S. cerevisiae

J Med Vet Mycol, 1989, 27(6), 397 - 406
Genetic and physiological analysis of azole sensitivity in Saccharomyces cerevisiae; Kenna S et al.; Ketoconazole and fluconazole are azole antifungal agents which inhibit cytochrome P-450 mediated sterol C14 demethylation during ergosterol biosynthesis . We report on the activity of these antifungals on a variety of Saccharomyces cerevisiae strains grown under differing conditions known to affect cyt P-450 levels . Only slight increases in resistance to azoles were observed under conditions which induce the yeast cyt P-450 from undetectable levels . Strain variation was observed, with some strains exhibiting a fungicidal, and others a fungistatic response . Two cyt P-450 deficient mutants examined exhibited resistance to treatment with fluconazole and ketoconazole . This was attributed, at least in part, to an additional defect in sterol delta 5,6 desaturation and possibly to reduced cellular levels of azole drug.

Adv Enzyme Regul, 1989, 28, 283 - 306
6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase from Saccharomyces cerevisiae; Hofmann E et al.; In permeabilized yeast cells 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase are studied during growth . It is shown that in yeast at least two fructose 2,6-bisphosphate degrading enzyme activities occur, differing in pH profile and in their substrate affinities . The activities of 6-phosphofructo-2-kinase and of fructose-2,6-bisphosphatases drop in the exponential and the transition phase while the activity of the alkaline phosphatases steadily increases . In the stationary phase the activities of 6-phosphofructo-2-kinase and of the low Km fructose-2,6-bisphosphatase increase again . Yeast 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase were purified and separated from each other . The purified 6-phosphofructo-2-kinase was found to exhibit a very high specific activity (1.3 U/mg) . The enzyme is efficiently inhibited by ATP . The ATP inhibition is most pronounced at low concentrations of magnesium and fructose-6-phosphate . Phosphoenolpyruvate and sn-glycerol 3-phosphate are inhibitors of the enzyme . The high-affinity yeast fructose-2,6-bisphosphatase releases inorganic phosphate from the 2-position of fructose 2,6-bisphosphate . It displays hyperbolic kinetics towards fructose 2,6-bisphosphate (Km = 0.3 microM) and is strongly inhibited by fructose 6-phosphate . The inhibition is counteracted by sn-glycerol 3-phosphate . The enzyme is shown to be inactivated by cAMP-dependent phosphorylation and reactivated by the action of protein phosphatase 2A.

Braz J Med Biol Res, 1989, 22(2), 171 - 7
Fructose 2,6-bisphosphate and trehalose metabolism in Saccharomyces cerevisiae; Panek AD et al.; 1 . A regulatory mutant of Saccharomyces (fdp) unable to activate fructose 1,6-bisphosphatase presented a normal response to the glucose and fructose signals as measured by trehalase activation, indicating that the inability of the strain to grow on these sugars is caused by a defect located beyond membrane interactions . 2 . In vivo experiments with a mutant strain bearing a phosphoglucoisomerase gene (pgil-delta) deletion showed that activation of trehalase and deactivation of the tehalose-6-phosphate synthase complex occurred to the same extent whether glucose or fructose was used as signal . 3 . These results suggest that fructose-2,6-bisphosphate is not involved in the interconversion of forms of the enzymes of trehalose metabolism . Furthermore, when fructose-2,6-bisphosphate was assayed on trehalose synthesizing activity using cell-free extracts and partially purified preparations of the complex, no effect was observed . 4 . We conclude that regulation by cAMP fulfills the requirements for control of trehalose levels in Saccharomyces.

Mol Biol (Mosk), 1989 Jan-Feb, 23(1), 279 - 85
{Isolation of nucleotide sequences, possessing enhancer functions from the Saccharomyces cerevisiae genetic library}; Polishchuk AG et al.; We propose a method for isolation of enhancer-like sequences from a yeast genomic library . The method was used to identify DNA inserts capable of increasing bacterial bla gene expression when located downstream from the transcription initiation site . Plasmids carrying different fragments of one such insert were used to localize the enhancer-like function on a 1.2 kb fragment.

Genes Dev, 1989 Jan, 3(1), 38 - 48
Functional analysis of the papilloma virus E2 trans-activator in Saccharomyces cerevisiae; Lambert PF et al.; The papilloma virus E2 transcriptional trans-activator is representative of a class of transcriptional modulators that activate transcription through direct binding to cis-acting DNA sequences . In this study we measured the capacity for this mammalian virus factor to function in Saccharomyces cerevisiae . When expressed in the yeast, the bovine papilloma virus E2 trans-activator could stimulate transcription from a yeast promoter having E2 DNA-binding sites present in cis . Whereas a single E2 DNA-binding site was sufficient for trans-activation, a strong cooperative effect was observed with two E2 DNA-binding sites . The level of trans-activation was dependent on the position of the E2 DNA-binding sites in relation to the yeast promoter, with the maximal effect demonstrated when the binding sites were positioned upstream . Deleted E2 proteins, lacking part of the trans-activation or DNA-binding domains, failed to activate transcription in yeast, similar to their behavior in mammalian cells . Replacement of the amino-terminal region of the E2 trans-activation domain with a synthetic amphipathic helix partially restored the trans-activation function; however, it did not result in a molecule that exhibited cooperativity between neighboring E2 DNA-binding sites.

Biomed Biochim Acta, 1989, 48(7), 403 - 11
Kinetics of 6-phosphofructo-2-kinase from Saccharomyces cerevisiae: inhibition of the enzyme by ATP; Bedri A et al.; 6-Phosphofructo-2-kinase (PFK-2) was purified from yeast and separated from fructose-2,6-biphosphatase (FBPase-2) . The purification procedure involved polyethylene glycol fractionation followed by chromatography on DEAE-Sephacel . PFK-2 and FBPase-2 were copurified in these steps . Separation of the two enzymes resulted from Sephacryl S-300 Blue chromatography . Then, PFK-2 was chromatographed on CM-Sephadex and eluted with a gradient of KCl . Finally, PFK-2 was rechromatographed at CM-Sephadex and specifically eluted with fructose 6-phosphate . PFK-2 (specific activity 1.3 U/mg) was purified about 25,000-fold . The enzyme is inhibited by ATP which is particularly pronounced at low concentrations of magnesium and fructose 6-phosphate . Phosphoenolpyruvate and sn-glycerol 3-phosphate are inhibitors of the enzyme.

J Bacteriol, 1989 Jan, 171(1), 230 - 7
The REV1 gene of Saccharomyces cerevisiae: isolation, sequence, and functional analysis; Larimer FW et al.; The REV1 gene of Saccharomyces cerevisiae is required for normal induction of mutations by physical and chemical agents . We have determined the sequence of a 3,485-base-pair segment of DNA that complements the rev1-1 mutant . Gene disruption was used to confirm that this DNA contained the REV1 gene . The sequenced segment contains a single long open reading frame, which can encode a polypeptide of 985 amino acid residues . The REV1 transcript is 3.1 kilobase pairs in length . Frameshift mutations introduced into the open reading frame yielded a Rev-phenotype . A base substitution, encoding Gly-193 to Arg-193, was found in this open reading frame in rev1-1 . Deletion mutants, lacking segments of the 5' region of REV1, had intermediate mutability relative to REV1 and rev1-1; a complete deletion exhibited lower mutability than rev1-1 . REV1 is not an essential gene . An in-frame fusion of the 5' end of the REV1 open reading frame to the lacZ gene produced beta-galactosidase activity constitutively . The predicted REV1 protein is hydrophilic, with a predicted pI of 9.82 . No homologies to RAD1, RAD2, RAD3, RAD7, or RAD10 proteins were noted . A 152-residue internal segment displayed 25% identity with UMUC protein.

Acta Microbiol Pol, 1989, 38(2), 97 - 106
Nuclear mutants of Saccharomyces cerevisiae affected in bc1 complex; Orlowska-Matuszewska G et al.; Three nuclear mutants, affected to various degrees with respect to cytochrome b are described . Detailed genetical study revealed that each mutant strain carried a single gene mutation; in complementation test the three mutations proved to be non-allelic . The measurements of enzymatic activities strongly suggest that the bc1 complex is the target of the mutations.

Acta Microbiol Pol, 1989, 38(2), 143 - 52
Effects of C8 alkoxymethylene trimethylammonium chloride on Saccharomyces cerevisiae; Misiewicz MH et al.; The influence of the C8 alkoxymethylene trimethyloammonium chloride on the growth of Saccharomyces cerevisiae and activity of mitochondria was studied . It was shown that the compound at low concentration inhibited growth on glycerol medium, but considerably higher concentration is involved in the inhibition of growth on glucose medium . C8-ATC also exerted another inhibitory effect on genotypically different yeast strains: it appeared that rho- strain is more sensitive than rho+ strain . C8-ATC compound was not capable itself of inducing petite mutations, but is able of retarding the petite inducing activity of the mutagen ethidium bromide . The result pointed out the role of mitochondria in the expression of sensitivity to the investigated compound.

Carlsberg Res Commun, 1989, 54(3), 85 - 97
Substrate specificity of proteinase yscA from saccharomyces cerevisiae; Dreyer T; Proteinase yscA is an intracellular aspartic proteinase located in the lysosome-like vacuole of the yeast cell . The specificity towards denatured protein substrates was determined by separation and identification of cleavage products after digestions with proteinase yscA, and compared to that obtained with pepsin used under similar conditions . Proteinase yscA is more selective towards the peptide bonds it cleaves than pepsin, but shows the same preference for large hydrophobic residues on both sides of the cleaved bond as pepsin and lysosomal cathepsin D . Phe, Leu and Glu are favoured in substrate subsite P1 and Phe, Ile, Leu and Ala in P'1, whereas Val is unfavoured in P'1 . The implications for the role of proteinase yscA as hydrolase maturase are discussed.

Vopr Virusol, 1989 Jan-Feb, 34(1), 69 - 72
{Changes in the parameters of nonspecific protection of mice upon inoculation of double-stranded RNA from Saccharomyces cerevisiae yeasts}; Verevkina KN et al.; The results of the study of the effect of double-stranded (ds) RNA from killer yeasts Sac . cerevisiae on the parameters of nonspecific protection are presented . Inoculation of dsRNA in a dose of 5 mg/kg intramuscularly was shown to induce interferon production, to increase body temperature, the activity of adrenal cortex, phagocytic activity of macrophages of the peritoneal exudate and blood neutrophils, the level of lysozyme in the blood, and the activity of macrophage acid phosphatase . The increase in the functional activity of the system components was observed at similar intervals, within 3-6 hours after administration of the preparation . The mutual induction of factors of nonspecific protection and their cooperative participation in the formation of antiviral resistance under the effect of dsRNA is discussed.

Gene, 1988 Dec 30, 74(2), 543 - 7
Potential DNA-binding domains in the RAD18 gene product of Saccharomyces cerevisiae; Chanet R et al.; The RAD18 gene of Saccharomyces cerevisiae is involved in the error-prone DNA repair . Its nucleotide sequence, as reported here, predicts an open reading frame of 1461 nt which corresponds to a protein of 487 amino acids, with an Mr of 55,237 . This protein has three putative zinc fingers, two acidic regions and a nucleotide-binding domain, suggesting that it is a nucleic acid-binding protein with a possible regulatory role.

Biochem Biophys Res Commun, 1988 Dec 30, 157(3), 1182 - 9
Two different protein kinase activities phosphorylate Ras2 protein in Saccharomyces cerevisiae; Sreenath TL et al.; In this report, we show that Ras2 protein in the yeast Saccharomyces cerevisiae is phosphorylated in vivo by protein kinase(s) and the phosphorylation is stable . Ras2 protein is phosphorylated by cAMP dependent protein kinase and by an additional protein kinase activity which is independent of cAMP levels.

J Biol Chem, 1988 Dec 25, 263(36), 19480 - 7
A second transport ATPase gene in Saccharomyces cerevisiae; Schlesser A et al.; A second transport ATPase gene from Saccharomyces cerevisiae has been identified by hybridization to a PMA1 probe and sequenced . The gene called PMA2 encodes a polypeptide of Mr = 102,157, which, with the exception of the 144 amino-terminal residues, is highly homologous to the structural gene PMA1 for the H+-ATPase . It is localized on the chromosome XVI at 16.7 centimorgan from gal4 and is not essential for haploid growth . Comparison between the upstream, noncoding DNA regions of PMA1 and PMA2 indicates that the two genes are controlled differently . The extensive amino acid sequence homology with the fungal H+-ATPases described so far indicates that the PMA2-encoded protein is also able to function as a H+ pump . This is supported by the observation that in pma1 mutants with reduced plasma membrane ATPase activity, disruption of the PMA2 gene confers the ability to grow under alkaline pH conditions . Slower development of diploids is also observed on normal minimal medium after bilateral disruption of PMA2 in the two parents.

J Biol Chem, 1988 Dec 25, 263(36), 19748 - 57
The sn-1,2-diacylglycerol ethanolaminephosphotransferase activity of Saccharomyces cerevisiae . Isolation of mutants and cloning of the EPT1 gene; Hjelmstad RH et al.; A colony autoradiographic assay was used to identify nine Saccharomyces cerevisiae mutants defective in in situ ethanolaminephosphotransferase activity (ept mutants) . Genetic analysis revealed five complementation groups . The EPT1 gene was cloned by complementation of ept1 using a yeast genomic library and was localized to a 2.1-kilobase region of DNA . An ept1 deletional mutant was constructed and introduced into the chromosome by integrative transformation . The ethanolaminephosphotransferase activities in membranes prepared from ept1 and ept2 mutants were reduced 30- to 90-fold and 2- to 3-fold compared with wild-type activity, respectively; the other ept mutants had activities similar to wild type . In strains transformed with a multicopy EPT1-bearing plasmid, a 22- to 33-fold overproduction of ethanolaminephosphotransferase activity was observed . The sn-1,2-diacylglycerol cholinephosphotransferase activities in membranes prepared from ept1 mutants were reduced 3.5- to 7-fold . In contrast to the residual CMP-sensitive cholinephosphotransferase activity observed in cpt1 mutants (Hjelmstad, R . H., and Bell, R . M . (1987) J . Biol . Chem . 262, 3909-3917), the residual cholinephosphotransferase activity of ept1 mutants was CMP-insensitive . The cholinephosphotransferase activities in strains bearing the EPT1 gene on multicopy plasmids were elevated 13- to 23-fold and were CMP-sensitive . The data indicate that 1) the cloned EPT1 gene most likely represents the structural gene for the yeast ethanolaminephosphotransferase, 2) the EPT1 gene product possesses both ethanolamine- and cholinephosphotransferase activities, and 3) the EPT1 gene is nonessential for growth.

J Biol Chem, 1988 Dec 25, 263(36), 19468 - 74
Analysis and in vivo disruption of the gene coding for adenylate kinase (ADK1) in the yeast Saccharomyces cerevisiae; Konrad M; The gene (designated ADK1) encoding the so-called cytosolic adenylate kinase of the yeast Saccharomyces cerevisiae was isolated using a single mixed oligonucleotide hybridization probe designed from the published amino acid sequence . ADK1 was found to be identical to an adenylate kinase gene recently isolated by an approach entirely different from ours (Magdolen, V., Oechsner, U., and Bandlow, W . (1987) Curr . Genet . 12, 405-411) . The gene resides on yeast chromosome IV adjacent to the histone gene H2A-1 . Southern blot analysis revealed only one copy of the gene, and no other related yeast DNA sequences were detected . By gene disruption it is shown that the ADK1 gene is needed for normal cell proliferation but is not essential for cell viability . Immunological studies confirmed the absence of the ADK1 gene product in mutant cells; in extracts of total cellular protein, however, there were still about 10% of the wild-type enzymatic activity present . This indicates the existence of two or more adenylate kinase isozymes in yeast . From preliminary 31P NMR measurements on suspensions of yeast cells, a significant decrease in the level of nucleoside triphosphates was found in the mutant strain carrying the disrupted and partially deleted ADK1 locus.

Nucleic Acids Res, 1988 Dec 23, 16(24), 11507 - 20
Cloning and characterization of the Saccharomyces cerevisiae CDC6 gene; Lisziewicz J et al.; The yeast cell division cycle gene CDC6 was isolated by complementation of a temperature-sensitive cdc6 mutant with a genomic library . The amino acid sequence of the 48 kDalton CDC6 gene product, as deduced from DNA sequence data, includes the three consensus peptide motifs involved in guanine nucleotide binding and GTPase activity, a target site for cAMP-dependent protein kinase and a carboxy-terminal domain related to metallothionein sequences . A plasmid-encoded CDC6-beta-galactosidase hybrid protein was located at the plasma membrane by indirect immunofluorescence . Disruption experiments indicate that the CDC6 gene product is essential for mitotic growth.

Biochim Biophys Acta, 1988 Dec 22, 946(2), 227 - 34
Lipid topology and physical properties of the outer mitochondrial membrane of the yeast, Saccharomyces cerevisiae; Sperka-Gottlieb CD et al.; The outer membrane of yeast mitochondria was studied with respect to its lipid composition, phospholipid topology and membrane fluidity . This membrane is characterized by a high phospholipid to protein ratio (1.20) . Like other yeast cellular membranes the outer mitochondrial membrane contains predominantly phosphatidylcholine (44% of total phospholipids), phosphatidylethanolamine (34%) and phosphatidylinositol (14%) . Cardiolipin, the characteristic phospholipid of the inner mitochondrial membrane (13% of total phospholipids) is present in the outer membrane only to a moderate extent (5%) . The ergosterol to phospholipid ratio is higher in the inner (7.0 wt%) as compared to the outer membrane (2.1 wt.%) . Attempts to study phospholipid asymmetry by selective degradation of phospholipids of the outer leaflet of the outer mitochondrial membrane failed, because isolated right-side-out vesicles of this membrane became leaky upon treatment with phospholipases . Selective removal of phospholipids of the outer leaflet with the aid of phospholipid transfer proteins and chemical modification with trinitrobenzenesulfonic acid on the other hand, gave satisfactory results . Phosphatidylcholine and phosphatidylinositol are more or less evenly distributed between the two sides of the outer mitochondrial membrane, whereas the majority of phosphatidylethanolamine is oriented towards the intermembrane space . The fluidity of mitochondrial membranes was determined by measuring fluorescence anisotropy using diphenylhexatriene (DPH) as a probe . The lower anisotropy of DPH in the outer as compared to the inner membrane, which is an indication for an increased lipid mobility in the outer membrane, was attributed to the higher phospholipid to protein and the lower ergosterol to phospholipid ratio . The data presented here show, that the outer mitochondrial membrane, in spite of its close contact to the inner membrane, is distinct not only with respect to its protein pattern, but also with respect to its lipid composition and physical membrane properties.

