosylated and nonglycosylated acid phosphatases were almost the same . These results suggested that 1) the conformation of the nonglycosylated acid phosphatase secreted at low temperatures is probably identical with that of the glycosylated one, and 2) the conformation of acid phosphatase is very important for its secretion . The rate of intracellular transport of nonglycosylated acid phosphatase is about one-fourth that of the glycosylated enzyme, indicating that glycosylation facilitates the transport of acid phosphatase proteins.

Genes Dev, 1988 Feb, 2(2), 160 - 72
Depletion of Saccharomyces cerevisiae ribosomal protein L16 causes a decrease in 60S ribosomal subunits and formation of half-mer polyribosomes; Rotenberg MO et al.; We constructed yeast strains containing deletion-insertion null alleles of the RPL16A or RPL16B genes encoding the 60S ribosomal subunit protein L16 to determine the role of L16 in the synthesis and function of ribosomes . Strains lacking a functional RPL16A gene grow as rapidly as wild type, whereas those containing a null allele of RPL16B grow more slowly than wild type . RNA analysis using RPL16 probes revealed that both RPL16 genes are transcribed and that RPL16B transcripts accumulate to twice the level of RPL16A transcripts . No evidence was obtained for the occurrence of dosage compensation at the level of RPL16 mRNA accumulation in either mutant . Strains lacking both RPL16 genes are apparently inviable, demonstrating that L16 is an essential yeast ribosomal protein . Introduction of an extra copy of either RPL16 gene into rpl16b mutants restored wild-type growth rates, indicating that the two forms of the L16 protein are interchangeable . rpl16 mutants are deficient in 60S ribosomal subunits relative to 40S subunits . 43S preinitiation complexes accumulate in half-mer polyribosomes in the absence of sufficient 60S subunits . We postulate that the slow-growth phenotype of rpl16 mutants results from the perturbation of initiation of protein synthesis.

Mol Cell Biol, 1988 Feb, 8(2), 945 - 54
Comparison of the structure and cell cycle expression of mRNAs encoded by two histone H3-H4 loci in Saccharomyces cerevisiae; Cross SL et al.; The haploid genome of Saccharomyces cerevisiae contains two nonallelic sets of histone H3 and H4 gene pairs, termed the copy I and copy II loci . The structures of the mRNA transcripts from each of these four genes were examined by nuclease protection and primer extension mapping . For each gene, several species of mRNAs were identified that differed in the lengths of their 5' and 3' untranslated regions . The cell cycle accumulation pattern of the H3 and H4 mRNAs was determined in cells from early-exponential-growth cultures fractionated by centrifugal elutriation . The RNA transcripts from all four genes were regulated with the cell division cycle, and transcripts from the nonallelic gene copies showed tight temporal coordination . Cell cycle regulation did not depend on selection of a particular histone mRNA transcript since the ratio of the multiple species from each gene remained the same across the division cycle . Quantitative measurements showed significant differences in the amounts of mRNA expressed from the two nonallelic gene sets . The mRNAs from the copy II H3 and H4 genes were five to seven times more abundant than the mRNAs from the copy I genes . There was no dosage compensation in the steady-state levels of mRNA when either set of genes was deleted . In particular, there was no increase in the amount of copy I H3 or H4 transcripts in cells in which the high-abundance copy II genes were deleted.

Mol Cell Biol, 1988 Feb, 8(2), 938 - 44
Suppression of chromosomal mutations affecting M1 virus replication in Saccharomyces cerevisiae by a variant of a viral RNA segment (L-A) that encodes coat protein; Uemura H et al.; For the maintenance of "killer" M1 double-stranded RNA in Saccharomyces cerevisiae, more than 30 chromosomal genes are required . The requirement for some of these genes can be completely suppressed by a cytoplasmic element, {B} (for bypass) . We have isolated a mutant unable to maintain {B} (mab) and found that it is allelic to MAK10, one of the three chromosomal MAK genes required for the maintenance of L-A . The heat curing of {B} always coincided with the loss of L-A . To confirm that {B} is located on L-A, we purified viral particles containing either L-A or M1 from strains with or without {B} activity and transfected these purified particles into a strain which did not have either L-A or M1 . The transfectants harboring L-A and M1 from a {B} strain showed the {B} phenotype, but the transfectants with L-A and M1 from a {B-o} strain did not show the {B} phenotype . Furthermore, the transfectants having L-A from a {B} strain and M1 from a {B-o} strain also showed the {B} phenotype . Therefore, we concluded that {B} is a property of a variant of L-A . In the transfection experiment, we also proved that the superkiller phenotype of the {B} strain is a property of L-A and that L-A with {B} activity can maintain a higher copy number of M1 regardless of the source of M1 viruslike particles . These data suggest that MAK genes whose mutations are suppressed by {B} are concerned with the protection of M1 (+) single-stranded RNA or the formation of M1 viruslike particles and that an L-A with more efficient production of M1 viruslike particles can completely dispense with the requirement for those MAK genes.

Mol Cell Biol, 1988 Feb, 8(2), 912 - 22
The SPS100 gene of Saccharomyces cerevisiae is activated late in the sporulation process and contributes to spore wall maturation; Law DT et al.; We previously described the use of a differential hybridization screen of a genomic DNA library of Saccharomyces cerevisiae to identify sporulation-specific (SPS) genes (A . Percival-Smith and J . Segall, Mol . Cell . Biol . 4:142-150, 1984) . This initial screen identified 14 SPS genes that are first expressed 6 to 8 h after transfer of cells to sporulation medium . Accumulation of transcripts corresponding to these genes becomes maximal at 8 to 12 h of sporulation, the time at which meiotic events are nearing completion, and by 15 h of sporulation, transcript levels are beginning to decrease . In the present study two additional SPS genes, first expressed at 12 h of sporulation, were isolated . The steady-state level of transcripts corresponding to these two genes, termed SPS100 and SPS101, remains unchanged from 15 to 35 h, a time coincident with spore wall maturation . The nature of the putative 34.2-kilodalton protein encoded by the SPS100 gene is consistent with its being a component of the glycoprotein matrix of the spore wall; the protein contains a potential signal sequence and cleavage site and numerous sites for potential glycosylation . A MATa sps100/MAT alpha sps100 strain was found to be indistinguishable from the wild-type strain when assessed for efficiency of ascus formation and spore viability . However, a more detailed analysis of the mutant strain revealed that the SPS100 gene product serves a protective role during the early stages of spore wall formation . The time at which resistance to ether could first be detected in developing spores was delayed by 5 h in the mutant strain relative to the wild-type strain . This phenotype is presumably a reflection of a defect in spore wall maturation . This study has confirmed that temporally distinct classes of sporulation-specific genes are sequentially activated during the process of meiosis and spore formation and has shown that the SPS100 gene, identified on the basis of its developmental-specific expression pattern, contributes to spore development.

Mol Cell Biol, 1988 Feb, 8(2), 664 - 73
ATR1, a Saccharomyces cerevisiae gene encoding a transmembrane protein required for aminotriazole resistance; Kanazawa S et al.; In Saccharomyces cerevisiae, 3-amino-1,2,4-triazole (aminotriazole) competitively inhibits the activity of imidazoleglycerolphosphate dehydratase, the product of the HIS3 gene . Wild-type strains are able to grow in the presence of 10 mM aminotriazole because they induce the level of imidazoleglycerolphosphate dehydratase . However, strains containing gcn4 mutations are unable to grow in medium containing aminotriazole because they lack the GCN4 transcriptional activator protein necessary for the coordinate induction of HIS3 and other amino acid biosynthetic genes . Here, we isolated a new gene, designated ATR1, which when present in multiple copies per cell allowed gcn4 mutant strains to grow in the presence of aminotriazole . In wild-type strains, multiple copies of ATR1 permitted growth at extremely high concentrations of aminotriazole (80 mM), whereas a chromosomal deletion of ATR1 caused growth inhibition at very low concentrations (5 mM) . When radioactive aminotriazole was added exogenously, cells with multiple copies of ATR1 accumulated less aminotriazole than wild-type cells, whereas cells with the atr1 deletion mutation retained more aminotriazole . Unlike the mammalian mdr or yeast PDR genes that confer resistance to many drugs, ATR1 appears to confer resistance only to aminotriazole . Genetic analysis, mRNA mapping, and DNA sequencing revealed that (i) the primary translation product of ATR1 contains 547 amino acids, (ii) ATR1 transcription is induced by aminotriazole, and (iii) the ATR1 promoter region contains a binding site for the GCN4 activator protein . The deduced amino acid sequence suggests that ATR1 protein is very hydrophobic with many membrane-spanning regions, has several potential glycosylation sites, and may contain an ATP-binding site . We suggest that ATR1 encodes a membrane-associated component of the machinery responsible for pumping aminotriazole (and possibly other toxic compounds) out of the cell.

Mutat Res, 1988 Feb, 204(2), 239 - 49
Mitotic crossing-over by anticancer drugs in Saccharomyces cerevisiae strain D5; Ferguson LR et al.; Treatment with an anticancer drug causing mitotic crossing-over could lead to expression of recessive genes, previously masked in a heterozygote . Used clinically, such drugs might cause an increased risk of cancer in cases of familial tumours, such as Wilm's tumour or retinoblastoma . Potentially, novel forms of drug resistance could also be unmasked by such a recombinogenic event . We have estimated the extent of this potential problem in current clinical drugs by comparing a range of antitumour agents for ability to cause mitotic crossing-over in Saccharomyces cerevisiae strain D5 . We have compared these data with ability to cause an increase in total aberrant colonies in the same experiments . Although many of the agents known to cause point mutation also have some ability for mitotic crossing-over, there are also point mutagens which have little recombinogenic potential . Conversely, some effective recombinogens appear to be either very specific or rather ineffective point mutagens . Although the most generally effective agents in the present experiments were alkylating agents, several other types of drug including DNA-cutting agents, topoisomerase inhibitors, other DNA-binding drugs and antimetabolites may stimulate mitotic crossing-over . None of the mitotic inhibitors or the DNA minor groove binding drugs tested caused recombinogenic events . It would seem that the ability to induce mitotic crossing-over is an important endpoint in its own right . Assays for this event might provide an important complement to other assays commonly required for registration of new pharmaceuticals.

Proc Natl Acad Sci U S A, 1988 Feb, 85(3), 743 - 6
Specific interaction between a Saccharomyces cerevisiae protein and a DNA element associated with certain autonomously replicating sequences; Eisenberg S et al.; We have isolated a protein from Saccharomyces cerevisiae that binds specifically to a nucleotide sequence associated with the autonomously replicating sequence (ARS) ARS120, located in the telomeric region of a yeast chromosome . "Footprinting" analysis revealed that a 26-base-pair DNA sequence, 5'-CAAGTGCCGTGCATAATGATGTGGGT-3', was protected by this protein from DNase I digestion . A plasmid containing 48 direct tandem repeats of this oligonucleotide was constructed and used to affinity-purify the binding activity . The purified protein, OBF1 (origin binding factor), showed specific binding to ARS120 . The 26-base-pair OBF1-protected sequence was sufficient for the recognition and binding of the protein, since the mobility of a DNA fragment containing the synthetic binding site was retarded in agarose gels when incubated with OBF1 . By performing competition experiments with a number of different ARSs, we showed that OBF1 binds tightly to some but not all ARSs . Interestingly, OBF1 does not appear to have a discernible affinity for ARS1 or the ARSs associated with mating type loci, HML alpha and HMRa, which are substrates for a DNA-binding activity reported by others . Since OBF1 appears to bind to DNA associated with a number of ARSs, we suggest that this protein may have a function related to ARS activity, perhaps in the initiation of DNA replication at selected ARSs.

Mol Cell Biol, 1988 Feb, 8(2), 822 - 7
SPT3 is required for normal levels of a-factor and alpha-factor expression in Saccharomyces cerevisiae; Hirschhorn JN et al.; Mutations in the Saccharomyces cerevisiae SPT3 gene were previously found to cause suppression of Ty and delta insertion mutations in 5'-noncoding regions of genes . This suppression likely results from the fact that SPT3 is required for transcription initiation in delta sequences . Other additional phenotypes of spt3 mutants, including a mating defect, suggest that SPT3 is required for normal levels of expression of other genes . We analyzed the mating defect in spt3 mutants and showed that the levels of transcripts of the three major mating pheromone genes, MF alpha 1, MFa1, MFa2, were all reduced . The reduction in expression of these genes in spt3 mutants was not due to expression of a silent mating type cassette . Furthermore, we showed that the spt3 mating defect was manifest at the levels of both cellular fusion and nuclear fusion.

Mol Gen Genet, 1988 Feb, 211(2), 260 - 5
Genetic characterization of hyperresistance to formaldehyde and 4-nitroquinoline-N-oxide in the yeast Saccharomyces cerevisiae; Mack M et al.; The hyperresistance to 4-nitroquinoline-N-oxide (4-NQO) and formaldehyde (FA) of yeast strains transformed with the multi-copy plasmids pAR172 and pAR184, respectively, is due to the two genes, SNQ and SFA, which are present on these plasmids . Restriction analysis revealed the maximal size of SFA as 2.7 kb and of SNQ as 2.2 kb, including transcription control elements . The presence of the smallest 2.7 kb subclone carrying SFA increased hyperresistance to formaldehyde fivefold over that of the original pAR184 isolate . No such increase in hyperresistance to 4-NQO was seen with the smaller subclones of the pAR172 isolate . Disruption of the SFA gene led to a threefold increase in sensitivity to FA as compared with the wild type . Expression of gene SNQ introduced on a multi-copy vector into haploid yeast mutants rad2, rad3, and snm1 did not complement these mutations that block excision repair.

Microbiologia, 1988 Feb, 4(1), 61 - 4
{Distribution of potassium and sodium in the vacuole and cytoplasm of Saccharomyces cerevisiae}; Ortega MD; In Na+ grown yeast cells the vacuole is the main Na+ reservoir, maintaining higher Na+/K+ ratios than those in the cytoplasm . This asymmetric distribution may enhance Na+ tolerance . However, in growing cells the effect is of low significance.

J Gen Microbiol, 1988 Feb, 134 ( Pt 2), 333 - 7
A study of the role of the hexose monophosphate pathway with respect to fatty acid biosynthesis in sporulation of Saccharomyces cerevisiae; Dickinson JR et al.; 13C NMR was used to study the pattern of label incorporation from {2-13C}acetate into trehalose during sporulation in Saccharomyces cerevisiae . A wild-type strain and a strain homozygous for the zwf1 mutation (which affects glucose-6-phosphate dehydrogenase) were used . In the wild-type it was possible to deduce the cycling of glucose 6-phosphate around the hexose monophosphate pathway whilst in the mutant strain this did not occur . The requirement of the hexose monophosphate pathway for providing NADPH for fatty acid biosynthesis was examined using 13C NMR and GC/MS . The wild-type strain produced a typical profile of fatty acids with palmitoleic acid being the most abundant whereas the mutant contained only one-quarter the amount of total fatty acid . As zwf1 homozygous diploids are able to sporulate this indicates that the large amount of fatty acid biosynthesis observed in sporulation of wild-type strains is not essential to the process.

Curr Genet, 1988 Feb, 13(2), 113 - 24
Arginine repression of the Saccharomyces cerevisiae ARG1 gene . Comparison of the ARG1 and ARG3 control regions; Crabeel M et al.; The Saccharomyces cerevisiae ARG1 gene coding for argininosuccinate synthetase has been isolated and the nucleotide sequence of both its control region and of its amino terminal end coding region determined . The startpoint of transcription was established by S1-mapping and reverse transcriptase procedures . Northern blot hybridizations showed that whereas arginine-specific repression reduced the enzyme activity fivefold, it did not reduce the steady state level of the corresponding messenger in proportion; by analogy with the coregulated ARG3 gene, this result suggests a post-transcriptional regulatory mechanism . In contrast, proportionally between enzyme activity and mRNA content was observed under conditions where general amino acid control (known to be transcriptional) was operating . Comparing the 5' untranscribed domains of ARG1 and ARG3 revealed a first region of homology between the TATA box and the transcription startpoint . In this region a 10 bp (ARG3) or 11 bp (ARG1) central box is flanked by two segments which, by mutation, have been shown to be part of the ARG operator (Crabeel et al . 1985) . The repressor is assumed to bind at this primary target site prior to establishing contacts with the proximal part of the nascent mRNA molecule (Crabeel et al . 1985) . By in vitro directed deletion mutagenesis we show that the central conserved box of ARG3 is not essential for arginine-specific repression to occur . Another region of homology was found in the leader part of the messenger RNA; deletion of this region causes a small reduction in ARG3 expression but also does not alter regulation . Neither of these two regions are thus part of the primary repressor target site . In addition, in terms of post-transcriptional regulation, the latter result indicates that no sequence specificity is required in the RNA recognition step.

J Bacteriol, 1988 Feb, 170(2), 708 - 13
Regulation of nitrogen assimilation in Saccharomyces cerevisiae: roles of the URE2 and GLN3 genes; Courchesne WE et al.; Mutations in the GLN3 gene prevented a normal increase in the NAD-glutamate dehydrogenase and glutamine synthetase levels in glutamate-grown Saccharomyces cerevisiae cells, whereas mutations in the URE2 gene resulted in high levels of these enzymes in glumate- and glutamine-grown cells . A ure2 gln3 double mutant had low levels of glutamate dehydrogenase and glutamine synthetase in cells grown on glutamate and glutamine; thus, gln3 mutations were epistatic to the ure2 mutations . The results suggest that the GLN3 product is capable of promoting increases in enzyme levels in the absence of a functional URE2 product and that the URE2 product antagonizes the GLN3 product . The URE2 and GLN3 genes were also found to regulate the level of arginase activity . This regulation is completely independent of the regulation of arginase by substrate induction . The activities of glutamate dehydrogenase, glutamine synthetase, and arginase were higher in cells grown on glutamate as the nitrogen source than they were in cells grown under a nitrogen-limiting condition . It had previously been shown that the levels of these enzymes can be increased by glutamine deprivation . We propose that the URE2-GLN3 system regulates enzyme synthesis, in response to glutamine and glutamate, to adjust the intracellular concentration of ammonia so as to maintain glutamine at the level required for optimal growth.

Genetics, 1988 Feb, 118(2), 203 - 12
Isolation and analysis of a novel class of suppressor of Ty insertion mutations in Saccharomyces cerevisiae; Fassler JS et al.; Using a new scheme for the isolation of suppressor of Ty insertion mutations (spt mutations) in yeast, we have identified six new SPT genes . Mutations in two of these genes, SPT13 and SPT14, exhibit a novel suppression pattern: suppression of complete Ty insertion mutations, but not of solo delta insertion mutations . Transcriptional analysis shows that spt13- and spt14-mediated suppression of Ty insertion mutations is the result of an elevation in the levels of adjacent gene transcription . In spite of the failure of these mutations to suppress solo delta insertion mutations, they do cause changes in transcription of at least one solo delta insertion mutation . In addition, spt13 and spt14 mutations are epistatic to mutations in certain other SPT genes that do suppress solo delta insertion mutations . These results suggest that the SPT13 and SPT14 gene products may act via sequences in both the delta and epsilon regions of Ty elements . Finally, mutations in SPT13 cause sporulation and mating defects and SPT14 is essential for growth, suggesting that these two genes have important roles in general cellular functions.

Mol Cell Biol, 1988 Feb, 8(2), 875 - 83
Nematode repetitive DNA with ARS and segregation function in Saccharomyces cerevisiae; Felsenstein KM et al.; Several members of a repetitive DNA family in the nematode Caenorhabditis elegans have been shown to express ARS and centromeric function in Saccharomyces cerevisiae . The repetitive family, denoted CeRep3, consists of dispersed repeated elements about 1 kilobase in length, present 50 to 100 times in the nematode genome . Three elements were sequenced and found to contain DNA sequences homologous to yeast ARS and CEN consensus sequences . Nematode DNA segments containing these repeats were tested for ARS and CEN (or SEG) function after ligation to shuttle vectors and introduction into yeast cells . Such nematode segments conferred ARS function to the plasmid, as judged by an increased frequency of transformation compared with control plasmids without ARS function . Some, but not all, also conferred to the plasmid increased mitotic stability, increased frequency of 2+:2- segregation in meiosis, and decreased plasmid copy number . These effects are similar to those of yeast centromeric DNA . In view of these results, we suggest that the CeRep3 repetitive family may have replication and centromeric functions in C . elegans.

Mol Cell Biol, 1988 Feb, 8(2), 655 - 63
The HAP3 regulatory locus of Saccharomyces cerevisiae encodes divergent overlapping transcripts; Hahn S et al.; Activation of the CYC1 upstream activation site, UAS2, and transcription of several other genes encoding respiratory functions requires the product of the regulatory gene HAP2 . We report here the isolation and characterization of a second UAS2 regulatory gene, HAP3 . Like mutations in HAP2, a mutation in HAP3 abolishes the activity of UAS2 and prevents growth on nonfermentable carbon sources . The HAP3 gene was cloned and, surprisingly, was found to encode two divergently transcribed, overlapping transcripts: a 570-base RNA and a 3-kilobase (kb) RNA . Chromosomal disruption experiments defined the critical region for HAP3 function to a 1.3-kb segment in which the two transcripts overlap . Analysis of the HAP3 DNA sequence showed that the 570-base transcript could encode a protein of 144 amino acids . Synthesis of the 144-amino-acid protein under regulatory control in vivo demonstrated that this protein is essential for activity of UAS2 as well as for growth on nonfermentable carbon sources . The largest open reading frame in the critical region of the 3-kb transcript is only 86 amino acids . Using site-directed mutagenesis, we demonstrated that the 86-amino-acid open reading frame was not involved in UAS2 activity . The possible role of this 3-kb antisense RNA in HAP3 expression or function is discussed.

Mol Cell Biol, 1988 Feb, 8(2), 647 - 54
Mutational analysis of upstream activation sequence 2 of the CYC1 gene of Saccharomyces cerevisiae: a HAP2-HAP3-responsive site; Forsburg SL et al.; We analyzed upstream activation sequence 2 (UAS2), one of two independent UAS elements in the CYC1 gene of Saccharomyces cerevisiae . Deletions and linker scanning mutations across the 87 base pairs previously defined as UAS2 showed two separate functional elements required for full activity . Region 1, from -230 to -200, contains the principal activation site and responds to the trans-acting regulatory loci HAP2 and HAP3 . A portion of region 1 is homologous to two other HAP2-HAP3-responsive UASs and includes the G----A transition mutation UP1, which increases UAS2 activity . This consensus sequence TNATTGGT bears striking similarity to several CAAT box sequences of higher cells . Region 2, from -192 to -178, substantially enhances the activity of region 1, yet has little activity by itself . These regions bind distinct proteins found in crudely fractionated yeast extracts.

J Bacteriol, 1988 Feb, 170(2), 828 - 33
Regulation of phosphatidylinositol kinase activity in Saccharomyces cerevisiae; Holland KM et al.; The effects of growth phase and carbon source on membrane-associated phosphatidylinositol kinase in cell extracts of Saccharomyces cerevisiae were examined . Phosphatidylinositol kinase activity increased 2- and 2.5-fold in glucose- and glycerol-grown cells, respectively, in the stationary phase as compared with the exponential phase of growth . The increase in phosphatidylinositol kinase activity in the stationary phase of growth correlated with an increase in the relative amounts of phosphatidylinositol 4-phosphate, the product of the reaction . The increase in phosphatidylinositol kinase activity was not due to the presence of water-soluble effector molecules in cell extracts as indicated by mixing experiments . Phosphatidylinositol kinase activity decreased in cell extracts of exponential-phase cells preincubated under phosphorylation conditions which favor cyclic AMP-dependent protein kinase activity . Phosphatidylinositol kinase activity was not affected in cell extracts of stationary-phase cells preincubated under phosphorylation conditions.

Biophys J, 1988 Feb, 53(2), 193 - 203
Ionophore properties of a synthetic alpha-helical transmembrane fragment of the mitochondrial H+ ATP synthetase of Saccharomyces cerevisiae . Comparison with alamethicin; Molle G et al.; A 22-amino acid polypeptide was synthesized to model the central transmembrane segment of subunit 8 of the H+ ATP synthetase of Saccharomyces cerevisiae and to test ionophore properties . Solid-phase synthesis was conducted on benzhydrilamino resin, and purification followed by high pressure liquid chromatography allowed the isolation of the pure product whose NH2 terminal was acetylated and whose molecular weight determined by Fast Atomic Bombardment was the expected 2,666 . The infrared spectrum of this peptide in the solid state reveals a fully alpha-helical conformation, whereas in low dielectric constant solvents the alpha-helical content is 60%, as determined by circular dichroism studies . Macroscopic current-voltage curves displayed by different planar lipid bilayers (monomyristoleoyl-glycerol and phosphatidylethanolamine) doped with this peptide suggest a weakly voltage-dependent conductance . Only one conductance level is observed in any given single-channel conductance experiment . However, a series of experiments shows a distribution of conductance states, most often 440 or 3,000 pS, and occasionally 80, 1,200, or 6,500 pS . This behavior contrasts with the usual behavior of alamethicin, chosen as a model of "aggregating-helices" ionophore and whose conductance fluctuates continually between substates, through uptake and release of monomers . Nevertheless, alamethicin too can display, under certain conditions, long-lived and mono-level conductance states similar to those reported here for the newly synthesized peptide . These properties could possibly be explained by the formation of large domains of helical rods with a set of allowed and independent ionic pathways.

Biochim Biophys Acta, 1988 Jan 29, 952(2), 238 - 43
The binding of glucose and nucleotides to hexokinase from Saccharomyces cerevisiae; Woolfitt AR et al.; The binding of glucose, ADP and AdoPP{NH}P, to the native PII dimer and PII monomer and the proteolytically-modified SII monomer of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from Saccharomyces cerevisiae was monitored at pH 6.7 by the concomitant quenching of protein fluorescence . The data were analysed in terms of Qmax, the maximal quenching of fluorescence at saturating concentrations of ligand, and {L}0.5, the concentration of ligand at half-maximal quenching . No changes in fluorescence were observed with free enzyme and nucleotide alone . In the presence of saturating levels of glucose, Qmax induced by nucleotide was between 2 and 7%, and {L}0.5 was between 0.12 and 0.56 mM, depending on the nucleotide and enzyme species . Qmax induced by glucose alone was between 22 and 25%, while {L}0.5 was approx . 0.4 mM for either of the monomeric hexokinase forms and 3.4 for PII dimer . In the presence of 6 mM ADP or 2 mM AdoPP{NH}P, Qmax for glucose was increased by up to 4% and {L}0.5 was diminished 3-fold for hexokinase PII monomer, 6-fold for SII monomer, and 15-fold for PII dimer . The results are interpreted in terms of nucleotide-induced conformational change of hexokinase in the presence of glucose and synergistic binding interactions between glucose and nucleotide.

Biochem Biophys Res Commun, 1988 Jan 29, 150(2), 794 - 801
Human lysozyme: sequencing of a cDNA, and expression and secretion by Saccharomyces cerevisiae; Yoshimura K et al.; A cDNA encoding human lysozyme was isolated from a human placenta cDNA library . The cDNA was 1.5 kb in size and coded for a signal peptide consisting of 18 amino acids and mature lysozyme . The amino acid sequence of the mature lysozyme, deduced from the nucleotide sequence, was identical with the published sequence . In the 3'-noncoding region of the cDNA, an Alu sequence was found in the reverse orientation . In a protein coding region, the human lysozyme cDNA shows 60.1% and 51.3% similarity with chicken lysozyme and human alpha-lactalbumin cDNAs, respectively . When the cDNA was expressed in Saccharomyces cerevisiae, an active and correctly processed human lysozyme was secreted efficiently into the culture medium.

Eur J Biochem, 1988 Jan 15, 171(1-2), 417 - 24
Primary structure of the uracil transport protein of Saccharomyces cerevisiae; Jund R et al.; We present in this paper the nucleotidic sequence of the FUR4 gene encoding the uracil permease in the yeast Saccharomyces cerevisiae . The deduced amino acid sequence of the permease has 633 residues; it consists of many hydrophobic stretches, only the N-terminal and C-terminal ends of the protein (about 100 and 50 amino acids respectively) being mostly hydrophilic . No N-terminal hydrophobic signal peptide is present, although it is shown in this work that the biosynthesis of the uracil permease goes through the secretion/glycosylation pathway . Using the results of three different methods, allowing the prediction of transmembrane alpha helices in proteic sequences, we drew a model of folding of the permease in the membrane.

J Biol Chem, 1988 Jan 15, 263(2), 917 - 24
DNA polymerase III from Saccharomyces cerevisiae . I . Purification and characterization; Bauer GA et al.; Yeast cells from a wild type or protease-deficient strain were lysed in the absence or presence of protease inhibitors and the extracts analyzed by analytical high pressure liquid chromatography on diethylaminoethyl silica gel . Conditions that inhibited protease action caused elution of a novel DNA polymerase activity at a position in the gradient distinct from the elution positions of both DNA polymerase I and II . In large scale purifications, this DNA polymerase, called DNA polymerase III, copurified with a single-stranded DNA dependent 3'-5' exonuclease activity, exonuclease III, to near homogeneity . Glycerol gradient centrifugation partially dissociated the complex to yield two peaks of exonuclease III activity, one at 7.7 S together with the DNA polymerase, and one at 4.0 S without polymerase activity . Gel filtration indicated that the complex has a molecular mass greater than 400 kDa . Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the complex consists of several subunits: 140, 62, 55, and 53 kilodaltons, some of which may be proteolysis products . The exonuclease component of the complex can excise single nucleotide mismatches providing a base-paired primer-template which can be elongated by the DNA polymerase . Under replication conditions, the complex exhibits a measurable turnover rate of dTTP to dTMP and it contains no primase activity . The enzymatic activities of the 3'-5' exonuclease are consistent with a proofreading function during in vivo DNA replication . A second exonuclease activity, exonuclease IV, separated from the complex late in the purification scheme . It degrades both single-stranded and double-stranded DNA in the 5'----3' direction.

Eur J Biochem, 1988 Jan 15, 171(1-2), 171 - 6
Control of enzyme synthesis in the lysine biosynthetic pathway of Saccharomyces cerevisiae . Evidence for a regulatory role of gene LYS14; Ramos F et al.; Several enzymes of the lysine pathway of Saccharomyces cerevisiae were found to respond to an induction mechanism mediated by the product of gene LYS14 in the presence of 2-aminoadipate semialdehyde, an intermediate of this pathway . This novel regulatory mechanism appears independent of the specific repression by lysine and of the general control of amino acid biosynthesis . Genes LYS1, LYS9 and LYS14 have been cloned and their DNAs used to assay the corresponding messenger RNAs . The results suggest that the induction mechanism, as well as the specific and general regulations, operate at the transcriptional level . The synthesis of saccharopine dehydrogenase (glutamate-forming), previously shown to require the unlinked genes LYS9 and LYS14, is also affected by the induction mechanism . The leaky auxotrophic behaviour of lys14 mutants is explained by the low basal level of expression of LYS9, the structural gene of this enzyme, in the absence of induction by 2-aminoadipate semialdehyde.