Biochim Biophys Acta, 1988 Dec 22, 946(2), 328 - 36
Chitin synthetase activity is bound to chitosomes and to the plasma membrane in protoplasts of Saccharomyces cerevisiae; Flores Martinez A et al.; The sub-cellular distribution of chitin synthetase was studied in homogenates of Saccharomyces cerevisiae protoplasts . Use of a mild disruption method minimized rupture of vacuoles and ensuing contamination of subcellular fractions by vacuolar proteinases . After fractionation of whole or partially purified homogenates through an isopycnic sucrose gradient chitin synthetase activity was found to be distributed between two distinct particulate fractions with different buoyant density and particle diameter . When whole homogenates were used, about 52% of the chitin synthetase loaded was localized in a microvesicular population identified as chitosomes (diameter 40-110 nm; buoyant density (d) = 1.146 g/cm3) . Another vesicular population containing 26% of the activity was identified as plasma membrane vesicles because of its large mean diameter (260 nm), its high buoyant density (d = 1.203 g/cm3) and by the presence of the vanadate-sensitive ATPase activity . Moreover, after surface labeling of protoplasts with 3H-concanavalin A, the label cosedimented with the presumed plasma membrane vesicles . There was a negligible cross-contamination of the chitosome fraction by yeast plasma membrane markers . In both the plasma membrane and the chitosome fractions, the chitin synthetase was stable and essentially zymogenic . Activation of the chitosome fraction produces microfibrils 100-250 nm in length . Our results support the idea that chitosomes do not originate by plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin synthetase in protoplasts of Saccharomyces cerevisiae.

EMBO J, 1988 Dec 20, 7(13), 4323 - 34
A novel nucleoskeletal-like protein located at the nuclear periphery is required for the life cycle of Saccharomyces cerevisiae; Hurt EC; In order to study the role of nucleoskeletal components for nuclear and cell division in the budding yeast Saccharomyces cerevisiae, we have employed a combined biochemical/genetic approach . We have identified a peripheral nuclear protein which appears to be located both at the nuclear membrane and the spindle pole body . The gene has been cloned and subsequently shown to be essential for cell growth . The DNA sequence of the gene has been determined . As deduced from the nucleotide sequence, the gene potentially codes for a novel 86 kd protein with a highly repetitive and conserved nine amino acid sequence motive in the middle part of the protein . The flanking amino- and carboxy-terminal regions have similarities to intermediate filaments and calcium binding proteins, respectively . It appears that the 86 kd protein is a regulated nucleoskeletal-like protein (NSP1) involved in the process of nuclear and/or cell division . The affinity-purified antibody against the yeast NSP1 protein stained the nucleus and centrosomes of mammalian MDCK (Madin Darby canine kidney) cells in indirect immunofluorescence.

Biochim Biophys Acta, 1988 Dec 20, 951(2-3), 335 - 43
Characterisation of the CDC7 gene product of Saccharomyces cerevisiae as a protein kinase needed for the initiation of mitotic DNA synthesis; Bahman M et al.; The product of the CDC7 gene of Saccharomyces cerevisiae, which is needed for the initiation of mitotic DNA synthesis, has homology with known and putative protein kinases . This homology is confined to the kinase catalytic domain, which has a unique organisation in CDC7 . To demonstrate that, nonetheless, CDC7 protein has kinase activity, the gene was subcloned under the control of the SP6 promoter . Protein synthesised by transcription and translation in vitro was capable of transferring 32P from {gamma-32P}ATP to histone . This activity was not dependent on Ca2+ or cyclic nucleotides . A mutation of CDC7 constructed in vitro, in which the organisation of the kinase catalytic domain was converted to that found in all other similar enzymes, was unable to function in vivo, as judged by its inability to complement the cdc7-1 allele . This suggests that the abnormal structure of the CDC7 catalytic domain is a key element in the cellular function of this protein in initiating DNA synthesis.

Biochim Biophys Acta, 1988 Dec 20, 951(2-3), 322 - 9
Three ribonucleases H and a reverse transcriptase from the yeast, Saccharomyces cerevisiae; Wintersberger U et al.; From the yeast, Saccharomyces cerevisiae, three proteins exhibiting ribonuclease H activity were isolated . These proteins differ in molecular weights and enzymatic properties . The two smaller ones, RNAase H(55) and RNAase H(42) are immunologically and structurally related to each other . Neither reacts with antibodies against the largest one, RNAase H(70) . Highly purified preparations of RNAase H(70) contain two polypeptides (Mr 70,000 and 160,000) and display reverse transcriptase activity . Deletion of part of the gene for the 160 kDa polypeptide results in mutants possessing about twice the amount of DNA as do wild-type cells . DNA polymerase stimulating activity resides in the 70,000 polypeptide . The processivity of yeast DNA polymerase A(I) does not change in presence of that protein . Possible functions of RNAases H are discussed.

Gene, 1988 Dec 15, 73(1), 153 - 61
The isolation, characterization and nucleotide sequence of the phosphoglucoisomerase gene of Saccharomyces cerevisiae; Tekamp-Olson P et al.; We have isolated the gene which encodes the glycolytic enzyme phosphoglucoisomerase (PGI) from the yeast Saccharomyces cerevisiae by functional complementation of a yeast mutant deficient in PGI activity with DNA from a wild-type yeast genomic library . The cloned gene has been localized by hybridization of specific DNA fragments to total yeast poly(A)+ RNA and by complementation of the mutant phenotype with subclones . The gene is expressed as an abundant mRNA of 1.9-kb and encodes a protein of 554 amino acids with an Mr of 61310 . The nucleotide sequence of the gene as well as the 5' and 3' flanking regions are presented . The predicted PGI amino acid sequence shows a high degree of homology with the sequence predicted for human and mouse neuroleukin, a putative neurotropic factor . The codon usage within the coding region is very restricted, characteristic of a highly expressed yeast gene.

Gene, 1988 Dec 15, 73(1), 113 - 20
Competitive expression of two heterologous genes inserted into one plasmid in Saccharomyces cerevisiae; Egel-Mitani M et al.; Plasmids were constructed which contained two expression units encoding single-chain insulin precursors . Surprisingly, the total amount of insulin precursor produced was similar to that produced from plasmids containing a single expression unit . In this system, therefore, two expression cassettes can be brought to compete for the limited ability of the yeast cell for synthesis and secretion . Using genes encoding B(1-29)-A(1-21) and B(1-29)-Ala-Ala-Lys-A-(1-21), the slightly different precursors could be quantified individually after separation by high-performance liquid chromatography from the culture supernatant . The two-cassette system allowed a sensitive and well controlled comparison of parameters important for optimal expression of a heterologous gene in Saccharomyces cerevisiae . The system was used to compare two promoter constructions and also to evaluate the position of expression cassettes in the plasmid . Finally the codon usage in the gene to be expressed was found to influence its ability to compete for expression.

J Biol Chem, 1988 Dec 15, 263(35), 18897 - 903
Purification and characterization of phosphatidylinositol kinase from Saccharomyces cerevisiae; Belunis CJ et al.; The membrane-associated phospholipid biosynthetic enzyme phosphatidylinositol kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified 8,000-fold from Saccharomyces cerevisiae . The purification procedure included Triton X-100 solubilization of microsomal membranes, DE-52 chromatography, hydroxylapatite chromatography, octyl-Sepharose chromatography, and two consecutive Mono Q chromatographies . The procedure resulted in the isolation of a protein with a subunit molecular weight of 35,000 that was 96% of homogeneity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Phosphatidylinositol kinase activity was associated with the purified Mr 35,000 subunit . Maximum phosphatidylinositol kinase activity was dependent on magnesium ions and Triton X-100 at pH 8 . The true Km values for phosphatidylinositol and MgATP were 70 microM and 0.3 mM, and the true Vmax was 4,750 nmol/min/mg . The turnover number for the enzyme was 166 min-1 . Results of kinetic and isotopic exchange reactions indicated that phosphatidylinositol kinase catalyzed a sequential Bi Bi reaction mechanism . The enzyme bound to phosphatidylinositol prior to ATP and phosphatidylinositol 4-phosphate was the first product released in the reaction . The equilibrium constant for the reaction indicated that the reverse reaction was favored in vitro . The activation energy for the reaction was 31.5 kcal/mol, and the enzyme was thermally labile above 30 degrees C . Phosphatidylinositol kinase activity was inhibited by calcium ions and thioreactive agents . Various nucleotides including adenosine and S-adenosylhomocysteine did not affect phosphatidylinositol kinase activity.

Gene, 1988 Dec 10, 72(1-2), 151 - 60
Messenger RNA degradation in Saccharomyces cerevisiae; Brown AJ et al.; The analysis of 17 functional mRNAs and two recombinant mRNAs in the yeast Saccharomyces cerevisiae suggests that the length of an mRNA influences its half-life in this organism . The mRNAs are clearly divisible into two populations when their lengths and half-lives are compared . Differences in ribosome loading amongst the mRNAs cannot account for this division into relatively stable and unstable populations . Also, specific mRNAs seem to be destabilized to differing extents when their translation is disrupted by N-terminus-proximal stop codons . The analysis of a mutant mRNA, generated by the fusion of the yeast PYK1 and URA3 genes, suggests that a destabilizing element exists within the URA3 sequence . The presence of such elements within relatively unstable mRNAs might account for the division between the yeast mRNA populations . On the basis of these, and other previously published observations, a model is proposed for a general pathway of mRNA degradation in yeast . This model may be relevant to other eukaryotic systems . Also, only a minor extension to the model is required to explain how the stability of some eukaryotic mRNAs might be regulated.

J Mol Biol, 1988 Dec 5, 204(3), 593 - 606
Saccharomyces cerevisiae protein involved in plasmid maintenance is necessary for mating of MAT alpha cells; Passmore S et al.; We previously reported the isolation of yeast mutants that seem to affect the function of certain autonomously replicating sequences (ARSs) . These mutants are known as mcm for their defect in the maintenance of minichromosomes . We have now characterized in more detail one ARS-specific mutation, mcm1-1 . This Mcm1 mutant has a second phenotype; MAT alpha mcm1-1 strains are sterile . MCM1 is non-allelic to other known alpha-specific sterile mutations and, unlike most genes required for mating, it is essential for growth . The alpha-specific sterile phenotype of the mcm1-1 mutant is manifested by its failure to produce a normal amount of the mating pheromone, alpha-factor . In addition, transcripts of the MF alpha 1 and STE3 genes, which encode the alpha-factor precursor and the alpha-factor receptor, respectively, are greatly reduced in this mutant . These and other properties of the mcm1-1 mutant suggest that the MCM1 protein may act as a transcriptional activator of alpha-specific genes . We have cloned, mapped and sequenced the wild-type and mutant alleles of MCM1, which is located on the right arm of chromosome XIII near LYS7 . The MCM1 gene product is a protein of 286 amino acid residues and contains an unusual region in which 19 out of 20 residues are either aspartic or glutamic acid, followed by a series of glutamine tracts . MCM1 has striking homology to ARG80, a regulatory gene of the arginine metabolic pathway located about 700 base-pairs upstream from MCM1 . A substitution of leucine for proline at amino acid position 97, immediately preceding the polyanionic region, was shown to be responsible for both the alpha-specific sterile and minichromosome-maintenance defective phenotypes of the mcm1-1 mutant.

J Mol Biol, 1988 Dec 5, 204(3), 561 - 8
Role of neighbouring bases and assessment of strand specificity in ethylmethanesulphonate and N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis in the SUP4-o gene of Saccharomyces cerevisiae; Kohalmi SE et al.; A total of 318 forward mutations induced by ethylmethanesulphonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in the SUP4-o gene of the yeast Saccharomyces cerevisiae was characterized by DNA sequence analysis . Only base-pair substitutions were detected among the mutations examined and, for both agents, the majority (greater than 96%) were G.C to A.T . transitions . The remaining changes included A.T to G.C transitions and transversions at G.C sites . For EMS, two of the transversions were accompanied by nearby G.C to A.T transitions . There was considerable overlap of the sites within the SUP4-o gene that were mutated by EMS and MNNG and of the sites that each agent failed to mutate . However, EMS and MNNG mutagenesis differed with respect to the frequencies at which mutations were recovered at G.C pairs where the guanine is flanked (5') by a purine or pyrimidine . EMS exhibited no preference for either type of site, whereas a G.C site was 12-fold or fivefold more likely to be mutated by MNNG if preceded by a 5' adenine or guanine, respectively, than if flanked by a 5' pyrimidine . Finally, neither EMS nor MNNG mutagenesis showed a preference for G.C sites having the guanine on the non-transcribed strand.

J Biol Chem, 1988 Dec 5, 263(34), 18236 - 40
Structure of Saccharomyces cerevisiae mating hormone a-factor . Identification of S-farnesyl cysteine as a structural component; Anderegg RJ et al.; Mating type a cells of the yeast Saccharomyces cerevisiae produce a mating hormone, the a-factor, that we have previously characterized as a very hydrophobic, modified dodecapeptide (Betz, R., Crabb, J . W., Meyer, H . E., Wittig, R., and Duntze, W . (1987) J . Biol . Chem . 262, 546-548) . We have investigated the molecular structure in detail using mass spectrometry and proton NMR spectrometry of the intact hormone and authentic component molecules . Tandem mass spectrometry confirms the previously determined peptide sequence of the hormone and shows that it contains additional structural components with masses of 205 and 15 daltons . These were identified by proton NMR and mass spectrometry as a farnesyl (C15H25) residue and a terminal methyl ester group . The farnesyl moiety is attached to the sulfur atom of the carboxyl-terminal cysteine residue, as revealed by NMR of synthetic S-farnesyl cysteine methyl ester . The stereochemical configuration of the farnesyl moiety was determined to be trans,trans by comparison of gas chromatography retention times, mass spectra, and NMR spectra with those of standards . These results define the structure of a-factor as: (Sequence: see text) . Replacement of the farnesyl by a methyl group leads to a partial reduction in specific biological activity of the a-factor, whereas hydrolysis of the carboxyl-terminal methyl ester causes a complete loss of activity.

FEBS Lett, 1988 Dec 5, 241(1-2), 55 - 9
Irreversible inactivation of Saccharomyces cerevisiae fructose-1,6-bisphosphatase independent of protein phosphorylation at Ser11; Rose M et al.; The fructose-1,6-bisphosphatase gene was used with multicopy plasmids to study rapid reversible and irreversible inactivation after addition of glucose to derepressed Saccharomyces cerevisiae cells . Both inactivation systems could inactivate the enzyme, even if 20-fold over-expressed . The putative serine residue, at which fructose-1,6-bisphosphatase is phosphorylated, was changed to an alanine residue without notably affecting the catalytic activity . No rapid reversible inactivation was observed with the mutated enzyme . Nonetheless, the modified enzyme was still irreversibly inactivated, clearly demonstrating that phosphorylation is an independent regulatory circuit that reduces fructose-1,6-bisphosphatase activity within seconds . Furthermore, irreversible glucose inactivation was not triggered by phosphorylation of the enzyme.

Mol Cell Biol, 1988 Dec, 8(12), 5410 - 6
Role of SGP2, a suppressor of a gpa1 mutation, in the mating-factor signaling pathway of Saccharomyces cerevisiae; Nakayama N et al.; Loss of function of GPA1, which encodes a guanine-nucleotide-binding protein, arrests the cell at the G1 phase and allows it to mate, suggesting that the gpa1 mutation spontaneously exerts an intracellular signal that mimics the action of mating factor . We have cloned the SGP2 gene, which was first identified as a secondary mutation that allowed a gpa1::HIS3 mutant to grow and to show a non-cell-type-specific sterile phenotype . Disruption of SGP2 confers temperature-sensitive growth and a-specific sterile phenotypes, characteristics similar to those conferred by the dpr1 (ram) mutation, a suppressor of RAS2Val-19 . The following observations indicate that SGP2 and DPR1 are in fact identical . (i) The cloned SGP2 complements both the temperature-sensitive growth and the a-specific sterility of the dpr1 mutant and can be integrated into the chromosomal DPR1 locus . (ii) The cloned DPR1, in turn, complements the ability of sgp2 to suppress the lethality of gpa1::HIS3 . (iii) The dpr1 mutation suppresses the growth defect of gpa1::HIS3, and the dpr1 gpa1::HIS3 strain shows a non-cell-type-specific sterile phenotype . (iv) sgp2 is closely linked to the dpr1 locus . The DPR1 product has been shown to be responsible for processing and fatty acid acylation of a-factor and RAS proteins at their carboxyl termini . Therefore, the SGP2 (DPR1) product may be involved in membrane localization of an essential component in the mating-factor signaling pathway.

Genetics, 1988 Dec, 120(4), 899 - 907
Identification and characterization of mutations affecting sporulation in Saccharomyces cerevisiae; Smith LM et al.; Mutations affecting the synthesis of the sporulation amyloglucosidase were isolated in a homothallic strain of Saccharomyces cerevisiae, SCMS7-1 . Two were found, both of which were deficient in sporulation at 34 degrees . One, SL484, sporulated to 50% normal levels at 30 degrees but less than 5% at 34 degrees or 22 degrees . The other, SL641, failed to sporulate at any temperature . Both mutants were blocked before premeiotic DNA synthesis, and both complemented spo1, spo3, and spo7 . Genetic analysis of the mutation in SL484 indicated linkage to TRP5 and placed the gene 10 map units from TRP5 on chromosome VII . A plasmid containing an insert which complements the mutation in SL484 fails to complement SL641 . We therefore conclude that these two mutations are in separate genes and we propose to call these genes SPO17 and SPO18 . These two genes are (with SPO7, SPO8, and SPO9) among the earliest identified in the sporulation pathway and may interact directly with the positive and negative regulators RME and IME.

Biochem Int, 1988 Dec, 17(6), 1099 - 106
Inactivation of Saccharomyces cerevisiae glucose-6-phosphate dehydrogenase by diethylpyrocarbonate; Kim YS et al.; Glucose-6-phosphate dehydrogenase purified from Saccharomyces cerevisiae is rapidly inactivated by diethylpyrocarbonate at pH 6.8 and 30 degrees C with a concomitant increase in absorbance at 242 nm . The second-order rate constant for inactivation was calculated to be 487.8 M-1 min-1 . The pH dependence of inactivation suggests the involvement of an amino acid residue having a pKa of 6.77 . These results indicate that the inactivation is due to the modification of a histidine residue(s) . In the presence of substrate, glucose-6-phosphate or NADP+, the rate of inactivation is decreased, indicating that the essential histidine residue(s) is located at the active site, possibly at the region of overlap of substrates at the binding site.

Mol Cell Biol, 1988 Dec, 8(12), 5166 - 78
Evidence for cooperation between cells during sporulation of the yeast Saccharomyces cerevisiae; Jakubowski H et al.; Diploid Saccharomyces cerevisiae cells heterozygous for the mating type locus (MATa/MAT alpha) undergo meiosis and sporulation when starved for nitrogen in the presence of a poor carbon source such as potassium acetate . Diploid yeast adenine auxotrophs sporulated well at high cell density (10(7) cells per ml) under these conditions but failed to differentiate at low cell density (10(5) cells per ml) . The conditional sporulation-deficient phenotype of adenine auxotrophs could be complemented by wild-type yeast cells, by medium from cultures that sporulate at high cell density, or by exogenously added adenine (or hypoxanthine with some mutants) . Adenine and hypoxanthine in addition to guanine, adenosine, and numerous nucleotides were secreted into the medium, each in its unique temporal pattern, by sporulating auxotrophic and prototrophic yeast strains . The major source of these compounds was degradation of RNA . The data indicated that differentiating yeast cells cooperate during sporulation in maintaining sufficiently high concentrations of extracellular purines which are absolutely required for sporulation of adenine auxotrophs . Yeast prototrophs, which also sporulated less efficiently at low cell density (10(3) cells per ml), reutilized secreted purines in preference to de novo-made purine nucleotides whose synthesis was in fact inhibited during sporulation at high cell density . Adenine enhanced sporulation of yeast prototrophs at low cell density . The behavior of adenine auxotrophs bearing additional mutations in purine salvage pathway genes (ade apt1, ade aah1 apt1, ade hpt1) supports a model in which secretion of degradation products, uptake, and reutilization of these products is a signal between cells synchronizing the sporulation process.

J Biochem (Tokyo), 1988 Dec, 104(6), 908 - 11
On the size and the role of a free cytosolic pool of acidic ribosomal proteins in yeast Saccharomyces cerevisiae; Mitsui K et al.; A small but distinct amount of yeast acidic ribosomal proteins A1/A2 was detected in cytosol by immunoblotting on a two-dimensional gel electrophoretogram, while 38 kDa acidic protein A0 was not detected . The free forms of A1/A2 in the cytosol were eluted in gel filtration at the molecular mass of about 30 kDa under non-denaturation conditions, suggesting that they exist as a dimer or timer without association with A0 . The amount of free A1/A2 was determined by immunoblotting to be 0.3% of the ribosome-bound A1/A2 in yeast . The time course of incorporation of radioactive amino acid showed that the cytosolic free A1/A2 are labeled more rapidly with high specific radioactivity than the ribosome-bound A1/A2 . This result suggested that some of the cytosolic A1/A2, if not all, are newly-synthesized proteins which are ready for incorporation into cytoplasmic ribosomes.

Genetics, 1988 Dec, 120(4), 923 - 34
Mutations in elongation factor EF-1 alpha affect the frequency of frameshifting and amino acid misincorporation in Saccharomyces cerevisiae; Sandbaken MG et al.; A mutational analysis of the eukaryotic elongation factor EF-1 alpha indicates that this protein functions to limit the frequency of errors during genetic code translation . We found that both amino acid misincorporation and reading frame errors are controlled by EF-1 alpha . In order to examine the function of this protein, the TEF2 gene, which encodes EF-1 alpha in Saccharomyces cerevisiae, was mutagenized in vitro with hydroxylamine . Sixteen independent TEF2 alleles were isolated by their ability to suppress frameshift mutations . DNA sequence analysis identified eight different sites in the EF-1 alpha protein that elevate the frequency of mistranslation when mutated . These sites are located in two different regions of the protein . Amino acid substitutions located in or near the GTP-binding and hydrolysis domain of the protein cause suppression of frameshift and nonsense mutations . These mutations may effect mistranslation by altering the binding or hydrolysis of GTP . Amino acid substitutions located adjacent to a putative aminoacyl-tRNA binding region also suppress frameshift and nonsense mutations . These mutations may alter the binding of aminoacyl-tRNA by EF-1 alpha . The identification of frameshift and nonsense suppressor mutations in EF-1 alpha indicates a role for this protein in limiting amino acid misincorporation and reading frame errors . We suggest that these types of errors are controlled by a common mechanism or closely related mechanisms.

Yeast, 1988 Dec, 4(4), 257 - 69
Genetic control of chromosome stability in the yeast Saccharomyces cerevisiae; Kouprina NYu et al.; We have identified four new genetic loci: CHL2 (on chromosome XII), CHL3 (on chromosome XII); CHL4 (on chromosome IV), and CHL5 (on chromosome IX), controlling mitotic transmission of yeast chromosomes . The frequency of loss of chromosomes is 10-100-fold higher in chl5, chl2, chl3 and chl4 mutants than observed in wild-type strains . The mutants also show unstable maintenance of artificial circular minichromosomes with various chromosomal replicators (ARS) and one of the centromeric loci (CEN3, CEN4, CEN5 or CEN6) . The instability of minichromosomes in the chl5, chl2, and chl4 mutants is due to the loss of minichromosomes in mitosis (1:0 segregation) . In the chl3 mutant the instability of artificial minichromosomes is due to nondisjunction (2:0 segregation) . The CHL3 gene therefore appears to affect the segregation of chromosomes during cell division.

J Bacteriol, 1988 Dec, 170(12), 5877 - 82
Role of cell wall in Saccharomyces cerevisiae mutants resistant to Hg2+; Ono B et al.; Hg2+-resistant mutants were isolated from Saccharomyces cerevisiae . Although they were very much like the parental strains in terms of colony-forming ability, they grew faster than the parental strains in the presence of sublethal doses of Hg2+ . The Hg2+-resistant mutations were dominant . They were centromere linked and were divided into two groups by means of recombination; one of the mutations, designated HGR1-1, was mapped on chromosome IV because of its linkage to the TRP1 locus . The Hg2+-resistant mutants took up Hg2+ as much as, or slightly more than, the parental strains did . The mutants and parental strains retained only about 5 and 15%, respectively, of the cell-associated Hg2+ after removal of the cell wall; therefore, the mutants had less spheroplast-associated Hg2+ than did the parental strains . These results indicate that the cell wall plays an important role in protection against Hg2+ by acting as an adsorption filter and that the mutations described confer Hg2+ resistance by increasing the Hg2+-binding capacity of the cell wall.

Mol Cell Biol, 1988 Dec, 8(12), 5555 - 60
Multistress resistance of Saccharomyces cerevisiae is generated by insertion of retrotransposon Ty into the 5' coding region of the adenylate cyclase gene; Iida H; Heat shock-resistant mutants, which were isolated by their ability to withstand lethal heat treatment, were characterized . Resistance was demonstrated to be a consequence of insertion of retrotransposon Ty into either the 5' coding or noncoding region, close to the putative initiation codon of the adenylate cyclase gene CYR1 (or CDC35) . These heat shock-resistant mutants contained about threefold lower adenylate cyclase activity than wild-type strains . The mutants were also observed to be resistant to other stresses such as UV light and ethanol . These results demonstrate that multistress resistance, which may confer a survival advantage to yeast cells, can be generated by transposition of a Ty element into CYR1.

Yeast, 1988 Dec, 4(4), 241 - 7
Membrane transport in an osmotically fragile mutant of Saccharomyces cerevisiae; Kotyk A et al.; Transport properties of the osmotically fragile strain VY1160 of Saccharomyces cerevisiae were compared with those of the parent S288c strain . Mediated diffusion of 6-deoxy-D-glucose was practically unaffected; membrane-potential-dependent transport of D-glucosamine was very much depressed in the fragile strain . The H+-driven transport of L-lysine and L-proline, as well as that of the hitherto uninvestigated D-glucose-6-phosphate, were also very depressed . 2-Deoxy-D-glucose transport displayed slightly different kinetic parameters . Primary H+ extrusion by the plasma membrane H-ATPase was not diminished although the ATP-splitting activity was depressed by about 50% . The overall proton-motive force (pmf) of the fragile mutant at pH 5.5 was only 20 mV while in the parent strain it was 108 mV . In parallel with this, spontaneous acidification of the external medium (a CO2-associated event) was only about 2% of that in the parent strain . The defect in this, together with the inability to stimulate transport protein synthesis by glucose, may account for the generally poorer transport performance of the fragile mutant.

J Biol Chem, 1988 Nov 25, 263(33), 17663 - 71
Circular dichroism and fluorescence studies on protein synthesis initiation factor eIF-4E and two mutant forms from the yeast Saccharomyces cerevisiae; McCubbin WD et al.; Circular dichroism studies have shown that eukaryotic initiation factor 4E contains low amounts of alpha-helix; the main elements of secondary structure are beta-sheets/turns and aperiodic regions . Interactions with cap analogs are accompanied by small but reproducible changes in overall secondary structure, which may also involve more significant perturbations of localized regions containing certain phenylalanine residues . Dissociation constants for interactions with nucleotides have been established from fluorescence titrations . Results show that the (N-7) methylated guanosine nucleotides bound more strongly than their nonmethylated counterparts . Involvement of a key tryptophan residue in the cap binding site was suggested . Additional studies with two cap binding mutant forms of the protein, designated SK-4 (W----75----L) and SK-6 (W----115----L), confirmed and extended these observations . Fluorescence melting experiments indicated that binding of cap analogs stabilized the protein against thermal perturbation and demonstrated subtle differences in folding between the wild-type and mutant forms of the protein . These subtle differences in folding may account for the observed loss in cap specificity of both mutant forms.

Arch Biochem Biophys, 1988 Nov 15, 267(1), 38 - 45
Affinity labeling of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase with the 2',3'-dialdehyde derivative of ATP; Saavedra C et al.; Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase {ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49} is completely inactivated by the 2',3'-dialdehyde derivative of ATP (oATP) in the presence of Mn2+ . The dependence of the pseudo-first-order rate constant on reagent concentration indicates the formation of a reversible complex with the enzyme (Kd = 60 +/- 17 microM) prior to covalent modification . The maximum inactivation rate constant at pH 7.5 and 30 degrees C is 0.200 +/- 0.045 min-1 . ATP or ADP plus phosphoenolpyruvate effectively protect the enzyme against inactivation . oATP is a competitive inhibitor toward ADP, suggesting that oATP interacts with the enzyme at the substrate binding site . The partially inactivated enzyme shows an unaltered Km but a decreased V as compared with native phosphoenolpyruvate carboxykinase . Analysis of the inactivation rate at different H+ concentrations allowed estimation of a pKa of 8.1 for the reactive amino acid residue in the enzyme . Complete inactivation of the carboxykinase can be correlated with the incorporation of about one mole of {8-14C}oATP per mole of enzyme subunit . The results indicate that oATP can be used as an affinity label for yeast phosphoenolpyruvate carboxykinase.

Science, 1988 Nov 4, 242(4879), 759 - 61
The effect of histone gene deletions on chromatin structure in Saccharomyces cerevisiae; Norris D et al.; As a way of studying nucleosome assembly and maintenance in Saccharomyces cerevisiae, mutants bearing deletions or duplications of the genes encoding histones H2A and H2B were analyzed . Previous genetic analysis had shown that only one of these mutants exhibited dramatic and pleiotropic phenotypes . This mutant was also the only one that contained disrupted chromatin, suggesting that the original phenotypes were attributable to alterations in chromosome structure . The chromatin disruption in the mutant, however, did not extend over the entire genome, but rather was localized to specific regions . Thus, while the arrangement of nucleosomes over the HIS4 and GAL1 genes, the telomeres, and the long terminal repeats (delta sequences) of Ty retrotransposons appeared essentially normal, nucleosomes over the CYH2 and UBI4 genes and the centromere of chromosome III were dramatically disrupted . The observation that the mutant exhibited localized chromatin disruptions implies that the assembly or maintenance of nucleosomes differs over different parts of the yeast genome.

Curr Genet, 1988 Nov, 14(5), 405 - 12
Identification of an upstream activation site in the pyruvate decarboxylase structural gene (PDC1) of Saccharomyces cerevisiae; Butler G et al.; The upstream region of the Saccharomyces cerevisiae pyruvate decarboxylase structural gene, PDC1, has been isolated and fused to the indicator gene Escherichia coli lacZ . 1.2 kb of the upstream region has been sequenced . The PDC1-lacZ fusion has been integrated at the ura3-52 locus in the yeast genome, and has a basal level of expression on ethanol . On glucose media this level is increased 30-50 fold . An upstream activation site, UASpdc, between 793 and 535 bp upstream from the ATG of PDC1, which mediates the response to glucose has been identified by deletion analysis . The UASpdc contains a consensus RPG box, originally identified in ribosomal protein genes (Leer et al . 1985) . The function of UASpdc is independent of distance from the ATG . There is also an upstream repressing sequence located between 535 and 385 bp upstream from the translational start of PDC1.

J Gen Microbiol, 1988 Nov, 134 ( Pt 11), 3049 - 60
Osmotic adjustment and the accumulation of organic solutes in whole cells and protoplasts of Saccharomyces cerevisiae; Meikle AJ et al.; In the presence of a suitable carbon source, whole cells and protoplasts of Saccharomyces cerevisiae synthesized glycerol as a compatible organic solute in response to increased external osmotic pressure . Boyle-van't Hoff plots showed that protoplasts, and non-turgid cells, exhibited a linear relationship between volume and the external osmotic pressure (i.e . they behaved as near-ideal osmometers), and that both protoplasts and cells have a component which is not osmotically responsive--the non-osmotic volume (NOV) . Glycerol levels in whole cells and protoplasts were elevated by increased external osmotic pressure over a similar time-scale to the period of exponential cell growth, reaching a maximum value at 6-12 h and declining thereafter . This suggests that the restoration of turgor pressure in whole cells was not the sole regulator of glycerol accumulation . Stationary phase whole cells had negligible levels of intracellular glycerol after growth in a medium of raised osmotic pressure . However, intracellular trehalose synthesis in these cells began earlier and reached a higher maximum level than in basal medium . Once exponential growth had stopped, cell turgor and internal osmotic pressure decreased somewhat . These new, lower values may be determined by the extent of trehalose accumulation in stationary phase cells.

Biochemistry, 1988 Nov 1, 27(22), 8393 - 400
Gene sequence and primary structure of mitochondrial malate dehydrogenase from Saccharomyces cerevisiae; Thompson LM et al.; The nucleotide sequence was determined for a 1.5-kilobase genomic fragment containing the mitochondrial malate dehydrogenase gene (MDH1) of Saccharomyces cerevisiae . The open-reading frame encodes a precursor form of the mature enzyme containing an amino-terminal extension of 17 amino acid residues . In vitro translation experiments confirm that the initial translation product of MDH1 is larger than the mature polypeptide . Transcription of MDH1 initiates at several sites from 83 to 97 nucleotides 5' of the translational start site . Alignment of the amino acid sequence for the mature yeast enzyme with those for mammalian mitochondrial and for Escherichia coli malate dehydrogenases reveals polypeptides of very similar sizes with identical amino acids at 54% and 48% of the residue positions, respectively . The amino acid sequences of the yeast and mammalian mitochondrial targeting sequences are similar but less related than the mature polypeptides . The yeast MDH1 gene is shown to reside on chromosome XI.

Genetika, 1988 Nov, 24(11), 1935 - 9
{Effect of DNA methylation on transformation of Saccharomyces cerevisiae}; Lebenka AIu; To study the action of methylation on transformation efficiency, pA2 was methylated in vitro with M . Sau3A, M . AluI, M . MspI, M . HpaII, M . PvuII, M . BcnI, M . MvaI and transformed into Saccharomyces cerevisiae . The results suggest that efficiency of transformation depends on specificity of methylation, the nature of methylcytosine (m5C or m4C).

Genetics, 1988 Nov, 120(3), 681 - 95
Isolation and characterization of conditional-lethal mutations in the TUB1 alpha-tubulin gene of the yeast Saccharomyces cerevisiae; Schatz PJ et al.; Microtubules in yeast are functional components of the mitotic and meiotic spindles and are essential for nuclear movement during cell division and mating . We have isolated 70 conditional-lethal mutations in the TUB1 alpha-tubulin gene of the yeast Saccharomyces cerevisiae using a plasmid replacement technique . Of the 70 mutations isolated, 67 resulted in cold-sensitivity, one resulted in temperature-sensitivity, and two resulted in both . Fine-structure mapping revealed that the mutations were located throughout the TUB1 gene . We characterized the phenotypes caused by 38 of the mutations after shifts of mutants to the nonpermissive temperature . Populations of temperature-shifted mutant cells contained an excess of large-budded cells with undivided nuclei, consistent with the previously determined role of microtubules in yeast mitosis . Several of the mutants arrested growth with a sufficiently uniform morphology to indicate that TUB1 has at least one specific role in the progression of the yeast cell cycle . A number of the mutants had gross defects in microtubule assembly at the restrictive temperature, some with no microtubules and some with excess microtubules . Other mutants contained disorganized microtubules and nuclei . There were no obvious correlations between these phenotypes and the map positions of the mutations . Greater than 90% of the mutants examined were hypersensitive to the antimicrotubule drug benomyl . Mutations that suppressed the cold-sensitive phenotypes of two of the TUB1 alleles occurred in TUB2, the single structural gene specifying beta-tubulin.

Curr Genet, 1988 Nov, 14(5), 413 - 8
Inheritance of chromosome length polymorphisms in Saccharomyces cerevisiae; Ono B et al.; Although Saccharomyces cerevisiae strains generally have similar chromosomal band patterns as revealed by pulsed field gel electrophoresis, individual bands often move slightly differently from one strain to the other . Surveying strains from our stock collection, we found that nearly all the bands of a certain pair of strains differed in their mobility . Some of these chromosome length polymorphisms segregated in a 2:2 ratio, indicating that they resulted from single structural alterations (i.e . additions or deletions) . One of these was mapped on the right arm of chromosome I . Others did not segregate in a simple 2:2 ratio . That is, there were progenies which had bands not present in either parent . We suggest that these new bands are the products of recombination between homologous chromosomes having two or more structural alterations.

Mol Gen Genet, 1988 Nov, 214(3), 396 - 404
The base-alteration spectrum of spontaneous and ultraviolet radiation-induced forward mutations in the URA3 locus of Saccharomyces cerevisiae; Lee GS et al.; A forward mutation system has been developed to obtain rapidly clonable mutants at the URA3 locus in yeast by means of selection for 5-fluoroorotic acid resistance . We have used this system to determine base changes in 35 spontaneous and 34 ultraviolet radiation-induced ura3 base substitution mutants . Other mutants (frameshift, deletion, duplication, replacement) were detected as well . Evidence is reported which suggests cyclobutane dimers are the principal mutagenic lesions induced by UV radiation in stationary phase cells of the yeast Saccharomyces cerevisiae . Since most of the induced lesions are at 5'-TT-3' sites, the results suggest that the "A-rule", preferential insertion of adenine residues opposite poorly pairing sites in DNA, does not apply for yeast cells irradiated in stationary phase, whereas the spontaneous mutation data indicate that the A-rule applies for cells in logarithmic phase . Most of the spontaneous mutations are transversions . UV-induced transitions and transversions occur at approximately equal frequencies.

Mol Cell Biol, 1988 Nov, 8(11), 4991 - 9
GAL11 protein, an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae; Suzuki Y et al.; Normal function of the GAL11 gene is required for maximum production of the enzymes encoded by GAL1, GAL7, and GAL10 (collectively termed GAL1,7,10) in Saccharomyces cerevisiae . Strains bearing a gal11 mutation synthesize these enzymes at 10 to 30% of the wild-type level in the induced state . In a DNA-RNA hybridization experiment, the gal11 effect was shown to be exerted at the transcription level . Yeast cells bearing the gal11 mutation were shown to grow on glycerol plus lactate more slowly than the wild type . We isolated recombinant plasmids carrying the GAL11 gene by complementation of the gal11 mutation . When the GAL11 locus was disrupted by insertion of the URA3 gene, the resulting yeast cells (gal11::URA3) exhibited phenotypes almost identical to those of the gal11 strains, with respect to both galactose utilization and growth on nonfermentable carbon sources . Deficiency of Gal4, the major transcription activator for GAL1,7,10, was epistatic over the gal11 defect . The Gal11 deficiency lowered the expression of GAL2 but not that of MEL1 or GAL80; expression of these genes is also known to be dependent on GAL4 function . We determined the nucleotide sequence of GAL11, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine) . An alpha-helix-beta-turn-alpha-helix structure was found in a distal part of the predicted amino acid sequence . A possible role of the GAL11 product in the regulation of galactose-inducible genes is discussed.

Mol Cell Biol, 1988 Nov, 8(11), 4949 - 57
Copy number and partition of the Saccharomyces cerevisiae 2 micron plasmid controlled by transcription regulators; Veit BE et al.; The 2 micron plasmid of Saccharomyces cerevisiae is maintained by the action of plasmid-encoded gene products that control copy number and promote equipartition of plasmid copies at cell division . We show that the REP1 and REP2 plasmid-encoded gene products are master regulators that act in concert to autoregulate the level of their own transcripts and to regulate transcript levels of the FLP gene that promotes plasmid copy amplification . REP1 and REP2 are also shown to repress transcription at REP3, the cis-acting site essential for plasmid equipartitioning . We propose a model in which REP3 acts by dislodging transcription apparatuses that otherwise cause plasmid molecules to adhere to the mother nucleus and segregate asymmetrically . On the basis of their ability to generate specific chromatin structures, we also propose that the REP1 and REP2 gene products interact with different specific sequences found iterated in the 2 micron plasmid.