J Biol Chem, 1988 Jan 15, 263(2), 925 - 30
DNA polymerase III from Saccharomyces cerevisiae . II . Inhibitor studies and comparison with DNA polymerases I and II; Burgers PM et al.; The newly identified yeast DNA polymerase III was compared to DNA polymerases I and II and the mitochondrial DNA polymerase . Inhibition by aphidicolin (I50) of DNA polymerases I, II, and III was 4, 6, and 0.6 micrograms/ml, respectively . The mitochondrial enzyme was insensitive to the drug . N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate strongly inhibited DNA polymerase I (I50 = 0.3 microM), whereas DNA polymerase III was less sensitive (I50 = 80 microM) . Conditions that allowed proteolysis to proceed during the preparation of extracts converted DNA polymerase II from a sensitive form (I50 = 2.4 microM) to a resistant form (I50 = 2 mM) . The mitochondrial DNA polymerase is insensitive (I50 greater than 5 mM) . With most other inhibitors tested (N-ethylmaleimide, heparin, salt) only small differences were observed between the three nuclear DNA polymerases . Polyclonal antibodies to DNA polymerase III did not inhibit DNA polymerases I and II, nor were those polymerases recognized by Western blotting . Monoclonal antibodies to DNA polymerase I did not crossreact with DNA polymerases II and III . The results show that DNA polymerase III is distinct from DNA polymerase I and II.

Biochim Biophys Acta, 1988 Jan 13, 937(1), 81 - 7
The influence of ATP on sugar uptake mediated by the constitutive glucose carrier of Saccharomyces cerevisiae; Schuddemat J et al.; The glucose carrier of Saccharomyces cerevisiae transports the phosphorylatable sugars glucose, mannose, fructose and 2-deoxy-D-glucose (2-dGlc) and the non-phosphorylatable sugar 6-deoxy-D-glucose (6-dGlc) . Reduction of the ATP concentration by, for example, incubating cells with antimycin A, results in a decrease in uptake of 2-dGlc and fructose . These uptake velocities can be increased again by raising the ATP level . These results establish a role of ATP in sugar transport . Transport of glucose and mannose is less affected by changes in the ATP concentration than 2-dGlc and fructose uptake, while the 6-dGlc transport is independent of the amount of ATP in the cells . Also, reduction of the kinase activity by incubation with xylose diminished transport of 2-dGlc and fructose, while the uptake of glucose and mannose remained unchanged . It is discussed that these results are due to transport-associated phosphorylation with ATP as substrate and the hexokinases and the glucokinase as phosphorylating enzymes.

J Biol Chem, 1988 Jan 5, 263(1), 454 - 60
Replicase of L-A virus-like particles of Saccharomyces cerevisiae . In vitro conversion of exogenous L-A and M1 single-stranded RNAs to double-stranded form; Fujimura T et al.; Virus-like particles that contain L-A double-stranded RNA are known to have transcriptase activity whose product is L-A single-stranded plus RNA . In low salt conditions, these particles release their double-stranded RNA and can then use added plus L-A or plus M1 single-stranded RNAs as templates to synthesize their respective double-stranded RNAs . The reaction requires dialyzed L-A virus-like particles as the source of the enzyme, a partially purified cell extract (host factor(s)), added single-stranded RNA as a template, and polyethylene glycol 6000, along with four NTPs . Crude host factor extracts prepared from mak3 or mak10ta mutants also support the reaction as effectively as that from a wild type strain, while a crude extract prepared from a pet18 mutant grown under the nonpermissive conditions is less effective . Template specificity of the in vitro reaction is the same as that expected for the enzyme reaction in vivo . Plus L-A and plus M1 single-stranded RNAs, but not 18 S rRNA, are converted to their respective double-stranded RNAs with net RNA synthesis . The newly synthesized strand of M1 double-stranded RNA is a full-length minus strand . This demonstration of replicase activity in the mature L-A virus-like particles which contain L-A double-stranded RNA is consistent with our previous L-A double-stranded RNA replication model; the difference between the mature L-A virus-like particles and L-A double-stranded RNA-synthesizing particles (expected to be replication intermediates in vivo) is just that the former contain L-A double-stranded RNA, while the latter contain L-A plus single-stranded RNA.

J Biol Chem, 1988 Jan 5, 263(1), 45 - 51
Characterization and function of catalytic subunit alpha of H+-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae . A study with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; Uchida E et al.; Subunit alpha (Mr 89,000) from vacuolar membrane H+-translocating adenosine triphosphatase of the yeast Saccharomyces cerevisiae was found to bind 8-azido{alpha-32P}adenosine triphosphate . Labeling by this photosensitive ATP derivative was saturable with an apparent dissociation constant of 10(-6) to 10(-5) M and decreased in the presence of ATP and ADP . The enzyme was inactivated by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), with about 1 microM causing half-maximal inactivation in the neutral pH range . This inactivation was prevented by the presence of ATP, ADP, or adenosyl-5'-yl imidodiphosphate (AMP-PNP) . The original activity was restored by treating the inactivated enzyme with 2-mercaptoethanol . Kinetic and chemical studies of the inactivation showed that the activity was lost on chemical modification of a single tyrosine residue per molecule of the enzyme . When the enzyme was inactivated with {14C}NBD-Cl, subunit alpha was specifically labeled, and this labeling was completely prevented by the presence of ATP, GTP, ADP, or AMP-PNP . From these results, it was concluded that subunit alpha of yeast vacuolar H+-ATPase has a catalytic site that contains a single, essential tyrosine residue . The kinetics of single site hydrolysis of {gamma-32P}ATP (Grubmeyer, C., Cross, R . L., and Penefsky, H . S . (1982) J . Biol . Chem . 257, 12092-12100) indicated the formation of an enzyme-ATP complex and subsequent hydrolysis of bound ATP to ADP and Pi at the NBD-Cl-sensitive catalytic site . NBD-Cl inactivated the single site hydrolysis and inhibited the formation of an enzyme-ATP complex . Dicyclohexylcarbodiimide did not affect the single site hydrolysis, but inhibited the enzyme activity under steady-state conditions.

Genetics, 1988 Jan, 118(1), 41 - 7
UGA suppressors in Saccharomyces cerevisiae: allelism, action spectra and map positions; Ono BI et al.; Sixty independent UGA suppressors of Saccharomyces cerevisiae have been studied . They are dominant and are divided into 16 groups (loci) by recombination . Suppressors representing these loci are divided into two classes by action spectra; four in class 1 (a broad action spectrum) and 12 in class 2 (a narrow action spectrum) . Class 1 suppressors are less frequent in terms of not only total number but also number per locus than class 2 suppressors, indicating difference in either or both mutation frequency and selective pressure between suppressors of the two classes . Two of the class 1 suppressors, SUP152 and SUP161, do not recombine with SUP28 and SUP33, leucine-inserting UAA suppressors, respectively, indicating that they are mutations in genes coding for tRNA(Leu)UUA . Of the remaining two class 1 suppressors, SUP160 which causes lethality in the psi+ cytoplasm is mapped on chromosome XV very close to the centromere, and SUP165 on the right arm of chromosome XIV 44 cM distal to lys9 . Of the class 2 suppressors, ten do not recombine with one or another of previously known UGA suppressors . The remaining two class 2 suppressors, SUP154 and SUP155, are mapped on the left and right arms of chromosome VII, respectively.

Mol Biol (Mosk), 1988 Jan-Feb, 22(1), 224 - 30
{Comparative analysis of the structure of double-stranded RNA from killer strains of Saccharomyces cerevisiae}; Amosova OA et al.; Double-stranded RNAs (M and L molecules) of two strains of the killer system Saccharomyces cerevisiae M437 (wild type) and ski-5 (superkiller mutant) were studied by means of electron microscopy and high resolution thermal melting . The M molecules of the ski-5 mutant were by 100 b.p . shorter than those of M437 . L molecules were of the same length for both strains . Analysis of the differential melting curves of L molecules showed that L molecules differ significantly in their nucleotide sequences, whereas M molecules were practically identical . It was found that M molecules contained a long AU region: that of M molecules of M 437 was 170-180 b.p . long and contained almost no GC pairs, whereas the AU region of M molecules of the ski-5 mutant was three times shorter and contained GC pairs.

Environ Mol Mutagen, 1988, 11(4), 497 - 508
Induction of mitotic chromosome loss in the diploid yeast Saccharomyces cerevisiae D61.M by genotoxic carcinogens and tumor promoters; Albertini S et al.; Three genotoxic carcinogens and eight tumor promoters were tested for induction of aneuploidy, specifically chromosome loss, in Saccharomyces cerevisiae D61.M . This is a heterozygous diploid yeast strain that permits the scoring of segregants expressing three linked recessive markers (cyhR2, ade6, and leu1), two of which (ade6 and leu1) are located close to the centromere on opposite arms of chromosome VII . The centromere marker leu was routinely checked, and a positive control (bavistan) was run with every experiment . The three genotoxic carcinogens aflatoxin B1, benzo(a)pyrene, and 7,12-dimethylbenz(a)anthracene did not induce aneuploidy, independent of the presence or absence of an exogenous metabolic activation system (rat liver homogenate; S9) . Four of the eight tumor promoters tested induced chromosome loss but not mitotic recombination or mutation: cholic acid, lithocholic acid, phenobarbital, and saccharin . Diethylstilbestrol (DES) led to positive as well as to negative results in several independent experiments . In the case of the positive experiment, DES also induced putative recombinants . Three tumor promoters induced neither chromosome loss nor mitotic recombination: anthralin, 4,4'-dichloro-diphenyl-ethane (DDT) and gamma-hexachlorcyclohexane (lindane) . From our experiments it can be concluded that the hypothesis put forward by Parry et al . {Nature; 294:263-265}, according to which tumor promoters induce chromosome loss in yeast, is not correct in a general sense . In our set of eight tumor promoters, only one half distinctly induced chromosome loss.

Folia Microbiol (Praha), 1988, 33(1), 34 - 7
A rapid preparation of plasmid DNA from Saccharomyces cerevisiae; Brozmanova J et al.; A procedure for extraction of plasmid DNA from Saccharomyces cerevisiae is described . The plasmid DNA of interest is extracted together with 2-micron circular DNA naturally occurring in many yeast strains . Spheroplasts are lyzed at alkaline pH which denatures linear but not covalently closed circular (CCC) DNA . The CCC DNA is recovered by ethanol precipitation and can be detected by gel electrophoresis or used for routine bacterial transformation.

Curr Genet, 1988, 13(1), 37 - 40
Allelism between pso1-1 and rev3-1 mutants and between pso2-1 and snm1 mutants in Saccharomyces cerevisiae; Cassier-Chauvat C et al.; In the yeast Saccharomyces cerevisiae, allelism between the pso1-1 and the rev3-1 mutants on the one hand and the pso2-1 and snm1 mutants on the other, is demonstrated by the comparison of phenotypes, complementation tests and meiotic segregation analysis.

Curr Genet, 1988, 13(1), 21 - 3
A colony procedure for transformation of Saccharomyces cerevisiae; Keszenman-Pereyra D et al.; A rapid and simple yeast transformation procedure has been developed using colonies on agar plates . Saccharomyces cerevisiae SHY3 cells were picked up from colonies on YPD plates grown freshly or stored at 4 degrees C and incubated with M13RK9-T DNA at 30 degrees C for 1-2 h in a solution of Li+, Ca2+, Mg2+, triacetin and polyethylene glycol . About 3,500 transformants were obtained per microgram of double stranded M13RK9-T DNA . Unlike the existing spheroplast techniques, single stranded M13RK9-T DNA transformed intact cells below one-hundredth frequency of the duplex form.

Environ Mol Mutagen, 1988, 11(3), 323 - 31
Effect of treatment medium on induction of aneuploidy by nocodazole in Saccharomyces cerevisiae; Taylor-Mayer RE et al.; While studying ways to improve responsiveness of Saccharomyces cerevisiae strain D61.M to agents that induce aneuploidy, we noted that nocodazole, which strongly induces aneuploidy when yeast cells are treated in yeast extract-peptone-dextrose (YEPD) medium, had no effect when a synthetic complete (SC) medium was used . Further study revealed that the presence of peptone was necessary for induction . Other aneuploidy-inducing agents, including ethyl acetate, acetone, and methyl benzimidazole-2-yl-carbamate (MBC), were equally active in either medium . Benomyl, which degrades to MBC, was less active in SC than in YEPD medium.

Radiobiologiia, 1988 Jan-Feb, 28(1), 134 - 7
{Genetic effects of the decay of radionuclide products of nuclear fission in Saccharomyces cerevisiae cells . Lethal effects of the decay of 89Sr incorporated in cells of different ploidies and radiosensitivity}; Korolev VG et al.; Decay of 89Sr incorporated in yeast cells produces a pronounced inactivating effect . The transmutation mainly contributes (about 80%) to cell inactivation . Haploid cells are more sensitive to 89Sr disintegration than diploid and tetraploid ones . A radiosensitive mutant XRS2, that is particularly sensitive to the transmutation effect of radionuclides, has proved to be sensitive to 89Sr transmutation as well . At the same time, another radiosensitive mutant, rad 54, does not virtually differ from the wild-type strain by its sensitivity to 89Sr decay.

Mol Gen Genet, 1988 Jan, 211(1), 168 - 75
Regulation of the TRP4 gene of Saccharomyces cerevisiae at the transcriptional level and functional analysis of its promotor; Furter R et al.; The TRP4 gene of Saccharomyces cerevisiae, which encodes anthranilate phosphoribosyl transferase (E.C . 2.4.2.18), is subject to the general control of amino acid biosynthesis . The regulation takes place at the transcriptional level by increasing the amount of initiation and not by changing the stability of mRNA . We have observed a change in the utilization of TRP4 mRNA start sites, depending on whether cells were grown under repressing or derepressing conditions . The function of promoter elements has been tested by deletion analysis with a plasmid-encoded TRP4 gene . A routinely practicable method was used for copy-number calibration of plasmids based on 2 micron DNA . Promoter structures and spacing problems in the TRP4 promoter region are discussed.

Proc Natl Acad Sci U S A, 1988 Jan, 85(2), 534 - 8
Generation of telomere-length heterogeneity in Saccharomyces cerevisiae; Shampay J et al.; Chromosome ends in the lower eukaryotes terminate in variable numbers of tandem, simple DNA repeats . We tested predictions of a model in which these telomeric repeats provide a substrate for the addition of more repeats by a terminal transferase-like mechanism that, in concert with DNA polymerase and primase, effectively counterbalances the loss of DNA due to degradation or incomplete replication . For individual chromosome ends in yeast, the mean length of any given telomere was shown to vary between different clonal populations of the same strain and to be determined by the initial length of that telomere in the single cell giving rise to the clone . This type of variation was independent of the major yeast recombination pathway . The length heterogeneity at each telomeric end increased with additional rounds of cell division or DNA replication . Lengths of individual telomeres within a single clone varied independently of each other . Thus, this clonal variability is distinct from genetic regulation of chromosome length, which acts on all chromosome ends coordinately . These in vivo phenomena suggest that lengthening and shortening activities act on yeast telomeres during each round of replication.

Environ Mol Mutagen, 1988, 11(1), 31 - 40
Aneuploidy induction in Saccharomyces cerevisiae by two solvent compounds, 1-methyl-2-pyrrolidinone and 2-pyrrolidinone; Mayer VW et al.; A number of solvent compounds that were tested in Saccharomyces cerevisiae were potent inducers of aneuploidy, although they did not induce any other genetic effects . As an extention of these earlier findings, 1-methyl-2-pyrrolidinone was tested and was found to induce aneuploidy . Several structurally related compounds were also tested; 2-pyrrolidinone induced aneuploidy, but succinimide, pyrrolidine, 1-methylpyrrolidine, 1-methyl-3-pyrrolidinol, and 2-pyrrolidineethanol did not . Maleimide and its N-hydroxy, N-methyl, and N-ethyl derivatives were also negative for aneuploidy induction.

Mutat Res, 1988 Jan, 197(1), 59 - 66
Effects of excess thymidylate on thymidylate low-requiring strains of Saccharomyces cerevisiae: high mutagenicity and absence of DNA strand breaks; Bender E et al.; dTMP exposure concentrations of 0.1 mM or higher are genotoxic in exponentially growing cells of thymidylate low-requiring mutants of Saccharomyces cerevisiae . Mutagenicity of excess dTMP is highest in an exposure concentration 10-fold of that needed for external supplementation of endogenously blocked thymidylate synthesis . Still higher dTMP concentrations are primarily cytotoxic . The canavanine forward-mutation system shows excess dTMP to be as potent a mutagen as irradiation by ultraviolet light . Mutagenicity of excess dTMP, however, differs from that of direct DNA-attacking mutagens in that it is highest in the absence of significant toxicity . Alkaline sucrose gradient centrifugation shows that excess dTMP does not induce significant numbers of DNA single- or double-strand breaks, while conditions of thymidylate deprivation lead to DNA-strand breaks and thymineless death.

Mol Cell Biol, 1988 Jan, 8(1), 361 - 70
Saccharomyces cerevisiae SUP53 tRNA gene transcripts are processed by mammalian cell extracts in vitro but are not processed in vivo; Ganguly S et al.; We describe the results of our studies of expression of a Saccharomyces cerevisiae amber suppressor tRNA(Leu) gene (SUP53) in mammalian cells in vivo and in cell extracts in vitro . Parallel studies were carried out with the wild-type (Su-) tRNA(Leu) gene . Extracts from HeLa or CV1 cells transcribed both tRNA(Leu) genes . The transcripts were processed correctly at the 5' and 3' ends and accurately spliced to produce mature tRNA(Leu) . Surprisingly, when the same tRNA(Leu) genes were introduced into CV1 cells, only pre-tRNAs(Leu) were produced . The pre-tRNAs(Leu) made in vivo were of the same size and contained the 5'-leader and 3'-trailer sequences as did pre-tRNAs(Leu) made in vitro . Furthermore, the pre-tRNAs(Leu) made in vivo were processed to mature tRNA(Leu) when incubated with HeLa cell extracts . A tRNA(Leu) gene from which the intervening sequence had been removed yielded RNAs that also were not processed at either their 5' or 3' termini . Thus, processing of pre-tRNA(Leu) in CV1 cells is blocked at the level of 5'- and 3'-end maturation . One possible explanation of the discrepancy in the results obtained in vivo and in vitro is that tRNA biosynthesis in mammalian cells involves transport of pre-tRNA from the site of its synthesis to a site or sites where processing takes place, and perhaps the yeast pre-tRNAs(Leu) synthesized in CV1 cells are not transported to the appropriate site.

Mol Cell Biol, 1988 Jan, 8(1), 309 - 20
Identification of a DNA segment that is necessary and sufficient for alpha-specific gene control in Saccharomyces cerevisiae: implications for regulation of alpha-specific and a-specific genes; Jarvis EE et al.; STE3 mRNA is present only in Saccharomyces cerevisiae alpha cells, not in a or a/alpha cells, and the transcript level increases about fivefold when cells are treated with a-factor mating pheromone . Deletions in the 5' noncoding region of STE3 defined a 43-base-pair (bp) upstream activation sequence (UAS) that can impart both modes of regulation to a CYC1-lacZ fusion when substituted for the native CYC1 UAS . UAS activity required the alpha 1 product of MAT alpha, which is known to be required for transcription of alpha-specific genes . A chromosomal deletion that removed only 14 bp of the STE3 UAS reduced STE3 transcript levels 50- to 100-fold, indicating that the UAS is essential for expression . The STE3 UAS shares a 26-bp homology with the 5' noncoding sequences of the only other known alpha-specific genes, MF alpha 1 and MF alpha 2 . We view the homology as having two components--a nearly palindromic 16-bp "P box" and an adjacent 10-bp "Q box." A synthetic STE3 P box was inactive as a UAS; a perfect palindrome P box was active in all three cell types . We propose that the P box is the binding site for a transcription activator, but that alpha 1 acting via the Q box is required for this activator to bind to the imperfect P boxes of alpha-specific genes . Versions of the P box are also found upstream of a-specific genes, within the binding sites of the repressor alpha 2 encoded by MAT alpha . Thus, the products of MAT alpha may render gene expression alpha or a-specific by controlling access of the same transcription activator to its binding site, the P box.

Mol Cell Biol, 1988 Jan, 8(1), 210 - 25
Two DNA-binding factors recognize specific sequences at silencers, upstream activating sequences, autonomously replicating sequences, and telomeres in Saccharomyces cerevisiae; Buchman AR et al.; Two DNA-binding factors from Saccharomyces cerevisiae have been characterized, GRFI (general regulatory factor I) and ABFI (ARS-binding factor I), that recognize specific sequences within diverse genetic elements . GRFI bound to sequences at the negative regulatory elements (silencers) of the silent mating type loci HML E and HMR E and to the upstream activating sequence (UAS) required for transcription of the MAT alpha genes . A putative conserved UAS located at genes involved in translation (RPG box) was also recognized by GRFI . In addition, GRFI bound with high affinity to sequences with the (C1-3A)-repeat region at yeast telomeres . Binding sites for GRFI with the highest affinity appeared to be of the form 5'-(A/G)(A/C)ACCCANNCA(T/C)(T/C)-3', where N is any nucleotide . ABFI-binding sites were located next to autonomously replicating sequences (ARSs) at controlling elements of the silent mating type loci HMR E, HMR I, and HML I and were associated with ARS1, ARS2, and the 2 micron plasmid ARS . Two tandem ABFI binding sites were found between the HIS3 and DED1 genes, several kilobase pairs from any ARS, indicating that ABFI-binding sites are not restricted to ARSs . The sequences recognized by ABFI showed partial dyad-symmetry and appeared to be variations of the consensus 5'-TATCATTNNNNACGA-3' . GRFI and ABFI were both abundant DNA-binding factors and did not appear to be encoded by the SIR genes, whose products are required for repression of the silent mating type loci . Together, these results indicate that both GRFI and ABFI play multiple roles within the cell.

J Bacteriol, 1988 Jan, 170(1), 266 - 71
Structure and transcription of the allantoate permease gene (DAL5) from Saccharomyces cerevisiae; Rai R et al.; We determined the nucleotide sequence of the DAL5 gene, which encodes a component of the allantoate transport system . Translation of the sequence revealed that the DAL5 gene product is highly hydrophobic . It possesses an alternating motif of hydrophilic sequences that can potentially be folded into alpha-helices and hydrophobic sequences that can potentially be folded into beta-pleated sheets . These are expected characteristics of an integral membrane protein, which correlate well with DAL5 gene function . S1 protection fragments generated by DAL5 transcripts exhibited high heterogeneity over a 30-base-pair range . This pattern of fragments was not affected by growth conditions of the cells or the conditions of the assay.

J Mol Evol, 1988, 27(4), 341 - 50
Evolutionary relationship and secondary structure predictions in four transport proteins of Saccharomyces cerevisiae; Weber E et al.; The comparison of the amino acid sequences of four yeast transport proteins indicates that there is a questionable relatedness between the uracil permease (FUR4) and the purine-cytosine permease (FCY2), whereas the arginine permease (CAN1) and the histidine permease (HIP1) clearly originated from a common molecular ancestor . The analysis of the primary structure of these transport proteins by two methods of secondary structure predictions suggests the presence of 9-12 membrane-spanning alpha-helices in each polypeptide chain . These results are concordant in that 90% of the alpha-helices were determined by both methods to be at the same positions . In the aligned sequences HIP1 and CAN1, the postulated membrane-spanning alpha-helices often start at corresponding sites, even though the overall sequence similarity of the two proteins is only 30% . In the aligned DNA coding sequences of CAN1 and HIP1, synonymous nucleotide substitutions occur with very similar frequencies in regions where the replacement substitution (changing the amino acids) frequencies are widely different . Moreover, our data suggest that the replacement substitutions can be considered as neutral in the N-terminal segment, whereas the other regions are subject to a conservative selective pressure because, if compared to a random drift, the replacement substitutions are underrepresented.

Folia Microbiol (Praha), 1988, 33(4), 285 - 91
Uptake of L-lysine by a double mutant of Saccharomyces cerevisiae; Garcia JC et al.; A gap1 can1 mutant of Saccharomyces cerevisiae with a single lysine transport system remaining was used to study detailed kinetics of this transport . Its half-saturation constant was 78 mumol per litre, its maximum rate of transport was 0.29 mumol L-lysine per g dry matter per minute, both parameters being lower by more than an order of magnitude in comparison with the GAP system . The pH optimum lay at very acid values of about 3, the temperature dependence without any transition point showed an activation energy of 48 kJ/mol . The transport was inhibited by common metabolic inhibitors (3'-chlorophenylhydrazonomalononitrile, antimycin, 2-deoxy-D-glucose, sodium arsenate) as well as by a membrane-active one (uranyl nitrate) . The specificity of the system was extremely high, none of the natural amino acids acting as competitor to L-lysine . The maximum accumulation ratio attained (at about 5 mg dry matter per mL) was 100: 1-120: 1, in agreement with the measured protonmotive force under the assumption of 1 H+ ion being transported with 1 lysine molecule . The ratio decreased with increasing external concentration of lysine to as little as 4: 1 at 1 mmol lysine per litre . It also decreased with increasing suspension density and it was at extremely low suspension densities (0.2 mg dry matter per mL) that ratios of as much as 500: 1 were reached . Application of group-specific inhibitors showed that the active site of the carrier contains an essential histidine residue.

Folia Microbiol (Praha), 1988, 33(4), 281 - 4
Effect of ethanol on the specific transport system for L-lysine in Saccharomyces cerevisiae; Garcia JC et al.; Preincubation of resting cells of Saccharomyces cerevisiae double mutant can1 gap1 (with a single transport system for L-lysine) with metabolic substrates stimulated subsequent uptake of lysine . While in the wild type the stimulation is connected primarily with carrier protein synthesis (delayed, cycloheximide-inhibitable effect) in the mutant an immediate tapping of an energy source (antimycin-inhibited) is practically solely involved.

Biochem J, 1988 Jan 1, 249(1), 163 - 70
Purification and properties of an extracellular glucoamylase from a diastatic strain of Saccharomyces cerevisiae; Kleinman MJ et al.; The extracellular glucoamylase from certain strains of Saccharomyces cerevisiae can be purified from culture medium by a simple chromatographic procedure . The native enzyme is heavily glycosylated and has an Mr of about 250,000, but gel filtration indicates the existence of oligomers of larger size . Dissociation yields a form of Mr about 70,000 . The glucoamylase is rich in serine and threonine and in aspartic acid plus asparagine, and has a pI of 4.62 and a pH optimum of 4.5-6.5 . The thermostability and resistance to denaturants of the yeast enzyme is compared with those of two other fungal glucoamylases . Kinetic data for the yeast enzyme and a variety of substrates is presented; the enzyme is particularly ineffective in cleaving alpha-(1----6)-glycosidic bonds.

Proc Natl Acad Sci U S A, 1988 Jan, 85(1), 55 - 9
The Saccharomyces cerevisiae BAR1 gene encodes an exported protein with homology to pepsin; MacKay VL et al.; Saccharomyces cerevisiae a cells secrete an extracellular protein, called "barrier" activity, that acts as an antagonist of alpha factor, the peptide mating pheromone produced by mating-type alpha cells . We report here the DNA sequence of BAR1, the structural gene for barrier activity . The deduced primary translation product of 587 amino acids has a putative signal peptide, nine potential asparagine-linked glycosylation sites, and marked sequence similarity of the first two-thirds of the protein with pepsin-like proteases . Barrier activity was abolished by in vitro mutation of an aspartic acid predicted from this sequence homology to be in the active site . Therefore, barrier protein is probably a protease that cleaves alpha factor . The sequence similarity suggests that the first two-thirds of the barrier protein is organized into two distinct structural domains like those of the pepsin-like proteases . However, the BAR1 gene product has a third carboxyl-terminal domain of unknown function; deletion of at least 166 of the 191 amino acids of this region has no significant effect on barrier activity.

Folia Microbiol (Praha), 1988, 33(6), 447 - 52
Ethanol tolerance of Saccharomyces cerevisiae and its relationship to lipid content and composition; Ghareib M et al.; Saccharomyces cerevisiae strain 14-12 is a highly ethanol-tolerant organism . It can grow in the presence of 13% ethanol but growth is completely prevented at 14% ethanol . A relationship was detected between yeast lipids and ethanol tolerance . A gradual decrease of lipid content was recorded as the concentration of supplemented ethanol increased . Moreover, free fatty acids were comparatively decreased in these lipid extracts . When separately added to media with 14% ethanol different lipids produced varied stimulatory effects on yeast growth . Maximum yield of yeast growth was obtained at 14% ethanol in the presence of lecithin, palmitic acid and cholesterol . Yeast lipids produced in the presence of these fractions are characterized by a relatively high percentage of free fatty acids . The change in the percentage of free fatty acids was shown to be the controlling factor in ethanol tolerance.

Acta Biochim Pol, 1988, 35(2), 105 - 18
Protein composition of Saccharomyces cerevisiae mitochondrial ribosomes; Mieszczak M et al.; Protein composition of mitochondrial ribosomes of the yeast Saccharomyces cerevisiae was analysed by two-dimensional electrophoresis . The small (37S) mitoribosomal subunit contains 36 different polypeptides with molecular weights ranging from 10,000 to 60,000 . The large (50S) subunit is composed of 41 proteins with molecular weights from 10,000 to 43,000 . The molecular weights of mitoribosomal small and large subunits are 1.85 MDa and 2.35 MDa, respectively . Proteins represent 60-62% and 42-45% of the total mass of 37S and 50S subunits respectively . On the basis of the protein content and molecular weights of individual proteins we conclude that all mitoribosomal proteins are present in the mitoribosome in equimolar proportions.

J Basic Microbiol, 1988, 28(5), 335 - 42
Construction of promoter-probe vectors for Candida maltosa, a n-alkane-assimilating yeast, using the LEU2 gene of Saccharomyces cerevisiae; Takagi M et al.; For the purpose of isolation of promoter regions which are regulated by a carbon source in the medium in an n-alkane-assimilating yeast, Candida maltosa, two promoter-probe vectors were constructed . Each of them consists of the LEU2 gene of Saccharomyces cerevisiae whose 5'-noncoding region was trimmed with BAL31, an autonomously replicating sequence isolated from C . maltosa genome (the TRA region) which we have previously isolated, and the pBR322 sequence . One of them, pPLC2, having the TATA box, lacks the regulatory sequence ("sequence L") of the LEU2 gene, and the other, pPLC1, lacks both the TATA box and sequence L . Using pPLC1 as a short-gun cloning vector in C . maltosa, many promoter regions which were active when glucose was present in the medium as a carbon source were obtained from the genome of C . maltosa . The sizes of the inserted fragments of two of them were determined . (In this paper, a promoter region refers to a promoter which includes a TATA box, plus a regulatory sequence such as an UAS (upstream activating sequence)-like sequence).

Folia Microbiol (Praha), 1988, 33(5), 372 - 6
The irreversibility of thiamin transport in Saccharomyces cerevisiae; Ruml T et al.; Neither exit nor counterflow efflux of thiamin, taken up previously by an active transport, were found in Saccharomyces cerevisiae, in either the wild type or a mutant with a lower rate of thiamin phosphorylation . Complete inhibition of thiamin phosphorylation by oxythiamin did not lead to any release of thiamin taken up by the cell.

Folia Microbiol (Praha), 1988, 33(4), 292 - 7
Increased sterol formation in Saccharomyces cerevisiae . Analysis of cell components and ultrastructure of vacuoles; Behalova B et al.; In Saccharomyces cerevisiae nitrogen limitation under aerobic conditions (low specific growth rate) provokes an enhanced synthesis of sterols . Analysis of east cultures during the enhanced sterol biosynthesis showed a temporary decrease of protein content and a simultaneous increase in polysaccharide and lipid levels . This was reflected in the ultrastructure of cells where numerous lipid globules (spherosomes, oleosomes) appeared around extensive membrane-bound compartments containing membrane vesicles and lipoprotein material . Electronograms showed that such compartments were formed between the layers of endoplasmic reticulum and belonged to the vacuome phase of the yeast cell . It appears that vacuoles formed in yeast during enhanced synthesis of sterols have a storage rather than a lysosomal function.

Nahrung, 1988, 32(4), 311 - 8
Structure of protein solutions . Part 2 . Solutions of total protein of yeast Saccharomyces cerevisiae; Soloshenko VM et al.; Water systems formed by total protein of yeast Saccharomyces cerevisiae and model systems on the basis of unfractionated casein were studied by viscosimetry and electron microscopy . Methodologically, both methods offered a possibility of distinguishing several quantitatively different concentration intervals in protein solutions under different conditions . Viscosimetry gave the most informative results at the concentration intervals where the solutions approached the true ones, while electron microscopy did so when supermolecular formations prevailed . In comparing the data obtained by these two methods the following conclusions were drawn: At low temperatures (4-10 degrees C) yeast protein was in molecular-dissociated condition . Association in the system starts at 10 degrees C, and becomes enhanced with a rise in temperature . A temperature rise to 25-30 degrees C promotes a more compact packing of polypeptide chains; further heating leads to high-temperature denaturation . Lipids induce covering of polypeptide chains and prevent associations, thus leading to an increase in the system's thermostability.

Prog Clin Biol Res, 1988, 268B, 85 - 90
Development of a heterologous gene expression system for the Na,K-ATPase subunits in the yeast Saccharomyces cerevisiae; Horowitz B et al.; cDNA fragments coding for the alpha and beta subunits of the Na,K-ATPase were separately ligated into the yeast expression vector YEp1PT in both the sense (YEpNKA(+)) and anti-sense (YEpNKA(-)) orientations with respect to the promoter . The recombinant plasmids were introduced into Saccharomyces cerevisiae strain UT4 by transformation . Total RNA from the transformed strains was isolated and analyzed by Northern hybridization . The resulting autoradiogram revealed strong signals indicative of a high level of transcriptional expression of both subunits in both orientations of the cDNA . 35S-Methionine labeled extracts were immunoprecipitated with antibodies specific for the beta subunit . A beta subunit translation product was produced from YEp beta NKA(+) but not from YEp beta NKA(-) . Experiments to detect an alpha specific translation product are in progress.

Mol Microbiol, 1988 Jan, 2(1), 89 - 99
Sequence of the nucleoprotein gene from a virulent British field isolate of transmissible gastroenteritis virus and its expression in Saccharomyces cerevisiae; Britton P et al.; Subgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA which was sequenced . Two non-overlapping open reading frames (ORFs) were identified . The largest, encoding a polypeptide of 382 amino acids (relative molecular mass (Mr) 43,483), was shown to be the viral nucleoprotein gene . The second ORF, found 3' to the larger ORF, encodes a polypeptide of 78 amino acids (Mr 9068) which has yet to be assigned to a viral product . The nucleoprotein gene was expressed in yeast cells under the control of two types of yeast promoters: the constitutive PGK promoter, and the inducible GAL1 promoter . Yeast cells containing recombinant plasmids, with the nucleoprotein gene in the correct orientation, produced a polypeptide of Mr 47,000, identical to the viral product, that reacted with a specific monoclonal antibody.

Mol Gen Genet, 1988 Jan, 211(1), 155 - 9
Amplification of plasmid copy number by thymidine kinase expression in Saccharomyces cerevisiae; Zealey GR et al.; A 2 micron circle-based chimaeric plasmid containing the yeast LEU2 and the Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) genes was constructed . Transformants grown under selective conditions for the LEU2 gene harboured the plasmid at about 15 copies per cell whilst selection for the HSV-1 TK gene led to an increase to about 100 copies per cell . Furthermore, the plasmid copy number could be controlled by the stringency of selection for the TK gene, and the increase in TK gene dosage was reflected in an increase in intracellular thymidine kinase activity . The mitotic stability of the plasmid in "high-copy" and "low-copy" number cells was determined . "High-copy" number cells showed a greater mitotic stability . The relationship of TK expression to plasmid copy number may be useful for the isolation of plasmid copy number mutants in yeast and the control of heterologous gene expression.

Mol Cell Biol, 1988 Jan, 8(1), 505 - 10
SRA5 encodes the low-Km cyclic AMP phosphodiesterase of Saccharomyces cerevisiae; Wilson RB et al.; sra5 mutations in Saccharomyces cerevisiae were previously shown to suppress the inefficient growth of ras2 strains on nonfermentable carbon sources and to result in deficient low-Km cyclic AMP (cAMP) phosphodiesterase activity . We have cloned SRA5 by complementation . It maps to the right arm of chromosome XV, tightly linked to PRT1, and its sequence matches the sequence of PDE2, encoding the low-Km cAMP phosphodiesterase . Disruptions of SRA5 allowed ras1 ras2 strains to grow either on rich media supplemented with cAMP or on minimal media without exogenous cAMP . sra5 strains failed to survive prolonged nitrogen starvation in the presence of exogenous cAMP.

Acta Microbiol Pol, 1988, 37(3-4), 271 - 80
Biological effect of alkoxymethylene trimethylammonium chlorides on yeast Saccharomyces cerevisiae; Skala J et al.; Six compounds of the group of quaternary ammonium salts have been tested for their biological activity using yeasts as a biological system . They have an inhibitory effect on respiration, cell growth and amino acid transport . A destroying action on protoplast regeneration and respiration has been also observed . The studied chemicals appear to have very pleiotropic action, focused on a damage of mitochondrial and cell plasma membranes.

Eur J Biochem, 1987 Dec 30, 170(1-2), 241 - 6
Secretion of biologically active porcine prophospholipase A2 by Saccharomyces cerevisiae . Use of the prepro sequence of the alpha-mating factor; van den Bergh CJ et al.; The cDNA coding for porcine pancreatic prophospholipase A2 (proPLA) has been cloned and expressed in Saccharomyces cerevisiae . Expression and secretion of proPLA could only be obtained after fusing the proPLA to the prepro sequence of the yeast alpha-mating factor . Upon secretion, the fusion protein was cleaved by the KEX2 protease yielding a 140-amino-acid zymogen-like form of the phospholipase A2 . This protein was purified in high yield by ion-exchange chromatography . Limited proteolysis with trypsin cleaved the 'zymogen' to yield active phospholipase A2, which was indistinguishable from the authentic porcine pancreatic enzyme . These results show that a protein with a disulphide bridge content as high as 7 per 124 amino acid residues can be correctly processed by the yeast secretory apparatus.

Eur J Biochem, 1987 Dec 30, 170(1-2), 217 - 24
Influence of different 5'-flanking sequences of tRNA genes on their in vivo transcription efficiencies in Saccharomyces cerevisiae; Dingermann T et al.; We have investigated the influence of 5'-flanking sequences on the in vivo transcription activities in yeast . Since eukaryotic tRNA genes belong to multi-copy gene families monitoring of the activity of a particular tRNA gene is not possible . We therefore used two different tRNA genes from the cellular slime mould Dictyostelium discoideum which are efficiently transcribed and processed in vivo in yeast . The original 5'-flanking sequences of the two tRNA genes were replaced by random plasmid sequences . The modified tRNA genes were introduced into Saccharomyces cerevisiae and bulk tRNAs from the transformants were analyzed for the presence and the relative number of Dictyostelium tRNA gene transcripts . Substantial differences of steady-state levels of RNA transcribed were detected dependent on the 5'-flanking sequence of the tRNA gene . Minute structural changes, such as inserting two additional nucleotides in front of a tRNA gene, can lead to drastic activity changes . The efficiency of tRNA gene transcription can be conferred by sequences located more than 40 nucleotides upstream from the 5' end of the mature tRNA coding region.

J Biol Chem, 1987 Dec 25, 262(36), 17659 - 67
Purification and characterization of an endo-exonuclease from Saccharomyces cerevisiae that is influenced by the RAD52 gene; Chow TY et al.; An endo-exonuclease has been purified from logarithmically growing cells of the yeast Saccharomyces cerevisiae . Identification and purification of this nuclease was facilitated by its being precipitable with an antibody raised against a previously described Neurospora crassa endo-exonuclease (Resnick, M . A., Chow, T . Y.-K . Nitiss, J., and Game, J . C . (1984) Cold Spring Harbor Symp . Quant . Biol . 49, 639-649 and T . Y.-K . Chow and M . A . Resnick (1988) Mol . Gen . Genet., in press) . The enzyme which was purified to near homogeneity was composed of a molecular weight 72,000 monomer . The single-strand nuclease activity is endonucleolytic and nonprocessive, whereas the double-strand DNase activity is exonucleolytic and weakly processive . Both nuclease activities have a pH optimum of 7.5, require Mg2+ or Mn2+ but not Zn2+ or Ca2+, are not inhibited by ATP, and exhibit the same kinetics of heat inactivation . Although this protein is not the product of the RAD52 gene, the greatly reduced amounts in rad52 mutants implicate the enzyme in repair and recombination processes in both mitotic and meiotic cells.

J Biol Chem, 1987 Dec 25, 262(36), 17260 - 3
Vacuolar ion channel of the yeast, Saccharomyces cerevisiae; Wada Y et al.; Ionic flux is most likely to regulate the chemiosmotic potential differences across vacuolysosomal membranes in animal, plant, and fungal cells . We found a membrane potential-dependent cation channel in yeast vacuolar membrane and characterized its several features by an electrophysiological method using artificial planar bilayer membranes incorporated with isolated yeast vacuolar membrane vesicles . This ion channel conducts K+ (single channel conductance, 435 pS in 0.3 M KCl) and several other monovalent cations (Cs+, Na+, and Li+) with broad selectivity, but does not conduct Cl- . The opening of this channel is regulated by the membrane potential and the presence of calcium ion on the cytoplasmic face . These characteristics suggested that the vacuolar cation channel functions as one of essential components for formation and regulation of the chemical and electrical potential differences across the vacuolar membrane.

Nucleic Acids Res, 1987 Dec 23, 15(24), 10299 - 309
Negative regulators of the PHO system in Saccharomyces cerevisiae: isolation and structural characterization of PHO85; Uesono Y et al.; One of the negative regulators of the PHO system of Saccharomyces cerevisiae, PHO85, has been isolated by transformation and complementation of a pho85 strain . The complementing activity was delimited within a 1258 bp DNA segment and this region has been sequenced . The largest open reading frame found in this region can encode a protein of 302 amino acid residues . A pho85 mutant resulted from disruption of the chromosomal counterpart of the open reading frame described above . Therefore, we concluded that the gene we have cloned is PHO85 . This result also indicates that PHO85 is nonessential . Northern analysis revealed that the size of the PHO85 message is 1.1 kb . No similarity was found between the putative amino acid sequences of two negative regulators, the PHO80 and PHO85 proteins.

Science, 1987 Dec 18, 238(4834), 1713 - 6
The structure of sister minichromosome DNA before anaphase in Saccharomyces cerevisiae; Koshland D et al.; The role of DNA topology in holding sister chromatids together before anaphase was investigated by analyzing the structure of a small circular minichromosome in cell cycle (cdc) mutants of the yeast Saccharomyces cerevisiae . In the majority of cells arrested after S phase but before anaphase, sister minichromosome molecules are not topologically interlocked with each other . The analysis of the ploidy of minichromosomes in cells that are released from arrest demonstrates that the sister molecules are properly segregated when the cell cycle block is removed . Therefore, sister minichromosome molecules need not remain topologically interlocked until anaphase in order to be properly segregated, and topological interlocking of sister DNA molecules apparently is not the primary force holding sister chromatids together.

Biochem Biophys Res Commun, 1987 Dec 16, 149(2), 431 - 6
Engineering of the hydrophobic segment of the signal sequence for efficient secretion of human lysozyme by Saccharomyces cerevisiae; Yamamoto Y et al.; To elucidate the structure-function relationship of the signal sequence for the secretion of human lysozyme by Saccharomyces cerevisiae, we have systematically engineered the hydrophobic segment using the signal sequence of chicken lysozyme . Replacement of Cys 10 with leucine caused a 1.6 times increase in the secretion of human lysozyme . An idealized signal sequence L10 in which 10 consecutive leucines were distributed from the 3rd to the 12th position was 1.8 times as effective as the native sequence . L10 can be generalized as Ln = Met-Arg-(Leu)n-Pro-Leu-Ala-Ala-Leu-Gly, where n = 10 . We have also studied the secretory capability of Ln, where n = 6,8,12, and 14, and found that the length, as well as hydrophobicity, of the hydrophobic segment is an important factor in the secretion of human lysozyme by yeast.

Eur J Biochem, 1987 Dec 15, 169(3), 477 - 86
Molecular cloning and characterization of the gene encoding cholinephosphate cytidylyltransferase in Saccharomyces cerevisiae; Tsukagoshi Y et al.; 1 . The structural gene for cholinephosphate cytidylyltransferase (CCT) was isolated from a Saccharomyces cerevisiae genomic library by means of complementation in a mutant of the yeast defective in the enzyme . The cloned DNA restored both the growth and cholinephosphate cytidylyltransferase activity of the mutant . Whereas the enzyme of the mutant was thermolabile, the enzyme produced by the transformant was indistinguishable in heat stability from that produced by the wild type . 2 . Strains carrying a multicopy recombinant plasmid overproduced cholinephosphate cytidylyltransferase . The overproduction of the enzyme brought about an increase in the synthesis of CDPcholine in the transformant, but there was no increase in the overall rate of phosphatidylcholine synthesis . 3 . The cloned DNA was subcloned into a 2.5-kb DNA fragment . The nucleotide sequence which contained CCT was determined by the dideoxy chain-termination method . The sequence contained an open reading frame capable of encoding a protein of 424 amino acid residues with a calculated relative molecular mass of 49,379.31 . Northern blot analysis showed that this DNA segment is transcribed in yeast cells and the length of the transcript is consistent with the putative translation product . 4 . Hydropathy analysis according to Kyte and Doolittle indicated that the primary translation product contains extended hydrophilic stretches in its N- and C-terminal regions . 5 . The primary translation product contains a region showing local sequence homology with nucleotidyl-transfer enzymes such as DNA polymerase (Escherichia coli), CDPdiacylglycerol pyrophosphatase (E . coli), 3-deoxy-manno-octulosonate cytidylyltransferase (E . coli) and DNA ligase (T4 phage), suggesting that these five enzymes are evolutionarily related . Statistically significant sequence homology was also noted between the human c-fos gene product and the enzyme.

FEBS Lett, 1987 Dec 10, 225(1-2), 259 - 63
Trehalose accumulates in Saccharomyces cerevisiae during exposure to agents that induce heat shock response; Attfield PV; The storage disaccharide, trehalose, is accumulated in yeast during a temperature shift from 30 to 45 degrees C . The response peaks at 90 min and is transient since levels of trehalose decline rapidly in cells returned to 30 degrees C . Storage of trehalose is inhibited when cells are incubated in the presence of acridine orange or ethidium bromide prior to and during temperature shift, suggesting a requirement for de novo RNA synthesis . Accumulation of trehalose occurs when cells are exposed to either ethanol, copper sulphate or hydrogen peroxide at 30 degrees C, indicating that the phenomenon may be a general response to physiological stress . Parallels are drawn between the trehalose accumulation response and the heat shock response in yeast.

Biochim Biophys Acta, 1987 Dec 7, 926(3), 205 - 14
Effects of growth state and amines on cytoplasmic and vacuolar pH, phosphate and polyphosphate levels in Saccharomyces cerevisiae: a 31P-nuclear magnetic resonance study; Greenfield NJ et al.; The vacuoles of logarithmic and stationary stage cells were compared by 31P-NMR with regard to pH, orthophosphate (Pi) content and average size of polyphosphate . The vacuoles of stationary cells had lower pH, higher Pi content, and polyphosphates of longer average chain length, although total polyphosphate content was about the same as in logarithmic cells . The lower vacuolar pH in stationary cells was the major cause of a larger cytoplasmic-vacuolar pH gradient . Addition of NH4Cl, (NH4)2SO4, methylamine or amantadine at pH 8 to cells in either stage caused an increase in both cytoplasmic and vacuolar pH, with little or no change in the cytoplasmic-vacuolar pH gradient . However, the administration of ammonium salts to the cells at pH 8.0 resulted in rapid hydrolysis of the intravacuolar polyphosphate to tripolyphosphate and Pi, with attendant redistribution of Pi between the vacuolar and cytoplasmic compartments.

Yeast, 1987 Dec, 3(4), 243 - 53
Mapping of the Saccharomyces cerevisiae CDC3, CDC25, and CDC42 genes to chromosome XII by chromosome blotting and tetrad analysis; Johnson DI et al.; CDC3, CDC25 and CDC42 were localized to chromosome XII by hybridizing the cloned genes to Southern blots of chromosomes separated by orthogonal-field-alternation gel electrophoresis . Meiotic tetrad analyses further localized these genes to the region distal to the RDN1 locus on the right arm of the chromosome . The STE11 gene, which had previously been mapped to chromosome XII (Chaleff and Tatchell, 1985), was found to be tightly linked to ILV5 . The data suggest a map order of CEN12-RDN1-CDC42-(CDC25-CDC3)-(ILV5- STE11)-URA4 . Certain oddities of the data set raise the possibility that there may be constraints on the patterns of recombination in this region of chromosome XII.

J Gen Microbiol, 1987 Dec, 133 ( Pt 12), 3355 - 63
Tubulin and actin topology during zygote formation of Saccharomyces cerevisiae; Hasek J et al.; The topology of tubulin and actin during mating of Saccharomyces cerevisiae was analysed by fluorescence microscopy with the monoclonal anti-tubulin antibody Tu01 and rhodamine-labelled phalloidin . Preconjugatory cells displayed an asymmetric distribution of the microtubule and actin cytoskeleton and an overall polarization of the cells preceding cell fusion . Prior to karyogamy, the haploid spindle pole bodies were associated with abundant cytoplasmic microtubules . Budding zygotes revealed the same tubulin and actin patterns as vegetative cells . Treatment of the mating mixture with the microtubule inhibitor nocodazole (10 micrograms ml-1) did not prevent polarization and fusion of haploids, zygote formation and emergence of the first zygotic bud . In marked contrast, the migration of the nucleus in preconjugatory cells as well as nuclear migration and fusion within the zygotes was unequivocally blocked by the action of the drug . It is suggested that the problem of the morphogenesis of mating should be approached by considering interactions at the cell periphery.

Genetika, 1987 Dec, 23(12), 2128 - 37
{Genetic analysis of the polyauxotrophy of the early mitotic progeny of Saccharomyces cerevisiae zygotes . III . The behavior of the chromosome-III markers in polyauxotrophic clones and their mitotic and meiotic segregants}; Stolbova AV; Cloning and segregation analysis of polyauxotrophic (PA) progeny, as well as the study of diploid segregants, revealed an unusual state of the diploid genome in these strains . Analysis of linkage markers of chromosome III in all crosses may be expected to illuminate relationships between the homologous chromosomes . All PA strains were assigned to two major classes . In the class of PA strains with recombinant chromosome III, the effect of approachment of third linkage markers was noted . In other strains, transposition of genetic marker metA1 to chromosome III and its linkage to leu2 were discovered . The present study has demonstrated that the unusual state of the diploid genome of PA strains induces multiple nonspecific alterations in chromosome relationship and structure . It is likely that these alterations are the consequence of disturbance in mitotic apparatus of cell.

Mol Cell Biol, 1987 Dec, 7(12), 4441 - 52
Functional domains of SIR4, a gene required for position effect regulation in Saccharomyces cerevisiae; Marshall M et al.; The product of the Saccharomyces cerevisiae SIR4 gene, in conjunction with at least three other gene products, prevents expression of mating-type genes resident at loci at either end of chromosome III, but not of the same genes resident at the MAT locus in the middle of the chromosome . To address the mechanism of this novel position effect regulation, we have conducted a structural and genetic analysis of the SIR4 gene . We have determined the nucleotide sequence of the gene and found that it encodes a lysine-rich, serine-rich protein of 152 kilodaltons . Expression of the carboxy half of the protein complements a chromosomal nonsense mutation of sir4 but not a complete deletion of the gene . These results suggest that SIR4 protein activity resides in two portions of the molecule, but that these domains need not be covalently linked to execute their biological function . We also found that high-level expression of the carboxy domain of the protein yields dominant derepression of the silent loci . This anti-Sir activity can be reversed by increased expression of the SIR3 gene, whose product is normally also required for maintaining repression of the silent loci . These results are consistent with the hypothesis that SIR3 and SIR4 proteins physically associate to form a multicomponent complex required for repression of the silent mating-type loci.

Mol Cell Biol, 1987 Dec, 7(12), 4390 - 9
Protease B of the lysosomelike vacuole of the yeast Saccharomyces cerevisiae is homologous to the subtilisin family of serine proteases; Moehle CM et al.; The PRB1 gene of Saccharomyces cerevisiae encodes the vacuolar endoprotease protease B . We have determined the DNA sequence of the PRB1 gene and the amino acid sequence of the amino terminus of mature protease B . The deduced amino acid sequence of this serine protease shares extensive homology with those of subtilisin, proteinase K, and related proteases . The open reading frame of PRB1 consists of 635 codons and, therefore, encodes a very large protein (molecular weight, greater than 69,000) relative to the observed size of mature protease B (molecular weight, 33,000) . Examination of the gene sequence, the determined amino-terminal sequence, and empirical molecular weight determinations suggests that the preproenzyme must be processed at both amino and carboxy termini and that asparagine-linked glycosylation occurs at an unusual tripeptide acceptor sequence.

Mol Cell Biol, 1987 Dec, 7(12), 4225 - 37
Replication and segregation of plasmids containing cis-acting regulatory sites of silent mating-type genes in Saccharomyces cerevisiae are controlled by the SIR genes; Kimmerly WJ et al.; In Saccharomyces cerevisiae, two cis-acting regulatory sites called E and I flank the silent mating-type gene, HMRa, and mediate SIR-dependent transcriptional repression of the a1-a2 promoters . It has been shown previously that the E and I sites have plasmid replicator (ARS) activity . We show in this report that the ARS activity of the E and I sites is governed by the SIR genotype of the cell . In wild-type cells, a plasmid carrying the E site from HMRa (HMR E) in the vector YIp5 exhibited very high mitotic stability at a copy number of approximately 25 per cell . However, in sir2, sir3, or sir4 mutants, plasmids with HMR E had the low mitotic stability characteristic of plasmids containing ARS1, a SIR-independent replicator . Elevated mitotic stability of plasmids that carry HMR E is due to a segregation mechanism provided by SIR and HMR E . In sir2 and sir4 mutants, the plasmid copy number was significantly lowered, suggesting that these gene products also participate in the replication of plasmids carrying HMR E . The phenotype of point mutations introduced at an 11-base-pair ARS consensus sequence present at HMR E indicated that this sequence is functional but not absolutely required for autonomous replication of the plasmid and that it is not required for SIR-dependent mitotic stabilization . A plasmid carrying both a centromere and HMR E exhibited reduced mitotic stability in wild-type cells . This destabilization appeared to be due to antagonism between the segregation functions provided by the centromere and by HMR E.

Mol Gen Genet, 1987 Dec, 210(2), 307 - 13
The Saccharomyces cerevisiae MET3 gene: nucleotide sequence and relationship of the 5' non-coding region to that of MET25; Cherest H et al.; In Saccharomyces cerevisiae, the expression of several genes implicated in methionine biosynthesis is co-regulated by a specific negative control . To elucidate the molecular basis of this regulation, we have cloned two of these genes, MET3 and MET25 . The sequence of MET25 has already been determined (Kerjan et al . 1986) . Here, we report the nucleotide sequence of the MET3 gene along with its 5' and 3' flanking regions . Plasmids bearing different deletions upstream of the transcribed region of MET3 were constructed . They were introduced into yeast cells and tested for their ability to complement met3 mutations and to respond to regulation by exogenous methionine . The regulatory region was located within a 100 bp region . The sequence of this regulatory region was compared with that of MET25 . A short common sequence which occurs 250-280 bp upstream of the translation initiation codon of the gene was found . This sequence is a good candidate for the cis-acting regulatory element.

Mol Gen Genet, 1987 Dec, 210(2), 277 - 81
CAN1-SUC2 gene fusion studies in Saccharomyces cerevisiae; Hoffmann W; Various gene fusions between the arginine permease and invertase have been constructed in order to obtain information about whether parts of the CAN1 gene product can induce secretion of biologically active invertase missing its own signal sequence . A construction containing 30 N-terminal amino acid residues of the CAN1 gene product fused to invertase was not secreted . When the CAN1 portion was elongated to 477 or 560 amino acid residues, secretion of the fusion proteins was observed . A fusion lacking 59 amino acids at the amino-terminal end of the arginine permease was also secreted . These results indicate that the amino-terminal end of the arginine permease is neither sufficient nor essential for membrane insertion; instead this enzyme should contain an internal targeting sequence facilitating secretion . Some general implications on the biosynthesis and topology of membrane proteins are also discussed as well as the homology with histidine permease.

Genetics, 1987 Dec, 117(4), 619 - 31
Evidence for related functions of the RNA genes of Saccharomyces cerevisiae; Last RL et al.; The yeast genes RNA2-RNA11 are necessary for splicing of nuclear intron-containing pre-mRNAs . We investigated the relationships among these genes by asking whether increased expression of one RNA gene leads to suppression of the temperature-sensitive lethality of a mutation in any other RNA gene . The presence of extra plasmid-borne copies of the RNA3 gene relieves the lethality of temperature-sensitive rna4 mutations . A region of the yeast genome (SRN2) is described that suppresses temperature-sensitive rna2 mutations when it is present on either medium or high-copy number plasmids . Neither suppression occurs via a bypass of RNA gene function since null alleles of rna2 and rna4 are not suppressed by elevated dosage of SRN2 and RNA3, respectively . These results suggest that the SRN2 and RNA2 gene products have related functions, as do the RNA3 and RNA4 gene products.

EMBO J, 1987 Dec 1, 6(12), 3833 - 9
Specific accessory sequences in Saccharomyces cerevisiae introns control assembly of pre-mRNAs into spliceosomes; Newman A; In experiments involving deletion and rearrangement of intron sequences two small regions of the intron in the yeast CYH2 ribosomal protein gene were found to play important roles in splicing of the pre-mRNA . One element lies downstream of the 5' splice site, and the other is upstream of the branchpoint sequence UACUAAC . Deletion of the element upstream of the branchpoint prevents spliceosome formation and blocks splicing in vivo and in vitro . Deletion of the element downstream of the 5' splice site does not on its own block splicing but rescues spliceosome formation and splicing of pre-mRNA lacking the element upstream of the branchpoint . These elements correspond to two regions of sequence complementarity which are a conserved feature of the introns in yeast pre-mRNAs . Mixing and matching of the elements from the ACT1 and CYH2 gene introns showed that these elements can cooperate in an intron-specific fashion to control spliceosome assembly.

DNA, 1987 Dec, 6(6), 529 - 37
Primary structure of the P450 lanosterol demethylase gene from Saccharomyces cerevisiae; Kalb VF et al.; We have sequenced the structural gene and flanking regions for lanosterol 14 alpha-demethylase (14DM) from Saccharomyces cerevisiae . An open reading frame of 530 codons encodes a 60.7-kDa protein . When this gene is disrupted by integrative transformation, the resulting strain requires ergosterol and, as expected, grows only in the absence of oxygen . The deduced amino acid sequence of 14DM includes a hydrophobic segment near the amino terminus which may be a transmembrane domain . The deduced sequence has been compared with those of eight other eukaryotic P450s, each from a different family within the P450 superfamily . These comparisons indicate that this yeast gene is the first member of a new P450 family, P450LI . The P450, designated P450LIA1, is more closely related to mammalian P450s than to the bacterial P450cam . In fact, both the yeast P450 and several mammalian P450s have equivalent alignment scores when each is compared with the bovine P450scc . Matrix comparisons of the amino acid sequence of this P450 with those of mammalian P450s reveal three conserved regions . The DNA region 5' to the structural 14DM gene includes poly(dA:dT) sequences and a repeating hexamer sequence.

Arch Biochem Biophys, 1987 Dec, 259(2), 589 - 96
Characterization of the Saccharomyces cerevisiae cis-prenyltransferase required for dolichyl phosphate biosynthesis; Adair WL Jr et al.; The prenyltransferase involved in the biosynthesis of dolichyl phosphate has been characterized in Saccharomyces cerevisiae . Although the enzyme is predominantly membrane-bound, a significant percentage was found in the soluble fraction . The prenyltransferase preferentially utilizes farnesyl pyrophosphate as the allylic substrate and isopentenyl pyrophosphate as cosubstrate with half-maximal velocities obtained at 25 and 6.7 microM, respectively . The enzymatic activity is sensitive to sulfhydryl reagents and is inhibited by all detergents tested, except 3-{(3-cholamidopropyl)dimethylammonio}-1-propanesulfonate at concentrations less than 5 mM . The product of the reaction has been characterized as an alpha-unsaturated polyprenyl pyrophosphate, containing 12-15 isoprene units, approximately two isoprene units shorter than the endogenous yeast dolichyl phosphate . The stereochemistry of addition of isoprene units by the prenyltransferase was shown to be cis by a comparison of the HPLC retention time for a pentadecaprenyl phosphate derived from the in vitro reaction product with that for an authentic mixture of alpha-cis- and alpha-trans-pentadecaprenyl phosphates.

Proc Natl Acad Sci U S A, 1987 Dec, 84(24), 8839 - 43
Accurate initiation at RNA polymerase II promoters in extracts from Saccharomyces cerevisiae; Lue NF et al.; A yeast nuclear extract supported transcription from the CYC1 and PYK1 promoters . Transcription was initiated in vitro at or near sites used in vivo . Deletion of "TATA" sequences abolished the reaction . alpha-Amanitin (10 micrograms/ml) and chloride (100 mM) were highly inhibitory.

J Bacteriol, 1987 Dec, 169(12), 5622 - 5
Production of heat shock protein is independent of cell cycle blockage in the yeast Saccharomyces cerevisiae; Barnes CA et al.; In response to certain environmental stresses, cells display a response characterized by the production of heat shock proteins . In this study we showed that blockage of cells of the yeast Saccharomyces cerevisiae at specific points in the mitotic cell cycle was not in itself a stress that induced the production of heat shock proteins . Nevertheless, cell cycle blockage did not preclude a normal heat shock response in arrested cells subjected to elevated temperatures.