Mol Cell Biol, 1988 Nov, 8(11), 4936 - 48
Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases; Robinson JS et al.; Using a selection for spontaneous mutants that mislocalize a vacuolar carboxypeptidase Y (CPY)-invertase fusion protein to the cell surface, we identified vacuolar protein targeting (vpt) mutants in 25 new vpt complementation groups . Additional alleles in each of the eight previously identified vpt complementation groups (vpt1 through vpt8) were also obtained . Representative alleles from each of the 33 vpt complementation groups (vpt1 through vpt33) were shown to exhibit defects in the sorting and processing of several native vacuolar proteins, including the soluble hydrolases CPY, proteinase A, and proteinase B . Of the 33 complementation groups, 19 were found to contain mutant alleles that led to extreme defects . In these mutants, CPY accumulated in its Golgi complex-modified precursor form which was secreted by the mutant cells . Normal protein secretion appeared to be unaffected in the vpt mutants . The lack of significant leakage of cytosolic markers from the vpt mutant cells indicated that the vacuolar protein-sorting defects associated with these mutants do not result from cell lysis . In addition, the observation that the precursor rather than the mature forms of CPY, proteinase A, proteinase B were secreted from the vpt mutants was consistent with the fact that mislocalization occurred at a stage after Golgi complex-specific modification, but before final vacuolar sorting of these enzymes . Vacuolar membrane protein sorting appeared to be unaffected in the majority of the vpt mutants . However, a subset of the vpt mutants (vpt11, vpt16, vpt18, and vpt33) was found to exhibit defects in the sorting of a vacuolar membrane marker enzyme, alpha-mannosidase . Up to 50% of the alpha-mannosidase enzyme activity was found to be mislocalized to the cell surface in these vpt mutants . Seven of the vpt complementation groups (vpt3, vpt11, vpt15, vpt16, vpt18, vpt29, and vpt33) contained alleles that led to a conditional lethal phenotype; the mutants were temperature sensitive for vegetative cell growth . This temperature-sensitive phenotype has been shown to be recessive and to cosegregate with the vacuolar protein-sorting defect in each case . Tetrad analysis showed that vpt3 mapped to the right arm of chromosome XV and that vpt15 mapped to the right arm of chromosome II . Intercrosses with other mutants that exhibited defects in vacuolar protein sorting or function (vpl, sec, pep, and end mutants) revealed several overlaps among these different sets of genes . Together, these data indicate that more than 50 gene products are involved, directly or indirectly, in the process of vacuolar protein sorting.

Mol Cell Biol, 1988 Nov, 8(11), 4927 - 35
Organization of replication of ribosomal DNA in Saccharomyces cerevisiae; Linskens MH et al.; Using recently developed replicon mapping techniques, we have analyzed the replication of the ribosomal DNA in Saccharomyces cerevisiae . The results show that (i) the functional origin of replication colocalizes with an autonomously replicating sequence element previously mapped to the nontranscribed spacer region, (ii) only a fraction of the potential origins are utilized in a single S phase, and (iii) the replication forks moving counter to the direction of transcription of the 37S precursor RNA stop at or near the termination site of transcription . Consequently, most ribosomal DNA is replicated unidirectionally by forks moving in the direction of transcription and most replicons are larger than the repeat unit . The significance of this finding for the replication of abundantly transcribed genes is discussed.

Mol Cell Biol, 1988 Nov, 8(11), 4792 - 8
Gene dosage alteration of L2 ribosomal protein genes in Saccharomyces cerevisiae: effects on ribosome synthesis; Lucioli A et al.; In Saccharomyces cerevisiae, the genes coding for the ribosomal protein L2 are present in two copies per haploid genome . The two copies, which encode proteins differing in only a few amino acids, contribute unequally to the L2 mRNA pool: the L2A copy makes 72% of the mRNA, while the L2B copy makes only 28% . Disruption of the L2B gene (delta B strain) did not lead to any phenotypic alteration, whereas the inactivation of the L2A copy (delta A strain) produced a slow-growth phenotype associated with decreased accumulation of 60S subunits and ribosomes . No intergenic compensation occurred at the transcriptional level in the disrupted strains; in fact, delta A strains contained reduced levels of L2 mRNA, whereas delta B strains had almost normal levels . The wild-type phenotype was restored in the delta A strains by transformation with extra copies of the intact L2A or L2B gene . As already shown for other duplicated genes (Kim and Warner, J . Mol . Biol . 165:79-89, 1983; Leeret al., Curr . Genet . 9:273-277, 1985), the difference in expression of the two gene copies could be accounted for via differential transcription activity . Sequence comparison of the rpL2 promoter regions has shown the presence of canonical HOMOL1 boxes which are slightly different in the two genes.

Mol Cell Biol, 1988 Nov, 8(11), 4675 - 84
DAF1, a mutant gene affecting size control, pheromone arrest, and cell cycle kinetics of Saccharomyces cerevisiae; Cross FR; The mating pheromone alpha-factor arrests Saccharomyces cerevisiae MATa cells in the G1 phase of the cell cycle . Size control is also exerted in G1, since cells do not exit G1 until they have attained a critical size . A dominant mutation (DAF1-1) which causes both alpha-factor resistance and small cell size (volume about 0.6-fold that of the wild type) has been isolated and characterized genetically and by molecular cloning . Several alpha-factor-induced mRNAs were induced equivalently in daf1+ and DAF1-1 cells . The DAF1-1 mutation consisted of a termination codon two-thirds of the way through the daf1+ coding sequence . A chromosomal deletion of DAF1 produced by gene transplacement increased cell volume about 1.5-fold; thus, DAF1-1 may be a hyperactive or deregulated allele of a nonessential gene involved in G1 size control . Multiple copies of DAF1-1 also greatly reduced the duration of the G1 phase of the cell cycle.

Mol Cell Biol, 1988 Nov, 8(11), 4651 - 8
ROX1 encodes a heme-induced repression factor regulating ANB1 and CYC7 of Saccharomyces cerevisiae; Lowry CV et al.; The ROX1 gene encodes a product implicated in the regulation of heme-repressed and heme-induced genes in Saccharomyces cerevisiae . The gene has been cloned and shown to code for a 1.4-kilobase transcript . The cloned gene was used to construct a null mutant to determine the role of ROX1 in regulating the expression of several heme-regulated genes . Constitutive expression of ANB1 (a heme-repressed gene) was observed in the null strain, indicating that ROX1 codes for a repressor or a facilitator of repression . Enhancement of expression of CYC7 in the null strain indicated that the ROX1 factor is required for repression of CYC7 to its normal low level of expression, consistent with evidence that CYC7 has a hybrid heme-induced, heme-repressed regulatory mechanism . The null mutation had only a slight negative effect on expression of the heme-induced genes CYC1 and tr-1 (a heme-induced homolog of ANB1), suggesting that the ROX1 factor is not directly involved in their regulation despite the existence of an unusual rox1 mutation (rox1-a1) causing constitutive expression of this group . The respiratory competence of the null mutant indicates that ROX1 is not a respiratory factor . ROX1 expression was found to be induced by heme, indicating that the heme repression of ANB1 and its family is the result of a cascade in which heme induces a repression factor which keeps the family of heme-repressed genes inactive during aerobic growth . The rox1-a1 allele had earlier been shown to cause constitutive expression of the family of heme-induced respiratory genes . This allele was found to cause constitutive expression of the ROX1 transcript itself, indicating that ROX1 is in the major heme-induced regulon.

Mol Cell Biol, 1988 Nov, 8(11), 4642 - 50
Characterization of two telomeric DNA processing reactions in Saccharomyces cerevisiae; Murray AW et al.; We have investigated two reactions that occur on telomeric sequences introduced into Saccharomyces cerevisiae cells by transformation . The elongation reaction added repeats of the yeast telomeric sequence C1-3A to telomeric sequences at the end of linear DNA molecules . The reaction worked on the Tetrahymena telomeric sequence C4A2 and also on the simple repeat CA . The reaction was orientation specific: it occurred only when the GT-rich strand ran 5' to 3' towards the end of the molecule . Telomere elongation occurred by non-template-directed DNA synthesis rather than any type of recombination with chromosomal telomeres, because C1-3A repeats could be added to unrelated DNA sequences between the CA-rich repeats and the terminus of the transforming DNA . The elongation reaction was very efficient, and we believe that it was responsible for maintaining an average telomere length despite incomplete replication by template-directed DNA polymerase . The resolution reaction processed a head-to-head inverted repeat of telomeric sequences into two new telomeres at a frequency of 10(-2) per cell division.

Mol Cell Biol, 1988 Nov, 8(11), 4616 - 24
Transcriptional regulation of the cell cycle-dependent thymidylate synthase gene of Saccharomyces cerevisiae; McIntosh EM et al.; We have previously shown that transcript levels expressed from the yeast TMP1 gene fluctuate periodically during the yeast cell cycle . However, it was not known whether periodic expression resulted from a regulatory mechanism acting at the level of transcription or a regulatory mechanism acting at the level of cell cycle stage-dependent changes in the stability of the TMP1 transcript . In this report we now show that the periodic expression of TMP1 transcript is primarily controlled at the level of its transcription by sequences which are upstream of its transcription initiation sites . We also localized the upstream sequences necessary for periodic transcription to a 150-base-pair region and show that this region encodes an element(s) with the properties of a periodic upstream activating sequence . The regulatory region defined in this study apparently does not contain consensus sequences similar to those reported for the cell cycle-regulated HO endonuclease or for the histone H2A and H2B genes of Saccharomyces cerevisiae.

J Bacteriol, 1988 Nov, 170(11), 5396 - 400
Glucose uptake in Saccharomyces cerevisiae grown under anaerobic conditions: effect of null mutations in the hexokinase and glucokinase structural genes; McClellan CJ et al.; Glucose uptake was investigated in a set of isogenic strains carrying a single glucose kinase structural gene, the other two kinase genes having been rendered nonfunctional through the construction of null mutations . Any one of the three kinases was sufficient for growth and glucose utilization aerobically or anaerobically . Under anaerobic conditions, substrate inhibition and regulation of carrier activity varied and depended upon the particular kinase present in the cell.

J Bacteriol, 1988 Nov, 170(11), 5375 - 7
Relationship between low- and high-affinity glucose transport systems of Saccharomyces cerevisiae; Ramos J et al.; The high-affinity glucose transport process in Saccharomyces cerevisiae whole cells was regulated by catabolite repression and inactivation . The low-affinity process was constitutive, and its activity was inhibited in proportion to the extent of derepression of the high-affinity process . The latter finding suggests that there is some regulatory relationship between the two processes.

Proc Natl Acad Sci U S A, 1988 Nov, 85(22), 8516 - 20
Localization of chitin synthetase in cell-free homogenates of Saccharomyces cerevisiae: chitosomes and plasma membrane; Leal-Morales CA et al.; We describe an improved method for fractionating cell-free extracts of Saccharomyces cerevisiae to separate its membranous components by a combination of isopycnic and velocity sedimentations . These procedures were used to examine the subcellular distribution of chitin synthetase (chitin-UDP acetylglucosaminyltransferase; EC 2.4.1.16) in homogenates from exponentially growing walled cells of a wild-type strain of yeast . Chitin synthetase (Chs1) activity was mainly found in two distinct vesicle populations of nearly equal abundance but with markedly different buoyant densities and particle diameters . One population contained 45-65% of the total chitin synthetase and was identified as chitosomes because of microvesicular size (median diameter = 61 nm) and characteristic low buoyant density (1.15 g/ml); it also lacked 1,3-beta-glucan synthetase activity . The second population (35-55%) was identified as plasma membrane because of its high buoyant density (1.22 g/ml), large vesicle size (median diameter = 252 nm), and presence of vanadate-sensitive ATPase . This fraction cosedimented with the main peak of 1,3-beta-glucan synthetase . A third, minor population of chitin synthetase particles was also detected . Essentially all of the chitin synthetase in the two vesicle populations was zymogenic; therefore, we regard these vesicles as precursors of the final active form of chitin synthetase whose location in the cell has yet to be unequivocally determined.

Genetics, 1988 Nov, 120(3), 671 - 80
Identification of REO1, a gene involved in negative regulation of COX5b and ANB1 in aerobically grown Saccharomyces cerevisiae; Trueblood CE et al.; In Saccharomyces cerevisiae, the COX5a and COX5b genes constitute a small gene family that encodes two forms of cytochrome c oxidase subunit V, Va and Vb, either of which can provide a function essential for cytochrome c oxidase activity and respiration . In aerobically grown wild-type yeast cells, Va is the predominant form of subunit V . The COX5b gene alone does not produce enough Vb to support a respiration rate sufficient to allow growth on nonfermentable carbon sources . By selecting for mutations that increase the respiratory capacity of a strain deleted for COX5a, we have identified a gene that is involved in negative regulation of COX5b expression under aerobic growth conditions . Each of four independently isolated reo1 mutations are shown to be recessive, unlinked to COX5b, but dependent on COX5b for phenotypic expression . The mutations define a single complementation and linkage group: designated as REO1 for regulator of expression of oxidase . reo1 mutations increase expression of COX5b in aerobically grown cells, but not in anaerobically grown cells, where expression is already elevated . These mutations have no effect on COX5a, the other member of this small gene family which is positively regulated by heme and oxygen . The REO1 gene does play a role in repression of ANB1, a gene that is normally repressed under aerobic but not anaerobic conditions . Neither rox1 or rox3 mutations, which have previously been shown to increase ANB1 expression, are in the same complementation group as reo1 mutations.

Mol Cell Biol, 1988 Nov, 8(11), 4773 - 9
Inositol regulates phosphatidylglycerolphosphate synthase expression in Saccharomyces cerevisiae; Greenberg ML et al.; The enzyme phosphatidylglycerolphosphate synthase (PGPS; CDPdiacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase; EC 2.7.8.5) catalyzes the committed step in the synthesis of cardiolipin, a phospholipid found predominantly in the mitochondrial inner membrane . To determine whether PGPS is regulated by cross-pathway control, we analyzed PGPS expression under conditions in which the regulation of general phospholipid synthesis could be examined . The addition of inositol resulted in a three- to fivefold reduction in PGPS expression in wild-type cells in the presence or absence of exogenous choline . The reduction in enzyme activity in response to inositol was seen in minutes, suggesting that inactivation or degradation of the enzyme plays an important role in inositol-mediated repression of PGPS . In cho2 and opi3 mutants, which are blocked in phosphatidylcholine synthesis, inositol-mediated repression of PGPS did not occur unless choline was added to the media . Three previously identified genes that regulate general phospholipid synthesis, INO2, INO4, and OP11, did not affect PGPS expression . Thus, ino2 and ino4 mutants, which are unable to derepress biosynthetic enzymes involved in general phospholipid synthesis, expressed wild-type levels of PGPS activity under derepressing conditions . PGPS expression in the opi1 mutant, which exhibits constitutive synthesis of general phospholipid biosynthetic enzymes, was fully repressed in the presence of inositol and partially repressed even in the absence of inositol . These results demonstrate for the first time that an enzymatic step in cardiolipin synthesis is coordinately controlled with general phospholipid synthesis but that this control is not mediated by the same genetic regulatory circuit.

Mol Cell Biol, 1988 Nov, 8(11), 4608 - 15
Genetic evidence for promoter competition in Saccharomyces cerevisiae; Hirschman JE et al.; The his4-912 delta mutation is an insertion of the long terminal repeat (delta) of the yeast retrotransposon Ty into the HIS4 promoter region, such that the delta is 97 base pairs upstream of the HIS4 transcription initiation site . Strains carrying the his4-912 delta allele are His- at 23 degrees C; this phenotype can be reversed either by growth at 37 degrees C or by mutations in trans-acting SPT genes . Under conditions in which his4-912 delta confers a His- phenotype . HIS4 transcription initiates at the delta initiation site, rather than at the HIS4 initiation site, producing a longer, nonfunctional transcript . Under conditions in which the strain is His+, transcription initiates at the wild-type HIS4 initiation site . To understand how transcription is balanced between the delta and HIS4 promoters, we have selected for cis-acting suppressors of his4-912 delta . Two classes defined by six independent mutations restore synthesis of a functional HIS4 transcript . The first class is an A-to-G base change 1 base upstream of the proposed delta TATA sequence . These mutants do not synthesize the delta-initiated transcript; instead, they synthesize only the wild-type HIS4 transcript . The second class of mutations alters base pairs surrounding the functional HIS4 TATA sequence . The two strongest His+ mutants of this class synthesize the wild-type HIS4 transcript at levels consistent with their His+ phenotype . Surprisingly, these two mutants also have a reduced level of the delta-initiated transcript relative to the his4-912 delta parent . Analysis of these mutants indicates that the level of transcription from one promoter can affect the level of transcription from the other promoter and suggests that delta and HIS4 transcription signals compete for initiation of transcription from each site.

Genes Dev, 1988 Nov, 2(11), 1476 - 85
The RAD6 protein of Saccharomyces cerevisiae polyubiquitinates histones, and its acidic domain mediates this activity; Sung P et al.; The RAD6 gene of the yeast Saccharomyces cerevisiae is required for post-replication repair of UV-damaged DNA, DNA damage-induced mutagenesis, and sporulation . Here we demonstrate that the protein encoded by the RAD6 gene, previously shown to be a ubiquitin-conjugating (E2) enzyme, multiply ubiquitinates histones H2A and H2B efficiently to give products containing as many as seven or more molecules of ubiquitin . We also show that the highly acidic 23-residue RAD6 carboxy-terminal tail domain, which contains a total of 20 acidic residues, is essential for the histone-polyubiquitinating activity . Because the RAD6 polyacidic tail is required for the sporulation function but not for the DNA repair and induced mutagenesis functions of RAD6, the present observations suggest that the histone-polyubiquitinating activity of RAD6 protein is essential for sporulation but not for DNA repair and induced mutagenesis . Attachment of multiple molecules of ubiquitin to histones by RAD6 protein may serve to target the histones for degradation via the ubiquitin-dependent proteolytic system or to alter chromatin structure . The in vitro system for synthesizing polyubiquitinated histones described herein provides a means for investigating these possibilities.

Eur J Biochem, 1988 Nov 1, 177(2), 285 - 93
cDNA cloning and functional expression in yeast Saccharomyces cerevisiae of beta-naphthoflavone-induced rabbit liver P-450 LM4 and LM6; Pompon D; A cDNA library was constructed from liver mRNA of a beta-naphthoflavone-induced rabbit . Two clones pLM4-1 and pLM6-1 containing 2.2-kbp inserts that hybridized at low stringincy with a mouse P1 P-450 probe were selected . The clone pLM4-1 was fully sequenced and found to contain a full-length cDNA coding for cytochrome P-450 LM4 . Partial sequence and restriction mapping made it possible to identify pLM6-1 as coding for the major part of cytochrome P-450 LM6 . Cloned LM4-1 cDNA was reformed by deletion of the 5' and 3' non-coding regions before insertion into yeast expression vectors PYe DP1/10 . A similar operation was performed on pLM6-1 cDNA after replacement of the missing N-terminus-coding sequences by homologous sequences form the pLM4-1 clone resulting in a chimeric cytochrome P-450 coding sequence . Expression of cloned rabbit cytochrome P-450 into transformed yeast was optimized by studying the effect of the nature of the DNA sequence just preceding the initiation codon on the level of cytochrome P-450 production . Yeast synthesized cytochromes P-450 were characterized by immunoblotting, spectra and catalytic activity determinations . Cloned cytochrome P-450 LM4 was found by all criteria to be identical to the authentic rabbit one . The chimeric cytochrome P-450 that contains the 143 N-terminal amino acids of cytochrome P-450 LM4 and the remaining 375 amino acids of cytochrome P-450 LM6 was found to exhibit most of the authentic cytochrome P-450 LM6 catalytic properties . Enzymatic and evolutionary implications of these results are discussed.