Mol Cell Biol, 1987 Dec, 7(12), 4431 - 40
Proline utilization in Saccharomyces cerevisiae: sequence, regulation, and mitochondrial localization of the PUT1 gene product; Wang SS et al.; The PUT1 gene of Saccharomyces cerevisiae, believed to encode proline oxidase, has been completely sequenced and contains an open reading frame capable of encoding a polypeptide of 476 amino acids in length . The amino terminus of the protein deduced from the DNA sequence has a characteristic mitochondrial import signal; two PUT1-lacZ gene fusions were constructed that produced mitochondrially localized beta-galactosidase in vivo . The transcription initiation and termination sites of the PUT1 mRNA were determined . By using a PUT1-lacZ gene fusion that makes a cytoplasmic beta-galactosidase, the regulation of the PUT1 gene was studied . PUT1 is inducible by proline, responds only slightly to carbon catabolite repression, and is not regulated by the cytochrome activator proteins HAP1 and HAP2 . The PUT1 gene is under oxygen regulation; expression in anaerobically grown cells is 10-fold lower than in aerobically grown cells . Oxygen regulation is abolished when cells are respiratory deficient . PUT1 expression in a {rho-} strain grown either aerobically or anaerobically is as high as that seen in a {rho+} strain grown aerobically . Studies on PUT1 promoter deletions define a region between positions -458 and -293 from the translation initiation site that is important for full expression of the PUT1 gene and required for oxygen regulation.

Mol Cell Biol, 1987 Dec, 7(12), 4204 - 10
Trans-acting regulatory mutations that alter transcription of Saccharomyces cerevisiae histone genes; Osley MA et al.; Using a Saccharomyces cerevisiae strain containing an integrated copy of an H2A-lacZ fusion gene, we screened for mutants which overexpressed beta-galactosidase as a way to identify genes which regulate transcription of the histone genes . Five recessive mutants with this phenotype were shown to contain altered regulatory genes because they had lost repression of HTA1 transcription which occurs upon inhibition of chromosome replication (D . E . Lycan, M . A . Osley, and L . Hereford, Mol . Cell . Biol . 7:614-621, 1987) . Periodic transcription was affected in the mutants as well, since the HTA1 gene was transcribed during the G1 and G2 phases of the cell cycle, periods in the cell cycle when this gene is normally not expressed . A similar loss of cell cycle-dependent transcription was noted for two of the three remaining histone loci, while the HO and CDC9 genes continued to be expressed periodically . Using isolated promoter elements inserted into a heterologous cycl-lacZ fusion gene, we demonstrated that the mutations fell in genes which acted through a negative site in the TRT1 H2A-H2B promoter.

J Bacteriol, 1987 Dec, 169(12), 5518 - 22
Heat-induced accumulation and futile cycling of trehalose in Saccharomyces cerevisiae; Hottiger T et al.; Heat shock resulted in rapid accumulation of large amounts of trehalose in Saccharomyces cerevisiae . In cultures growing exponentially on glucose, the trehalose content of the cells increased from 0.01 to 1 g/g of protein within 1 h after the incubation temperature was shifted from 27 to 40 degrees C . When the temperature was readjusted to 27 degrees C, the accumulated trehalose was rapidly degraded . In parallel, the activity of the trehalose-phosphate synthase, the key enzyme of trehalose biosynthesis, increased about sixfold during the heat shock and declined to the normal level after readjustment of the temperature . Surprisingly, the activity of neutral trehalase, the key enzyme of trehalose degradation, also increased about threefold during the heat shock and remained almost constant during recovery of the cells at 27 degrees C . In pulse-labeling experiments with {14C}glucose, trehalose was found to be turned over rapidly in heat-shocked cells, indicating that both anabolic and catabolic enzymes of trehalose metabolism were active in vivo . Possible functions of the heat-induced accumulation of trehalose and its rapid turnover in an apparently futile cycle during heat shock are discussed.

Mol Cell Biol, 1987 Dec, 7(12), 4522 - 34
Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae; Ng R et al.; Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA . The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry) . Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site . This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences . An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3 . In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters . Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays . The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.

Mol Cell Biol, 1987 Dec, 7(12), 4169 - 77
Pheromonal regulation and sequence of the Saccharomyces cerevisiae SST2 gene: a model for desensitization to pheromone; Dietzel C et al.; Strains of both haploid mating types containing sst2 mutations are altered in response to pheromone; MATa sst2 cells are supersensitive to alpha-factor, and MAT alpha sst2 cells are supersensitive to a-factor . This phenotype suggests that SST2 encodes a component of the pheromone response pathway that is common to both mating types . We have cloned the SST2 gene by isolation of multicopy plasmids that complement the sst2-1 mutation . One such plasmid contained a 4.5-kilobase HindIII fragment that was able to complement the sst2-1 mutation in high or low copy number, integrated at the SST2 locus, and resulted in an sst2 phenotype when disrupted, indicating that this fragment contained the SST2 gene . We identified the functional region of the complementing DNA fragment by transposon mutagenesis . Sequencing of this fragment identified an open reading frame encoding 698 amino acids at a position that correlated well with the functional region . Expression of an Sst2-beta-galactosidase fusion was haploid specific and induced by exposure to pheromone . We discuss a model in which induction of the SST2 product results in inhibition of a component of the pheromone response pathway, resulting in desensitization to pheromone.

Mol Gen Genet, 1987 Dec, 210(3), 413 - 8
Mutant regulatory subunit of 3',5'-cAMP-dependent protein kinase of yeast Saccharomyces cerevisiae; Yamano S et al.; Four mutants with amino acid substitution(s) at or near the putative phosphorylation site (Arg142 Arg143 Thr144 Ser145) of the regulatory subunit of cAMP-dependent protein kinase were obtained by site-directed mutagenesis . Three mutants, BCY1A1a145 (Ser145 to Ala), BCY1His143 (Arg143 to His) and BCY1Asn144, Ala145 (Thr144 to Asn and Ser145 to Ala) complemented a bcy1 mutant, whereas BCY1Gly143 (Arg143 to Gly) did not . In addition, mutant, BCY1Asn144, Ala145 exhibited a dominant cold-sensitive phenotype, which can be most easily explained by the functional alteration of the regulatory subunit of cAMP-dependent protein kinase by the mutations . Analyses of these mutant genes revealed that phosphorylation of the regulatory subunit is not a prerequisite for the regulation of the cAMP-dependent protein kinase activity in responding to the cAMP level.

Genetics, 1987 Dec, 117(4), 645 - 55
Identifying mutations in duplicated functions in Saccharomyces cerevisiae: recessive mutations in HMG-CoA reductase genes; Basson ME et al.; The two yeast genes for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, HMG1 and HMG2, each encode a functional isozyme . Although cells bearing null mutations in both genes are inviable, cells bearing a null mutation in either gene are viable . This paper describes a method of screening for recessive mutations in the HMG1 gene, the gene encoding the majority of HMG-CoA reductase activity in the cell . This method should be applicable to the isolation of mutations in other recovered in HMG1 . These mutations exhibited intragenic complementation: one allele is in one complementation group and three alleles are in a second complementation group . Assays of HMG-CoA reductase activity indicated that the point mutations destroy most if not all of the activity encoded by HMG1 . Intragenic complementation occurred with partial restoration of enzymatic activity . HMG1 was mapped to the left arm of chromosome XIII near SUP79, and HMG2 was mapped to the right arm of chromosome XII near SST2 . A slight deleterious effect of a null mutation in either HMG-CoA reductase gene was detected by a co-cultivation experiment involving the wild-type strain and the two single mutants.

Genetics, 1987 Dec, 117(4), 633 - 43
Intrachromosomal recombination in Saccharomyces cerevisiae: reciprocal exchange in an inverted repeat and associated gene conversion; Willis KK et al.; Intrachromosomal gene conversion has not shown a strong association with reciprocal exchanges . However, reciprocal exchanges do occur between intrachromosomal repeats . To understand the relationship between reciprocal exchange and gene conversion in repeated sequences the recombination behavior of an inverted repeat was studied . We have found that in one orientation a single copy of the kanr gene of the bacterial transposon Tn903 flanked by part of the inverted repeats IS903 does not give G418 resistance in Saccharomyces cerevisiae . A reciprocal exchange in the IS903 repeats inverts the kanr gene, which then gives G418 resistance in a single copy . Using this as a selection for intrachromosomal reciprocal exchange we have introduced multiple restriction site heterologies into the IS903 repeats and examined the crossover products for associated gene conversions . Approximately 50% of crossovers, both in mitosis and meiosis, were associated with a gene conversion . This suggests that these crossovers result from an intermediate that gives a gene conversion in 50% of the events, that is, both reciprocal exchange and gene conversion between repeated sequences have a common origin . The data are most consistent with a heteroduplex mismatch repair mechanism.

Proc Natl Acad Sci U S A, 1987 Dec, 84(24), 8951 - 5
RAD3 protein of Saccharomyces cerevisiae is a DNA helicase; Sung P et al.; The Saccharomyces cerevisiae RAD3 gene, which is required for cell viability and excision repair of damaged DNA, encodes an 89-kDa protein that has a single-stranded DNA-dependent ATPase activity . We now show that the RAD3 protein also possesses a helicase activity that unwinds duplex regions in DNA substrates constructed by annealing DNA fragments of 71-851 nucleotides to circular, single-stranded M13 DNA . The DNA helicase activity is dependent on the hydrolysis of ATP, has a pH optimum of approximately 5.6, and is inhibited by antibodies raised against a truncated RAD3 protein produced in Escherichia coli . The RAD3 helicase translocates along single-stranded DNA in the 5'----3' direction . The direction of RAD3 helicase movement is consistent with the possibility that it unwinds DNA duplexes in advance of the replication fork during DNA replication.

Proc Natl Acad Sci U S A, 1987 Dec, 84(23), 8553 - 7
High-frequency deletion between homologous sequences during retrotransposition of Ty elements in Saccharomyces cerevisiae; Xu H et al.; By following the fates of genetically marked Ty elements, we observed a very high frequency (80-90%) of deletion between directly repeated marker sequences during transposition . From blot hybridization analyses of Ty RNA and DNA species found in the Ty virus-like particles, we determined that the deletion events occurred during or immediately after reverse transcription of Ty RNA but before integration of Ty DNA . The results suggest that the Ty reverse-transcription machinery can recognize homologous sequences in the template . This capacity may be utilized in the replication and recombination processes of retrotransposons and retroviruses.

Nucleic Acids Res, 1987 Nov 25, 15(22), 9309 - 24
Identification of a new promoter within the tRNA gene cluster of the mitochondrial DNA of Saccharomyces cerevisiae; Backer JS et al.; We have identified a new promoter within the tRNA gene cluster of Saccharomyces cerevisiae mitochondrial DNA . It is located upstream of the gene encoding the leucyl tRNA . It conforms to the consensus sequence of other yeast mitochondrial promoters, ATATAAGTA . It serves as a site for the initiation of transcription in vivo and in vitro.

Biochemistry, 1987 Nov 17, 26(23), 7483 - 92
31P NMR saturation-transfer measurements in Saccharomyces cerevisiae: characterization of phosphate exchange reactions by iodoacetate and antimycin A inhibition; Campbell-Burk SL et al.; 31P nuclear magnetic resonance (NMR) saturation-transfer (ST) techniques have been used to measure steady-state flows through phosphate-adenosine 5'-triphosphate (ATP) exchange reactions in glucose-grown derepressed yeast . Our results have revealed that the reactions catalyzed by glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK) and by the mitochondrial ATPase contribute to the observed ST . Contributions from these reactions were evaluated by performing ST studies under various metabolic conditions in the presence and absence of either iodoacetate, a specific inhibitor of GAPDH, or the respiratory chain inhibitor antimycin A . Intracellular phosphate (Pi) longitudinal relaxation times were determined by performing inversion recovery experiments during steady-state ATP gamma saturation and were used in combination with ST data to determine Pi consumption rates . 13C NMR and O2 electrode measurements were also conducted to monitor changes in rates of glucose consumption and O2 consumption, respectively, under the various metabolic conditions examined . Our results suggest that GAPDH/PGK-catalyzed Pi-ATP exchange is responsible for antimycin-resistant saturation transfer observed in anaerobic and aerobic glucose-fed yeast . Kinetics through GAPDH/PGK were found to depend on metabolic conditions . The coupled system appears to operate in a unidirectional manner during anaerobic glucose metabolism and bidirectionally when the cells are respiring on exogenously supplied ethanol . Additionally, mitochondrial ATPase activity appears to be responsible for the transfer observed in iodoacetate-treated aerobic cells supplied with either glucose or ethanol, with synthesis of ATP occurring unidirectionally.

Eur J Biochem, 1987 Nov 16, 169(1), 193 - 200
Functional analysis of the regulatory region adjacent to the cargB gene of Saccharomyces cerevisiae . Nucleotide sequence, gene fusion experiments and cis-dominant regulatory mutation analysis; Degols G; In Saccharomyces cerevisiae the expression of the cargB gene (coding for ornithine aminotransferase) is submitted to dual regulation: an induction by allophanate and a specific induction process by arginine . We have determined the nucleotide sequence of the cargB gene along with its 5' region . The coding portion of the gene encodes a protein of 423 amino acid residues with a calculated Mr value of 46049 . To characterize further the regulatory mechanisms modulating the expression of the gene we have analyzed fusions of several fragments of the 5' non-coding region to lacZ, compared the 5' sequences of the cargA (coding for arginase) and cargB coregulated genes and determined the nature of two constitutive cis-dominant mutations affecting the arginine control . These approaches allowed us to define three domains in the 5' non-coding region . The upstream one is implicated in the induction by allophanate . The two other domains are involved in the specific control by arginine; the target of the ARGR gene products, that mediate a positive regulation by arginine, lies upstream of another site where a repression by the CARGRI molecule occurs . The constitutive cargB+O- mutations are located in this repressor domain . The 5' non-coding region of cargA presents the same two-domain organization . These two domains contain three sequences homologous to the cargA and cargB 5' regions.

J Cell Sci, 1987 Nov, 88 ( Pt 4), 431 - 9
Isolation of morphologically intact mitochondrial nucleoids from the yeast, Saccharomyces cerevisiae; Miyakawa I et al.; Mitochondrial nucleoids (mt-nucleoids) of the yeast, Saccharomyces cerevisiae, were isolated from spheroplasts of stationary phase cells and their structure and organization were investigated by fluorescence microscopy, electron microscopy, and biochemical techniques . Isolated mt-nucleoids were spherical or ovoid and 0.3-0.6 micron in diameter, and were about the same size and shape as those observed in the cell by the DAPI staining technique . Measurement of DNA content of mt-nucleoids, using a video-intensified microscope system, after DAPI staining revealed that a mt-nucleoid in spheroplasts from stationary phase cells contains, on average, 3.9 mtDNA molecules and an isolated mt-nucleoid contains, on average, 3.1 . Negatively stained electron micrographs showed that mt-nucleoids consist of particles 20-50 nm in diameter . SDS-polyacrylamide gel electrophoresis of mt-nucleoids detected 20 species of polypeptides in the molecular weight range from 10 X 10(3) to 70 X 10(3) . Acid-urea/SDS two-dimensional electrophoresis of acid extract from mt-nucleoids indicated that a polypeptide of 20 X 10(3) is the only major polypeptide with basic property like histones.

J Gen Microbiol, 1987 Nov, 133 ( Pt 11), 3229 - 36
Iron uptake by the yeast Saccharomyces cerevisiae: involvement of a reduction step; Lesuisse E et al.; Among several parameters affecting the rate and amount of iron uptake by Saccharomyces cerevisiae, the oxidation state of iron appeared to be determinant . Iron presented as Fe(II) was taken up faster than Fe(III) and the kinetic parameters were different . Iron was taken up by the cells from different ferric chelates, at rates that did not depend on their stability constants, and uptake was strongly inhibited by an iron(II)-trapping reagent like ferrozine . Iron was physiologically reduced by a transplasmamembrane redox system, which was induced in iron-deficient conditions . We propose that iron must be reduced to be taken up by the cells in the same way as other divalent cations.

Anal Biochem, 1987 Nov 1, 166(2), 328 - 34
Isolation of secretory vesicles from Saccharomyces cerevisiae; Holcomb CL et al.; Purification of secretory vesicles from Saccharomyces cerevisiae has been hindered because these organelles normally represent a small proportion of cellular membranes . In the yeast secretory mutant sec1, secretory vesicles accumulate intracellularly in large quantities . Using a sec1 strain we have devised a procedure for the partial purification of these vesicles . The purification employs differential and density gradient centrifugations and an electrophoretic separation of membranes . The fractions obtained from this procedure are enriched for secretory vesicles at least fivefold over other cellular membranes . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane fractions reveals a distinct set of polypeptides associated with secretory vesicles.

Mol Cell Biol, 1987 Nov, 7(11), 4122 - 4
Towards determination of the structure of the Saccharomyces cerevisiae a-factor: an acylated pentadecapeptide blocks a-factor activity; Becker JM et al.; Putative a-factor peptides YIIKGVFWADP, YIIKGVFWANP, YIIKGLFWADP, YIIKGLFWANP, YIIKGVFWDPA, and YIIKGVFWDPACVIA and several peptide derivatives were synthesized and were found to be inactive in growth arrest assays, yet they blocked the activity of biological a-factor . Antagonism was greatest with YIIKGVFWDPAC(palmitoyl)VIA . Thus, the structure of a-factor may be a lipopeptide resembling this palmitoylated pentadecapeptide.

Mol Cell Biol, 1987 Nov, 7(11), 3888 - 98
Genetic and biochemical characterization of clathrin-deficient Saccharomyces cerevisiae; Payne GS et al.; Clathrin is important but not essential for yeast cell growth and protein secretion . Diploid Saccharomyces cerevisiae cells heterozygous for a clathrin heavy-chain gene (CHC1) disruption give rise to viable, slow-growing, clathrin heavy-chain-deficient meiotic progeny (G . Payne and R . Schekman, Science 230:1009-1014, 1985) . The possibility that extragenic suppressors account for growth of clathrin-deficient cells was examined by deletion of CHC1 from haploid cell genomes by single-step gene transplacement and independently by introduction of a centromere plasmid carrying the complete CHC1 gene into diploid cells before eviction of a chromosomal CHC1 locus and subsequent tetrad analysis . Both approaches yielded clathrin-deficient haploid strains . In mutants missing at least 95% of the CHC1 coding domain, transcripts related to CHC1 were not detected . The time course of invertase modification and secretion was measured to assess secretory pathway functions in the viable clathrin-deficient cells . Core-glycosylated invertase was converted to the mature, highly glycosylated form at equivalent rates in mutant and wild-type cells . Export of mature invertase from mutant cells was delayed but not prevented . Abnormal vacuoles, accumulated vesicles, and Golgi body-derived structures were visualized in mutant cells by electron microscopy . We conclude that extragenic suppressors do not account for the viability of clathrin-deficient cells and, furthermore, that many standard laboratory strains can sustain a CHC1 disruption . Clathrin does not appear to mediate protein transfer from the endoplasmic reticulum to the Golgi body but may function at a later stage of the secretory pathway.

J Virol, 1987 Nov, 61(11), 3543 - 9
Expression of hepatitis B virus middle and large surface antigen genes in Saccharomyces cerevisiae; Imamura T et al.; The hepatitis B virus genome carries the surface antigen (SAg) gene and an open reading frame that encodes two SAg-related polypeptides: SAg with a 55-amino-acid N-terminal extension polypeptide and SAg with a 174-amino-acid N-terminal extension polypeptide . These are termed middle S and large S, respectively . These polypeptides or their glycosylated derivatives have been detected in Dane particles, but their chemical and biological properties have remained largely unknown because of their limited availability . We attempted to produce these proteins in Saccharomyces cerevisiae by placing the coding regions under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene . Yeast cells carrying middle S and large S coding sequences produced 33,000- and 42,000-dalton products, respectively, each of which reacted with anti-S antibody and bound to polymerized human serum albumin, in accordance with the known properties of pre-S proteins from particles in human sera (K . H . Heermann, U . Goldmann, W . Schwartz, T . Seyffarth, H . Baumgarten, and W . H . Gerlich, J . Virol . 52:396-402, 1984; A . Machida, S . Kishimoto, H . Ohnuma, K . Baba, Y . Ito, H . Miyamoto, G . Funatsu, K . Oda, S . Usuda, S . Togami, T . Nakamura, Y . Miyakawa, and M . Mayumi, Gastroenterology 86:910-918, 1984) . The middle S polypeptide is glycosylated and can be assembled into particles whose size and density are similar to those of SAg . However, this polypeptide was highly susceptible to proteolytic degradation into 29,000- and 26,000-dalton polypeptides, of which only the former retained the binding activity to polymerized albumin . The large S polypeptides are nonglycosylated, relatively stable, and do not seem to assemble into particles by themselves.

Genetics, 1987 Nov, 117(3), 429 - 35
Evidence for positive regulation of the proline utilization pathway in Saccharomyces cerevisiae; Brandriss MC; A mutation has been identified that prevents Saccharomyces cerevisiae cells from growing on proline as the sole source of nitrogen, causes noninducible expression of the PUT1 and PUT2 genes, and is completely recessive . In the put3-75 mutant, the basal level of expression (ammonia as nitrogen source) of PUT1-lacZ and PUT2-lacZ gene fusions as measured by beta-galactosidase activity is reduced 4- and 7-fold, respectively, compared with the wild-type strain . Normal regulation is not restored when the cells are grown on arginine as the sole nitrogen source and put3-75 cells remain sensitive to the proline analog, L-azetidine-2-carboxylic acid, indicating that the block is not at the level of transport of the inducer, proline . In a cross between the put3-75 strain and the semidominant, constitutive mutation PUT3c-68, only parental ditype tetrads were found, indicating allelism of the two mutations . Further support for allelism derives from the comparison of enzyme levels in heteroallelic and heterozygous diploid strains . The constitutive allele appears to be fully dominant to the noninducible allele but only partially dominant to the wild type, suggesting an interaction between the wild-type and PUT3c-68 gene products . The PUT3 gene maps on chromosome XI, about 5.7 cM from the centromere . The phenotypes of alleles of the PUT3 gene, either recessive and noninducible (the put3-75 phenotype) or semidominant and constitutive (the PUT3c-68 phenotype), and their pleiotropy suggest that the PUT3 gene product is a positive activator of the proline utilization pathway.

Proc Natl Acad Sci U S A, 1987 Nov, 84(22), 8031 - 4
Physiological levels of normal tRNA(CAGGln) can effect partial suppression of amber mutations in the yeast Saccharomyces cerevisiae; Weiss WA et al.; A number of ciliated protozoa are known to read the stop codons UAA and UAG as sense codons that specify glutamine during protein synthesis . In considering evolutionary mechanisms for this curious divergence from the standard genetic code, we propose the existence of progenitor tRNAs for glutamine that can weakly suppress UAA and UAG codons . It has been previously shown that multicopy plasmids that overexpress normal tRNA(CAAGln) and tRNA(CAGGln) genes from the yeast Saccharomyces cerevisiae can partially suppress a number of yeast ochre and amber mutations, respectively . In the present study we show that the tRNA(CAGGln) gene can also function as a weak amber suppressor when expressed in cells at physiological levels . This observation is consistent with a role of tRNA(CAGGln) as an evolutionary progenitor of tRNAs that strongly decode UAG codons.

J Biochem (Tokyo), 1987 Nov, 102(5), 1089 - 100
Primary structure and product characterization of the Saccharomyces cerevisiae CHO1 gene that encodes phosphatidylserine synthase; Kiyono K et al.; An open reading frame of 828 base pairs was found in the CHO1 gene region of Saccharomyces cerevisiae by nucleotide sequencing analysis . Its enhanced expression with the aid of the PHO5 regulatory sequence resulted in an overproduction of a protein with a molecular weight of approximately 30,000, which in turn was converted by proteolysis to active phosphatidylserine synthase, whose molecular weight was approximately 23,000 . The larger protein was concluded to be the primary product of the CHO1 gene, since its amino-terminal sequence was identical to that deduced from the nucleotide sequence of the above open reading frame, except for the terminal methionine residue . A partial homology in primary structures was noticed between this yeast enzyme and phosphatidylglycerophosphate synthase of Escherichia coli which also uses CDP-diacylglycerol as a substrate . The overproduced phosphatidylserine synthase in both microsomal and extensively purified fractions displayed two different Km values for L-serine, i.e., 0.14 mM at low L-serine concentrations and 9.5 mM at high L-serine concentrations . This may indicate a negatively cooperative regulation of this enzyme activity or the presence of two active components with different affinities for L-serine.

Genes Dev, 1987 Nov, 1(9), 931 - 7
Rigorous feedback control of cAMP levels in Saccharomyces cerevisiae; Nikawa J et al.; We have isolated and characterized normal and mutant alleles of many of the genes of the RAS/adenylyl cyclase pathway of the yeast Saccharomyces cerevisiae . Manipulation of those genes has revealed a system for feedback control that can modulate cAMP levels over at least a 10,000-fold range . The feedback control depends upon the activity of the cAMP-dependent protein kinases and requires the presence of the CDC25 and RAS proteins . The capacity for such dramatic control of cAMP levels raises fundamental questions about the normal mechanism of action of the cAMP signaling system in yeast.

J Bacteriol, 1987 Nov, 169(11), 4941 - 5
MKT1, a nonessential Saccharomyces cerevisiae gene with a temperature-dependent effect on replication of M2 double-stranded RNA; Wickner RB; The MKT1 gene was defined by recessive alleles present in many laboratory strains of Saccharomyces cerevisiae that result in loss of M2 double-stranded RNA at temperatures above 30 degrees C if L-A-HN double-stranded RNA is present but not if L-A-H is present . I mapped MKT1 near TOP2 and isolated the gene by chromosome walking from TOP2 . The gene location was defined by deletions, and a 2.8-kilobase transcript corresponding to the gene was detected . The recessive natural-variant mutations are not deletions as judged by Southern blots, but deletions of the MKT1 gene constructed in vitro and used to replace the normal gene surprisingly resulted in the same phenotype as that of the mkt1 natural variants, namely, a temperature-dependent maintenance of M2 double-stranded RNA . Thus the MKT1 gene product is only needed for M2 replication or maintenance at temperatures above 30 degrees C and if L-A-HN is present . The temperature dependence does not reflect the thermolability of a mutant gene product, as had previously been thought, nor does L-A double-stranded RNA need MKT1, as previously hypothesized . MKT1 may be involved in the process of packaging M2 double-stranded RNA . MKT1 is dispensable for host cell growth, mating, meiosis, and spore germination.

J Cell Biol, 1987 Nov, 105(5), 1981 - 7
Apparent endocytosis of fluorescein isothiocyanate-conjugated dextran by Saccharomyces cerevisiae reflects uptake of low molecular weight impurities, not dextran; Preston RA et al.; Concurrent with Riezman's report (Riezman, H . 1985, Cell . 40:1001-1009) that fluid-phase endocytosis of the small molecule Lucifer yellow occurs in the yeast Saccharomyces cerevisiae, Makarow (Makarow, M . 1985 . EMBO {Eur . Mol . Biol . Organ.} J . 4:1861-1866) reported the endocytotic uptake of 70-kD FITC-dextran (FD) and its subsequent compartmentation into the yeast vacuole . Samples of FD synthesized and purified here failed to label yeast vacuoles under conditions that allowed labeling using commercial FD . Chromatography revealed that the commercial FD was heavily contaminated with at least three low molecular weight fluorescent compounds . Dialysis was ineffective for removing the contaminants . After purification (Sephadex G25, ethanol extraction), commercial FD was incapable of labeling vacuoles . Extracts of cells labeled with partially purified FD contained FITC, not FD, based on Sephadex and thin layer chromatography . In either the presence or absence of unlabeled 70-kD dextran, authentic FITC (10 micrograms/ml) was an effective labeling agent for vacuoles . The rapid kinetics (0.28 pmol/min per 10(6) cells at pH 5.5) and the pH dependence of FITC uptake suggest that the mechanism of FITC uptake involves diffusion rather than endocytosis . In view of these results, labeling experiments that use unpurified commercial FD should be interpreted with caution.

Nucleic Acids Res, 1987 Oct 26, 15(20), 8439 - 50
Metal-binding, nucleic acid-binding finger sequences in the CDC16 gene of Saccharomyces cerevisiae; Icho T et al.; The CDC16 gene is involved in the process of chromosome segregation in mitosis and a cdc16ts mutant accumulates the predominant microtubule-associated protein at the nonpermissive temperature . We find that the CDC16 gene open reading frame (ORF) is capable of encoding a protein whose calculated molecular weight and pI are 94,967 and 6.60, respectively . This hypothetical protein contains 16 cysteine residues; five are clustered at the N-terminal, 4 are placed about 3 residues apart in the middle of the peptide, and 3 are located close to the C-terminal . Each of these could form a metal-binding, nucleic acid-binding domain, suggesting this protein acts either as a repressor of the microtubule-associated protein gene or as a component necessary for spindle elongation, possibly interacting with the DNA . The start of the CDC16 ORF is only 95 bp downstream from the end of the MAK11 ORF . In this region there are two TATA boxes in tandem, but there is no room for a UAS or other regulatory sequences . An ATG is present 5 bp upstream of the start of the large ORF . Its frame terminates after only two amino acids.

J Biol Chem, 1987 Oct 25, 262(30), 14563 - 70
Purification and characterization of CDP-diacylglycerol synthase from Saccharomyces cerevisiae; Kelley MJ et al.; The membrane-associated phospholipid biosynthetic enzyme CDP-diacylglycerol synthase (CTP:phosphatidate cytidylyltransferase, EC 2.7.7.41) was purified 2,300-fold from Saccharomyces cerevisiae . The purification procedure included Triton X-100 solubilization of mitochondrial membranes, CDP-diacylglycerol-Sepharose affinity chromatography, and hydroxylapatite chromatography . The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Radiation inactivation of mitochondrial associated and purified CDP-diacylglycerol synthase suggested that the molecular weight of the native enzyme was 114,000 . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme preparation yielded two subunits with molecular weights of 56,000 and 54,000 . Antibodies prepared against the purified enzyme immunoprecipitated CDP-diacylglycerol synthase activity and subunits . CDP-diacylglycerol synthase activity was dependent on magnesium ions and Triton X-100 at pH 6.5 . Thio-reactive agents inhibited activity . The activation energy for the reaction was 9 kcal/mol, and the enzyme was thermally labile above 30 degrees C . The Km values for CTP and phosphatidate were 1 and 0.5 mM, respectively, and the Vmax was 4,700 nmol/min/mg . Results of kinetic and isotopic exchange reactions suggested that the enzyme catalyzes a sequential Bi Bi reaction mechanism.

Biochem J, 1987 Oct 15, 247(2), 377 - 84
The role of the metal ion in the mechanism of the K+-activated aldehyde dehydrogenase of Saccharomyces cerevisiae; Dickinson FM et al.; The effect of K+ on assays of the enzyme was studied and it appears that the activation occurs slowly by a two-step process . Kinetic measurements suggest that the enzyme-catalysed reaction can proceed slowly (0.4%) in the complete absence of K+ . The enzyme exhibits a K+-activated esterase activity, which is further activated by NAD+ or NADH . Stopped-flow studies indicated that the principal effect of K+ on the dehydrogenase reaction is to accelerate a step (possibly acyl-enzyme hydrolysis) associated with a fluorescence and small absorbance transient that occurs after hydride transfer and before NADH dissociation from the terminal E-NADH complex . The variation of activity of the enzyme with pH was studied . An enzyme group with pKa approx . 7.1 apparently promotes enzyme activity when in its alkaline form.