Gene, 1988 Oct 30, 70(2), 303 - 12
A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae; Elledge SJ et al.; A simple assay called the sectoring shuffle was developed to monitor the mutational state of essential genes cloned into yeast centromeric plasmids . The essence of this assay is the creation of a conditional phenotype, colony color sectoring, for an essential gene in the absence of conditional thermosensitive or cold-sensitive alleles of that gene . This allows the quick determination of the mutational state of a cloned essential gene by observing its effect on the sectoring phenotype of the tester strain . During the course of this work we developed a family of 20 Escherichia coli-yeast shuttle vectors, pUN plasmids, containing ARS1 CEN4 and a variety of selectable markers as well as the SUP11 gene which can act as a color marker in the proper background . These vectors are compact and have been very useful for the sectoring-shuffle assay and for gene analysis in general . This paper describes these vectors, the sectoring shuffle and several applications of sectoring phenotypes.

J Biol Chem, 1988 Oct 25, 263(30), 15342 - 7
Secretion of somatostatin by Saccharomyces cerevisiae . Correct proteolytic processing of pro-alpha-factor-somatostatin hybrids requires the products of the KEX2 and STE13 genes; Bourbonnais Y et al.; Somatostatin is a 14-amino-acid peptide hormone that is proteolytically excised from its precursor, prosomatostatin, by the action of a paired-basic-specific protease . Yeast (Saccharomyces cerevisiae Mat alpha) synthesizes an analogous peptide hormone precursor, pro-alpha-factor, which is proteolytically processed by at least two separate proteases, the products of the KEX2 and STE13 genes, to generate the mature bioactive peptide . Expression in yeast of recombinant DNAs encoding hybrids between the proregion of alpha-factor and somatostatin results in proteolytic processing of the chimeric precursors and secretion of mature somatostatin . To determine if the chimeras were processed by the same enzymes that cleave endogenous pro-alpha-factor, the hybrid DNAs were introduced into kex2 and ste13 mutants, and the secreted proteins were analyzed . Expression of the pro-alpha-factor-somatostatin hybrids in kex2 mutant yeast resulted in secretion of a high molecular weight hyperglycosylated precursor . No mature somatostatin was secreted, and there was no proteolytic cleavage at the Lys-Arg processing site . Similarly, in ste13 yeast, only somatostatin molecules containing the (Glu-Ala)3 spacer peptide at the amino terminus were secreted . Our results demonstrate that in yeast processing mutants, the behavior of the chimeric precursors with respect to proteolytic processing was exactly as that of endogenous pro-alpha-factor . We conclude that the same enzymes that generate mature alpha-factor proteolytically process hybrid precursors . This suggests that structural domains of the proregion rather than the mature peptide are recognized by the processing proteases.

Eur J Biochem, 1988 Oct 15, 177(1), 207 - 11
Localization of the synthesis of very-long-chain fatty acid in mitochondria from Saccharomyces cerevisiae; Bessoule JJ et al.; The localization of the mitochondrial elongation activities ('elongases') from Saccharomyces cerevisiae has been investigated . It was shown, using carboxyatractyloside in the incubation mixture, that synthesis of very-long-chain fatty acids probably occurred outside the matrix and, by fractionation experiments, that elongases are membrane-bound enzymes . The solubilization of the outer membrane by digitonin showed that three elongating activities are correlated with a marker of the outer membrane and not with an inner membrane marker . A further partial purification of the outer membrane showed that elongases are present in the outer membrane of mitochondria.

J Biol Chem, 1988 Oct 15, 263(29), 14970 - 7
In addition to RNase H(70) two other proteins of Saccharomyces cerevisiae exhibit ribonuclease H activity; Karwan R et al.; Two ribonucleases H (RNases H) were purified to apparent homogeneity from the yeast Saccharomyces cerevisiae . The enzymes were separated from the previously described yeast ribonuclease H (RNase H(70), Karwan, R., Blutsch, H., and Wintersberger, U . (1983) Biochemistry 22, 5500-5507) by chromatography on Mono Q and blue-Sepharose columns and from each other on a Mono S column . The two proteins, RNase H(55) of molecular weight around 55,000 and RNase H(42) of molecular weight around 42,000, exhibit distinct enzymatic properties: RNase H(55) acts as a 5'-exonuclease of low specific activity and produces predominantly monoribonucleotides from the synthetic hybrid poly(rA)-poly(dT) . RNase H(42) efficiently releases oligoribonucleotides from the same substrate . Polyclonal antibodies against these proteins do not cross-react with RNase H(70), and thus, these two RNases H probably do not represent proteolytic breakdown products of RNase H(70) . Peptide maps obtained by total digestion of RNase H(55) and RNase H(42) with trypsin reveal several common peptides and, therefore, suggest that the two enzymes are related to each other . We tentatively conclude that RNase H(55) is proteolytically processed to RNase H(42) in vivo.

J Biol Chem, 1988 Oct 15, 263(29), 14757 - 63
Glycoprotein biosynthesis in Saccharomyces cerevisiae . Purification of the alpha-mannosidase which removes one specific mannose residue from Man9GlcNAc; Jelinek-Kelly S et al.; A soluble form of the specific alpha-mannosidase from Saccharomyces cerevisiae, which catalyzes the following reaction, was purified at least 100,000-fold by conventional chromatography procedures: (Formula: see text) . The purified enzyme migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band of about 60 kDa in the absence of reducing agent, and as two bands of about 44.5 kDa and 22.5 kDa in the presence of reducing agent . The apparent molecular weight of the soluble enzyme is about 75,000 by gel filtration on Sephacryl S-200 . The specific alpha-mannosidase does not require the addition of divalent cation for activity, but it is inhibited by Tris, EDTA, Mn2+, Co2+, Zn2+, and Mg2+ . The inhibition caused by EDTA can be reversed completely by Ca2+ and partially by Mg2+, but not by other divalent cations . The soluble alpha-mannosidase arises from a larger hydrophobic form of the enzyme which is found in the detergent phase during partition in Triton X-114 . The formation of the soluble enzyme, which is recovered in the aqueous phase during partition in Triton X-114, is time- and temperature-dependent and is prevented by pepstatin, but not by other protease inhibitors . These results indicate that the purified soluble alpha-mannosidase represents the catalytically active domain of the enzyme which has been proteolytically released from its membrane-bound form.

J Biol Chem, 1988 Oct 15, 263(29), 14948 - 55
Purification and characterization of an N alpha-acetyltransferase from Saccharomyces cerevisiae; Lee FJ et al.; N alpha-Acetyltransferase, which catalyzes the transfer of an acetyl group from acetyl coenzyme A to the alpha-NH2 group of proteins and peptides, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked . The enzyme was purified 4,600-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, hydroxylapatite, DE52 cellulose, and Affi-Gel blue . The Mr of the native enzyme was estimated to be 180,000 +/- 10,000 by gel filtration chromatography, and the Mr of each subunit was estimated to be 95,000 +/- 2,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The enzyme has a pH optimum near 9.0, and its pI is 4.3 as determined by chromatofocusing on Mono-P . The enzyme catalyzed the transfer of an acetyl group to various synthetic peptides, including human adrenocorticotropic hormone (ACTH) (1-24) and its {Phe2} analogue, yeast alcohol dehydrogenase I (1-24), yeast alcohol dehydrogenase II (1-24), and human superoxide dismutase (1-24) . These peptides contain either Ser or Ala as NH2-terminal residues which together with Met are the most commonly acetylated NH2-terminal residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H . (1985) Eur . J . Biochem . 152, 523-527) . Yeast enolase, containing a free NH2-terminal Ala residue, is known not to be N alpha-acetylated in vivo (Chin, C . C . Q., Brewer, J . M., and Wold, F . (1981) J . Biol . Chem . 256, 1377-1384), and enolase (1-24), a synthetic peptide mimicking the protein's NH2 terminus, was not acetylated in vitro by yeast acetyltransferase . The enzyme did not catalyze the N alpha-acetylation of other synthetic peptides including ACTH(11-24), ACTH(7-38), ACTH(18-39), human beta-endorphin, yeast superoxide dismutase (1-24) . Each of these peptides has an NH2-terminal residue which is rarely acetylated in proteins (Lys, Phe, Arg, Tyr, Val, respectively) . Among a series of divalent cations, Cu2+ and Zn2+ were demonstrated to be the most potent inhibitors . The enzyme was inactivated by chemical modification with diethyl pyrocarbonate and N-bromosuccinimide.

J Biol Chem, 1988 Oct 15, 263(29), 14812 - 8
Separate genes encode functionally equivalent ADP/ATP carrier proteins in Saccharomyces cerevisiae . Isolation and analysis of AAC2; Lawson JE et al.; Genetic and biochemical analysis of Saccharomyces cerevisiae containing a disruption of the nuclear gene (AAC1) encoding the mitochondrial ADP/ATP carrier has revealed a second gene for this protein . The second gene, designated AAC2, has been isolated by genetic complementation and sequenced . AAC2 contains a 954-base pair open reading frame coding for a protein of 318 amino acids which is highly homologous to the AAC1 gene product except that it is nine amino acids longer at the NH2 terminus . The two yeast genes are highly conserved at the level of DNA and protein and share identity with the ADP/ATP carriers from other organisms . Both genes complement an ADP/ATP carrier defect (op1 or pet9) . However, the newly isolated gene AAC2 need be present only in one or two copies while the previously isolated AAC1 gene must be present in multiple copies to support growth dependent on a functional carrier protein . This gene dosage-dependent complementation combined with the high degree of conservation suggest that these two functionally equivalent genes may be differentially expressed.

FEBS Lett, 1988 Oct 10, 238(2), 329 - 32
Alterations in the cleavage site of the signal sequence for the secretion of human lysozyme by Saccharomyces cerevisiae; Nagahora H et al.; The amino acids corresponding to the cleavage site of a hybrid preprotein containing a chicken lysozyme signal and a mature portion of human lysozyme were altered . The processing of mutant signals of -3Pro and -3Asp/-1Ala decreased remarkably, while that of -2Pro was 75% of that of the native signal . The major cleavage site of -3Pro was the same as that of the native signal, but that of the -2Pro and -3Asp/-1Ala signals was shifted one residue closer to the N-terminal side than the original site . The cleavage of the -2Pro signal, which was identical to the native processing of pheasant prelysozyme, suggested that the signal peptidases in yeast and bird are similar.

Biochim Biophys Acta, 1988 Oct 7, 971(3), 255 - 65
Effective stage in the cell cycle for control of the budding direction of cdc mutants of Saccharomyces cerevisiae using electric stimulus; Matsuoka H et al.; Cell division cycle (cdc) mutants of Saccharomyces cerevisiae were used to determine the most effective stage for the directional control of cell budding using an electric stimulus . The selected mutants were cdc 35 and cdc 28, which could be reversibly arrested before spindle pole body satellite formation (SPBSF) and spindle pole body duplication (SPBD), respectively . The budding direction (theta) was defined so that the direction parallel to that of the electric field was 0 degree . Considering the symmetry of the experimental conditions, the range of theta was defined as 0-90 degrees . The electric stimulus applied in the present study was alternating pulses (pulse height, +/- 15 V; pulse width at half pulse height, 5 microseconds; frequency; 10 kHz) . The peak height of the cross membrane potential was estimated as 472 mV, which was sufficient to induce considerable strain in the cell membrane . In the case of cdc 35, the 95% confidence interval (95% CI) of the budding direction was 7-25 degrees when subjected to electric stimulus, while the 95% CI of the budding direction without electric stimulus was 35-57 degrees . In the case of cdc 28, 95% CI values of the budding direction with and without electric stimulus were 1229 degrees and 23-56 degrees, respectively . These results demonstrate that the stage after SPBD is effective for the directional control of yeast cell budding using an electric stimulus . Simultaneously, an electric stimulus reduced the cell budding time of both the cdc mutants used . Therefore, the electric stimulus was also effective in promoting cell cycle progression under the present conditions.

Mol Cell Biol, 1988 Oct, 8(10), 4533 - 6
Efficiency of translation initiation by non-AUG codons in Saccharomyces cerevisiae; Clements JM et al.; The quantitative levels of initiation of protein synthesis at codons other than AUG were determined with a CYC7-lacZ fused gene in the yeast Saccharomyces cerevisiae . AUG was the only codon which efficiently initiated translation, although some non-AUG codons allowed initiation at very low efficiency, below 1% of the normal level . Since translation initiates at codons other than AUG in at least two wild-type genes from eucaryotes, other factors presumably play a role in enhancing the activity of non-AUG codons.

Mol Gen Genet, 1988 Oct, 214(2), 271 - 7
Domains of the Saccharomyces cerevisiae CDC25 gene controlling mitosis and meiosis; Munder T et al.; The cell division cycle gene CDC25 was replaced by various disrupted and deleted mutant copies . Mutants disrupted at a central position of the gene, or lacking 532 residues within the amino-terminal half of the gene product grow normally in glucose, but not in acetate media, and they fail to sporulate as homozygous diploids . Disruptions or deletions within the carboxy-terminal half are lethal, except for the deletion of the 38 carboxy-terminal residues, which are required for sporulation but not for growth in glucose or acetate media . It is concluded that distinct domains of the CDC25 gene product are involved in the control of mitosis and/or meiosis.

Curr Genet, 1988 Oct, 14(4), 337 - 44
The glucose-and ethanol-dependent regulation of PDC1 from Saccharomyces cerevisiae are controlled by two distinct promoter regions; Kellermann E et al.; A 870 bp promoter fragment of the PDC1 gene that includes the carbon source dependent regulatory regions was investigated using 5' and 3' promoter deletions . The results indicate that glucose and ethanol regulation of PDC1 transcription are independently controlled by distinct cis-acting regions . The consensus sequence AAATCGATA may play a role in this regulation, while the sequence (ATCA)AACCT may be important in transcription initiation.

Curr Genet, 1988 Oct, 14(4), 325 - 9
Construction and characterization of a haploid strain of Saccharomyces cerevisiae that completely lacks all genomic CYH2 sequences; Miles DJ et al.; A diploid strain of the yeast Saccharomyces cerevisiae has been constructed that has one copy of the ribosomal protein gene CYH2 completely deleted and replaced with the TRP1 gene using the method of Rothstein (1983) . There are only small differences in growth rate and no detectable difference in steady state level of CYH2 mRNA between the diploid that is heterozygous for the CYH2 deletion and the parent diploid with two normal copies of this gene . This suggests that the diploid must partially compensate for the loss of one CYH2 gene . Tetrad dissection shows that haploid spores lacking the CYH2 gene cannot germinate . The lethality of this deletion can be rescued by a CYH2 cDNA on a low copy vector . Haploids which lack the genomic copy of the CYH2 gene, but contain a plasmid copy of the CYH2 cDNA are able to grow normally . These CYH2 deleted yeast haploids should be useful to analyze mutationally altered CYH2 genes and genes homologous to CYH2 from other organisms without interference from a genomic copy.

Appl Biochem Biotechnol, 1988 Oct, 19(1), 27 - 32
Studies on Saccharomyces cerevisiae carrying the plasmid pCYG4 related with ammonia assimilation . Batch experiments; Lima Filho JL et al.; Batch culture experiments of three different strains of Saccharomyces cerevisiae have been carried out . The first strain was transformed by a plasmid pCYG4, which carries the glutamate dehydrogenase (NADP-GDH, E.C . 1.4.14) gene conferring an 11-fold increase in activity . The second was transformed by the same plasmid, but without NADP-GDH, and the third was the wild type . The specific growth rates of the two recombinant DNA strains were below that of the wild type, which can be related to extra plasmid protein production.

Genetics, 1988 Oct, 120(2), 367 - 77
Different types of recombination events are controlled by the RAD1 and RAD52 genes of Saccharomyces cerevisiae; Klein HL; Intrachromosomal recombination within heteroallelic duplications located on chromosomes III and XV of Saccharomyces cerevisiae has been examined . Both possible orientations of alleles have been used in each duplication . Three recombinant classes, gene conversions, pop-outs and triplications, were recovered . Some of the recombinant classes were not anticipated from the particular allele orientation of the duplication . Recovery of these unexpected recombinants requires the RAD1 gene . These studies show that RAD1 has a role in recombination between repeated sequences, and that the recombination event is a gene conversion associated with a crossover . These events appear to involve very localized conversion of a heteroduplex region and are distinct from RAD52 mediated gene conversion events . Evidence is also presented to suggest that most recombination events between direct repeats are intrachromatid, not between sister chromatids.

Mol Cell Biol, 1988 Oct, 8(10), 4370 - 80
Direction of chromosome rearrangements in Saccharomyces cerevisiae by use of his3 recombinational substrates; Fasullo MT et al.; We used the his3 recombinational substrates (his3 fragments) to direct large interchromosomal (translocations) and intrachromosomal (deletions and tandem duplications) rearrangements in the yeast Saccharomyces cerevisiae . In strains completely deleted for the wild-type HIS3 gene, his3 fragments, one containing a deletion of 5' amino acid coding sequences and the other containing a deletion of 3' amino acid coding sequences, were first placed at preselected sites by homologous recombination . His+ revertants that arose via spontaneous mitotic recombination between the two his3 fragments were selected . This strategy was used to direct rearrangements in both RAD52+ and rad52 mutant strains . Translocations occurred in the RAD52+ genetic background and were characterized by orthogonal field alternating gel electrophoresis of yeast chromosomal DNA and by standard genetic techniques . An unexpected translocation was also identified in which HIS3 sequences were amplified . Two types of tandem duplications of the GAL(7, 10, 1) locus were also directed, and one type was not observed in rad52 mutants . Recombination mechanisms are discussed to account for these differences.

Mol Cell Biol, 1988 Oct, 8(10), 3997 - 4008
Isolation and characterization of temperature-sensitive mutations in RPA190, the gene encoding the largest subunit of RNA polymerase I from Saccharomyces cerevisiae; Wittekind M et al.; The isolation and characterization of temperature-sensitive mutations in RNA polymerase I from Saccharomyces cerevisiae are described . A plasmid carrying RPA190, the gene encoding the largest subunit of the enzyme, was subjected to in vitro mutagenesis with hydroxylamine . Using a plasmid shuffle screening system, five different plasmids were isolated which conferred a temperature-sensitive phenotype in haploid yeast strains carrying the disrupted chromosomal RPA190 gene . These temperature-sensitive alleles were transferred to the chromosomal RPA190 locus for mapping and physiology experiments . Accumulation of RNA was found to be defective in all mutant strains at the nonpermissive temperature . In addition, analysis of pulse-labeled RNA from two mutant strains at 37 degrees C showed that the transcription of rRNA genes was decreased, while that of 5S RNA was relatively unaffected . RNA polymerase I was partially purified from several of the mutant strains grown at the nonpermissive temperature and was shown to be deficient when assayed in vitro . Fine-structure mapping and sequencing of the mutant alleles demonstrated that all five mutations were unique . The rpa190-1 and rpa190-5 mutations are tightly clustered in region I (S.S . Broyles and B . Moss, Proc . Natl . Acad . Sci . USA 83:3141-3145, 1986), the putative zinc-binding region that is common to all eucaryotic RNA polymerase large subunits . The rpa190-3 mutation is located between regions III and IV, and a strain carrying it behaves as a mutant that is defective in the synthesis of the enzyme . This mutation lies within a previously unidentified segment of highly conserved amino acid sequence homology that is shared among the largest subunits of eucaryotic nuclear RNA polymerases . Another temperature-sensitive mutation, rpa190-2, creates a UGA nonsense codon.

EMBO J, 1988 Oct, 7(10), 3255 - 62
Mitochondrial RNA polymerase of Saccharomyces cerevisiae: composition and mechanism of promoter recognition; Schinkel AH et al.; Mitochondrial RNA polymerase of Saccharomyces cerevisiae consists of two different proteins: a core RNA polymerase of 145 kd and a specificity factor of 43 kd, which contributes the capacity to recognize promoters of the various genes encoded in the mitochondrial genome . We purified both components by SDS-PAGE, followed by renaturation to the active state . The two components were used either singly or in combination to study their interactions with promoter-containing DNA fragments . The core component showed random and weak interaction with DNA, the specificity factor none at all, whereas both components together specifically bound to a promoter . In DNase I footprinting experiments, promoter-bound RNA polymerase protected a short region of DNA flanked by hypersensitivity sites and centred around the position at which RNA synthesis starts . The initial phase of transcription gave rise to specific changes in this footprint: the upstream border remained at the same position up to synthesis of a 4-nt RNA chain, whereas at the downstream border progressive disappearance of hypersensitivity sites took place.

Proc Natl Acad Sci U S A, 1988 Oct, 85(20), 7546 - 50
Cloning and nucleotide sequence of the gene for dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae; Niu XD et al.; A 537-base cDNA encoding a portion of Saccharomyces cerevisiae dihydrolipoamide acetyltransferase (acetyl-CoA:dihydrolipoamide S-acetyltransferase, EC 2.3.1.12) was isolated from a lambda gt11 yeast cDNA library by immunoscreening . This cDNA was subcloned and used as a probe to screen a lambda gt11 yeast genomic DNA library . Two overlapping clones were used to determine the complete sequence of the acetyltransferase gene . The composite sequence has an open reading frame of 1446 nucleotides encoding a presequence of 28 amino acids and a mature protein of 454 amino acids (Mr = 48,546) . The deduced amino acid sequence contains the experimentally determined amino acid sequences of the amino terminus and two internal peptide fragments of the acetyltransferase . Hybridization analysis of yeast genomic DNA showed that the gene has a single copy . A 915-base segment of the acetyltransferase gene hybridized to a yeast mRNA of approximately equal to 1.6 kilobases . Analysis of the deduced amino acid sequence of the dihydrolipoamide acetyltransferase revealed a multidomain structure similar to those reported for the corresponding acetyltransferases from Escherichia coli and rat liver, and extensive sequence similarity among the three enzymes . However, the yeast enzyme contains only one lipoyl domain, in contrast to three lipoyl domains reported for the E . coli enzyme and apparently two for the rat liver enzyme.

Mutat Res, 1988 Oct, 206(2), 209 - 16
Genetic effects of chlorinated ethylenes in the yeast Saccharomyces cerevisiae; Koch R et al.; The chlorinated ethylenes 1,1-dichloroethylene (vinylidene chloride), trans-1,2-dichloroethylene, trichloroethylene, and tetrachloroethylene (perchloroethylene) were assayed for their ability to induce mitotic gene conversion and point mutation as well as mitotic aneuploidy in diploid strains of the yeast Saccharomyces cerevisiae . From strain D7 late logarithmic-phase cells grown in 20% glucose liquid medium, containing a high level of cytochrome P-450, as well as stationary-phase cells combined with an exogenous metabolic activating system (S9) were used, in order to activate the chlorinated compounds and to produce electrophilic mutagenic intermediates . Only 1,1-dichloroethylene exhibited a dose-dependent genetic activity, while the other ethylenes did not . The 2 ways of metabolic activation were compared and were found to cause approximately the same effect . In contrast to the findings with strain D7, vinylidene chloride, trans-1,2-dichloroethylene, and trichloroethylene induced, without metabolic activation, mitotic chromosomal malsegregation in strain D61.M . The presence of liver homogenate as an activating system did not enhance the respective frequencies of chromosome loss . In the case of tetrachloroethylene, sufficient data have not become available, since this compound showed a highly toxic effect towards yeast cells, decreasing the rate of surviving cells to less than 30% at a concentration of 9.8 mM.

Mutat Res, 1988 Oct, 201(2), 413 - 21
Investigations of aneuploidy-inducing chemical combinations in Saccharomyces cerevisiae; Mayer VW et al.; For several years we have been investigating combinations of chemicals for their ability to induce aneuploidy . Earlier published results indicated that combinations of certain chemicals showed a potentiation effect while other combinations did not . We have continued to explore this phenomenon and report additional findings in this communication . Combinations of ethyl acetate and methyl ethyl ketone showed a potentiation effect as did 1-methyl-2-pyrrolidinone-nocodazole combinations . Combinations that did not show a potentiation effect were 2-pyrrolidinone-nocodazole and 1-methyl-2-pyrrolidinone-ethyl acetate . We also found that nocodazole, which is a potent inducer of aneuploidy in yeast extract-peptone-dextrose (YEPD) medium but not in synthetic complete (SC) medium, showed a potentiation effect with ethyl acetate in SC medium . This effect in SC medium is similar to that previously reported for nocodazole with ethyl acetate in YEPD medium . When nocodazole was dissolved in 1-methyl-2-pyrrolidinone as a concentrated stock solution, a potentiation effect occurred even at low concentrations of the solvent.

J Cell Biol, 1988 Oct, 107(4), 1409 - 26
Functions of microtubules in the Saccharomyces cerevisiae cell cycle; Jacobs CW et al.; We used the inhibitor nocodazole in conjunction with immunofluorescence and electron microscopy to investigate microtubule function in the yeast cell cycle . Under appropriate conditions, this drug produced a rapid and essentially complete disassembly of cytoplasmic and intranuclear microtubules, accompanied by a rapid and essentially complete block of cellular and nuclear division . These effects were similar to, but more profound than, the effects of the related drug methyl benzimidazole carbamate (MBC) . In the nocodazole-treated cells, the selection of nonrandom budding sites, the formation of chitin rings and rings of 10-nm filaments at those sites, bud emergence, differential bud enlargement, and apical bud growth appeared to proceed normally, and the intracellular distribution of actin was not detectably perturbed . Thus, the cytoplasmic microtubules are apparently not essential for the establishment of cell polarity and the localization of cell-surface growth . In contrast, nocodazole profoundly affected the behavior of the nucleus . Although spindle-pole bodies (SPBs) could duplicate in the absence of microtubules, SPB separation was blocked . Moreover, complete spindles present at the beginning of drug treatment appeared to collapse, drawing the opposed SPBs and associated nuclear envelope close together . Nuclei did not migrate to the mother-bud necks in nocodazole-treated cells, although nuclei that had reached the necks before drug treatment remained there . Moreover, the double SPBs in arrested cells were often not oriented toward the budding sites, in contrast to the situation in normal cells . Thus, microtubules (cytoplasmic, intranuclear, or both) appear to be necessary for the migration and proper orientation of the nucleus, as well as for SPB separation, spindle function, and nuclear division.

J Bacteriol, 1988 Oct, 170(10), 4838 - 45
High-affinity glucose transport in Saccharomyces cerevisiae is under general glucose repression control; Bisson LF; Saccharomyces cerevisiae mutants defective in growth on low glucose concentration (lgn mutants) were isolated and screened for abnormal glucose transport . Nine complementation groups were identified, falling into two broad groups: those unable to significantly derepress high-affinity (low-Km) glucose uptake (lgn1, lgn4, lgn5, lgn7, and lgn8), and those with elevated repressed levels of high-affinity uptake that either derepress to normal or near normal levels of high-affinity uptake with loss of low-affinity transport (lgn2 and lgn3) or derepress only slightly, appearing to have an intermediate yet constitutive level of high-affinity transport (lgn6 and lgn9) . Further analysis of the lgn mutations revealed pleiotropic phenotypes most consistent with the true defect being in regulation or expression of glucose repression and derepression . The kinetics of glucose uptake in strains carrying known mutations preventing derepression of glucose-repressible functions (snf1, snf2, snf4, and snf6) demonstrated that three of these mutations (snf1, snf4, and snf6) were similarly defective in derepression of high-affinity glucose uptake . The snf2 and snf5 mutations had no apparent effect on glucose uptake . Two mutations resulting in constitutive expression of glucose-repressible functions, cid1 and reg1, resulted in constitutive expression of high-affinity glucose uptake . These data support the conclusion that high-affinity glucose uptake in Saccharomyces cerevisiae is under general glucose repression control . The implications of other properties of these mutants are discussed.

J Biochem (Tokyo), 1988 Oct, 104(4), 526 - 30
Molecular cloning and expression of a gene for a factor which stabilizes formation of inhibitor-mitochondrial ATPase complex from Saccharomyces cerevisiae; Akashi A et al.; Stabilizing factor, a 9 kDa protein, stabilizes and facilitates formation of the complex between mitochondrial ATP synthase and its intrinsic inhibitor protein . A clone containing the gene encoding the 9 kDa protein was selected from a yeast genomic library to determine the structure of its precursor protein . As deduced from the nucleotide sequence, the precursor of the yeast 9 kDa stabilizing factor contains 86 amino acid residues and has a molecular weight of 10,062 . From the predicted sequence we infer that the stabilizing factor precursor contains a presequence of 23 amino acid residues at its amino terminus . We also used S1 mapping to determine the initiation site of transcription under glucose-repressed or derepressed conditions . These experiments suggest that transcription of this gene starts at three different sites and that only one of them is not affected by the presence of glucose.

Int J Radiat Biol, 1988 Oct, 54(4), 563 - 6
Survival of Saccharomyces cerevisiae after treatment with the restriction endonuclease Alu I; Winckler K et al.; Treatment of yeast cells proficient in the repair of radiation damage (Saccharomyces cerevisiae) with the restriction endonuclease Alu I leads to a positive dose-effect relationship between inactivation level and enzyme concentration . The data suggest an uptake of the active restriction enzyme into the cells and a relationship between induction of DNA double-strand breaks and cell killing.

Mol Gen Genet, 1988 Oct, 214(2), 218 - 23
A nuclear gene essential for mitochondrial replication suppresses a defect of mitochondrial transcription in Saccharomyces cerevisiae; Lisowsky T et al.; A genomic DNA fragment from yeast was isolated by transforming a temperature sensitive pet mutant . This mutant, pet-ts 798, has previously been characterized by its altered mitochondrial transcription apparatus . Subcloning and DNA sequencing of the genomic DNA fragment identified a reading frame responsible for the restoration of the pet-ts phenotype . The reading frame of 1023 bp is transcribed as an RNA of about 1100 nucleotides . The putative protein of 40 kDa possesses a hydrophobic amino-terminus and acidic and basic domains characteristic of recently described transcriptional activators . The inactivation of the functional gene by the introduction of an insertion fragment into the reading frame, leads to a stable pet phenotype . Further analysis of this mutant created by gene disruption makes clear that the respiratory defect is caused by the complete loss of mitochondrial DNA . Experimental evidence is given that the cloned gene acts as an intergenic suppressor of the mutant pet-ts 798 . Therefore, the isolated gene represents a new factor involved in the regulation of mitochondrial replication and transcription.

Mol Cell Biol, 1988 Oct, 8(10), 4537 - 40
Differential regulation of the two genes encoding Saccharomyces cerevisiae cytochrome c oxidase subunit V by heme and the HAP2 and REO1 genes; Trueblood CE et al.; In Saccharomyces cerevisiae, the COX5a and COX5b genes encode two forms of cytochrome c oxidase subunit V, Va and Vb . We report here that heme increases COX5a expression and decreases COX5b expression and that the HAP2 and REO1 genes are involved in positive regulation of COX5a and negative regulation of COX5b, respectively . Heme regulation of COX5a and COX5b may dictate which subunit V isoform is available for assembly into cytochrome c oxidase under conditions of high- and low-oxygen tension.

J Bacteriol, 1988 Oct, 170(10), 4808 - 15
Changes in activities of several enzymes involved in carbohydrate metabolism during the cell cycle of Saccharomyces cerevisiae; Van Doorn J et al.; Activity changes of a number of enzymes involved in carbohydrate metabolism were determined in cell extracts of fractionated exponential-phase populations of Saccharomyces cerevisiae grown under excess glucose . Cell-size fractionation was achieved by an improved centrifugal elutriation procedure . Evidence that the yeast populations had been fractionated according to age in the cell cycle was obtained by examining the various cell fractions for their volume distribution and their microscopic appearance and by flow cytometric analysis of the distribution patterns of cellular DNA and protein contents . Trehalase, hexokinase, pyruvate kinase, phosphofructokinase 1, and fructose-1,6-diphosphatase showed changes in specific activities throughout the cell cycle, whereas the specific activities of alcohol dehydrogenase and glucose-6-phosphate dehydrogenase remained constant . The basal trehalase activity increased substantially (about 20-fold) with bud emergence and decreased again in binucleated cells . However, when the enzyme was activated by pretreatment of the cell extracts with cyclic AMP-dependent protein kinase, no significant fluctuations in activity were seen . These observations strongly favor posttranslational modification through phosphorylation-dephosphorylation as the mechanism underlying the periodic changes in trehalase activity during the cell cycle . As observed for trehalase, the specific activities of hexokinase and phosphofructokinase 1 rose from the beginning of bud formation onward, finally leading to more than eightfold higher values at the end of the S phase . Subsequently, the enzyme activities dropped markedly at later stages of the cycle . Pyruvate kinase activity was relatively low during the G1 phase and the S phase, but increased dramatically (more than 50-fold) during G2 . In contrast to the three glycolytic enzymes investigated, the highest specific activity of the gluconeogenic enzyme fructose-1, 6-diphosphatase 1 was found in fractions enriched in either unbudded cells with a single nucleus or binucleated cells . The observed changes in enzyme activities most likely underlie pronounced alterations in carbohydrate metabolism during the cell cycle.

J Bacteriol, 1988 Oct, 170(10), 4727 - 31
Expression of the Saccharomyces cerevisiae PIS gene and synthesis of phosphatidylinositol in Escherichia coli; Nikawa J et al.; Expression of the Saccharomyces cerevisiae PIS gene encoding phosphatidylinositol synthase in Escherichia coli was achieved by inserting its coding sequence into lacZ on pUC8 . The fused gene encoded a phosphatidylinositol synthase whose amino-terminal three amino acids had been replaced by the amino-terminal five amino acids of E . coli beta-galactosidase . E . coli cells bearing this recombinant plasmid produced a significant level of phosphatidylinositol synthase in the presence of a lacZ inducer, isopropylthio-beta-D-galactopyranoside . When the culture medium was supplemented with myo-inositol and isopropylthio-beta-D-galactopyranoside, the cells accumulated a substantial amount of phosphatidylinositol in their membranes . When a saturating level of myo-inositol was added, phosphatidylinositol constituted about 4% of the total phospholipids . Phosphatidylinositol accumulation occurred at the expense of phosphatidylglycerol . The ratio of phosphatidylethanolamine to total acidic phospholipids remained constant . The growth rate of phosphatidylinositol-containing E . coli cells did not differ significantly from that of cells with the normal phospholipid composition.

J Bacteriol, 1988 Oct, 170(10), 4991 - 4
Bleomycin-induced DNA repair by Saccharomyces cerevisiae ATP-dependent polydeoxyribonucleotide ligase; Moore CW; In contrast to ligase-deficient (cdc9) Saccharomyces cerevisiae, which did not rejoin bleomycin-induced DNA breaks, ligase-proficient (CDC9) yeast cells eliminated approximately 90% of DNA breaks within 90 to 120 min after treatment . Experimental conditions restricted enzymatic removal of the unusual 3'-phosphoglycolate termini in DNA cleaved by bleomycin and involved doses producing equivalent numbers of DNA breaks or doses producing equivalent killing.

FEBS Lett, 1988 Sep 26, 238(1), 175 - 9
Amino-terminal sequence of a Saccharomyces cerevisiae nuclear protein, NHP6, shows significant identity to bovine HMG1; Kolodrubetz D et al.; Several nonhistone chromatin proteins (NHPs) have been isolated from Saccharomyces cerevisiae nuclei . They have molecular masses and amino acid compositions typical of the high mobility group (HMG) proteins from higher eukaryotic cells . Polyclonal antisera raised against two of the NHPs have been used in immunoblots of proteins from subcellular fractions of yeast to show that the NHPs are indeed nuclear . In addition, the amino-terminal amino acid sequences of several of the NHPs were determined . Importantly, the amino-terminal sequence of one of the proteins, NHP6, has significant (60%) identity with a stretch of amino acids in calf thymus HMG1.

Nucleic Acids Res, 1988 Sep 26, 16(18), 8869 - 86
Foreign transcriptional enhancers in yeast . II . Interplay of the polyomavirus transcriptional enhancer and Saccharomyces cerevisiae promoter elements; Ciaramella M et al.; In this paper, to further analyze the function of the polyoma enhancer in Saccharomyces cerevisiae, we use as reporter-genes derivatives of the yeast HIS3 gene flanked by two types of partially deleted promoters: in one, UAS elements are removed by deletion of sequences upstream of nt -80 (pGM3181) in the second both TATA boxes and UAS elements are removed by deletion of sequences upstream of nt -35 (pGM2809) . These constructs have been studied both as free plasmids and after integration at the TRP1 chromosomal locus . We find that in general the polyoma holoenhancer (A + B domains) elicits transcription from the physiological HIS3 RNA start sites when the native TATA boxes are present . In contrast, an altered enhancer B-domain from polyoma mutant Py-B78, although active when inserted downstream of the test-gene or when coupled to a pseudopromoter (Ciaramella et al, accompanying manuscript), does not work properly in concert with the native yeast TATA boxes . We describe experiments that suggest an important role for the foreign enhancer in RNA start-site selection in yeast.

J Biol Chem, 1988 Sep 25, 263(27), 13839 - 47
Intron sequence and structure requirements for tRNA splicing in Saccharomyces cerevisiae; Szekely E et al.; Predicted single-stranded structure at the 3' splice site is a conserved feature among intervening sequences (IVSs) in eukaryotic nuclear tRNA precursors . The role of 3' splice site structure in splicing was examined through hexanucleotide insertions at a central intron position in the Saccharomyces cerevisiae tRNA gene . These insertions were designed to alter the structure at the splice site without changing its sequence . Endonuclease cleavage of pre-tRNA substrates was then measured in vitro, and suppressor activity was examined in vivo . A precursor with fully double-stranded structure at the 3' splice site was not cleaved by endonuclease . The introduction of one unpaired nucleotide at the 3' splice site was sufficient to restore cleavage, although at a reduced rate . We have also observed that guanosine at the antepenultimate position provides a second consensus feature among IVSs in tRNA precursors . Point mutations at this position were found to affect splicing although there was no specific requirement for guanosine . These and previous results suggest that elements of secondary and/or tertiary structure at the 3' end of IVSs are primary determinants in pre-tRNA splice site utilization whereas specific sequence requirements are limited.