Biochim Biophys Acta, 1987 Oct 15, 915(3), 385 - 92
The presence of functional arginine residues in phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae; Malebran LP et al.; Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) is completely inactivated by phenylglyoxal and 2,3-butanedione in borate buffer at pH 8.4, with pseudo-first-order kinetics and a second-order rate constant of 144 min-1 X M-1 and 21.6 min-1 X M-1, respectively . Phosphoenolpyruvate, ADP and Mn2+ (alone or in combination) protect the enzyme against inactivation, suggesting that the modification occurs at or near to the substrate-binding site . Almost complete restoration of activity was obtained when a sample of 2,3-butanedione-inactivated enzyme was freed of excess modifier and borate ions, suggesting that only arginyl groups are modified . The changes in the rate of inactivation in the presence of substrates and Mn2+ were used to determine the dissociation constants for enzyme-ligand complexes, and values of 23 +/- 3 microM, 168 +/- 44 microM and 244 +/- 54 microM were found for the dissociation constants for the enzyme-Mn2+, enzyme-ADP and enzyme-phosphoenolpyruvate complexes, respectively . Based on kinetic data, it is shown that 1 mol of reagent must combine per enzyme active unit in order to inactivate the enzyme . Complete inactivation of the carboxykinase can be correlated with the incorporation of 3-4 mol {7-14C}phenylglyoxal per mol of enzyme subunit . Assuming a stoichiometry of 1:1 between phenylglyoxal incorporation and arginine modification, our results suggest that the modification of only two of the three to four reactive arginine residues per phosphoenolpyruvate carboxykinase subunit is responsible for inactivation.

Nucleic Acids Res, 1987 Oct 12, 15(19), 7975 - 89
The nucleotide sequence of the PRI1 gene related to DNA primase in Saccharomyces cerevisiae; Plevani P et al.; The PRI1 gene of Saccharomyces cerevisiae encodes for the p48 polypeptide of DNA primase . We have determined the nucleotide sequence of a 1,965 bp DNA fragment containing the PRI1 locus . The entire coding sequence of the gene lies within an open reading frame, and there are 409 amino acids in the single polypeptide protein if translation is assumed to start at the first ATG in this frame . The 5' and 3' end-points of PRI1 mRNA have been determined by S1 mapping and primer extension analysis . The primary structure and the codon usage of PRI1 suggest that this essential gene is poorly expressed in yeast cells.

Nucleic Acids Res, 1987 Oct 12, 15(19), 7951 - 62
The effects of alterations within the 3' untranslated region of the pyruvate kinase messenger RNA upon its stability and translation in Saccharomyces cerevisiae; Purvis IJ et al.; A 53 basepair deletion was constructed within the 3' untranslated region (3' UTR) of the yeast pyruvate kinase (PYK) gene borne upon a centromeric plasmid . Various modular assemblies of the pUC13 polylinker DNA (single unit = 44 bp) were used to replace the deleted region, and the effects of these modifications upon both transcript stability and translation ascertained in yeast . The use of a differential probing stratagem, based on the hybridisation of specific oligonucleotides to either pUC13 polylinker or unaltered PYK 3' UTR sequences, allowed for discrimination between mutant (plasmid borne) and wild-type (chromosomal) PYK transcripts . In no construct was there any significant alteration in mRNA stability, but translation of the PYK mRNA was severely curtailed by truncation of the 3' UTR or the presence of a strong hairpin-loop structure in the 3' UTR . A specific mutation in the N-terminal coding sequences, which created a premature termination codon in both a 3' 'tagged' PYK plasmid and a PYK/LacZ fusion gene, aborted the translation of a majority of their transcripts but left their chemical half-lives unaltered . This observation is at variance with some previously published data (Losson & Lacroute (1979) Proc Natl Acad Sci USA 76, 5134; Pelsey & Lacroute (1984) Curr Genet 8, 277), but is consistent with our own earlier observation that there is no obvious link between ribosome loading and mRNA stability in yeast (Santiago et al . (1986) Nucleic Acids Res 14, 8347) . Possible reasons for this disparity are discussed.

Nucleic Acids Res, 1987 Oct 12, 15(19), 7963 - 74
Translation and stability of an Escherichia coli beta-galactosidase mRNA expressed under the control of pyruvate kinase sequences in Saccharomyces cerevisiae; Purvis IJ et al.; Plasmids were assembled in which the coding region of the pyruvate kinase (PYK) gene of Saccharomyces cerevisiae was replaced by that of the B-galactosidase (LacZ) gene from Escherichia coli . Analysis of the resultant, chimaeric transcripts from low copy number, centromeric plasmids indicated that this substitution caused a dramatic reduction in the steady-state level of the messenger RNA (mRNA) . This fluctuation cannot be wholly accounted for by the 2-fold decrease in mRNA stability observed . This is consistent with the existence of a transcriptional Downstream Activation Site (DAS) within the PYK coding region, analogous to the DAS reported within the yeast phosphoglycerate kinase gene (PGK; Kingsman, S M et al . (1985) Biotech . Gen . Eng . Rev . 3, 377) . At these low levels of heterologous gene expression, comparison of the distribution of PYK and PYK/LacZ transcripts across polysome gradients revealed no significant effect mediated by their striking disparity in codon usage . Nevertheless, upon increasing B-galactosidase mRNA levels, via manipulation of plasmid copy number, a distinct decline in ribosome loading was observed for the heterologous PYK/LacZ transcript which was not mirrored by either endogenous PYK transcripts or other yeast mRNAs of high (Ribosomal protein 1) or moderate (Actin) codon bias . However, high levels of the PYK/LacZ mRNA did affect the translation of an endogenous mRNA with poor codon bias (TRP2) . The possible basis for this phenomenon is discussed.

J Cell Sci, 1987 Oct, 88 ( Pt 3), 273 - 81
Preliminary characterization of maturation-promoting factor from yeast Saccharomyces cerevisiae; Tachibana K et al.; It has been known for some time that maturation-promoting factor (MPF) appears in a wide variety of eukaryotic cells at M phase and exerts equal M-phase-promoting activity in both meiotic cells and mitotic cells in a non-specific manner . MPF was extracted from cdc20 mutant cells of the yeast Saccharomyces cerevisiae synchronized at M phase by incubation at the restrictive temperature . When injected into immature oocytes of Xenopus laevis, yeast MPF caused meiosis reinitiation in a dose-dependent manner and even in the presence of cycloheximide . Yeast MPF exerted its activity in starfish oocytes as well . MPF activity was obtained only from cells in M phase and not from G1, S or G2 phase cells, indicating cyclical changes during the yeast mitotic cell cycle . Preliminary characterization of yeast MPF revealed that its activity was associated with a heat-labile protein having a sedimentation coefficient value of 6 S . In contrast to the current assumption that MPF is a Ca-sensitive phosphoprotein stabilized by phosphorylated small molecules, such as ATP and Na-beta-glycerophosphate, the present study revealed that yeast MPF was still active even after treatment with either Ca2+ or alkaline phosphatase . Furthermore, it was found that yeast MPF and these phosphorylated small molecules were complementary in inducing reinitiation of meiosis, since the meiosis-reinitiating activity was detected only when both were present simultaneously and almost undetectable when either of them was present alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1987 Oct, 209(3), 458 - 66
The RAD3 gene of Saccharomyces cerevisiae: isolation and characterization of a temperature-sensitive mutant in the essential function and of extragenic suppressors of this mutant; Naumovski L et al.; Mutations in the RAD3 gene of Saccharomyces cerevisiae were generated by integration of a mutagenized incomplete copy of the cloned gene into wild-type cells . Integrants were mass screened for colonies with abnormal growth characteristics at 37 degrees C . A single temperature-sensitive mutant (rad3ts-1) was isolated and was shown to result from a missense mutation at codon 73 of the RAD3 gene . When shifted from 30 degrees C to 37 degrees C the strain undergoes only 2-4 cell doublings . This phenotype can be rescued by plasmids in which the essential function of the cloned RAD3 gene is intact, but not plasmids in which this function is inactivated . The mutant strain is weakly sensitive to ultraviolet (UV) radiation at restrictive temperatures . Measurement of RNA, DNA and protein synthesis at various times after shifting to restrictive temperatures does not show preferential inactivation of any one of these parameters and the temperature-sensitive mutation does not cause arrest at any specific phase of the cell cycle . The rad3ts-1 strain was transformed with multicopy plasmids from a normal yeast genomic library and two plasmids that partially suppress the temperature-sensitive phenotype were isolated . These suppressor genes (designated SRE1 and SRE2) are distinct from RAD3 and do not suppress the phenotype of several other temperature-sensitive mutants tested . Mutant strains carrying disruptions of the SRE1 gene are viable and are not sensitive to UV or gamma radiation.

Lipids, 1987 Oct, 22(10), 715 - 20
Sterol and phospholipid acyl chain alterations in Saccharomyces cerevisiae secretion mutants as a function of temperature stress; Low C et al.; Analyses of free sterol, steryl ester and fatty acid components from yeast secretion mutants indicated that free and esterified sterol remained relatively constant over a growth range of 24 C to 34 C . The saturated fatty acid components (16:0 and 18:0) increased while the unsaturated fatty acids (16:1 and 18:1) decreased as the growth temperature increased . In secretory mutants, fatty acid composition changes are more pronounced than in the wild-type strain . A shift toward increased saturated and decreased unsaturated fatty acid was observed when cells were subjected to a 2-hr temperature upshift to 37 C . Steady-state fluorescence anisotropy data indicated that modifications to the lipid component of yeast plasma membrane produced lipid thermotropic transitions that were 3 C to 6 C higher in yeast cells subjected to thermal stress.

Mol Cell Biol, 1987 Oct, 7(10), 3785 - 91
Sequences required for transcriptional initiation of the Saccharomyces cerevisiae CYC7 genes; Healy AM et al.; A series of BAL 31 deletions were constructed in the upstream region of the Saccharomyces cerevisiae CYC7 gene to determine sequences required for transcriptional initiation . These deletions identified the TATA box as an alternating A-T sequence at -160 and the initiation sequences as well as the spatial relationship between them . The TATA box was necessary for wild-type levels of expression of the CYC7 gene . Decreasing the distance between the TATA sequence and the initiation site did not alter gene expression, but the site of transcription was shifted 3'-ward . In most cases, transcription initiated at a number of sites, the 5'-most of which was the first suitable site greater than 45 base pairs 3' of the TATA sequence, suggesting a spatial relationship between these sequences . Consensus sequences previously proposed for initiation sites were evaluated with respect to the start sites identified in this study as well as the start sites of other yeast genes.

Mol Cell Biol, 1987 Oct, 7(10), 3678 - 87
Immunofluorescence localization of the Saccharomyces cerevisiae CDC12 gene product to the vicinity of the 10-nm filaments in the mother-bud neck; Haarer BK et al.; Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud (B . Byers and L . Goetsch, J . Cell Biol . 69:717-721, 1976) . Mutants defective in any of four genes (CDC3, CDC10, CDC11, and CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and cell-wall deposition and an inability to complete cytokinesis . We fused the cloned CDC12 gene to the Escherichia coli lacZ and trpE genes and used the resulting fusion proteins to raise polyclonal antibodies specific for the CDC12 gene product . In immunofluorescence experiments with affinity-purified antibodies, the neck region of wild-type and mutant cells stained in patterns consistent with the hypothesis that the CDC12 gene product is a constituent of the ring of 10-nm filaments . Without careful affinity purification of the CDC12-specific antibodies, these staining patterns were completely obscured by the staining of residual cell wall components in the neck by antibodies present even in the "preimmune" sera of all rabbits tested.

Mol Cell Biol, 1987 Oct, 7(10), 3637 - 45
Molecular analysis of SSN6, a gene functionally related to the SNF1 protein kinase of Saccharomyces cerevisiae; Schultz J et al.; Mutations in the SSN6 gene suppress the invertase derepression defect caused by a lesion in the SNF1 protein kinase gene . We cloned the SSN6 gene of Saccharomyces cerevisiae and identified its 3.3-kilobase poly(A)-containing RNA . Disruption of the gene caused phenotypes similar to, but more severe than, those caused by missense mutations: high-level constitutivity for invertase, clumpiness, temperature-sensitive growth, alpha-specific mating defects, and failure to homozygous diploids to sporulate . In contrast, the presence of multiple copies of SSN6 interfered with derepression of invertase . An ssn6 mutation was also shown to cause glucose-insensitive expression of a GAL10-lacZ fusion and maltase . The mating defects of MAT alpha ssn6 strains were associated with production of two a-specific products, a-factor and barrier, and reduced levels of alpha-factor; no deficiency of MAT alpha 2 RNA was detected . We showed that ssn6 partially restored invertase expression in a cyr1-2 mutant, although ssn6 was clearly not epistatic to cyr1-2 . We also determined the nucleotide sequence of SSN6, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine) . Possible functions of the SSN6 product are discussed.

Mol Cell Biol, 1987 Oct, 7(10), 3566 - 73
Roles of the 2 microns gene products in stable maintenance of the 2 microns plasmid of Saccharomyces cerevisiae; Reynolds AE et al.; We have examined the replication and segregation of the Saccharomyces cerevisiae 2 microns circle . The amplification of the plasmid at low copy numbers requires site-specific recombination between the 2 microns inverted repeat sequences catalyzed by the plasmid-encoded FLP gene . No other 2 microns gene products are required . The overexpression of FLP in a strain carrying endogenous 2 microns leads to uncontrolled plasmid replication, longer cell cycles, and cell death . Two different assays show that the level of Flp activity decreases with increasing 2 microns copy number . This regulation requires the products of the REP1 and REP2 genes . These gene products also act together to ensure that 2 microns molecules are randomly segregated between mother and daughter cells at cell division.

Mol Cell Biol, 1987 Oct, 7(10), 3473 - 81
The two gene pairs encoding H2A and H2B play different roles in the Saccharomyces cerevisiae life cycle; Norris D et al.; We have isolated Saccharomyces cerevisiae mutants bearing deletions of one or the other of the two divergently transcribed gene pairs encoding H2A and H2B . The deletions produced diverse effects on the yeast life cycle . Deletion of TRT1, one of the H2A-H2B gene pair sets, affected mitotic growth, sporulation, spore germination, the heat shock response, and exit from the stationary phase; deletion of TRT2, the other H2A-H2B gene pair set, had negligible effects on these same processes . Using a genetic complementation assay, we found that the differential effects of the deletions could be attributed to two features of the gene sets: first, the expression of the TRT1 gene pair, but not the TRT2 gene pair, could compensate for the absence of its partner; second, the protein subtypes encoded by the two gene pairs appear to have different functions in the heat shock response.

J Cell Biol, 1987 Oct, 105(4), 1527 - 38
Subcellular localization of a protein kinase required for cell cycle initiation in Saccharomyces cerevisiae: evidence for an association between the CDC28 gene product and the insoluble cytoplasmic matrix; Wittenberg C et al.; The product of the Saccharomyces cerevisiae gene CDC28, a protein kinase required for initiation of the cell division cycle, was localized within yeast cells . By using immunofluorescence methods, the CDC28 product was shown to be primarily cytoplasmic in distribution . The gene product was localized largely to the particulate fraction by differential centrifugation after mechanical disruption in aqueous buffers . The particulate association was not affected by the presence of nonionic detergent . To refine this localization further, a procedure was developed for the preparation of yeast cytoplasmic matrices which resemble the cytoskeletons of vertebrate cells on the basis of methodology, immunochemistry, and gross ultrastructure . A portion of the CDC28 product was found to be tightly associated with these detergent-insoluble cytoplasmic matrices by both immunofluorescence and immunoblotting procedures . Although, for technical reasons, precise quantitation was not possible, it is estimated that a minimum of 2-15% of the total CDC28 product pool is involved in the association with the insoluble matrix . Alcohol dehydrogenase, a soluble cytoplasmic protein, was found not to be associated with the cytoplasmic matrices at any detectable level, whereas, in contrast, approximately 10-40% of the total cellular actin, a bonafide cytoskeletal protein, was present in these structures . The proportion of CDC28 gene product associated with the particulate fraction, and perhaps the insoluble matrix, appears to be substantially decreased during the preparation of spheroplasts.

Genetics, 1987 Oct, 117(2), 203 - 12
Mutational analysis of meiotic and mitotic centromere function in Saccharomyces cerevisiae; Cumberledge S et al.; A centromere (CEN) in Saccharomyces cerevisiae consists of approximately 150 bp of DNA and contains 3 conserved sequence elements: a high A + T region 78-86 bp in length (element II), flanked on the left by a conserved 8-bp element I sequence (PuTCACPuTG), and on the right by a conserved 25-bp element III sequence . We have carried out a structure-function analysis of the element I and II regions of CEN3 by constructing mutations in these sequences and subsequently determining their effect on mitotic and meiotic chromosome segregation . We have also examined the mitotic and meiotic segregation behavior of ARS plasmids containing the structurally altered CEN3 sequences . Replacing the periodic tracts of A residues within element II with random A + T sequences of equal length increases the frequency of mitotic chromosome nondisjunction only 4-fold; whereas, reducing the A + T content of element II while preserving the length results in a 40-fold increase in the frequence of chromosome nondisjunction . Structural alterations in the element II region that do not decrease the overall length have little effect on the meiotic segregation behavior of the altered chromosomes . Centromeres containing a deletion of element I or a portion of element II retain considerable mitotic activity, yet plasmids carrying these same mutations segregate randomly during meiosis I, indicating these sequences to be essential for maintaining attachment of the replicated sister chromatids during the first meiotic division . The presence of an intact element I sequence properly spaced from the element III region is absolutely essential for proper meiotic function of the centromere.

Eur J Biochem, 1987 Oct 1, 168(1), 13 - 9
Purification and biochemical properties of calmodulin from Saccharomyces cerevisiae; Ohya Y et al.; Calmodulin from the yeast Saccharomyces cerevisiae was purified to complete homogeneity by hydrophobic interaction chromatography and HPLC gel filtration . The biochemical properties of the purified protein as calmodulin were examined under various criteria and its similarity and dissimilarity to other calmodulins have been described . Like other calmodulins, yeast calmodulin activated bovine phosphodiesterase and pea NAD kinase in a Ca2+-dependent manner, but its concentration for half-maximal activation was 8-10 times that of bovine calmodulin . The amino acid composition of yeast calmodulin was different from those of calmodulins from other lower eukaryotes in that it contained no tyrosine, but more leucine and had a high ratio of serine to threonine . Yeast calmodulin did not contain tryptophanyl or tyrosyl residues, so its ultraviolet spectrum reflected the absorbance of phenylalanyl residues, and had a molar absorption coefficient at 259 nm of 1900 M-1 cm-1 . Ca2+ ions changed the secondary structure of yeast calmodulin, causing a 3% decrease in the alpha-helical content, unlike its effect on other calmodulins . Antibody against yeast calmodulin did not cross-react with bovine calmodulin, and antibody against bovine calmodulin did not cross-react with yeast calmodulin, presumably due to differences in the amino acid sequences of the antigenic sites . It is concluded that the molecular structure of yeast calmodulin differs from those of calmodulins from other sources, but that its Ca2+-dependent regulatory functions are highly conserved and essentially similar to those of calmodulins of higher eukaryotes.

Proc Natl Acad Sci U S A, 1987 Oct, 84(19), 6815 - 9
Expression of a set of synthetic suppressor tRNA(Phe) genes in Saccharomyces cerevisiae; Masson JM et al.; Synthetic ochre and amber tRNA suppressor genes derived from the yeast tRNA(PheGAA) sequence have been constructed . They were efficiently transcribed in vitro and expressed in vivo via a synthetic expression cassette . tRNA(PheUUA) and tRNA(PheUUA) delta IVS (IVS = intervening sequence) are relatively inefficient ochre suppressors . They are toxic to the cell when expressed on a multicopy plasmid, and they do not suppress at all when present as single copies . The intron does not seem to have any effect on suppression . In contrast, the amber suppressor tRNA(PheCUA) delta IVS is efficient when expressed from a single-copy plasmid, while its efficiency is reduced on a multicopy vector.

J Bacteriol, 1987 Oct, 169(10), 4811 - 5
Pheromone induction of agglutination in Saccharomyces cerevisiae a cells; Terrance K et al.; a-Agglutinin, the cell surface sexual agglutinin of yeast a cells, was assayed by its ability to bind its complementary agglutinin, alpha-agglutinin . The specific binding of 125I-alpha-agglutinin to a cells treated with the sex pheromone alpha-factor was 2 to 2.5 times that of binding to a cells not treated with alpha-factor . Competition with unlabeled alpha-agglutinin revealed that the increased binding was due to increased cell surface expression of a-agglutinin, with no apparent change in the binding constant . The increase in site number was similar to the increase in cellular agglutinability . Increased expression of a-agglutinin followed the same kinetics as the increase in cellular agglutinability, with a 10-min lag followed by a 15- to 20-min response time . Induction kinetics were similar in cells in phases G1 and G2 of the cell cycle . Maximal expression levels were similar in cells treated with excess pheromone and in cells exposed to pheromone after destruction of constitutively expressed a-agglutinin.

Microbiologia, 1987 Oct, 3(3), 195 - 203
Purification of two exoglucanases secreted by Saccharomyces cerevisiae and partial characterization of their protein moieties; Ramirez M et al.; Growth of Saccharomyces cerevisiae cells in a synthetic buffered culture medium resulted in the secretion of high levels of two soluble exoglucanases which were purified by a procedure involving one (exglucanase II) and two (exoglucanase I) steps, respectively . Once treated with endoglucosaminidase H (Endo H) both enzymes behaved indistinguishably when analyzed by SDS-PAGE, high pressure liquid chromatography (HPLC) and ionic exchange chromatography . Exoglucanase I, the isoenzyme with higher carbohydrate content, exhibited a higher Km against laminarin and a higher thermal stability than exoglucanase II . However, once the enzymes were deglycosylated in vitro these parameters turned out to be identical . These results suggest that both exoglucanases share a very similar, if not identical protein portion and accordingly may be product of either the same gene or a family of related genes.

Mol Cell Biol, 1987 Oct, 7(10), 3857 - 61
The activation of adenylate cyclase by guanyl nucleotides in Saccharomyces cerevisiae is controlled by the CDC25 start gene product; Daniel J et al.; In the thermosensitive cdc25 start mutant of Saccharomyces cerevisiae, the regulation of adenylate cyclase by guanyl nucleotides was rapidly nullified when the enzyme was prepared from nonsynchronized cells shifted to the restrictive temperature . In agreement with previous in vivo complementation studies, this biochemical defect was fully suppressed by the expression of either the whole cloned CDC25 gene or its C-terminal portion . Moreover, membranes prepared from cdc25(Ts) cells grown at the permissive temperature evinced an altered regulation of adenylate cyclase by guanyl nucleotides . These results indicate that the CDC25 protein, together with RAS, is involved in the regulation of adenylate cyclase by guanyl nucleotides and raise the possibility that adenylate cyclase might form a ternary complex with RAS and CDC25.

J Bacteriol, 1987 Oct, 169(10), 4660 - 7
Allantoin transport in Saccharomyces cerevisiae is regulated by two induction systems; Cooper TG et al.; We show that the allantoin transport system of Saccharomyces cerevisiae responds to two induction systems, one mediated by allophanate or its analog oxalurate and the other mediated by allantoin or its analog hydantoin acetate . The effects of the two inducers were additive in strain M85 . Like other allantoin pathway genes, oxalurate-mediated induction of allantoin transport required a functional DAL81 gene product . Hydantoin acetate-mediated induction of the system, on the other hand, occurred normally in dal81 mutants . This suggests that induction was not only mediated by two separate inducers, but also involved different regulatory proteins . Induction is probably a transcriptionally regulated process, because addition of hydantoin acetate or oxalurate to the culture medium increased the steady-state levels of mRNA encoded by a gene required for allantoin transport (DAL4).

J Biol Chem, 1987 Sep 25, 262(27), 13359 - 65
Study of the kinetic and physical properties of the orotidine-5'-monophosphate decarboxylase domain from mouse UMP synthase produced in Saccharomyces cerevisiae; Langdon SD et al.; In mammals, the bifunctional protein UMP synthase contains the final two enzymatic activities, orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase (ODCase), for de novo biosynthesis of UMP . The plasmid pMEJ contains a cDNA for the ODCase domain of mouse Ehrlich ascites UMP synthase . The cDNA from pMEJ was joined to the Saccharomyces cerevisiae iso-1-cytochrome c (CYC1) promoter and the first four CYC1 coding nucleotides in the plasmid pODCcyc . ODCase-deficient yeast cells (HF200x1) transformed with pODCcyc expressed an active ODCase domain with a specific activity of 20 nmol/min/mg in cell extracts . The expressed ODCase domain has a lower affinity for the substrate orotidine 5'-monophosphate and the inhibitor 6-azauridine 5'-monophosphate than intact UMP synthase or an ODCase domain isolated after proteolysis of homogenous UMP synthase . Sucrose density gradient sedimentation experiments showed that the expressed ODCase domain forms a dimer in the presence of ligands which bind at the catalytic site . These studies support the existence of an ODCase structural domain which contains the ODCase catalytic site and a dimerization surface of UMP synthase, but the domain may not have the regulatory site required to form the altered dimer form.

Biochim Biophys Acta, 1987 Sep 25, 921(2), 205 - 12
Relationship between intracellular sterol content and sterol esterification and hydrolysis in Saccharomyces cerevisiae; Lewis TA et al.; The relationship between the supply of free sterol and the synthesis of steryl esters by an auxotroph of Saccharomyces cerevisiae has been examined in order to understand the role of cellular free sterol content in the regulatory interactions of sterol esterification . Our results show that the yeast cells must maintain an essential, low level of free sterol that is critical for growth . An additional, expandable pool of free sterol is maintained by the cells, provided there is adequate available sterol . As the quantity of sterol in the expandable pool increases, there is a progressively increasing rate of sterol esterification, which is consistent with the results from in vitro assays of acyl-Co A:ergosterol acyltransferase . Some increases in acyltransferase activity were insensitive to the protein synthetic inhibitor, cycloheximide . The data support the conclusion that sterol interconversion between the free and esterified forms is directed toward maintaining the essential amount of free sterol and that the activity of the relevant sterol enzymes in this organism are modulated in response to intracellular sterol content.

Science, 1987 Sep 18, 237(4821), 1484 - 7
Saccharomyces cerevisiae has a U1-like small nuclear RNA with unexpected properties; Siliciano PG et al.; Previous experiments indicated that only a small subset of the approximately equal to 24 small nuclear RNAs (snRNAs) in Saccharomyces cerevisiae have binding sites for the Sm antigen, a hallmark of metazoan small nuclear ribonucleoproteins (snRNPs) involved in pre-messenger RNA splicing . Antibodies from human serum to Sm proteins were used to show that four snRNAs (snR7, snR14, snR19, and snR20) can be immunoprecipitated from yeast extracts . Three of these four, snR7, snR14, and snR20, have been shown to be analogs of mammalian U5, U4, and U2, respectively . Several regions of significant homology to U1 (164 nucleotides) have now been found in cloned and sequenced snR19 (568 nucleotides) . These include ten out of ten matches to the 5' end of U1, the site known to interact with the 5' splice site of mammalian introns . Surprisingly, the precise conservation of this sequence precludes perfect complementarity between snR19 and the invariant yeast 5' junction (GTATGT), which differs from the mammalian consensus at the fourth position (GTPuAGT).

Biochem Biophys Res Commun, 1987 Sep 15, 147(2), 556 - 64
In vitro assay of squalene epoxidase of Saccharomyces cerevisiae; M'Baya B et al.; We describe a simple assay for measuring squalene epoxidase specific activity in Saccharomyces cerevisiae cell-free extracts, by using {14C} farnesyl pyrophosphate as substrate . Cofactor requirements for activity are FAD and NADPH or NADH, NADPH being the preferred reduced pyridine nucleotide . Squalene epoxidase activity is localized in microsomal fraction and no supernatant soluble factor is required for maximum activity . Microsomal fraction converted farnesyl pyrophosphate into squalene, squalene 2,3-epoxide and lanosterol, showing that squalene 2,3-epoxide-lanosterol cyclase is also a microsome-bound enzyme . We show also that squalene epoxidase activity is not inhibited by ergosterol or lanosterol, but that enzyme synthesis is induced by oxygen.

J Biol Chem, 1987 Sep 15, 262(26), 12752 - 8
Purification and properties of a nuclease from Saccharomyces cerevisiae that cleaves DNA at cruciform junctions; West SC et al.; An endonuclease specific for cruciform junctions has been purified from yeast cells treated with a DNA-damaging agent . The activity was followed through five chromatographic steps by assaying for the linearization of supercoiled plasmid DNA, which extrudes cruciform structures in vitro . The sites of cleavage on pColIR215 were sequenced, and nicks were located to positions symmetrically opposed across the cruciform junction . The products of cleavage were unit length linear duplexes that contained terminal hairpin loops . In contrast to pColIR215, the cleavage patterns of pXG540 plasmid DNA were found to be complex, and cuts were found up to 40 bases from an (A-T)34 sequence that extrudes into a cruciform . Little or no activity could be detected on single-stranded DNA, linear duplex DNA, or nicked circular duplex DNA . The nuclease was insensitive to RNase but was inactivated by treatment with proteinase K . Mg2+ was required as cofactor and could not be replaced by Mn2+, Ca2+, Co2+, or Cu2+ . The native molecular weight of the activity was approximately 200,000 as estimated by gel filtration.

J Biol Chem, 1987 Sep 5, 262(25), 12275 - 82
Mitochondrial and cytoplasmic fumarases in Saccharomyces cerevisiae are encoded by a single nuclear gene FUM1; Wu M et al.; Respiratory defective pet mutants of Saccharomyces cerevisiae assigned to complementation group G5 are deficient in fumarase . A representative mutant from this complementation group was used to clone a nuclear gene (FUM1) whose sequence encodes a protein homologous to bacterial fumarase . Based on the primary structure homology and the elevated levels of fumarase in transformants harboring FUM1 on a multicopy plasmid, this gene is concluded to code for yeast fumarase . In wild type yeast, fumarase is detected in both mitochondria and the soluble postribosomal protein fraction . Several lines of evidence indicate that the two compartmentally distinct fumarases are isoenzyme products of FUM1 . Mutations in FUM1 simultaneously abolish both activities . Transformation of a fumarase mutant with a plasmid containing FUM1 leads to increased fumarase activity in mitochondria and in the postribosomal supernatant fraction . Transformation of the same mutant with a plasmid construct in which the region of FUM1 coding for the amino-terminal 17 amino acids of fumarase is deleted results in a preferential increase of nonmitochondrial fumarase . Northern and S1 nuclease analysis of fumarase transcripts in wild type yeast and in a mutant transformed with FUM1 on an episomal plasmid indicate that the gene is transcribed from multiple start sites, some of which are located inside the coding sequence . The major transcript presumed to code for mitochondrial fumarase has a 5'-untranslated leader of 185 nucleotides . The most abundant shorter transcripts have 5' termini from 57 to 68 nucleotides downstream of the first ATG; their translation products lacking the amino-terminal mitochondrial import signal are proposed to target fumarase to the cytoplasm.

Mikrobiologiia, 1987 Sep-Oct, 56(5), 888 - 9
{Relation of fluctuations in the physiological traits of a continuous Saccharomyces cerevisiae culture to the cell division cycle}; Ivanitskaia IuG et al.; Such physiological characteristics of a continuous Saccharomyces cerevisiae culture as the rate of O2 uptake and CO2 evolution, the rate at which the medium was acidified, were found to vary . When the flow rate was increased from 0.05 to 0.1 h-1, the period of variations also rose (from 3 to 33 h) and two maxima were detected in the quantity of budding cells within one 33-h period of the variations . These findings indicate that the variations do not depend on changes in the physiology of the cell in the course of its division cycle.

Mikrobiologiia, 1987 Sep-Oct, 56(5), 797 - 804
{Cell wall permeability and export of proteins into the culture broth of Saccharomyces cerevisiae}; Tsiomenko AB et al.; The upper limit of Saccharomyces cerevisiae cell wall permeability is such that only molecules with a mass not greater than 2-3 kDa can pass across the cell wall . However, proteins with a much higher molecular mass are exported into the cultural broth when the organism is cultivated in a liquid medium with peptone . Under certain conditions, exogenous proteins interact with the cytoplasmic membrane . These as well as some other findings imply that the cell wall may have microplots with an anomalous permeability, via which the regulated exchange of macromolecules is realized between the intracellular and outer media.

J Gen Microbiol, 1987 Sep, 133 ( Pt 9), 2509 - 16
Proteolysis of hexokinase PII is not the triggering signal of carbon catabolite derepression in Saccharomyces cerevisiae; Fernandez MT et al.; The role of hexokinase PII in mediating carbon catabolite derepression in yeast has been examined . Hexokinase isoenzyme PII (EC 2.7.1.1) was partially degraded when protease inhibitors were omitted from the buffer used for preparation of cell-free extracts . The hexokinase PII inactivation induced by D-xylose was correlated with derepression of maltase (EC 3.2.1.20) in the wild-type strain Saccharomyces cerevisiae G-517 and in D.308.3, a strain that contains the cloned hexokinase PII gene on a multicopy plasmid . This inactivation was not correlated with the loss of hexokinase PII protein as assayed by immunoblotting . We conclude that during the derepression process there is no release of proteolytic peptides from hexokinase PII.

Prikl Biokhim Mikrobiol, 1987 Sep-Oct, 23(5), 703 - 5
{A method of determining the level of provitamin D in Saccharomyces cerevisiae with altered sterol metabolism}; Chichikalova MM et al.; A method is proposed that allows to choose among the polyene-resistant mutants of the yeasts Saccharomyces cerevisiae with changed biosynthesis of ergosterol promising producers of provitamin D4 and structural analogs of provitamin D3 . The method involves both UV-spectrophotometry and thin-layer chromatography . The results obtained are supported by the data of mass-spectrometry of sterols.

Prikl Biokhim Mikrobiol, 1987 Sep-Oct, 23(5), 613 - 6
{Effect of protein synthesis inhibitors on the activity of mitochondrial enzymes of Saccharomyces cerevisiae during desiccation}; Krallish IL et al.; The work deals with the effect of carbon sources, presence of protein synthesis inhibitors (cycloheximide and chloramphenicol) and dehydration regime on the enzyme activity of the dried yeast Saccharomyces cerevisiae . The yeast grown on molasses and dried by aeration demonstrated a notable increase of the NADH-dehydrogenase and succinate dehydrogenase activities as compared with the analogously treated yeast grown on ethanol . The latter showed a notable rise only in the activity of NADH-dehydrogenase during slow drying . Addition of protein synthesis inhibitors into the cultivation medium caused no decrease of activities of the above mentioned enzymes of the dried yeasts in any variant under study.

Genetika, 1987 Sep, 23(9), 1564 - 73
{Repair of plasmid DNA treated with 8-methoxypsoralen and long-wave UV light (lambda=365 nm) in wild type and mutant rad2 cells of Saccharomyces cerevisiae}; Fedorova IV et al.; The method of repeated irradiation has been used to study excision of 8-MOP monoadducts from plasmid and chromosomal DNA in cells of wild type and rad2 mutant of Saccharomyces cerevisiae . The measurement of kinetics of monoadduct removal from chromosomal DNA in intact and competent yeast cells showed that monoadducts were excised in both types of cells with normal repair, but this process was blocked in intact and competent cells of the rad2 mutant . The survival of pYF91 plasmid treated in vitro with 8-MOP plus near UV-light has been studied in the cells of the wild type and in incision-defective rad2 mutant by the measurement of cell transformation frequency . Episomic pYF91 plasmid used in these experiments contained the yeast nuclear LEU2 gene, a portion of 2 mkm DNA and DNA of bacterial plasmid pBR322 with resistance to ampicillin . The pYF91 plasmid was treated with 8-MOP plus near UV-light in vitro, then unbound 8-MOP was removed by dialysis . This DNA was used for transformation . The transformed yeast cells were irradiated repeatedly . The quantitative alteration of the yield of transformants, depending on the time of keeping these yeast cells in complete liquid medium at 30 degrees C, prior to repeated irradiation, allowed to measure the kinetics of monoadduct excision from plasmid DNA . It was shown that monoadducts were removed equally effectively from plasmid DNA introduced into cells of the wild type and rad2 mutant . Possibly, the repair system of both these strains provides excision of monoadducts from plasmid DNA, but this process is blocked in the rad2 mutant, relatively to monoadduct excision from chromosomal DNA.

Genetika, 1987 Sep, 23(9), 1555 - 63
{Mutants of the yeast Saccharomyces cerevisiae characterized by enhanced induced mutagenesis . III . Effect of the him mutation on the effectiveness and specificity of UF-induced mutagenesis}; Ivanov EL et al.; We have studied the influence of him1-1, him2-1, him3-1 and himX mutations on induction frequency and specificity of UV-induced adenine-dependent mutations in the yeast Saccharomyces cerevisiae . Him mutations do not render haploid cells more sensitive to the lethal action of UV-light; however, in him strains adenine-dependent mutations (ade1, ade2) were induced more frequently (1.5--2-fold), as compared to the HIM strain . An analysis of the molecular nature of ade2 mutants revealed that him1-1, him2-1 and himX mutations increase specifically the yield of transitions (AT----GC and GC----AT), whereas in the him3-1 strain the yield of transversions was enhanced as well . We suggest him mutations analysed to affect specific repair pathway for mismatch correction.

Arch Microbiol, 1987 Sep, 148(3), 208 - 12
Isolation and composition of the constitutive agglutinins from haploid Saccharomyces cerevisiae cells; Sijmons PC et al.; Sex-specific agglutinins from the cell surface of haploid cells of Saccharomyces cerevisiae (X2180, mta and mt alpha) were purified and analysed . The constitutive agglutinin from mta cells was extractable with 3 mM dithiothreitol . It was shown to be a glycoprotein (3% mannose) with an apparent Mr of 43,000 based on gel filtration, but in SDS-PAGE it behaved as a much smaller molecule (Mr between 18,000 and 26,000) . About one in three amino acids was a hydroxyamino acid . Its biological activity was resistant to boiling for 1 h, but sensitive to pronase . Intact mt alpha cells retained their agglutinability in the presence of dithiothreitol but limited trypsinizing released a biologically active agglutinin fragment . It had an apparent Mr of 320,000 (gel filtration) . When analysed by SDS-PAGE, a single diffuse band with an apparent Mr of 225,000 was observed . The protein was 94% (w/w) mannose with a trace of N-acetyl glucosamine . Its biological activity was almost completely lost after boiling for 1 h . Both agglutinins behaved as monovalent molecules and specifically inhibited the biological activity of both noninduced and pheromone-induced cells . Pheromone treatment of mta cells resulted in an apparent 32-fold increase in agglutinin activity at the cell surface, whereas pheromone treatment of mt alpha cells only doubled the apparent agglutinin activity.

Mutat Res, 1987 Sep, 189(1), 11 - 4
Base-substitution and frameshift mutagenesis by sodium chloride and potassium chloride in Saccharomyces cerevisiae; Parker KR et al.; Sodium chloride (NaCl) and potassium chloride (KCl) are both capable of inducing lethality and mutations when each is administered at a molarity of two for different lengths of time to logarithmic phase cells of the yeast Saccharomyces cerevisiae . Analysis of the revertants indicates that the reversions can be base substitutions, of both the transition and the transversion type, as well as frameshift mutations . At equal molarity, with the frequency of mutations as the criterion, KCl and NaCl are equally efficient in inducing all types of mutations.

Mutat Res, 1987 Sep, 180(1), 81 - 7
Mutants of Saccharomyces cerevisiae deficient in adenine phosphoribosyltransferase; Sahota A et al.; Spontaneous and ethyl methanesulfate induced mutants of Saccharomyces cerevisiae, with partial and complete deficiency of adenine phosphoribosyltransferase (APRT, EC 2.4.2.7), were isolated by selection for resistance to 8-azaadenine . Matings between totally deficient mutants and tester strain resulted in diploid heterozygotes that were sensitive to azaadenine . Upon sporulation and tetrad analysis, azaadenine resistance (and APRT deficiency) segregated as expected for a single Mendelian gene . Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) activity in the mutants was similar to that in the wild-type cells . There was no detectable activity of adenine aminohydrolase (EC 3.5.4.2) in the wild-type or mutant cells.

J Bacteriol, 1987 Sep, 169(9), 4384 - 7
Tunicamycin inhibition of epispore formation in Saccharomyces cerevisiae; Weinstock KG et al.; The ascopore wall of Saccharomyces cerevisiae was found to contain more protein, polymeric glucosamine, and beta-glucan than the vegetative cell wall, which was enriched in mannoprotein relative to ascospore walls . Tunicamycin inhibited sporulation, as judged by the absence of refractile ascospores visible by phase-contrast microscopy, but cells completed meiosis, as demonstrated by the presence of multinucleate asci . Such spores lacked the dense outer layer characteristic of normal spores . Thus, the tunicamycin effect was similar to that of glucosamine auxotrophy (W . L . Whelan and C . E . Ballou, J . Bacteriol . 124:1545-1557, 1975).

J Bacteriol, 1987 Sep, 169(9), 4041 - 8
Identification of the structural gene for dipeptidyl aminopeptidase yscV (DAP2) of Saccharomyces cerevisiae; Suarez Rendueles P et al.; Mutants of Saccharomyces cerevisiae lacking dipeptidyl aminopeptidase yscV were isolated from a strain already defective in dipeptidyl aminopeptidase yscIV, an enzyme with overlapping substrate specificity . The mutants were identified by a staining technique with the chromogenic substrate Ala-Pro-4-methoxy-beta-naphthylamide to screen colonies for the absence of the enzyme . One of the mutants had a thermolabile activity, indicating that it contained a structural gene mutation . The 53 mutants analyzed fell into one complementation group that corresponded to the yscV structural gene, DAP2 . The defect segregated 2:2 in meiotic tetrads, indicating a single chromosomal gene mutation, which was shown to be recessive . Diploids heterozygous for DAP2 displayed gene dosage effects with respect to yscV enzyme activity . The absence of dipeptidyl aminopeptidase yscV or the combined loss of both dipeptidyl aminopeptidases yscIV and yscV did not affect mitotic growth under rich or poor growth conditions . In contrast to the dipeptidyl aminopeptidase yscIV lesion (ste13), which leads to alpha sterility because strains secrete incompletely processed forms of the alpha-factor pheromone, the dipeptidyl aminopeptidase yscV lesion did not affect mating, and strains produced fully active alpha-factor pheromone . dap2 mutants did not show any obvious phenotype under a variety of conditions tested.

Mol Cell Biol, 1987 Sep, 7(9), 3185 - 93
Identification of sequence elements that confer cell-type-specific control of MF alpha 1 expression in Saccharomyces cerevisiae; Inokuchi K et al.; The MF alpha 1 gene of Saccharomyces cerevisiae, a major structural gene for mating pheromone alpha factor, is an alpha-specific gene whose expression is regulated by the mating-type locus . To study the role of sequences upstream of MF alpha 1 in its expression and regulation, we generated two sets of promoter deletions: upstream deletions and internal deletions . By analyzing these deletions, we have identified a TATA box and two closely related, tandemly arranged upstream activation sites as necessary elements for MF alpha 1 expression . Two upstream activation sites were located ca . 300 and 250 base pairs upstream of the MF alpha 1 transcription start points, which were also determined in this study . Each site contained a homologous 22-base-pair sequence, and both sites were required for maximum transcription level . The distance between the upstream activation sites and the transcription start points could be altered without causing loss of transcription efficiency, and the sites were active in either orientation with respect to the coding region . These elements conferred cell type-specific expression on a heterologous promoter . Analysis with host mating-type locus mutants indicates that these sequences are the sites through which the MAT alpha 1 product exerts its action to activate the MF alpha 1 gene . Homologous sequences with these elements were found in other alpha-specific genes, MF alpha 2 and STE3, and may mediate activation of this set of genes by MAT alpha 1.

Proc Natl Acad Sci U S A, 1987 Sep, 84(17), 6045 - 9
The RAD3 gene of Saccharomyces cerevisiae encodes a DNA-dependent ATPase; Sung P et al.; The RAD3 gene of the yeast Saccharomyces cerevisiae is required for excision repair of damaged DNA and for cell viability . A protein of approximately equal to 89 kDa was purified to near homogeneity from yeast strains harboring multicopy plasmids that overproduce RAD3 protein; this protein corresponds closely to the expected size of the RAD3 protein and cross-reacts with the antiserum raised against a truncated RAD3 protein produced in Escherichia coli . The purified RAD3 protein shows a single-stranded DNA-dependent ATPase activity that catalyzes hydrolysis of ATP to ADP and Pi . The ATPase activity was coincident with the RAD3 protein during purification and is inhibited by anti-RAD3 antibodies, indicating that the RAD3 gene encodes this activity.

Eur J Biochem, 1987 Sep 1, 167(2), 291 - 7
Amino acid substitutions in subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae . Sequence analysis of a series of revertants of an oli1 mit- mutant carrying an amino acid substitution in the hydrophilic loop of subunit 9; Willson TA et al.; This work concerns a biochemical genetic study of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae . Subunit 9, encoded by the mitochondrial oli1 gene, contains a hydrophilic loop connecting two transmembrane stems . In one particular oli1 mit- mutant 2422, the substitution of a positively charged amino acid in this loop (Arg39----Met) renders the ATPase complex non-functional . A series of 20 revertants, selected for their ability to grow on nonfermentable substrates, has been isolated from mutant 2422 . The results of DNA sequence analysis of the oli1 gene in each revertant have led to the recognition of three groups of revertants . Class I revertants have undergone a same-site reversion event: the mutant Met39 is replaced either by arginine (as in wild-type) or lysine . Class II revertants maintain the mutant Met39 residue, but have undergone a second-site reversion event (Asn35----Lys) . Two revertants showing an oligomycin-resistant phenotype carry this same second-site reversion in the loop region together with a further amino acid substitution in either of the two membrane-spanning segments of subunit 9 (either Gly23----Ser or Leu53----Phe) . Class III revertants contain subunit 9 with the original mutant 2422 sequence, and additionally carry a recessive nuclear suppressor, demonstrated to represent a single gene . The results on the revertants in classes I and II indicate that there is a strict requirement for a positively charged residue in the hydrophilic loop close to the boundary of the lipid bilayer . The precise location of this positive charge is less stringent; in functional ATPase complexes it can be found at either residue 39 or 35 . This charged residue is possibly required to interact with some other component of the mitochondrial ATPase complex . These findings, together with hydropathy plots of subunit 9 polypeptides from normal, mutant and revertant strains, led to the conclusion that the hydrophilic loop in normal subunit 9 extends further than previously suggested, with the boundary of the N-terminal membrane-embedded stem lying at residue 34 . The possibility is raised that the observed suppression of the 2422 mutant phenotype in class III revertants is manifested through an accommodating change in a nuclear-encoded subunit of the ATPase complex.

J Gen Microbiol, 1987 Sep, 133 ( Pt 9), 2497 - 501
Coordinated regulation of ammonium assimilation and carbon catabolism by glyoxylate in Saccharomyces cerevisiae; Gonzalez A et al.; The activities of citrate synthase (EC 4.1.3.7) and NADP+-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.4) of Saccharomyces cerevisiae were inhibited in vitro by glyoxylate . In the presence of glyoxylate, pyruvate and glyoxylate pools increased, suggesting that glyoxylate was efficiently transported and catabolized . Pyruvate accumulation also indicates that citrate synthase was inhibited . A decrease in the glutamate pool was also observed under these conditions . This can be attributed to an increased transamination rate and to the inhibitory effect of glyoxylate on NADP+-dependent GDH . Furthermore, the increase in the ammonium pool in the presence of glyoxylate suggests that NADP+-dependent GDH was being inhibited in vivo, since the activity of glutamine synthetase did not decrease under these conditions . We propose that the inhibition of both citrate synthase and NADP+-dependent GDH could form part of a mechanism that regulates the internal 2-oxoglutarate concentration.

J Gen Microbiol, 1987 Sep, 133 ( Pt 9), 2517 - 22
Regulation of gluconeogenic enzymes during the cell cycle of Saccharomyces cerevisiae growing in a chemostat; Arreguin de Lorencez M et al.; Oscillation of the activities of gluconeogenic enzymes (malate dehydrogenase, phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase) was observed during the cell cycle of chemostat cultures of Saccharomyces cerevisiae . Since ethanol is released by the cells at the beginning of the division cycle, its effect on enzyme expression was determined . Pulsing ethanol to a synchronously dividing yeast culture led to a prolongation of the metabolically active phase as indicated by the course of oxygen uptake and carbon dioxide production rates (concomitant ethanol and glucose assimilation) . Enzyme activities also remained elevated as long as ethanol was available to the cells . After a substrate shift from glucose to ethanol during cell division, ethanol was used without a lag phase and enzyme induction increased from the level reached at the point of the substrate change . The data confirmed that the small amount of ethanol produced when the cells begin active reproduction acts as an inducer of gluconeogenic enzymes.

Can J Microbiol, 1987 Sep, 33(9), 777 - 82
ESR study of copper(II) retention by entire cell, cells walls, and protoplasts of Saccharomyces cerevisiae; Kihn JC et al.; Copper retention by whole cells, protoplasts, and isolated cell walls of Saccharomyces cerevisiae was investigated in the absence of any energy source in the medium . The cell walls accounted only for a small fraction of the cation retention by whole cells . ESR results showed that copper was not bound only at the outer face of the plasma membrane, but it was also distributed in the plasma membrane and (or) in the cytoplasm . ESR studies also showed that, in all three systems, copper was chelated by peptides or proteins . The binding sites were formed by an amide and a strongly complexing ligand such as an amine . Their configuration depended upon pH: in slightly acidic conditions, copper was bound by the oxygen of the amide; at basic pH, NHCO became deprotonated and the negatively charged nitrogen bound to the metal.

Biochem J, 1987 Sep 1, 246(2), 375 - 86
The pentafunctional arom enzyme of Saccharomyces cerevisiae is a mosaic of monofunctional domains; Duncan K et al.; The nucleotide sequence of the Saccharomyces cerevisiae ARO1 gene which encodes the arom multifunctional enzyme has been determined . The protein sequence deduced for the pentafunctional arom polypeptide is 1588 amino acids in length and has a calculated Mr of 174555 . Functional regions within the polypeptide chain have been identified by comparison with the sequences of the five monofunctional Escherichia coli enzymes whose activities correspond with those of the arom multifunctional enzyme . The observed homologies demonstrate that the arom polypeptide is a mosaic of functional domains and are consistent with the hypothesis that the ARO1 gene evolved by the linking of ancestral E . coli-like genes.

Experientia, 1987 Aug 15, 43(8), 888 - 90
Occurrence of thiaminase II in Saccharomyces cerevisiae; Kimura Y et al.; It was found that cell-free extracts of Saccharomyces cerevisiae contain thiaminase II which hydrolyzes thiamine and thiamine analogs . The possible involvement of this enzyme and thiamine-synthesizing enzymes in thiamine production from thiamine antagonists is discussed.

J Biol Chem, 1987 Aug 15, 262(23), 10955 - 9
Purification and characterization of Saccharomyces cerevisiae mitochondrial elongation factor Tu; Rosenthal LP et al.; Yeast mitochondrial elongation factor Tu (EF-Tu) was purified 200-fold from a mitochondrial extract of Saccharomyces cerevisiae to yield a single polypeptide of Mr = approximately 47,000 . The factor was detected by complementation with Escherichia coli elongation factor G and ribosomes in an in vitro phenylalanine polymerization reaction . Mitochondrial EF-Tu, like E . coli EF-Tu, catalyzes the binding of aminoacyl-tRNA to ribosomes and possesses an intrinsic GTP hydrolyzing activity which can be activated either by kirromycin or by ribosomes . Kinetic and binding analyses of the interactions of mitochondrial EF-Tu with guanine nucleotides yielded affinity constants for GTP and GDP of approximately 5 and 25 microM, respectively . The corresponding affinity constants for the E . coli factor are approximately 0.3 and 0.003 microM, respectively . In keeping with these observations, we found that purified mitochondrial EF-Tu, unlike E . coli EF-Tu, does not contain endogenously bound nucleotide and is not stabilized by GDP . In addition, we have been unable to detect a functional counterpart to E . coli EF-Ts in extracts of yeast mitochondria and E . coli EF-Ts did not detectably stimulate amino acid polymerization with mitochondrial EF-Tu or enhance the binding of guanine nucleotides to the factor . We conclude that while yeast mitochondrial EF-Tu is functionally analogous to and interchangeable with E . coli EF-Tu, its affinity for guanine nucleotides and interaction with EF-Ts are quite different from those of E . coli EF-Tu.

FEBS Lett, 1987 Aug 10, 220(1), 177 - 80
Saccharomyces cerevisiae mRNA populations of different intrinsic stability in unstressed and heat shocked cells display almost constant m7GpppA:m7GpppG 5'-cap structure ratios; Piper PW et al.; The half-lives of mRNAs in yeast vary from about 1 to over 100 min . While mRNA stabilities must strongly influence overall gene expression in this organism, very little is known about how they are determined . Labellings of yeast cells were conducted to investigate whether the 5'-cap structures of yeast mRNAs might influence their stability . Variation of the pulse-labelling period from 7.5 min to 120 min did not have any major influence on the relative labelling of m7GpppA (A cap) and m7GpppG (G cap) in total polyadenylated RNA . Whether an mRNA has the A cap or the G cap does not therefore have a marked effect on its stability . During the heat shock response the relative labelling of A caps to G caps in total polyadenylated RNA also does not fluctuate appreciably . This indicates that cap structure alone does not determine the destabilisation of non-heat shock mRNAs and stabilisation of heat shock mRNAs during this stress response.

FEBS Lett, 1987 Aug 10, 220(1), 113 - 5
Rapid changes of heat and desiccation tolerance correlated with changes of trehalose content in Saccharomyces cerevisiae cells subjected to temperature shifts; Hottiger T et al.; The trehalose content of exponentially growing Saccharomyces cerevisiae cells rapidly increased in response to a temperature shift from 27 to 40 degrees C and decreased again when the temperature was shifted back from 40 to 27 degrees C . These changes were closely correlated with increases and decreases in the thermotolerance and desiccation tolerance of the cells . Our results support the hypothesis that trehalose functions as a protectant against heat and desiccation.

Biochim Biophys Acta, 1987 Aug 7, 902(1), 19 - 23
Effect of ruthenium red upon Ca2+ and Mn2+ uptake in Saccharomyces cerevisiae . Comparison with the effect of La3+; van der Pal RH et al.; The initial rate of both Ca2+ and Mn2+ uptake is inhibited by ruthenium red to about the same extent as by equivalent concentrations of La3+ . The inhibition of Ca2+ uptake, however, is relieved during further incubation with ruthenium red . On preincubating the cells with ruthenium red even a stimulation of divalent cation uptake can be found . Relieve of the inhibition of divalent cation uptake is accompanied by K+ efflux . Both ruthenium red and La3+ displace Ca2+ very effectively from binding sites at the cell surface . The inhibition of initial Ca2+ uptake is accompanied by a reduction in the binding of Ca2+.

J Biol Chem, 1987 Aug 5, 262(22), 10807 - 13
Construction and analysis of deletions in the amino-terminal extension of glutamine tRNA synthetase of Saccharomyces cerevisiae; Ludmerer SW et al.; GLN4 of Saccharomyces cerevisiae encodes an amino-terminal extension of 224 amino acids . This is connected to a polypeptide which is colinear with and 40% identical to Escherichia coli glutamine tRNA synthetase . We examined the potential significance of the amino-terminal extension . Two single base and five multiple base frame shift deletions were constructed in this segment . Each of these mutations is associated with a lethal phenotype . This suggests that the coding sequence for the amino-terminal extension is translated . It also implies that there are no translation restarts downstream of the coding region for the amino-terminal extension which produce active enzyme . Three internal deletions of various sizes, and which preserve the correct reading frame, were constructed in the coding region of the amino-terminal extension . Cells which harbor such in-frame deletions on a multi copy plasmid are viable, even when a deletion construct is the only source of GLN4-encoded activity . Extracts of cells which have one of these deletions have reduced, but measurable, glutamine tRNA synthetase activity . We conclude that the catalytic activity resides with the segment which is homologous to the E . coli enzyme and that the amino-terminal extension itself is dispensable for aminoacylation activity . Each of the internal in-frame deletion constructions is respiration-proficient . The amino-terminal extension, therefore, is not used for an essential mitochondrial function of the GLN4 gene product . Within the accuracy of the measurements, activities of four other aminoacyl-tRNA synthetases are not affected by the presence of a GLN4 internal deletion allele as the only source of GLN4-encoded activity . This suggests that the amino-terminal extension does not stabilize a complex which includes one or more of these four enzymes and whose activity depends on proper assembly of the complex.

Mol Cell Biol, 1987 Aug, 7(8), 2783 - 93
Identification and isolation of the gene encoding the small subunit of ribonucleotide reductase from Saccharomyces cerevisiae: DNA damage-inducible gene required for mitotic viability; Elledge SJ et al.; Ribonucleotide reductase catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis . The gene encoding the small subunit of ribonucleotide reductase was isolated from a Saccharomyces cerevisiae genomic DNA expression library in lambda gt11 by a fortuitous cross-reaction with anti-RecA antibodies . The cross-reaction was due to an identity between the last four amino acids of each protein . The gene has been named RNR2 and is centromere linked on chromosome X . The nucleotide sequence was determined, and the deduced amino acid sequence, 399 amino acids, shows extensive homology with other eucaryotic ribonucleotide reductases . Transplason mutagenesis was used to disrupt the RNR2 gene . A novel assay using colony color sectoring was developed to demonstrate visually that RNR2 is essential for mitotic viability . RNR2 encodes a 1.5-kilobase mRNA whose levels increase 18-fold after treatment with the DNA-damaging agent 4-nitroquinoline 1-oxide . CDC8 was also found to be inducible by DNA damage, but POL1 and URA3 were not inducible by 4-nitroquinoline 1-oxide . The expression of these genes defines a new mode of regulation for enzymes involved in DNA biosynthesis and sharpens our picture of the events leading to DNA repair in eucaryotic cells.

Mol Cell Biol, 1987 Aug, 7(8), 2680 - 90
Identification and regulation of a gene required for cell fusion during mating of the yeast Saccharomyces cerevisiae; McCaffrey G et al.; We have devised a screen for genes from the yeast Saccharomyces cerevisiae whose expression is affected by cell type or by the mating pheromones . From this screen we identified a gene, FUS1, whose pattern of expression revealed interesting regulatory strategies and whose product was required for efficient cell fusion during mating . Transcription of FUS1 occurred only in a and alpha cells, not in a/alpha cells, where it was repressed by a1 X alpha 2, a regulatory activity present uniquely in a/alpha cells . Transcription of FUS1 showed an absolute requirement for the products of five STE genes, STE4, STE5, STE7, STE11, and STE12 . Since the activators STE4, STE5, and STE12 are themselves repressed by a1 X alpha 2, the failure to express FUS1 in a/alpha cells is probably the result of a cascade of regulatory activities; repression of the activators by a1 X alpha 2 in turn precludes transcription of FUS1 . In addition to regulation of FUS1 by cell type, transcription from the locus increased 10-fold or more when a or alpha cells were exposed to the opposing mating pheromone . To investigate the function of the Fus1 protein, we created fus1 null mutants . In fus1 X fus1 matings, the cells of a mating pair adhered tightly and appeared to form zygotes . However, the zygotes were abnormal . Within the conjugation bridge the contained a partition that prevented nuclear fusion and mixing of organelles . The predicted sequence of the Fus1 protein (deduced from the FUS1 DNA sequence) and subcellular fractionation studies with Fus1-beta-galactosidase hybrid proteins suggest that Fus1 is a membrane or secreted protein . Thus, Fus1 may be located at a position within the cell where it is poised to catalyze cell wall or plasma membrane fusion.

Mol Gen Genet, 1987 Aug, 209(1), 142 - 8
A complex pattern of sensitivity to simple monofunctional alkylating agents exists amongst the rad mutants of Saccharomyces cerevisiae; Cooper AJ et al.; The radiation-sensitive rad mutants of the yeast Saccharomyces cerevisiae exhibit a complex pattern of sensitivity to simple monofunctional alkylating agents . The RAD1, RAD2, RAD4 and RAD14 genes of the RAD3 epistasis group are implicated in the repair of ethylations to DNA . The RAD3, RAD10 and RAD16 genes of this group are not involved . The RAD4 and RAD14 genes have a particular role in repair following exposure to those ethylating agents that preferentially alkylate oxygen, but not to those that preferentially ethylate nitrogen . The RAD1 and RAD2 genes are involved in the repair of damage induced by all the ethylating agents used except EMS . The mutants in this group that are sensitive to ENU were not sensitive to MNU, suggesting that nucleotide excision operates on ethylations but not on methylations . In the RAD6 group, the RAD6 and RAD18 genes are involved in DNA repair after exposure to all the alkylating agents tested, whereas RAD8 appears to have a role in the repair of O-alkylations but not N-alkylations . RAD9 operates in the repair of methylations and ethylations, but does not influence events after exposure to EMS . In the RAD52 group, the mutants tested were sensitive to ENU and DES . Thus some members of all three epistasis groups are involved in the repair of alkylations to DNA.

Genetika, 1987 Aug, 23(8), 1390 - 8
{Genetic analysis of polyauxotrophy and early mitotic progeny of Saccharomyces cerevisiae zygotes . II . Spore-forming segregants in the mitotic and meiotic progeny of polyauxotrophic clones}; Stolbova AV; This article continues the investigation of polyauxotrophic (PA) clones formed in early mitotic progeny of zygotes . Cloning and segregation analysis of PA progeny suggest an unusual state of diploid genome in these strains, which is expressed as elimination of the dominance effect of the wild allele and as suppression or conversion of either of two loci of mating type . In PA progeny, except for recombinant haploids, sporulating diploids and unstable clones were detected . The tetrad analysis of the diploids points to homozygotization for individual markers . Over-replication of diploid set of chromosomes, prior to meiosis, and replacement of the haploid nucleus (the product of meiosis) for the diploid nucleus may explain the appearance of sporulating segregants in the diploid meiotic progeny . Unstable segregants may be considered as heterokaryons with complex interaction of nuclei.

Genetika, 1987 Aug, 23(8), 1383 - 9
{Saccharomyces cerevisiae mutants characterized by increased induced mutagenesis . II . Genetic analysis of mutants}; Ivanov EL et al.; Induction of forward adenine-dependent (Ade+----Ade-) mutations by HAP was used to analyse genetically yeast mutants with enhanced induced mutagenesis . Three mutations studied in detail segregated as a single mendelian trait and composed independent complementation groups (HIM1, HIM2, HIM3) . the him1-1 mutation was centromere-linked, the him3-1 and him2-1 mutations being not . All three mutations did not show any cross-linkage . Uracil-DNA glycosylase activity was determined in crude cell extract from wild type strain and him mutants; no detectable differences were observed.