Eur J Biochem, 1988 Sep 15, 176(2), 377 - 84
Purification and characterization of a rotenone-insensitive NADH:Q6 oxidoreductase from mitochondria of Saccharomyces cerevisiae; de Vries S et al.; A mitochondrial NADH:Q6 oxidoreductase has been isolated from cells of Saccharomyces cerevisiae by a simple method involving extraction of the enzyme from the mitochondrial membrane with Triton X-100, followed by chromatography on DEAE-cellulose and blue Sepharose CL-6B . By this procedure a 2000-fold purification is achieved with respect to whole cells or a 150-fold purification with respect to the mitochondrion . The purified NADH dehydrogenase consists of a single subunit with molecular mass of 53 kDa as indicated by SDS/polyacrylamide gel electrophoresis . The enzyme contains FAD, non-covalently linked, as the sole prosthetic group with Em,7.6 = -370 mV and no iron-sulphur clusters . The enzyme is specific for NADH with apparent Km = 31 microM and was found to be inhibited by flavone (I50 = 95 microM), but not by rotenone or piericidin . The purified enzyme can use ubiquinone-2, -6 or -10, menaquinone, dichloroindophenol or ferricyanide as electron acceptors, but at different rates . The greatest turnover of NADH was obtained with ubiquinone-2 as acceptor (2500 s-1) . With the natural ubiquinone-6 this value was 500 s-1 . The NADH:Q2 oxidoreductase activity shows a maximum at pH 6.2, the NADH:Q6 oxidoreductase activity is constant between pH 4.5-9.0 . The amount of enzyme in the cell is subject to glucose repression; it increases slightly when cells, grown on glucose or lactate, enter the stationary phase . The experiments performed so far suggest that the enzyme purified in this study is the external NADH:Q6 oxidoreductase, bound to the mitochondrial inner membrane and that it is involved in the oxidation of cytosolic NADH . The relation of this enzyme with respect to various other NADH dehydrogenases from yeast and plant mitochondria is discussed.

Gene, 1988 Sep 7, 68(2), 249 - 57
Expression of chicken beta-actin in Saccharomyces cerevisiae; Karlsson R; Actin interacts with a number of so-called actin-binding proteins which participate at various stages of the cell motility process such as regulation of filament formation, assembly and disassembly of filaments, force generation and depolymerization . Gene technology makes a precise mapping of the interacting surfaces on the actin molecules possible by studying specifically designed actin mutants expressed in a suitable organism . In addition, the production of engineered actin will become increasingly important when the three-dimensional structure of actin is determined . Chicken beta-actin can be produced in large quantities in Escherichia coli but such actin shows only a limited biological activity and thus seems to be of minor interest in future studies of structure-function relationships of this molecule . To circumvent the problem of a denatured bacterial protein, the yeast Saccharomyces cerevisiae was chosen as an alternative organism to express actin . This paper describes the expression, isolation and characterization of the yeast-produced chicken beta-actin . From a 12-liter culture of yeast cells, 500 micrograms of polymerizable beta-actin was isolated.

Biochim Biophys Acta, 1988 Sep 7, 950(3), 385 - 94
The expression of functional ricin B-chain in Saccharomyces cerevisiae; Richardson PT et al.; Yeast cells transformed with plasmids containing ricin B-chain coding sequences expressed this heterologous protein . When ricin B-chain was expressed in a form which resulted in its deposition in the yeast cytosol it formed insoluble aggregates which were devoid of galactose-binding activity . In contrast, when DNA fusions were constructed, in which the B-chain coding sequence was preceded by either the preproalpha-factor leader sequence or the native preproricin signal sequence, the recombinant B-chain products were soluble and biologically active . Both the homologous yeast signal peptide and the heterologous plant signal peptide directed the expressed product into the lumen of the yeast endoplasmic reticulum . As a result, the recombinant B-chain products were processed at the N-terminus, glycosylated and folded into an active conformation, presumably stabilized by correct intrachain disulphide bond formation.

J Biol Chem, 1988 Sep 5, 263(25), 12571 - 5
Calculation of the three-dimensional structure of Saccharomyces cerevisiae cytochrome b inserted in a lipid matrix; Brasseur R; Cytochrome b is an integral membrane protein, which forms the core of the ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex . A computer-aided three-dimensional modeling procedure was carried out in four steps . First, the candidate hydrophobic helices were searched for throughout the protein primary sequence by a computer procedure based upon the method of Eisenberg; second, a secondary helical structure was imposed to the transmembrane peptides; third, the helical segments at a lipid-water interface were oriented, and finally the possible interactions between helices with similar properties were investigated . This procedure enabled the identification of nine hydrophobic segments, of which eight are membrane-spanning helices while one has amphipathic properties . Three hydrophilic receptor-binding domains were also identified . Based upon their hydrophobicity profiles, the transmembrane helices could be associated in pairs inside the lipid bilayer . In our folding model proposed for cytochrome b, all mutation sites are not only located on the same side of the membrane but are also in close proximity in the three-dimensional structure . Inhibitor resistance mutational sites which were recently characterized (di Rago, J.-P., and Colson, A.-M . (1988) J . Biol . Chem . 263, 12564-12570) have been located on this model . Moreover, the receptor-binding domains and the mutation sites are close neighbors in the three-dimensional spatial representation.

J Biol Chem, 1988 Sep 5, 263(25), 12564 - 70
Molecular basis for resistance to antimycin and diuron, Q-cycle inhibitors acting at the Qi site in the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae; di Rago JP et al.; The bc1 complex of the mitochondrial respiratory chain transfers electrons from ubiquinol to cytochrome c oxidase . Cytochrome b, a transmembranous protein, is thought to form a transmembrane electron circuit, transferring electrons between two ubiquinone redox sites, (Qi) and (Qo), respectively, near the inner and outer sides of the inner mitochondrial membrane . Antimycin and diuron appear to block cytochrome b oxidation-reduction at one ubiquinone site, presumably Qi . The cytochrome b gene is carried by the organelle DNA . Yeast mutants resistant to antimycin and diuron have been previously isolated and mapped to specific loci of the cytochrome b gene . In the present work the mutated amino acid residues from nine antimycin- and three diuron-resistant mutants have been identified by sequencing the relevant segments of the resistant cytochrome b gene . The sequencings were performed by primer extension in the presence of dideoxynucleotides on total mitochondrial RNA preparations using reverse transcriptase . Regions of the cytochrome b protein affecting the inhibitor and putative quinone-binding sites have been defined.

J Gen Microbiol, 1988 Sep, 134 ( Pt 9), 2481 - 91
Identification of a 31 kDa protein in Saccharomyces cerevisiae whose phosphorylation is controlled negatively by the CDC25 gene product; Tripp ML et al.; Phosphoprotein patterns in two mutants of Saccharomyces cerevisiae, cdc25-20(ts) and cdc25-20(ts) bcy1, were analysed by two-dimensional polyacrylamide gel electrophoresis . Comparison with the phosphoprotein patterns of the mutants cyr1-2(ts) and bcy1, analysed in a previous study, demonstrated not only that the CDC25 gene product is a positive element in the regulation of adenylyl cyclase activity, as suggested by recent studies, but that it is also a negative element in the phosphorylation of a 31 kDa protein (p31c and p31d), a protein whose phosphorylation is correlated with cell cycle arrest, and dephosphorylation with cell cycle initiation, respectively . Moreover, the phosphorylation phenotype of p31c and p31d suggests that the activity of the CDC25 protein is subject to feedback regulation by cAMP-dependent protein kinase, and that the CDC25 protein is a key element in an ammonium (NH+4) signal-response system.

J Gen Microbiol, 1988 Sep, 134 ( Pt 9), 2475 - 80
The cdc30 mutation in Saccharomyces cerevisiae affects phosphoglucose isomerase, the cell cycle and sporulation; Dickinson JR et al.; Spontaneous revertants of the cdc30 mutation in Saccharomyces cerevisiae simultaneously regained the ability to grow and divide at 36.5 degrees C on glucose-containing media along with a more thermostable phosphoglucose isomerase (PGI) . An independently isolated allele of cdc30 gave a similar phenotype to that previously described including temperature-sensitivity of PGI . Isoelectric focussing allowed the separation of two isoenzymes of PGI . These results all support the idea that two genes--PGI1 and CDC30--are responsible for PGI activity in yeast . Diploid strains homozygous for the cdc30 mutation sporulated poorly in potassium acetate irrespective of whether the cells had previously been cultured at a temperature that was permissive or restrictive for cell cycle progression . This was not surprising because a strain defective in PGI would not be expected to be able to complete the gluconeogenic events of sporulation.

Mol Gen Genet, 1988 Sep, 214(1), 162 - 4
PHO85, a negative regulator of the PHO system, is a homolog of the protein kinase gene, CDC28, of Saccharomyces cerevisiae; Toh-e A et al.; The product of the PHO85 gene, which encodes one of the negative regulatory factors of the PHO system in Saccharomyces cerevisiae, shows significant amino acid sequence homology with the CDC28 protein kinase . However, overexpressing PHO85 did not suppress the temperature sensitive phenotype of the cdc28-1 mutation . The nucleotide sequence of the PHO85 gene strongly suggests the presence of an intron near the sequence encoding the N-terminal region.

Mol Cell Biol, 1988 Sep, 8(9), 3918 - 28
Efficient repair of HO-induced chromosomal breaks in Saccharomyces cerevisiae by recombination between flanking homologous sequences; Rudin N et al.; Novel recombinational repair of a site-specific double-strand break (DSB) in a yeast chromosome was investigated . When the recognition site for the HO endonuclease enzyme is embedded in nonyeast sequences and placed between two regions of homology, expression of HO endonuclease stimulates recombination between the homologous flanking regions to yield a deletion, the apparent product of an intrachromosomal exchange between direct repeats . This deletion-repair event is very efficient, thus preventing essentially all the potential lethality due to the persistence of a DSB . Interestingly, unlike previous studies involving spontaneous recombination between chromosomal repeats, the recombination events stimulated by HO-induced DSBs are accompanied by loss of the sequences separating the homologous regions greater than 99.5% of the time . Repair is dependent on the RAD52 gene . The deletion-repair event provides an in vivo assay for the sensitivity of any particular recognition site to HO cleavage . By taking advantage of a galactose-inducible HO gene, it has been possible to follow the kinetics of this event at the DNA level and to search for intermediates in this reaction . Deletion-repair requires approximately 45 min and is inhibited when cycloheximide is added after HO endonuclease cleavage.

Mol Cell Biol, 1988 Sep, 8(9), 3777 - 83
Role of STE genes in the mating factor signaling pathway mediated by GPA1 in Saccharomyces cerevisiae; Nakayama N et al.; The ste mutants (ste2, ste4, ste5, ste7, ste11, and ste12) are insensitive to mating factors and are, therefore, sterile . Roles of the STE gene products in the GPA1-mediated mating factor signaling pathway were studied by using ste gpa1 double mutants . Mating efficiency of a ste2 mutant defective in the alpha-factor receptor increased 1,000-fold in a gpa1 background, while G1 arrest and aberrant morphology (shmoo) caused by gpa1 were not suppressed by ste2 . Furthermore, the steady-state level of the FUS1 transcript, which normally increases in response to mating factors, was also elevated when the GPA1 function was impaired . These results suggest that the GPA1 protein functions downstream of the STE2 receptor . Conversely, the sterility of ste4, ste5, ste7, ste11, and ste12 mutants was not suppressed by gpa1, but the lethal phenotype of gpa1 was suppressed by these ste mutations . Northern (RNA) blotting analysis revealed that the ste7, ste11, and ste12 mutations caused reductions of 50 to 70% in the steady-state levels of the GPA1 transcript, while ste4 had a slight effect and ste5 had no effect . This implies that the suppression by ste7, ste11, and ste12 could be due to reduced syntheses of additional components, including an effector, and that suppression by ste4 and ste5 may result from direct effects on the signaling pathway . The STE4, STE5, STE7, STE11, and STE12 products, therefore, appear to specify components of the signal transduction machinery, directly or indirectly, which function together with or downstream of GPA1.

Mol Cell Biol, 1988 Sep, 8(9), 3647 - 60
Structure and regulation of a nuclear gene in Saccharomyces cerevisiae that specifies MRP13, a protein of the small subunit of the mitochondrial ribosome; Partaledis JA et al.; MRP13 is defined by biochemical criteria as a 35-kilodalton small subunit protein of the yeast mitochondrial ribosome . The MRP13 gene was identified by immunological screening of a yeast genomic library in lambda gt11 and a functional copy of the gene has been cloned on a 2.2-kilobase BglII fragment . Sequencing of this fragment showed that the MRP13 coding region specifies a 324-amino-acid basic protein with a calculated Mr of 37,366 . Computer searches failed to reveal any significant sequence similarity to previously identified ribosomal proteins or to the sequences in the current National Biomedical Research Foundation data base . Cells carrying disrupted copies of MRP13 lacked the MRP13 protein but were not impaired in either mitochondrial protein synthesis or assembly of 37S ribosomal subunits, indicating that, like L29 and L30 in Escherichia coli (M . Lotti, E . R . Dabbs, R . Hasenbank, M . Stoffler-Meilicke, and G . Stoffler, Mol . Gen . Genet . 192:295-300, 1983), MRP13 is not essential for ribosome synthesis or function . Analysis of the sequence in the MRP13 5'-flanking region revealed the closely linked gene for the cytoplasmic ribosomal protein rp39A . The rp39A coding region began at nucleotide -846 and ended at -325 with respect to the MRP13 translational start . The steady-state levels of the MRP13 mRNA were determined in response to carbon catabolite repression, variation in the mitochondrial genetic background, and increased gene dosage of MRP13 . In {rho+} cells, transcript levels were repressed severalfold by growth in glucose compared with growth in either galactose or nonfermentable carbon sources . In respiratory-deficient strains ({rho0}, {mit-}), however, transcription appeared to be largely derepressed even in the presence of high concentrations of glucose . Despite high levels of the MRP13 transcripts in {rho0} cells, the MRP13 protein did not accumulate, suggesting that the protein is relatively unstable in the absence of ribosome assembly . Cells carrying the MRP13 gene on a multiple-copy plasmid overproduced the mRNA in rough proportion to the gene dosage and the protein in a significant but lesser amount . The results indicate that MRP13 expression is regulated predominantly at the transcriptional level in response to catabolite repression and the cellular capacity for respiration and, in addition, that protein levels appear to be modulated posttranscriptionally by degradation of free copies of the MRP13 protein.

Mol Cell Biol, 1988 Sep, 8(9), 3619 - 26
RAD1, an excision repair gene of Saccharomyces cerevisiae, is also involved in recombination; Schiestl RH et al.; The RAD1 gene of Saccharomyces cerevisiae is required for the incision step of excision repair of damaged DNA . In this paper, we report our observations on the effect of the RAD1 gene on genetic recombination . Mitotic intrachromosomal and interchromosomal recombination in RAD+, rad1, rad52, and other rad mutant strains was examined . The rad1 deletion mutation and some rad1 point mutations reduced the frequency of intrachromosomal recombination of a his3 duplication, in which one his3 allele is deleted at the 3' end while the other his3 allele is deleted at the 5' end . Mutations in the other excision repair genes, RAD2, RAD3, and RAD4, did not lower recombination frequencies in the his3 duplication . As expected, recombination between the his3 deletion alleles in the duplication was reduced in the rad52 mutant . The frequency of HIS3+ recombinants fell synergistically in the rad1 rad52 double mutant, indicating that the RAD1 and RAD52 genes affect this recombination via different pathways . In contrast to the effect of mutations in the RAD52 gene, mutations in the RAD1 gene did not lower intrachromosomal and interchromosomal recombination between heteroalleles that carry point mutations rather than partial deletions; however, the rad1 delta mutation did lower the frequency of integration of linear plasmids and DNA fragments into homologous genomic sequences . We suggest that RAD1 plays a role in recombination after the formation of the recombinogenic substrate.

Genetics, 1988 Sep, 120(1), 63 - 74
Mutational analysis of the GAL4-encoded transcriptional activator protein of Saccharomyces cerevisiae; Johnston M et al.; The GAL4 protein of Saccharomyces cerevisiae binds to DNA upstream of each of six genes and stimulates their transcription . To locate regions of the protein responsible for these processes, we identified and characterized 88 gal4 mutations selected in vivo to reduce the ability to GAL4 protein to activate transcription . These mutations alter two regions of GAL4 protein: the DNA binding domain, and the transcription activation domain . Some mutations in the DNA binding domain that abolish the ability of GAL4 protein to bind to DNA in vitro change amino acid residues proposed to form a zinc finger, confirming that this structure is indeed involved in DNA binding . Four different amino acid changes in the zinc finger appear to reduce (but not abolish) the affinity of GAL4 protein for zinc ions, thereby identifying some of the amino acids involved in forming the zinc-binding structure . Several other mutations that abolish the DNA binding activity of the protein alter the 20 amino acids adjacent to the zinc finger, suggesting that these residues are part of the DNA binding domain . Two amino acid changes in the region adjacent to the zinc finger also appear to affect the ability of GAL4 protein to bind zinc ions, suggesting that this region of the protein can influence the structure of the zinc binding domain . The transcription activation domain of GAL4 protein is remarkably resistant to single amino acid changes: only 4 of the 42 mutations that alter this region of the protein are of the missense type . This observation is consistent with other lines of evidence that GAL4 protein possesses multiple transcription activation domains with unusual sequence flexibility.

Can J Microbiol, 1988 Sep, 34(9), 1102 - 4
A physical method for separating Saccharomyces cerevisiae cells according to their ploidy; Vagvolgyi C et al.; A centrifugation technique, using genetically marked Saccharomyces cerevisiae strains, has been developed to separate Saccharomyces cerevisiae cells of different ploidy levels from exponential phase cultures . The method involves the conversion of yeast cells to protoplasts, the separation of the protoplasts on an osmotically stabilized Nycodenz gradient, and their regeneration . This type of selection may be of importance where selectable markers are not available.

Yeast, 1988 Sep, 4(3), 209 - 17
A non-conserved sequence in the 5'region of the CYH2 intron from Saccharomyces cerevisiae controls splicing efficiency of the pre-mRNA; Swida U et al.; The CYH2 gene from Saccharomyces cerevisiae containing one 510 bp intron is spliced inefficiently . We have shown previously that a non-conserved sequence within the intron is responsible for this low splicing efficiency . Using synthetic oligonucleotides comprising the identified region we show in this report that a very short region contains the specificity to act negatively on the splicing efficiency of the CYH2 gene . Furthermore, this sequence influences the splicing efficiency only when it is placed close to the 5' splice site of the gene . Investigations with chimeric CYH2/beta-actin genes show that this sequence acts independent from its natural surroundings . We propose that this sequence might interact with splicing factor(s).

Genetika, 1988 Sep, 24(9), 1525 - 38
{Nuclear and mitochondrial genes in the biogenesis of Saccharomyces cerevisiae mitochondria}; Sudarikov AB et al.; The mechanisms of interaction of nuclear and mitochondrial genes in biogenesis of mitochondria are discussed in this review . Brief characterization of yeast mitochondrial genes and their products is presented . The mechanism of nuclear and mitochondrial control of expression of the mosaic genes in mitochondria is described . The data on the processing of imported mitochondrial proteins synthesized on cytoplasmic ribosomes are presented . The possibility of existence of common proteins encoded for by common genes and possessing similar functions in the cytoplasm and mitochondria is discussed . A hypothesis is put forward considering the role of common proteins in coordination of nuclear and mitochondrial genes' expression in biogenesis of mitochondria.