Proc Natl Acad Sci U S A, 1987 Aug, 84(16), 5560 - 4
Purification and characterization of an activity from Saccharomyces cerevisiae that catalyzes homologous pairing and strand exchange; Kolodner R et al.; An activity that catalyzes the formation of joint molecules from linear M13mp19 replicative form DNA and circular M13mp19 viral DNA was purified 1000- to 2000-fold from mitotic Saccharomyces cerevisiae cells . The activity appeared to reside in a Mr 132,000 polypeptide . The reaction required that the substrates be homologous and also required Mg2+ . There was no requirement for ATP . The reaction required stoichiometric amounts of protein and showed a cooperative dependence on protein concentration . Electron microscopic analysis of the joint molecules indicated they were formed by displacement of one strand of the linear duplex by the single-stranded circular molecule . This analysis also showed that heteroduplex formation started at the 3'-homologous end of the linear duplex strand followed by extension of the hybrid region toward the 5'-homologous end of the linear duplex strand (3'-to-5' direction).

J Bacteriol, 1987 Aug, 169(8), 3521 - 4
Transcriptional regulation of the DAL5 gene in Saccharomyces cerevisiae; Rai R et al.; We demonstrate that the DAL5 gene, encoding a necessary component of the allantoate transport system, is constitutively expressed in Saccharomyces cerevisiae . Its relatively high basal level of expression did not increase further upon addition of allantoin pathway intermediates . However, steady-state DAL5 mRNA levels dropped precipitously when a repressive nitrogen source was provided . These control characteristics of DAL5 expression make this gene a good model with which to unravel the mechanism of nitrogen catabolite repression . Its particular advantage relative to other potentially useful genes derives from its lack of control by induction and hence the complicating effects of inducer exclusion.

J Bacteriol, 1987 Aug, 169(8), 3458 - 63
Partial purification and some properties of homoserine O-acetyltransferase of a methionine auxotroph of Saccharomyces cerevisiae; Yamagata S; A wild-type strain and six methionine auxotrophs of Saccharomyces cerevisiae were cultured in a synthetic medium supplemented with 0.1 mM L-cysteine or L-methionine and analyzed for the synthesis of homoserine O-acetyltransferase (EC 2.3.1.31) . Among them, four mutant strains exhibited enzyme activity in cell extracts . Methionine added to the synthetic medium at concentrations higher than 0.1 mM repressed enzyme synthesis in two of these strains . The enzyme was partially purified (3,500-fold) from an extract of a mutant strain through ammonium sulfate fractionation and chromatography on columns of DEAE-cellulose, Phenyl-Sepharose C1-4B, and Sephadex G-150 . The enzyme exhibited optimal pH at 7.5 for activity and at 7.8 for stability . The reaction product was ascertained to be O-acetyl-L-homoserine by confirming that it produced L-homocysteine in an O-acetyl-L-homoserine sulfhydrylase reaction . The Km for L-homoserine was 1.0 mM, and for acetyl coenzyme A it was 0.027 mM . The molecular weight of the enzyme was estimated to be approximately 104,000 by Sephadex G-150 column chromatography and 101,000 by sucrose density gradient centrifugation . The isoelectric point was at pH 4.0 . Of the hydroxy amino acids examined, the enzyme showed reactivity only to L-homoserine . Succinyl coenzyme A was not an acyl donor . In the absence of L-homoserine, acetyl coenzyme A was deacylated by the enzyme, with a Km of 0.012 mM . S-Adenosylmethionine and S-adenosylhomocysteine slightly inhibited the enzyme, but methionine had no effect.

Exp Cell Res, 1987 Aug, 171(2), 411 - 25
Alpha-factor inhibition of the rate of cell passage through the "start" step of cell division in Saccharomyces cerevisiae yeast: estimation of the division delay per alpha-factor.receptor complex; Moore SA; A highly sensitive, kinetically unambiguous assay for alpha-factor-induced delay of cell passage through the "start" step of cell division in yeast is presented . The assay employs perfusion with periodic microscopy to monitor the bud emergence kinetics on the 20% of cells within an exponentially growing population which exist prior to the alpha-factor execution point of start . The t1/2 for cell passage through start by this population of cells is 31 min in the absence of alpha-factor . The inhibition constant, KI, represents the alpha-factor concentration which produces a 50% inhibition of this rate and is equal to 2 X 10(-10) M . A second assay for maximal cell division arrest by alpha-factor on whole populations of cells is presented . This assay shows a maximum cell division arrest time of 125 +/- 5 h at saturating alpha-factor, and a K50 (that is, an alpha-factor concentration which produces a half-maximal response) of 2.5 X 10(-8) M . Both assays were performed in the effective absence of alpha-factor inactivation . Values of the dissociation constant KD and total number of receptors per cell which specifically mediate cell division arrest or delay were estimated to be 2.5 X 10(-8) M and 10(4), respectively . These estimates, along with the quantitative dose-response data for division arrest which are presented here, are consistent with each receptor.alpha-factor complex which is present on the cell at equilibrium producing a 43 +/- 10 s delay of cell passage through start . Surprisingly, this number is constant within twofold over the entire range of cellular division arrest responses to alpha-factor, that is, from a 1.9-fold inhibition of the rate of cell passage through start at 0.17 nM alpha-factor to a 125 +/- 5 h maximum arrest at saturating alpha-factor concentrations of greater than 170 nM . The possible significance of this observation toward the mechanism of alpha-factor-induced cell division arrest is discussed.

J Biochem (Tokyo), 1987 Aug, 102(2), 273 - 9
Single site catalysis of the F1-ATPase from Saccharomyces cerevisiae and the effect of inorganic phosphate on it; Konishi J et al.; The kinetical characteristics of ATP hydrolysis by mitochondrial F1-ATPase from Saccharomyces cerevisiae (yeast) have been studied under conditions where only a single catalytic site per enzyme molecule bound ATP . Four major features were observed, that is, fast ATP binding to the enzyme, slow product release from the enzyme, an equilibrium close to unity between ATP and products on the enzyme, and promotion of ATP hydrolysis on the second addition of a large excess of ATP (cold chase) . These are essentially the same as the kinetical characteristics observed for beef heart mitochondrial F1-ATPase, which were called as unisite catalysis by Grubmeyer et al . (Grubmeyer, C . et al . (1982) J . Biol . Chem . 257, 12092-12100), although the release of a hydrolysis product, Pi, from the yeast enzyme appeared to occur significantly faster than that from the beef enzyme, which resulted in a decreased extent of cold chase promotion of ATP hydrolysis of the yeast enzyme . The yeast F1-ATPase showed unisite catalysis even in the absence of Pi in the reaction mixtures, while it was reported for the beef F1-ATPase that the presence of Pi in the reaction mixture was essential for unisite catalysis (Penefsky, H.S . & Grubmeyer, C . (1984) in H+-ATPase (ATP Synthase) (Papa, S . et al., eds.) pp . 195-204, The ICSU Press) . Another difference in the Pi effect on the kinetics was that ATP hydrolysis was initiated without a lag time in the absence of Pi in the case of the yeast enzyme when a 1,000-fold molar excess of ATP per enzyme molecular was mixed with the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

J Gen Microbiol, 1987 Aug, 133 ( Pt 8), 2197 - 205
Regulation of the cAMP level in the yeast Saccharomyces cerevisiae: the glucose-induced cAMP signal is not mediated by a transient drop in the intracellular pH; Thevelein JM et al.; Addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae is known to cause a rapid, transient increase in the cAMP level, which lasts for 1-2 min and induces a cAMP-dependent protein phosphorylation cascade . The glucose-induced cAMP signal cannot be explained solely on the basis of an increased ATP level . Transient membrane depolarization and transient intracellular acidification have been suggested as possible triggers for the cAMP peak . Addition of glucose to cells in which the plasma membrane had been depolarized still produced the increase in the cAMP level excluding membrane depolarization as the possible trigger . Using in vivo 31P NMR-spectroscopy we followed phosphate metabolism and the time course of the drop in the intracellular pH after addition of glucose with a time resolution of 15 s . Under aerobic conditions the initial pH and ATP level were high . On addition of glucose, they both showed a rapid, transient drop, which lasted for about 30 s . Under anaerobic conditions, the initial pH and ATP level were low and on addition of glucose they both increased relatively slowly compared to aerobic conditions . Several conditions were found in which the pH drop which occurs under aerobic conditions could be blocked completely without effect on the cAMP signal or without completely preventing it: addition of NH4Cl together with glucose at high extracellular pH and addition of a low concentration of glucose before a high concentration . Also, when glucose was added twice to the same cells no consistent relationship was observed between the pH drop and the cAMP peak . These results appear to exclude transient intracellular acidification as the trigger for the cAMP signal . Hence, we conclude that the effect of glucose cannot be explained on the basis of effects known to be caused by the membrane depolarizing compounds which cause increases in the cAMP level . A new, more specific kind of interaction appears to be involved.

J Gen Microbiol, 1987 Aug, 133 ( Pt 8), 2191 - 6
Regulation of the cAMP level in the yeast Saccharomyces cerevisiae: intracellular pH and the effect of membrane depolarizing compounds; Thevelein JM et al.; Addition of plasma membrane depolarizing agents, such as dinitrophenol (DNP) and azide, to cells of Saccharomyces cerevisiae under aerobic conditions, is known to cause an increase in the cAMP level within 15 s . We found that both compounds lowered the intracellular pH (measured by in vivo 32P-NMR) drastically within the same time period . Plasma membrane depolarization, however, was much slower: DNP and azide had no effect on the membrane potential during, respectively, the first 2 min and the first 10 min after addition . Apparently, the intracellular pH of yeast is much more sensitive to perturbation than the membrane potential . The effect of both compounds on the cAMP level was highly dependent on the extracellular pH: when the latter was raised, the effect disappeared completely between pH 6 and 7 . A similar dependence on the extracellular pH was observed for the lowering of intracellular pH . Addition of organic acids, such as acetate and butyrate, at low pH and under aerobic conditions, also caused an immediate increase in the cAMP level and an immediate drop in the intracellular pH . These results suggest that agents such as DNP and azide do not raise the cAMP level in yeast cells because of their membrane depolarizing properties but because they lower the intracellular pH . Under anaerobic conditions, DNP, azide and organic acids were much less effective in increasing the cAMP level . Addition of a small amount of glucose, however, restored their capacity to enhance the cAMP level . This suggests that under anaerobic conditions and in the absence of glucose the ATP level is a limiting factor for cAMP synthesis.

Mol Gen Mikrobiol Virusol, 1987 Aug, (8), 19 - 25
{A model system for the study of repair of DNA double-strand breaks in Saccharomyces cerevisiae}; Glazunov AV et al.; The efficiency of "LiCl transformation" in Saccharomyces cerevisiae haploid cells by an autonomously replicating pLL12 plasmid carrying yeast LEU2 and LYS2 genes is increased (by an order or more) when the plasmid is linearized by the restriction endonuclease XhoI cleavage of a unique site in LYS2 gene . Transformants were selected on the medium lacking leucine . This phenomenon has been shown to be a result of recombinational repair of double-strand breaks (DSB) of plasmid DNA stimulated by a restriction endonuclease . The kinetic data have shown the process of plasmid DNA DSB repair to consist of two phases . The completion of the first phase occurs during an hour and the second phase occurs in 14-18 hours . DNA double-strand gaps (the deleted sequences of plasmid LYS2 gene in DSB region) with maximal length of 2-2.5 kb are repaired with the same efficiency as DSB . The genetic control of the recombinational repair of plasmid DNA DSB has been studied.

Mol Cell Biol, 1987 Aug, 7(8), 2947 - 55
Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins; Jong AY et al.; To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function . The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853 . There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins . At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein . Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site . Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins . Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins . In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus . Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded.

Mol Cell Biol, 1987 Aug, 7(8), 2728 - 34
Saccharomyces cerevisiae positive regulatory gene PET111 encodes a mitochondrial protein that is translated from an mRNA with a long 5' leader; Strick CA et al.; The yeast nuclear gene PET111 is required specifically for translation of the mitochondrion-coded mRNA for cytochrome c oxidase subunit II . We have determined the nucleotide sequence of a 3-kilobase segment of DNA that carries PET111 . The sequence contains a single long open reading frame that predicts a basic protein of 718 amino acids . The PET111 gene product is a mitochondrial protein, since a hybrid protein which includes the amino-terminal 154 amino acids of PET111 fused to beta-galactosidase is specifically associated with mitochondria . PET111 is translated from a 2.9-kilobase mRNA which, interestingly, has an extended 5'-leader sequence containing four short open reading frames upstream of the long open reading frame . These open reading frames exhibit an interesting pattern of overlap with each other and with the PET111 reading frame.

Mol Cell Biol, 1987 Aug, 7(8), 2708 - 17
LEU3 of Saccharomyces cerevisiae encodes a factor for control of RNA levels of a group of leucine-specific genes; Friden P et al.; Although the majority of genes for amino acid biosynthesis which have been examined are under general amino acid control, LEU1 and LEU2 of Saccharomyces cerevisiae respond specifically to leucine . We report here an analysis of LEU3, a putative leucine-specific regulatory locus . We show that LEU3 is necessary for expression of wild-type levels of LEU1- and LEU2-specific RNAs and, further, that the levels of LEU4-specific transcripts are also affected by LEU3 . We cloned LEU3 and showed by DNA sequence analysis that it contained an open reading frame of 886 amino acids . A striking feature of the predicted LEU3 protein was a cluster of acidic amino acids (19 of 20) located in the C-terminal half of the coding region . The protein also had a repeated cysteine motif which was conserved in a number of other yeast proteins implicated in gene regulation . We show that whole-cell extracts contained a LEU3-dependent DNA-binding activity that interacted with the 5' region of LEU2 . Subdivision of the LEU2 5' region established that the LEU3-dependent DNA-binding activity interacted with the segment which had the previously reported homology with LEU1.

Mol Cell Biol, 1987 Aug, 7(8), 2664 - 70
Phycomyces blakesleeanus TRP1 gene: organization and functional complementation in Escherichia coli and Saccharomyces cerevisiae; Revuelta JL et al.; We have cloned the gene encoding the TRPF and TRPC functions of Phycomyces blakesleeanus by complementation of the corresponding activities of Escherichia coli . TRPF also complemented a trpl mutation in Saccharomyces cerevisiae . As in other filamentous fungi, such as Neurospora and Aspergillus spp., the P . blakesleeanus TRPF and TRPC formed part of a trifunctional polypeptide encoded by a single gene (called TRP1) . Transcription of TRP1 in P . blakesleeanus did not appear to be regulated by light or by the nutritional status of the culture . The information on the structure and organization of a P . blakesleeanus gene derived from these studies should be useful in devising molecular genetic strategies to analyze the sensory physiology of this organism.

Mol Cell Biol, 1987 Aug, 7(8), 2653 - 63
Characterization of Saccharomyces cerevisiae genes encoding subunits of cyclic AMP-dependent protein kinase; Cannon JF et al.; Mutations in the SRA1 or SRA3 gene eliminate the requirement for either RAS gene (RAS1 or RAS2) in Saccharomyces cerevisiae . We cloned SRA1 and SRA3 and determined their DNA sequences . SRA1 encodes the regulatory subunit of the cyclic AMP (cAMP)-dependent protein kinase and therefore is identical to REG1 and BCY1 . This gene is not essential, but its deletion confers many traits: reduction of glycogen accumulation, temperature sensitivity, reduced growth rate on maltose and sucrose, inability to grow on galactose and nonfermentable carbon sources, and nitrogen starvation intolerance . SRA3 is homologous to protein kinases that phosphorylate serine and threonine and likely encodes the catalytic subunit of the cAMP-dependent protein kinase . The wild-type SRA3 gene either triplicated in the chromosome or on episomal, low-copy plasmids behaves like spontaneous dominant SRA3 mutations by suppressing ras2-530 (RAS2::LEU2 disruption), cdc25, and cdc35 mutations . These findings indicate that the yeast RAS genes are dispensable if there is constitutive cAMP-dependent protein kinase activity.

J Biol Chem, 1987 Jul 25, 262(21), 10127 - 33
The gene and the primary structure of ornithine decarboxylase from Saccharomyces cerevisiae; Fonzi WA et al.; The nucleotide sequence was determined for a 3-kilobase genomic fragment containing the ornithine decarboxylase gene of Saccharomyces cerevisiae . The fragment contained two open reading frames . Gene disruption localized the ornithine decarboxylase gene to a 1398-nucleotide open reading frame . Transcription of the yeast gene initiated at several sites 171 to 211 nucleotides 5' of the translational start site . The 3' end of the transcript extended approximately 300 nucleotides beyond the end of the ornithine decarboxylase coding region and contained two copies of the yeast ARS core sequence . Translation of the ornithine decarboxylase gene appeared to initiate at the first AUG condon of the open reading frame based upon translational fusions with the Escherichia coli beta-galactosidase gene . Since no introns were apparent, the 1398-nucleotide open reading frame was predicted to encode a 466-amino acid protein with a calculated Mr = 52,369 . The deduced protein differed significantly in size from previous reports on yeast ornithine decarboxylase, but was very similar in size to mammalian ornithine decarboxylase . When the predicted amino acid sequence of yeast ornithine decarboxylase was compared with that of the mouse enzyme, alignment of the sequences revealed that 40% of the amino acid residues were identical . Chou-Fasman predictions of the secondary structure of the two enzymes indicated that secondary structure was also highly conserved.

J Biol Chem, 1987 Jul 25, 262(21), 10114 - 9
Phosphorylation in vivo of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase at the cyclic AMP-dependent site; Rittenhouse J et al.; In vivo labeled fructose-1,6-bisphosphatase was immunopurified from yeast (Saccharomyces cerevisiae) cells that had been incubated in the presence of {32P} orthophosphate . Tryptic peptides from labeled enzyme were mapped by high performance liquid chromatography . Most of the radioactivity was found to be associated with the peptide Arg9 through Arg24, the same peptide which had been previously shown to be phosphorylated in vitro by cAMP-dependent protein kinase (Rittenhouse, J., Harrsch, P . B., Kim, J . N., and Marcus, F . (1986) J . Biol . Chem . 261, 3939-3943) . The amino acid sequence analysis suggests that phosphorylation occurs at the same site, Ser11 . We have also determined the extent of phosphorylation at Ser11 of fructose-1,6-bisphosphatase in yeast cultures growing under various nutritional conditions by measuring the relative amounts of phospho- and corresponding dephosphopeptides in tryptic digests . Significant levels of phosphorylation of the enzyme were found in yeast cultures grown under gluconeogenic conditions that varied from 0.15 to 0.50 mol of phosphate per mol of enzyme subunit . However, phosphate incorporation rapidly increased to greater than 0.8 mol after addition of glucose to these cultures . An alternative technique, based solely on enzyme activity measurements, was also developed to estimate the extent of fructose-1,6-bisphosphatase phosphorylation in yeast cultures . The results obtained with this technique agreed with those obtained by high performance liquid chromatography of tryptic peptides.

Nature, 1987 Jul 23-29, 328(6128), 362 - 4
The role of heteroduplex correction in gene conversion in Saccharomyces cerevisiae; Bishop DK et al.; Two different models have been proposed to explain the relative frequencies of the non-mendelian allelic segregations which are detected by tetrad analysis after meiosis in fungi . The first model maintains that 6:2 type tetrads result from correction of heteroduplexes containing mismatched sites and 5:3 type tetrads result from failure to correct mismatched sites . The second model suggests that 6:2 segregations result from the filling-in of double-strand gaps using information obtained from both strands of a homologous duplex . In this model 5:3 type tetrads result if the allele is included in the heteroduplex regions flanking the gap and the resulting mismatched nucleotides are not corrected . We have studied the correction of heteroduplex plasmid DNA in pms1 mutant strains of Saccharomyces cerevisiae, which are known to exhibit higher frequencies of 5:3 type tetrads and lower frequencies of 6:2 tetrads than wild-type strains . Our results suggest that the pms1 mutation causes a defect in mismatch correction, supporting the hypothesis that meiotic gene conversion in wild-type yeast cells often results from the correction of heteroduplex DNA.

Eur J Biochem, 1987 Jul 15, 166(2), 371 - 7
Nucleotide sequence of the ARG3 gene of the yeast Saccharomyces cerevisiae encoding ornithine carbamoyltransferase . Comparison with other carbamoyltransferases; Huygen R et al.; The complete nucleotide sequence of the ARG3 structural gene encoding the monomer of the trimeric ornithine carbamoyltransferase (OTCase) (EC 2.1.3.3) has been determined . It consists of 338 codons with a corresponding molecular mass of 37842 Da . Comparing OTCases from Escherichia coli, yeast, Aspergillus, rat and man emphasizes peculiarities of the yeast enzyme but also brings to light an important degree of conservation between these proteins . Comparing the various OTCases with E . coli aspartate carbamoyltransferase (ATCase) (EC 2.1.3.2) confirms the evolutionary relationship previously noted between the two types of carbamoyltransferases and points to residues probably involved in catalysis and structural folding in OTCases.

FEBS Lett, 1987 Jul 13, 219(1), 249 - 53
Ca2+/calmodulin-dependent protein kinase in Saccharomyces cerevisiae; Londesborough J et al.; Ca2+-dependent chromatography of soluble cytosolic proteins on calmodulin-Sepharose gave a fraction that exhibited Ca2+- and calmodulin-dependent phosphorylation of several polypeptides, including 60, 56 and 45 kDa species . At 0.2 microM beef calmodulin the phosphorylation was optimal at 3 microM free Ca2+, and at 80 microM free Ca2+ it was half-maximal at about 0.1 microM beef calmodulin . It is concluded that the fraction contains calmodulin-dependent protein kinase(s) which is (are) autophosphorylated or associated with substrates.

FEBS Lett, 1987 Jul 13, 219(1), 254 - 8
A TY1 element is inserted in the CYR1 control region of Saccharomyces cerevisiae strain AB320; Lenzen G et al.; Southern blotting using a 5'-proximal probe of the Saccharomyces cerevisiae CYR1 gene has revealed heterogeneity in laboratory strains . It is demonstrated that strain AB320 contains a Ty1 element inserted in the promoter region of CYR1 . The Ty1 orientation suggests that transcription of CYR1 is initiated downstream from the insertion region.

Biochim Biophys Acta, 1987 Jul 10, 901(1), 30 - 4
L-malic-acid permeation in resting cells of anaerobically grown Saccharomyces cerevisiae; Salmon JM; The study of permeation of L-malic acid in cells of Saccharomyces cerevisiae at pH 3.0 was carried out with (U-14C)-labelled L-malic acid . Resting cells were used in these experiments . They were previously anaerobically grown on glucose . This study showed that this transport is the result of two competitive mechanisms, one for the uptake and one for the efflux . The uptake mechanism seems to be a simple diffusion of the L-malic acid in a non-dissociated form . The efflux mechanism seems to be an active transport of L-malic acid that is very dependent on the temperature . At the steady state, the result of uptake and efflux mechanisms leads to an intracellular concentration which is twice or three times the extracellular concentration.

Arch Microbiol, 1987 Jul, 148(2), 88 - 94
Cell wall mannoproteins during the population growth phases in Saccharomyces cerevisiae; Valentin E et al.; Mannoproteins from cell walls of Saccharomyces cerevisiae synthesized at successive stages of the population growth cycle have been solubilized with Zymolyase and subsequently analyzed . The major change along the population cycle concerned a large size mannoprotein material; the size of the newly-synthesized molecules varied from 120,000-500,000 (mean of about 200,000) at early exponential phase to 250,000-350,000 (mean of about 300,000) at late exponential phase . These differences are due to modifications in the amount of N-glycosidically linked mannose residues, since the size of the peptide moiety was 90,000-100,000 at all growth stages and the level of O-glycosylation changed only slightly . After incubation of the purified walls with concanavalin A-ferritin and subsequent analysis by electron microscopy, labelling was localized at the external and internal faces of the walls . The middle space of these was labelled after digestion of the glucan network with Zymolyase, which demonstrate the presence of mannoproteins in close contact with the structural glucan molecules throughout the wall.

Mol Cell Biol, 1987 Jul, 7(7), 2568 - 77
Complex interactions among members of an essential subfamily of hsp70 genes in Saccharomyces cerevisiae; Werner-Washburne M et al.; Saccharomyces cerevisiae contains a large family of genes related to hsp70, the major heat shock-inducible gene of Drosophila melanogaster . One subfamily, identified by sequence homology, contains four genes, SSA1, SSA2, SSA3, and SSA4 (formerly YG100, YG102, YG106, and YG107, respectively) . Previous studies showed that strains containing mutations in SSA1 and SSA2 are temperature sensitive for growth . SSA4, which is normally heat inducible and not expressed during vegetative growth, is expressed at high levels in ssa1 ssa2 strains at 23 degrees C . We constructed mutations in SSA3 and SSA4 and analyzed strains carrying mutations in the four genes . Strains carrying mutations in SSA3 SSA4 or SSA3 and SSA4 were indistinguishable from the wild type . However, ssa1 ssa2 ssa4 strains were inviable . SSA3, like SSA4, is a heat-inducible gene that is not normally expressed at 23 degrees C . Nevertheless, an intact copy of SSA3 regulated by the constitutive SSA2 promoter was capable of rescuing a ssa1 ssa2 ssa4 strain . This indicates that SSA3 encodes a functional protein and that the SSA1, SSA2, SSA3, and SSA4 gene products are functionally similar.

Mol Cell Biol, 1987 Jul, 7(7), 2545 - 51
In vitro splicing of the terminal intervening sequence of Saccharomyces cerevisiae cytochrome b pre-mRNA; Gampel A et al.; A region of the Saccharomyces cerevisiae mitochondrial cytochrome b gene encompassing the entire terminal intron plus flanking exonic sequences has been cloned in an SP6 vector . A runoff transcript prepared from this construct as well as the native cytochrome b pre-mRNA containing the terminal intervening sequence were found to act as substrates for the autocatalytic excision of the intervening sequence in vitro . This reaction proceeds under conditions previously shown by Cech and co-workers to promote protein-independent excision of the Tetrahymena rRNA intervening sequence . The 5' and 3' termini of the excised intervening sequence, determined by S1 nuclease mapping and sequence analysis, are consistent with the known sequence of the cytochrome b mRNA . The same region of the cytochrome b gene from a yeast mutant, defective in splicing due to a mutation in a critical sequence inside the terminal intron, has also been cloned in an SP6 vector . The mutant transcript fails to self-splice in the in vitro assay . These observations provide strong presumptive evidence that in vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA occurs by an autocatalytic mechanism analogous to that shown for other group I introns . In vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA, however, exhibits complete dependence on a protein factor previously shown to be encoded by the nuclear gene CBP2.

Mol Cell Biol, 1987 Jul, 7(7), 2484 - 90
Increased copy number of the 5' end of the SPS2 gene inhibits sporulation of Saccharomyces cerevisiae; Percival-Smith A et al.; We found that the introduction into a yeast cell of a high-copy-number plasmid containing the 5' end of the SPS2 gene, a sporulation-specific gene of Saccharomyces cerevisiae, led to a reduction in the efficiency of spore formation . The plasmid pAP290, which contains the sequence from -138 to +152 of the SPS2 gene, caused a fivefold reduction in spore formation; the presence of the plasmid had no effect on transcription of the chromosomal SPS2 gene . A plasmid containing only the sequence upstream of the TATA box of the SPS2 gene (-350 to -68) was unable to inhibit the completion of sporulation, whereas the downstream sequence, from -70 to +404, although unable by itself to inhibit sporulation, could do so when provided with an upstream fragment containing the CYC1 upstream activation sequence . Deletion of 22 base pairs from pAP290, which introduced a frameshift after codon 17 of the SPS2 gene and reduced the open reading frame to 26 amino acids, generated a plasmid (pAP290 delta Pst) which could no longer inhibit sporulation . The SPS2 inserts of pAP290 and pAP290 delta Pst were found to direct equivalent levels of sporulation-specific transcription . We conclude from these results that the presence of both the SPS2 promoter (or a substitute promoter) and the initial coding sequence of the SPS2 gene is required in the high-copy-number plasmid to generate the asporogenous phenotype . We speculate that the accumulation of a protein containing the amino-terminal portion of the SPS2 gene product, synthesized from the transcripts of the truncated plasmid-borne copies of the SPS2 gene, prevents ascus formation.

Mol Cell Biol, 1987 Jul, 7(7), 2344 - 51
Fatty acylation is important but not essential for Saccharomyces cerevisiae RAS function; Deschenes RJ et al.; Two proteins in the yeast Saccharomyces cerevisiae that are encoded by the genes RAS1 and RAS2 are structurally and functionally homologous to proteins of the mammalian ras oncogene family . We examined the role of fatty acylation in the maturation of yeast RAS2 protein by creating mutants in the putative palmitate addition site located at the carboxyl terminus of the protein . Two mutations, Cys-318 to an opal termination codon and Cys-319 to Ser-319, were created in vitro and substituted in the chromosome in place of the normal RAS2 allele . These changes resulted in a failure of RAS2 protein to be acylated with palmitate and a failure of RAS2 protein to be localized to a membrane fraction . The mutations yielded a Ras2- phenotype with respect to the ability of the resultant mutants to grow on nonfermentable carbon sources and to complement ras1- mutants . However, overexpression of the ras2Ser-319 product yielded a Ras+ phenotype without a corresponding association of the mutant protein with the membrane fraction . We conclude that the presence of a fatty acyl moiety is important for localizing RAS2 protein to the membrane where it is active but that the fatty acyl group is not an absolute requirement of RAS2 protein function.

Mol Cell Biol, 1987 Jul, 7(7), 2309 - 15
Regulatory function of the Saccharomyces cerevisiae RAS C-terminus; Marshall MS et al.; Activating mutations (valine 19 or leucine 68) were introduced into the Saccharomyces cerevisiae RAS1 and RAS2 genes . In addition, a deletion was introduced into the wild-type gene and into an activated RAS2 gene, removing the segment of the coding region for the unique C-terminal domain that lies between the N-terminal 174 residues and the penultimate 8-residue membrane attachment site . At low levels of expression, a dominant activated phenotype, characterized by low glycogen levels and poor sporulation efficiency, was observed for both full-length RAS1 and RAS2 variants having impaired GTP hydrolytic activity . Lethal CDC25 mutations were bypassed by the expression of mutant RAS1 or RAS2 proteins with activating amino acid substitutions, by expression of RAS2 proteins lacking the C-terminal domain, or by normal and oncogenic mammalian Harvey ras proteins . Biochemical measurements of adenylate cyclase in membrane preparations showed that the expression of RAS2 proteins lacking the C-terminal domain can restore adenylate cyclase activity to cdc25 membranes.

Genetics, 1987 Jul, 116(3), 371 - 5
Chromosome specificity of polysomy promotion by disruptions of the Saccharomyces cerevisiae RNA1 gene; Atkinson NS et al.; Previously, we showed that a disruption of the yeast RNA1 gene with LEU2 sequences promotes polysomy for chromosome XIII . Here we demonstrate that this phenotype is due to sequences specific to the RNA1 gene and that the disruption allele does not affect nondisjunction of three other chromosomes or polysomy of a minichromosome . Hence polysomy appears to be restricted to chromosome XIII.

J Bacteriol, 1987 Jul, 169(7), 2932 - 7
Glucose transport in a kinaseless Saccharomyces cerevisiae mutant; Lang JM et al.; Wild-type Saccharomyces cerevisiae organisms contain three kinases which catalyze the phosphorylation of glucose: two hexokinase isozymes (PI and PII) and one glucokinase . Glucose transport measurements for triple-kinaseless mutants, which lack all three of these kinases, confirm that the kinases are involved in the low apparent Km transport process observed in metabolizing cells . Thus kinase-positive cells containing one or more of the three kinases exhibit biphasic transport kinetics with a low apparent Km (1 to 2 mM) and high apparent Km (40 to 50 mM) component . Triple-kinaseless cells, however, exhibit only the high apparent Km component of kinase-positive cells (60 mM) . Kinetic analysis of glucose transport in the triple-kinaseless cells shows that glucose is transported by a facilitated diffusion process which exhibits trans-stimulated equilibrium exchange and influx counterflow.

J Gen Microbiol, 1987 Jul, 133 ( Pt 7), 1675 - 84
Growth and metabolism of mannitol by strains of Saccharomyces cerevisiae; Quain DE et al.; Of 40 polyploid strains of Saccharomyces cerevisiae screened for growth on D-mannitol (5%, w/v), half grew well (5-20 mg dry biomass ml-1) . Certain of these strains were unable to grow on low concentrations of mannitol (1-2%, w/v) and others, initially unable to grow on mannitol, exhibited long-term adaptation to growth . An NAD+-dependent D-mannitol dehydrogenase (EC 1.1.1.67) was detected in mannitol-grown yeast . Growth was dependent on mitochondrial function and was obligately aerobic . Measurement of products of metabolism and respiratory activity indicated that growth on mannitol allows catabolite derepression.

J Biochem (Tokyo), 1987 Jul, 102(1), 181 - 9
A positive regulatory sequence of the Saccharomyces cerevisiae ENO1 gene; Uemura H et al.; ENO1-'lacZ fusions with various lengths of the ENO1 5'-flanking region were constructed on various types of yeast plasmid vectors . The fully expressed level of beta Gal directed by ENO1-'lacZ fusions differed depending on the type of vector, but on any type of vector, beta Gal activity was not greatly influenced by the carbon source in the medium . The 86-bp DNA region of ENO1 at position -487 to -402 upstream of the initiation codon, in which we had previously delimited the positive regulatory region of ENO1 (Uemura, H., Shiba, T., Paterson, M., Jigami, Y., & Tanaka, H . (1986) Gene 45, 67-75), exerted its function without requiring precise location with respect to the TATA box . The action of the positive regulatory region was not affected by its orientation . In addition, the substitution of the UASs of PHO5, encoding repressible acid phosphatase, with the regulatory region of ENO1 changed the expression of PHO5-'lacZ gene to constitutive, irrespective of the concentration of inorganic phosphate in the medium . Furthermore, the GCR1 gene cloned in a multicopy plasmid increased the expression of the ENO1-'lacZ fused genes.

J Bacteriol, 1987 Jul, 169(7), 2926 - 31
Glucose transport in vesicles reconstituted from Saccharomyces cerevisiae membranes and liposomes; Ongjoco R et al.; Glucose transport activity was reconstituted into liposomes by the freeze-thaw-sonication procedure from unextracted Saccharomyces cerevisiae membranes and preformed phospholipid liposomes . Fluorescence-dequenching measurements with octadecylrhodamine B chloride (R18)-labeled membranes showed that the yeast membrane lipids are diluted by the liposome lipids after the freeze-thaw-sonication procedure . At lipid-to-protein ratios greater than 75:1, vesicles with single transporters were formed . Reconstituted specific activity was increased at least twofold if the liposomes contained 50 mol% cholesterol . A further increase in specific activity, from 3- to 10-fold, was achieved by fractionation of the membranes on a Renografin gradient before reconstitution . Examination of the fractions from the Renografin gradient by sodium dodecyl sulfate-gel electrophoresis showed a parallel enrichment of glucose transport activity and a number of proteins including one with an apparent Mr of ca . 60,000, which might be the glucose transporter . Finally, preliminary kinetic analysis of glucose transport activity in vesicles reconstituted at a high lipid-to-protein ratio gave a Vmax of ca . 2.8 mumol/mg of protein per min at 23 degrees C and a Km of ca . 8 mM . The latter value corresponds to the kinase-independent, low-affinity component of glucose transport observed in wild-type cells.

Genetika, 1987 Jul, 23(7), 1141 - 8
{Cloning of the ADE2 gene of Saccharomyces cerevisiae and localization of the ARS-sequence}; Sasnauskas KV et al.; Mutational changes in ADE2 result in the accumulation of red pigment in cells, which serves as an indicator for the selection of mutants . This easily detectable phenotype of red-coloured colonies can account for the wide use of ade2 mutants in yeast genetics . ADE2 gene was cloned in a shuttle vector by complementing the ade2 mutation in the yeast . It was shown that the 2.2 kbp HindIII fragment of yeast DNA contains structural sequences of the ADE2 gene as well as the ARS sequence . Deletion analysis of the 5' end of the ADE2 gene showed the ARS sequence to be situated at the distal end of the 1 kbp HindIII fragment . Removal of the ARS sequence does not influence ADE2 gene complementation ability . Transformants containing the ADE2 gene comprised in their plasmids form white colonies . Loss of the plasmids results in colour change of colonies.

J Biol Chem, 1987 Jun 25, 262(18), 8901 - 9
Isolation and characterization of the nuclear gene encoding the Rieske iron-sulfur protein (RIP1) from Saccharomyces cerevisiae; Beckmann JD et al.; The nuclear gene encoding the Rieske iron-sulfur protein of the cytochrome bc1 complex of the mitochondrial respiratory chain has been isolated and characterized from Saccharomyces cerevisiae . We used a segment of the iron-sulfur protein gene from Neurospora crassa (Harnisch, U., Weiss, H., and Sebald, W . (1985) Eur . J . Biochem . 149, 95-99) to detect the yeast gene by Southern analysis . Five different but overlapping clones were then isolated by probing a yeast genomic library carried on YEp 13 by colony lift hybridization . Several approaches confirmed that the isolated DNA contained the gene for the Rieske iron-sulfur protein . The yeast gene, which contains no introns, can be expressed in Escherichia coli . A 900-base pair HindIII-EcoRI fragment was subcloned into pUC19 and directed the synthesis of immunodetectable protein . The gene was also identified by disruption of its chromosomal copy by homologous integration . A 400 base pair PstI-EcoRI fragment cloned adjacent to a HIS3 marker in pUC18 was used as an integrating vector . HIS+ transformants were obtained which were unable to grow on the nonfermentable carbon source glycerol . Southern analysis of the respiration deficient (gly-) strains confirmed that the chromosomal copy of the gene was disrupted, and immunoblots of extracts of the transformants indicated a lack of iron-sulfur protein . A respiration-deficient integrant was transformed to GLY+ by a 2-kilobase pair HindIII-BglII fragment, including a complete copy of the gene, carried on a multicopy episomal vector . Immunoblots with monoclonal antibodies to the iron-sulfur protein indicated overproduction of the protein in the complemented strain and revealed expression of approximately equal amounts of mature iron-sulfur protein and of a protein approximately 3 kDa larger than the mature protein in the complemented strain . A 1.2-kilobase pair segment of DNA from the clone which complemented the disrupted strains was sequenced and found to contain an open reading frame of 645 nucleotides, capable of encoding a 21,946-dalton protein . The gene is flanked by consensus signal sequences for initiation and termination which are common in yeast and is preceded by a possible upstream activating sequence . Amino acid sequence analysis of the amino-terminal end of the mature iron-sulfur protein agreed exactly with that predicted by the nucleotide sequence starting at Lys-31.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1987 Jun 25, 262(18), 8636 - 42
Expression in Escherichia coli of BCY1, the regulatory subunit of cyclic AMP-dependent protein kinase from Saccharomyces cerevisiae . Purification and characterization; Johnson KE et al.; The regulatory (R) subunit of cAMP-dependent protein kinase from the yeast Saccharomyces cerevisiae was expressed in Escherichia coli by engineering the gene for yeast R, BCY1, into an E . coli expression vector that contained a promoter from phage T7 . Oligonucleotide-directed mutagenesis was used to create an NdeI restriction site at the natural ATG of the yeast R . This facilitated construction of the T7 expression vector so that the sequence of the protein produced was identical to the natural R subunit . Yeast R was highly expressed in a soluble form . 20 mg of purified yeast R was obtained from 4 liters of E . coli . N-terminal amino acid sequencing revealed that the expressed protein began with the natural sequence . 60% of the molecules contained an N-terminal methionine, and 40% initiated with valine, the second amino acid of yeast R . The protein produced in E . coli migrated on a sodium dodecyl sulfate-polyacrylamide gel with an Mr of 52,000 . The yeast R bound 2 mol of cAMP/mol of R monomer with a Kd of 76 nM . The protein was treated with urea to remove bound cAMP . Sedimentation values before and after the urea treatment were identical (s20,w = 5.1) . Addition of purified R subunit to a preparation of yeast C subunit (TPK1) rendered catalytic activity cAMP-dependent with an activity ratio of 4.6 . The yeast R was autophosphorylated by yeast C to a level of 0.8 mol of phosphate/mol of R monomer . By these criteria, the R subunit produced in E . coli was structurally and functionally identical to the natural yeast R subunit and similar to mammalian type II R subunits.

Eur J Biochem, 1987 Jun 15, 165(3), 671 - 4
Internal acidification and cAMP increase are not correlated in Saccharomyces cerevisiae; Eraso P et al.; Addition of glucose to a yeast suspension can produce both an increase in the level of cAMP and a decrease in the intracellular pH . This observation led to the idea that internal acidification triggers the cAMP increase . We have tested this hypothesis using different approaches . To study the effect of sugar metabolism on internal pH we added to the yeast either glucose or a sugar, like xylose, that cannot be phosphorylated . We also utilized yeast strains lacking hexose kinases or phosphoglucose isomerase . We found that phosphorylation of the sugar added is a requisite for internal acidification but not for the cAMP increase . Internal acidification is due to an imbalance between the rate of the metabolic reactions that generate protons and the rate at which protons can be pumped out of the cell . We have manipulated the excretion of protons by using yeast harvested at different phases of growth and resuspended in a medium with or without added K+ . Addition of glucose produced a marked drop in internal pH only when the yeast was harvested in the stationary phase of growth and transferred to a medium without added K+ . In contrast an increase in cAMP was observed in all situations . We conclude that in yeast there is no correlation between internal acidification and cAMP increase.

Yeast, 1987 Jun, 3(2), 95 - 105
Tryptophan accumulation in Saccharomyces cerevisiae under the influence of an artificial yeast TRP gene cluster; Prasad R et al.; Plasmid pME559, carrying all five yeast TRP genes, was constructed . This plasmid is a yeast/Escherichia coli shuttle vector based on pBR322 and 2 micron-DNA sequences derived from plasmid pJDB207 . We studied in yeast (i) the stability of the plasmid under selective and non-selective conditions, (ii) expression of all five TRP genes and (iii) tryptophan accumulation in yeast transformants . These studies were conducted in comparison with an earlier construction, pME554, which differs from plasmid pME559 in the expression of the TRP1 gene and which carries the TRP2 wild type instead of the TRP2fbr mutant allele . For stable maintenance of the plasmids in yeast a selection was necessary . Plasmid pME559 displayed normal expression of all TRP genes, and enzyme levels on average 23-fold higher than in the wild type strain were found . In comparison, the maximal tryptophan flux observed in such a plasmid-carrying strain was about ten-fold higher than the maximal flux capacity in the wild type strain.

Yeast, 1987 Jun, 3(2), 107 - 15
Analysis of DNA sequences homologous with the ARS core consensus in Saccharomyces cerevisiae; Bouton AH et al.; We have previously identified an autonomously replicating segment (ARS) near the 3' end of the histone H4 gene at the copy-I H3-H4 locus . We have now searched for additional autonomously replicating segments and sequences homologous with the ARS core consensus sequence near the copy-II histone H4 gene and both of the histone H3 genes . No new ARS elements were identified by functional cloning assays . However, several matches to the ARS core consensus element were found within the DNA sequences of the copy-I and copy-II genes . An exact match to the ARS core consensus was identified in the region downstream from the copy-I histone H3 gene and a set of sequences with weak homology was also located within the copy-II region . However, restriction fragments including these sequences did not demonstrate ARS activity on a plasmid in transformed cells.

J Gen Microbiol, 1987 Jun, 133 ( Pt 6), 1471 - 80
Differential translational efficiency of the mRNAs isolated from derepressed and glucose repressed Saccharomyces cerevisiae; Soler AP et al.; Carbon catabolite derepression induced changes in the pool of yeast mRNAs translatable in a protein-synthesizing reticulocyte system . Competition experiments with globin mRNA showed that the mRNA population obtained from derepressed cells possessed a higher translational efficiency than mRNA from repressed cells . The mRNAs that could account for the high translational efficiency of the derepressed mRNA were not detected in cells growing in glucose-rich medium . Analysis of protein synthesis in the presence of 7-methylguanosine 5'-phosphate indicated that the initiation factors recognizing the 5'-terminal structure of capped messengers interacted with lower affinity with the repressed than with some specific derepressed mRNAs.

Mol Gen Genet, 1987 Jun, 208(1-2), 101 - 6
The effects of ADR1 and CCR1 gene dosage on the regulation of the glucose-repressible alcohol dehydrogenase from Saccharomyces cerevisiae; Denis CL; The dosage of the transcriptional activator ADR1 was varied in order to study the regulation of the glucose-repressible alcohol dehydrogenase (ADH II) from Saccharomyces cerevisiae . ADH II activity during glucose growth conditions was shown to increase linearly with increasing ADR1 gene dosage . In contrast, under derepressed growth conditions a 100-fold increase in ADR1 copy number resulted in only a 4-fold increase in ADH II expression . Saturation of ADH II gene expression by ADR1 under derepressed conditions was shown not to result from decreased ADR1 transcription . Increases in ADH2 gene dosage in conjunction with high ADR1 gene dosages resulted in increased ADH II activity, indicating that ADH2 was the limiting factor during derepression . Under glucose-repressed conditions the activator CCR1 was not required for ADR1 activity . During derepression increasing ADR1 dosage could partially compensate for a CCR1 defect . Increasing CCR1 gene dosage, however, had no effect on ADH2 expression regardless of the ADR1 allele present . These results suggest that CCR1 acts through ADR1 in controlling ADH2 expression . It was also observed that high numbers of ADR1, or a few copies of ADR1-5c, substantially increased the cell doubling time under ethanol growth conditions, indicating that increased ADR1 activity is toxic.

Vaccine, 1987 Jun, 5(2), 90 - 101
Antigenicity and immunogenicity of domains of the human immunodeficiency virus (HIV) envelope polypeptide expressed in the yeast Saccharomyces cerevisiae; Barr PJ et al.; Expression vectors were constructed for the production of various domains of the envelope gene product of the SF-2 isolate of human immunodeficiency virus (HIV) in the yeast Saccharomyces cerevisiae . Serum specimens from HIV seropositive blood donors reacted in immunoblot assays with recombinant polypeptides from both the gp120 and gp41 coding regions of env . Polypeptides from both domains were purified and injected into experimental animals . Antibodies raised in rabbits to env-2, a recombinant polypeptide representing the majority of the protein moiety of gp120, reacted with fully glycosylated native gp120 of HIV-SF2 virions . In addition, these env-2 antisera showed reactivity with viral gp120 of divergent HIV isolates . A 121 amino acid polypeptide (env-5), representing the region of gp41 stretching between the two hydrophobic domains of the protein, elicited antibodies in rabbits that reacted with glycosylated, native gp41 . Thus, selected domains of the HIV env gene expressed in genetically engineered yeast, are recognized by sera from HIV infected humans, elicit antibodies that react with native HIV glycoproteins and provide a source of envelope antigens for evaluation as potential subunit vaccines for HIV.

Mol Cell Biol, 1987 Jun, 7(6), 2155 - 64
Isolation and characterization of temperature-sensitive RNA polymerase II mutants of Saccharomyces cerevisiae; Himmelfarb HJ et al.; Three independent, recessive, temperature-sensitive (Ts-) conditional lethal mutations in the largest subunit of Saccharomyces cerevisiae RNA polymerase II (RNAP II) have been isolated after replacement of a portion of the wild-type gene (RPO21) by a mutagenized fragment of the cloned gene . Measurements of cell growth, viability, and total RNA and protein synthesis showed that rpo21-1, rpo21-2, and rpo21-3 mutations caused a slow shutoff of RNAP II activity in cells shifted to the nonpermissive temperature (39 degrees C) . Each mutant displayed a distinct phenotype, and one of the mutant enzymes (rpo21-1) was completely deficient in RNAP II activity in vitro . RNAP I and RNAP III in vitro activities were not affected . These results were consistent with the notion that the genetic lesions affect RNAP II assembly or holoenzyme stability . DNA sequencing revealed that in each case the mutations involved nonconservative amino acid substitutions, resulting in charge changes . The lesions harbored by all three rpo21 Ts- alleles lie in DNA sequence domains that are highly conserved among genes that encode the largest subunits of RNAP from a variety of eucaryotes; one mutation lies in a possible Zn2+ binding domain.

Mol Cell Biol, 1987 Jun, 7(6), 2128 - 33
Guanine nucleotide activation of, and competition between, RAS proteins from Saccharomyces cerevisiae; Field J et al.; In the yeast Saccharomyces cerevisiae, yeast RAS proteins are potent activators of adenylate cyclase . In the present work we measured the activity of adenylate cyclase in membranes from Saccharomyces cerevisiae which overexpress this enzyme . The response of the enzyme to added RAS2 proteins bound with various guanine nucleotides and their analogs suggests that RAS2 proteins are active in their GTP-bound form and are virtually inactive in their GDP-bound form . Also, active RAS2 protein is not inhibited by inactive RAS2, suggesting that the inactive form does not compete with the active form in binding to its effector.

Proc Natl Acad Sci U S A, 1987 Jun, 84(11), 3570 - 4
Expression of the Saccharomyces cerevisiae glycoprotein invertase in mouse fibroblasts: glycosylation, secretion, and enzymatic activity; Bergh ML et al.; Oligosaccharide processing is controlled by host- and protein-dependent factors . To increase our understanding of the relative contribution of those factors we studied the glycosylation of yeast invertase expressed in a heterologous system . Invertase synthesized in psi-2 cells (an NIH 3T3-derived packaging line) is secreted efficiently, enzymatically active, and heavily glycosylated . It was estimated that the protein contains 8 or 9 carbohydrate chains . Two classes can be observed, of an approximate size of 100-110 kDa and 115-130 kDa, respectively . The size differences are due to differences in glycosylation . The smaller class contains two high-mannose carbohydrate chains; the remainder is of the complex type, sialylated and most likely tri- or tetraantennary . This profile parallels the situation observed with invertase glycosylation in yeast, where 2 of 9 or 10 chains remain unprocessed . The larger size class of invertase expressed in mouse fibroblasts has a different profile, since it contains probably only complex-type glycans . There are no apparent differences, however, in the size of the protein backbone between the two size classes . When invertase is synthesized in the presence of the mannosidase inhibitor 1-deoxymannojirimycin, processing is blocked completely, since all glycans are susceptible to endo-beta-N-acetylglucosaminidase H . The glucosidase inhibitor 1-deoxynojirimycin does not inhibit processing completely . In both cases secretion of the protein is not affected . The glycosylation inhibitor tunicamycin prevents secretion of invertase completely when cells are cultured at 37 degrees C . At 26 degrees C, however, nonglycosylated invertase can be detected in the medium . These data suggest that glycosylation of invertase seems to be essential for the early steps of the secretory pathway but is less critical for later events.

J Bacteriol, 1987 Jun, 169(6), 2598 - 600
Ureidosuccinate is transported by the allantoate transport system in Saccharomyces cerevisiae; Turoscy V et al.; The regulatory characteristics exhibited by ureidosuccinate transport in Saccharomyces cerevisiae led us to hypothesize that this biosynthetic intermediate was transported via the degradative allantoate transport system . The hypothesis was verified by the finding that neither dal5 nor urep1 mutant strains could transport allantoate or ureidosuccinate . Mutations in the two loci were tightly linked and failed to complement one another, suggesting that they were allelic . The use of a common transport system for accumulation of both biosynthetic and degradative metabolites explains the paradoxical characteristics observed for control of ureidosuccinate and allantoate transport.

Microbiologia, 1987 Jun, 3(2), 101 - 6
Inhibition by alpha factor of the glucose-induced activation of regulatory trehalase in Saccharomyces cerevisiae; Arguelles JC et al.; The enzyme activity of the regulatory trehalase (alpha, alpha-trehalose glycohydrolase; EC 3.2.1.28) in stationary-phase cells of Saccharomyces cerevisiae increased upon addition of glucose to cell suspensions . Such increase was temporarily retarded in the presence of alpha factor in the medium . The transient inhibition required the joined action of the pheromone-like factor and the protease inhibitor N-alpha-p-tosyl-L-lysine chloromethyl ketone . The inhibition of the glucose-induced activation of trehalase by alpha factor lends support to the involvement of adenosine 3',5'-cyclic monophosphate in the enzyme activation in vivo.

Mol Gen Mikrobiol Virusol, 1987 Jun, (6), 42 - 5
{Expression of human leukocyte interferon A gene in Saccharomyces cerevisiae under the control of regulatory yeast elements PHO5, GAL1 and GAL10}; Nikoshkov AB et al.; A chromosomal gene for human leucocyte interferon A is expressed in Saccharomyces cerevisiae yeasts due to interaction of 5'-nontranslating region of the cloned interferon gene with the regulatory elements of yeast genes PHO5, GAL1 and GAL10 . Regulated systhesis of interferon was obtained in all cases . The level of interferon genes expression in case using GAL1 and GAL10 genes regulatory elements (5 X 10(5) and 5 X 10(6) u X l-1) correlated with the distances to their promoters . The highest yield of interferon (10(8) u X l-1) was obtained when the PHO5 gene regulatory elements were used.

Mol Cell Biol, 1987 Jun, 7(6), 2212 - 20
Elements involved in oxygen regulation of the Saccharomyces cerevisiae CYC7 gene; Zitomer RS et al.; The CYC7 gene of Saccharomyces cerevisiae encodes the minor species, iso-2, of the cytochrome c protein . Its expression is governed by two regulatory sequences upstream from the gene: a positive site which stimulates transcription 240 base pairs 5' from the protein-coding sequence (-240) and a negative site which inhibits transcription at -300 . In this study, the nature of the positive site and its relationship to the negative site has been investigated . Expression of the CYC7 gene is weakly inducible by oxygen . This effect was greatly enhanced by the semidominant CYP1-16 mutation in the trans-acting gene CYP1 . The weak oxygen regulation in wild-type cells and the enhanced induction in CYP1-16 mutants were found to be mediated through the positive site . A mutational analysis of this site implicated at least part of a tandem, direct repeat of 9 base pairs as essential for the functioning of this site . The relationship between the positive and negative sites was investigated by comparing the expression of the intact gene with that of derivatives lacking either one or the other site . The expression of the gene containing only the negative site was actually stimulated anaerobically, while the gene containing the positive site alone, although having higher expression aerobically than anaerobically, had higher anaerobic expression than did the intact gene . Thus, it appeared that the combination of the positive and negative sites suppressed anaerobic expression . A model which attempts to explain these properties of the two sites and account for the regulation of the expression of the intact gene is presented.

Mol Cell Biol, 1987 Jun, 7(6), 2180 - 7
Expression of Rous sarcoma virus transforming protein pp60v-src in Saccharomyces cerevisiae cells; Brugge JS et al.; The Rous sarcoma virus (RSV) pp60v-src protein was expressed in Saccharomyces cerevisiae cells either from a plasmid vector carrying the v-src gene or in yeast cells containing a single-copy v-src gene chromosomally integrated . In both yeast strains, v-src gene transcription is regulated by the galactose-inducible GAL10 promoter . Growth in galactose-containing medium resulted in constitutive expression of pp60v-src in the integrated strain and transient expression of higher levels of pp60v-src in the plasmid-bearing strain . The concentration of pp60v-src in the plasmid-bearing strain at its peak of expression was approximately threefold lower than that found in RSV-transformed mammalian cells . pp60v-src synthesized in yeast cells was phosphorylated in vivo on sites within the amino and carboxyl halves of the molecule . In immune complex kinase assays, the yeast pp60v-src was autophosphorylated on tyrosine and was able to phosphorylate exogenous substrates such as casein and enolase . The specific activity of pp60v-src synthesized in yeast cells was approximately 5- to 10-fold higher than that made in mammalian cells . Induction of pp60v-src caused the death of the plasmid-bearing yeast strain and transient inhibition of growth of the single-copy strain . Concomitantly, this induction resulted in high levels of tyrosine phosphorylation of yeast cell proteins . This indicates that pp60v-src functions as a tyrosine-specific phosphotransferase in yeast cells and suggests that hyperphosphorylation of yeast proteins is inimical to cell growth.

Mol Cell Biol, 1987 Jun, 7(6), 2141 - 7
Initiation of meiosis and sporulation in Saccharomyces cerevisiae does not require a decrease in cyclic AMP; Olempska-Beer Z et al.; Meiosis and sporulation of Saccharomyces cerevisiae are initiated in a guanine auxotroph by guanine deprivation (E . Bautz Freese, Z . Olempska-Beer, A . Hartig, and E . Freese, Dev . Biol . 102:438-451, 1984) . We used this condition to examine a hypothesis (K . Matsumoto, I . Uno, and T . Ishikawa, Cell 32:417-423, 1983) that initiation of meiosis requires a low level of cAMP . We found that, after guanine deprivation, the intracellular concentration of cAMP transiently decreased not more than 20% and not at all if the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) was added to the medium . Under these conditions, at least 76% of the cells sporulated in the absence of IBMX, and almost 100% sporulated in its presence . The sporulating cells continually excreted cAMP and utilized the gluconeogenic carbon source . The cells failed to sporulate efficiently and to form four-spored asci if simultaneously deprived of guanine and carbon . After guanine deprivation in glucose medium, sporulation remained suppressed and intracellular cAMP was unchanged . We conclude that, under conditions of guanine starvation, cAMP deficiency is not required for initiation of meiosis and sporulation, cAMP is produced in excess and excreted to the medium, the cells sporulate better if the cAMP concentration is increased by addition of IBMX, the cells require a gluconeogenic carbon source for complete and efficient sporulation, and suppression of sporulation by glucose is not mediated by cAMP.

Eur J Biochem, 1987 Jun 1, 165(2), 419 - 25
Nucleotide sequence of the gene encoding the 11-kDa subunit of the ubiquinol-cytochrome-c oxidoreductase in Saccharomyces cerevisiae; Maarse AC et al.; The nucleotide sequence of the gene encoding the 11-kDa subunit VIII of the ubiquinol-cytochrome-c oxidoreductase in Saccharomyces cerevisiae has been determined . The coding sequence has a length of 330 bp and is preceded at a distance of 361 bp by another reading frame, coding for a protein of as yet unknown function . The 11-kDa gene is transcribed independently of the URFx gene and transcription of both is sensitive to catabolite repression . Multiple 5' and 3' termini of transcripts of the gene for the 11-kDa subunit were identified by S1 nuclease protection analysis of DNA X RNA hybrids . The 5' termini map 52 +/- 2 and 60 +/- 2 nucleotides upstream of the initiation codon whereas the 3' termini map 336 +/- 2 and 350 +/- 2 nucleotides downstream of the stop codon . The subunit VIII reading frame encodes a protein with a molecular mass of 12.4 kDa and a polarity of 37.6% . It is predicted to contain a high content of beta-sheet segments, which may be capable of forming a barrel-like structure in a lipid bilayer . A comparison of the sequence with those of the small subunits of the beef heart complex reveals similarity with the 9.5-kDa subunit VII (core-linked protein) characterized by Borchart et al . (1986) FEBS Lett . 200, 81-86 . The significance of this is discussed.

Eur J Biochem, 1987 Jun 1, 165(2), 289 - 96
Molecular characterization of transposable-element-associated mutations that lead to constitutive L-ornithine aminotransferase expression in Saccharomyces cerevisiae; Degols G et al.; The cargB or CAR2 gene, coding for ornithine aminotransferase, was isolated by functional complementation of a cargB- mutation in Saccharomyces cerevisiae . It was used as a hybridization probe to analyse RNA and chromosomal DNA from four strains bearing cis-dominant regulatory mutations leading to constitutive, mating-type-dependent, ornithine aminotransferase synthesis . The four mutations appear to be insertions . Their size and restriction pattern suggested that they were transposable elements, Ty1 . All were inserted in the same orientation with respect to the cargB gene . We cloned the cargB gene with its associated insertion from two constitutive mutants (cargB+ Oh-1 and cargB+ Oh-2) . We determined the sequence of the cargB 5' region from the wild-type gene and from the two mutated genes . The DNA sequences of the extremities of the two insertions were very homologous but not identical and were similar to previously reported Ty1 element direct repeats (delta) . The same five-base-pair sequence, ATATA, was found at both ends of both Ty1 elements, indicating that both Ty1 were transposed to the same site . This site is located 115 base pairs upstream from the putative cargB coding region . The 5' end of cargB transcript as determined by S1 mapping was the same in the wild-type strain and in the four mutants . The cargB transcript was not detected in the wild-type strain grown under non-induced conditions, while under the same conditions it was present in all four mutants.

Proc Natl Acad Sci U S A, 1987 Jun, 84(12), 4156 - 60
SSC1, a member of the 70-kDa heat shock protein multigene family of Saccharomyces cerevisiae, is essential for growth; Craig EA et al.; The genome of the yeast Saccharomyces cerevisiae contains a family of genes related to the HSP70 genes (encoding the 70-kDa heat shock protein) of other eukaryotes . Mutations in two of these yeast genes (SSC1 and SSD1), whose expression is increased a few fold after temperature upshift, were constructed in vitro and substituted into the yeast genome in place of the wild-type alleles . No phenotypic effects of the mutation in SSD1 were detected . However, a functional SSC1 gene is essential for vegetative growth . This result, in conjunction with experiments involving mutations in other members of this multigene family, indicates that at least three distinct functions are carried out by genes of the HSP70 family.

Yeast, 1987 Jun, 3(2), 85 - 93
Cyclic AMP controls the switch between division cycle and resting state programs in response to ammonium availability in Saccharomyces cerevisiae; Boy-Marcotte E et al.; We have identified a mutation called rcal (for rescue by cAMP) which allows adenylate cyclase-deficient mutants to divide in the presence of cAMP . We took advantage of this rcal mutation to study the effect of externally added cAMP on the onset of the resting state when cells are starved for ammonium . We measured the resistance of the cells to zymolyase treatment as a parameter of the resting state . We observed that the onset of the resting state is reversibly blocked by cAMP . This inhibitory effect of cAMP is discussed together with the cAMP control of the start . This leads us to propose a model in which the cAMP level, controlled by the availability of nutrients, should trigger the choice between the entry of the cell into the resting state and the initiation of a new division cycle.

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