Curr Genet, 1988 Sep, 14(3), 191 - 9
High frequency FLP-independent homologous DNA recombination of 2 mu plasmid in the yeast Saccharomyces cerevisiae; Bruschi CV et al.; The purpose of this work is to identify and quantitate in vivo 2 mu plasmid FLP-independent recombination in yeast, using a nonselective assay system for rapid detection of phenotypic expression of the recombination events . A tester plasmid was constructed such that in vivo recombination between 2 mu direct repeat sequences produces the resolution of the plasmid into two circular DNA molecules . This recombinational event is detected as a phenotypic shift from red to white colonies, due to the mitotic loss of the plasmid portion containing the yeast ADE8 gene in a recipient ade1 ade2 ade8 genetic background . In the absence of the 2 mu FLP recombinase and/or its target DNA sequence, recombination is not abolished but rather continues at a high frequency of about 17% . This suggests that the FLP-independent events are mediated by the chromosomally-encoded general homologous recombination system . We therefore conclude that the totality of 2 mu DNA recombination events occurring in FLP+ cells is the contribution of both FLP-mediated and FLP-independent events.

Curr Genet, 1988 Sep, 14(3), 183 - 9
Cosmids carrying Aspergillus terreus DNA can integrate into Saccharomyces cerevisiae chromosome XII via recombination between yeast and foreign DNAs; Shoubochkina EA et al.; A genome clonotheque consisting of 25- to 40-kb Sau3AI fragments of Aspergillus terreus DNA was constructed in the episomal cosmid vector pES33 containing the yeast ARG4 gene . From the 475 transformants of cir0 yeast strain ESH-0, 23 stable Arg+ transformants were independently selected . Genetic and Southern analysis of these stable transformants showed that 39% arose as a result of recombination between cloned A . terreus DNA sequences and yeast chromosome XII . The recombination events most likely occurred in the regions of homology within the rDNA clusters of A . terreus and Saccharomyces cerevisiae.

Mutat Res, 1988 Sep, 201(1), 213 - 8
'Petite' mutagenesis in Saccharomyces cerevisiae by a series of 2,7-di-alkyl-substituted derivatives of proflavine with differing DNA-binding properties; Ferguson LR et al.; The ability of proflavine (3,6-diaminoacridine) and its 2,7-dimethyl, 2,7-diethyl, 2,7-diisopropyl and 2,7-di-tert.-butyl derivatives to induce the 'petite' mutation in Saccharomyces cerevisiae has been studied in relation to the DNA-binding properties of the compounds . The nature of the binding has been investigated by nuclear magnetic resonance techniques, and the results support and clarify earlier suggestions that the first 3 members of the series intercalate into DNA while the diisopropyl and di-tert.-butyl compounds do not . Toxicity of the drugs was primarily associated with their mode of DNA binding, but lipophilicity had an important secondary effect . It seems likely that the toxic properties of the more lipophilic DNA-intercalating members of the series mask their potential for 'petite' mutagenesis.

Mutat Res, 1988 Sep, 194(2), 151 - 63
The induction of rho- mutants by UV or gamma-rays is independent of the nuclear recombinational repair pathway in Saccharomyces cerevisiae; Heude M; In order to discover whether the nuclear recombinational repair pathway also acts on lesions induced in mitochondrial DNA (mtDNA), the possible role of the RAD50, -51, -52, -55 and -56 genes on the induction of rho- mutants by radiations was studied . Such induction appeared to be independent of this pathway . Nevertheless, an efficient induction of respiration-deficient mutants was observed in gamma-irradiated rad52 diploids . We demonstrate that these mutants do not result from a lack of mtDNA repair, but from chromosome losses induced by gamma-rays . Such an impairment of the respiratory ability of diploids by chromosome losses was effectively observed in the aneuploid progeny of unirradiated RAD+ cdc6 diploids incubated at the restrictive temperature.

Mol Cell Biol, 1988 Sep, 8(9), 3717 - 25
Adenovirus transcriptional regulatory regions are conserved in mammalian cells and Saccharomyces cerevisiae; Kornuc M et al.; The adenovirus early region 3 (E3) promoter is an early viral promoter which is strongly induced by the adenovirus transactivator protein E1A . DNase I footprinting with HeLa cell extracts has identified four factor-binding domains which appear to be involved in basal and E1A-induced transcriptional regulation . These binding domains may bind TATA region-binding factors (site I), the CREB/ATF protein (site II), the AP-1 protein (site III), and nuclear factor I/CTF (site IV) . Recently, it has been shown that the DNA-binding domain of transcription factor AP-1 has homology with the yeast transcription factor GCN4 and that the yeast transactivator protein GAL4 is able to stimulate transcription in HeLa cells from promoters containing GAL4-binding sites . These results suggest an evolutionary conservation of both transcription factors and the mechanisms responsible for transcriptional activation in Saccharomyces cerevisiae and higher eucaryotic organisms . To determine whether similar patterns of transcriptional regulation were seen with the E3 promoter in HeLa and yeast cells, the E3 promoter fused to the chloramphenicol acetyltransferase (cat) gene was cloned into a high-copy-number plasmid and stably introduced into yeast cells . S1 analysis revealed that similar E3 promoter mRNA start sites were found in yeast and HeLa cells . DNase I footprinting with partially purified yeast extracts revealed that four regions of the E3 promoter were protected . Several of these regions were similar to binding sites determined by using HeLa cell extracts . Oligonucleotide mutagenesis of these binding domains indicated their importance in the transcriptional regulation of the E3 promoter in yeast cells . These results suggest that similar cellular transcription factor-binding sites may be involved in the regulation of promoters in both yeast and mammalian cells.

Mol Gen Genet, 1988 Sep, 214(1), 165 - 9
Structure of the ARO3 gene of Saccharomyces cerevisiae; Paravicini G et al.; In Saccharomyces cerevisiae, the genes ARO3 and ARO4 encode isoenzymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase . Both genes are derepressed seven-fold under the general control of amino acid biosynthesis . A previously isolated 1.7 kb fragment containing the ARO3 gene and the 5'- and 3'-flanking regions was sequenced . The endpoints of the ARO3 transcript coding for a 370 amino acid protein were mapped by primer extension experiments and S1 nuclease digestion . Promoter elements involved in transcription initiation and responsible for the strong general control derepression response are discussed.

Int J Radiat Biol, 1988 Sep, 54(3), 367 - 80
Radiation-induced mating-type switching in the yeast Saccharomyces cerevisiae; Luggen-Holscher J et al.; Haploid yeast cells possess two different mating types which are controlled genetically by the MAT locus . Information of the opposite mating type is stored on the same chromosome but not expressed . Radiation may initiate a gene conversion event leading to 'mating-type switching' . This was studied by using X-rays and 254 nm ultraviolet light . X-ray-induced mating type switching shows an oxygen enhancement ratio of 2.9 which is higher than that for survival (1.8) and equals that for double-strand break induction . Mating-type switching by UV is not photoreactivable and depends on a functioning excision repair system . The results are compatible with the interpretation that mating type switching is initiated by a double-strand break in the MAT coding region.

Mol Cell Biol, 1988 Sep, 8(9), 3571 - 81
Transpositional competence and transcription of endogenous Ty elements in Saccharomyces cerevisiae: implications for regulation of transposition; Curcio MJ et al.; Transposition of Ty elements in the yeast Saccharomyces cerevisiae occurs through an RNA intermediate . Although Ty RNA accounts for 5 to 10% of the total polyadenylated RNA in a haploid cell, the transposition frequency is only 10(-7) to 10(-8) per gene . To determine whether Ty elements native to the yeast genome are transpositionally competent, two elements were fused to the GAL1 promoter and tested for their ability to transpose . These native elements, Ty1-588 and Ty2-117, transposed at high levels when the GAL1 promoter was induced . Three Ty's identified as spontaneous transpositions in specific target genes were also tested . Of these three, Ty2-917 and the previously characterized element Ty1-H3 were shown to be transpositionally competent . The third element, Ty1-H1, was transposition defective . In addition, we marked the chromosomal copy of Ty1-588 with the NEO gene and demonstrated that Ty1-588NEO was actively transcribed in yeast cells . Ty1-588NEO transcription was regulated by the SPT3 and MAT loci in the same manner as that observed for Ty's collectively . These results indicate that the yeast genome contains functional Ty elements . The presence of a transpositionally competent, actively transcribed element suggests that regulation of Ty transposition occurs at a posttranscriptional level.

Yeast, 1988 Sep, 4(3), 227 - 32
Substrate specificity of the phosphorylated fructose-1,6-bisphosphatase dephosphorylating protein phosphatase from Saccharomyces cerevisiae; Manhart A et al.; Enzymatic dephosphorylation of the phosphorylated forms of five different yeast enzymes has been studied: fructose-1,6-bisphosphatase, glycogen phosphorylase, neutral trehalase, NAD-glutamate dehydrogenase and 6-phosphofructo-2-kinase . Phosphorylated fructose-1,6-bisphosphatase and phosphorylated 6-phosphofructo-2-kinase were present in extracts of starved yeast cells which had been incubated for 10 min with glucose . Phosphorylated glycogen phosphorylase, neutral trehalase and NAD-glutamate dehydrogenase were obtained by incubation of yeast extract with ATP, cyclic AMP and Mg2+ . After incubation with commercially available preparations of alkaline phosphatase, all five phosphorylated enzymes studied showed the changes in catalytic activity that would be expected as a consequence of dephosphorylation . The recently purified yeast enzyme which dephosphorylates phosphorylated fructose-1,6-bisophosphatase (Horn and Holzer (1987) however, was found to be active only with the phosphorylated fructose-1,6-bisphosphatase, but not with the other four phosphorylated enzymes studied . By contrast, a crude extract from yeast showed dephosphorylating activity towards all five substrates . Substrate specificity with the five phosphorylated enzymes studied of different phosphoprotein phosphatases from yeast prepared by others is discussed.

J Bacteriol, 1988 Sep, 170(9), 3870 - 5
Selection of in vivo deletions in Saccharomyces cerevisiae; Bitoun R et al.; A general screening procedure has been devised for the selection of in vivo-generated deletions in haploid Saccharomyces cerevisiae cells . It is based on the introduction into a cyh2 host (resistant to the drug cycloheximide) of a tandemly duplicated CYH2 gene (a dominant allele, conferring sensitivity to cycloheximide), and subsequent selection for Cyhr derivatives . The duplicated CYH2 gene has been introduced on CEN ARS plasmids or integrated into chromosome II . A variable but significant proportion of the Cyhr derivatives of such transformants were deletion mutants in which both CYH2 copies had suffered deletions . Some of the deletions extended into sequences outside the tandemly duplicated CYH2 gene . A total of 61 independently selected deletions ranged in length from 3.1 to over 20 kilobases and had no obvious preferred endpoints . Restriction analysis showed that other frequently isolated Cyhr derivatives appeared to retain one of the two CYH2 copies . Such single-copy derivatives of CEN ARS plasmids did not contain a functional CYH2 gene . The frequency of true deletions in CEN ARS plasmids, of approximately 10(-7) per viable cell, was comparable in RAD52 and rad52 strains . Chromosomal deletions, which occurred at a frequency of approximately 10(-8) per viable cell, were observed only in rad52 hosts . Derivatives exhibiting an additional altered phenotype, such as the inactivation of a neighboring gene or, less frequently, the transcriptional activation of a previously silent gene, were isolated by screening deletion mutants . These results show that the method described can be used for in vivo deletion mapping or for the generation of gene fusions.

Mutat Res, 1988 Sep, 201(1), 9 - 16
Caffeine interactions with methyl methanesulphonate, hycanthone, benlate, and cadmium chloride in chromosomal meiotic segregation of Saccharomyces cerevisiae; Sora S et al.; Interactions of caffeine with chemicals known for their effects on chromosomal segregation during meiosis of Saccharomyces cerevisiae were studied . It appears that caffeine does interfere with the action of other compounds during the different phases of meiosis . Treatments with methyl methanesulphonate (MMS) and cadmium chloride (CdCl2) resulted in a synergistic effect consisting of an increase in the frequency of recombination . The greatest effects were found on the induction of diploid spores: MMS, hycanthone, and distamycin demonstrated strong, benlate little synergistic action . CdCl2 demonstrated antagonism to caffeine by counter-inhibiting its effect on the induction of diploids . Concerning disomic induction: caffeine reduced (or left unchanged) the effect on non-disjunction when MMS and hycanthone were used . Simple additive effects were caused in conjunction with distamycin, benlate, and (in small doses) CdCl2 . 2 mg of caffeine/ml in treatments with CdCl2 resulted in a very high frequency of disomic clones.

J Biol Chem, 1988 Aug 25, 263(24), 11948 - 53
Asparaginase II of Saccharomyces cerevisiae . Characterization of the ASP3 gene; Kim KW et al.; Purified preparations of asparaginase II of Saccharomyces cerevisiae exhibit two protein bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Cloning and sequencing of the ASP3 gene, and partial amino acid sequencing as asparaginase II, imply that both bands are encoded by ASP3 but have different N termini . Northern blot analysis using the cloned ASP3 gene as a probe indicates that nitrogen catabolite repression of asparaginase II is achieved by alteration in mRNA levels . Deletion of sequences greater than 600 base pairs upstream from the initiation AUG codon results in an altered response to certain nitrogen sources in strains containing the truncated gene.

J Biol Chem, 1988 Aug 25, 263(24), 11692 - 6
Biosynthesis of diphthamide in Saccharomyces cerevisiae . Partial purification and characterization of a specific S-adenosylmethionine:elongation factor 2 methyltransferase; Chen JY et al.; The inactivation of elongation factor 2 (EF-2) by diphtheria toxin requires the presence of a post-translationally modified histidine residue in EF-2 . This residue, diphthamide, has the structure 2-{3-carboxyamido-3-(trimethylammonio)propyl}histidine . The present work was undertaken to study the pathway of diphthamide biosynthesis using diphtheria toxin-resistant yeast mutants (Chen . J.-Y., Bodley, J . W., and Livingston, D . M . (1985) Mol . Cell . Biol . 5, 3357-3360) which are defective in diphthamide formation . We demonstrate here that one of these mutants (dph5) contains a toxin-resistant form of EF-2 which can be converted in vitro to a toxin-sensitive form through the action of an enzyme present in other yeast strains . Both this toxin-resistant EF-2 and its modifying enzyme have been partially purified and evidence is presented that the modifying enzyme is a specific S-adenosylmethionine:EF-2 methyltransferase . In vitro complementation to diphtheria toxin sensitivity required S-adenosylmethionine, and when partially purified components were incubated with {methyl-3H}S-adenosylmethionine, label was incorporated specifically into EF-2 . Hydrolysis of labeled EF-2 yielded diphthine (the unamidated form of diphthamide) and a single chromatographically separable labeling intermediate . We conclude that the S-adenosylmethionine:EF-2 methyltransferase adds at least the last two of the three methyl groups present in diphthine and that this modification is sufficient to create diphtheria toxin sensitivity . Evidence is also presented for the existence of an ATP-dependent amidating enzyme which catalyzes the final step in the biosynthesis of diphthamide in EF-2.

J Biol Chem, 1988 Aug 25, 263(24), 11675 - 82
Purification and properties of ferrochelatase from the yeast Saccharomyces cerevisiae . Evidence for a precursor form of the protein; Camadro JM et al.; Ferrochelatase was purified to homogeneity from yeast mitochondrial membranes and found to be a 40-kDa polypeptide with a pI at 6.3 . Fatty acids were absolutely necessary to measure the activity in vitro . The Michaelis constants for protoporphyrin IX (9 x 10(-8) M), ferrous iron (1.6 x 10(-7) M), and zinc (9 x 10(-6) M) were determined on purified enzyme preparations in the presence of dithiothreitol . However, the Km for zinc was lower when measured in the absence of dithiothreitol (K-m(Zn2+) = 2.5 x 10(-7) M, Km(protoporphyrin) unchanged) . The maximum velocities of the enzyme were 35,000 nmol of heme/h/mg of protein and 27,000 nmol of zinc-protoporphyrin/h/mg of protein . Antibodies against yeast ferrochelatase were raised in rabbits and used in studies on the biogenesis of the enzyme . Ferrochelatase is synthesized as a higher molecular weight precursor (Mr = 44,000) that is very rapidly matured in vivo to the Mr = 40,000 membrane-bound form . This precursor form of ferrochelatase was immunoprecipitated from in vitro translation (in a rabbit reticulocyte lysate system) of total yeast RNAs . The antibodies were used to characterize two yeast mutant strains deficient in ferrochelatase activity as being devoid of immunodetectable protein in vivo and ferrochelatase mRNA in vitro translation product . The N-terminal amino acid sequence of the purified protein has been established and was found to be frayed.

J Biol Chem, 1988 Aug 15, 263(23), 11145 - 9
Metabolic changes in Saccharomyces cerevisiae strains lacking citrate synthases; Kispal G et al.; The yeast, Saccharomyces cerevisiae, contains two citrate synthase isoenzymes, mitochondrial (CS1) and cytosolic (CS2) . In this study, we have examined the metabolic consequences of the absence of CS1, CS2, and both isoenzymes in the respective mutant strains CS1-, CS2-, and CS1-CS2- . No significant differences were found in the growth rates of the parental, CS1-, or CS2- strains when grown in the single carbon sources galactose, glycerol, lactate, pyruvate, or glutamate . However, in nonfermentable carbon sources, the lag period in growth of CS1- was approximately 4 times that of the parental strain and the CS2- mutant . This difference was found even in glutamate . The CS1- mutant failed to grow on acetate in either complete or minimal liquid medium . Total cellular citrate concentration in the CS1- compared to the parental strain was higher when the cells were grown in lactate or pyruvate . On these same substrates, the malate concentration was 2-fold higher in the CS1-mutant when compared to the parental or CS2- strains . The production of 14CO2 by CS1- from {1-14C}acetate was 36% and that from {2-14C}acetate was 9.2% of the amount from the parental or CS2- strains . The 14CO2 production from {1-14C}glutamate was 28% and 20% in CS1- and CS1-CS2-, respectively, compared to the parental strain . Since these results are not easily explained solely by the absence of mitochondrial citrate synthase enzyme, we also determined the activity of some other enzymes of the citric acid cycle and electron transport chain . We found decreased activity of pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and aconitase, while the rest of the citric acid cycle enzymes and oxidative enzymes did not change significantly . The same changes in enzyme activities were found in two different yeast strains carrying the same citrate synthase mutations.

FEBS Lett, 1988 Aug 15, 236(1), 195 - 200
Isolation and primary structure of the gene encoding fructose-1,6-bisphosphatase from Saccharomyces cerevisiae; Entian KD et al.; The gene encoding Saccharomyces cerevisiae fructose-1,6-bisphosphatase (FBP1) was isolated . Constructed fbp1::HIS3 null mutants were unable to grow with ethanol, and growth was restored after transformation with the cloned fbp gene . The gene codes for a protein of 347 amino acid residues with an Mr of 38131 . Homology with the pig kidney cortex and the sheep liver enzyme is 47.7% and 46.6%, respectively, within a central core of 328 amino acid residues . The cloned promoter size was 318 bp and allowed only low level expression of the gene . This indicates a positive activation site (UAS) upstream of the cloned DNA fragment.

Biochim Biophys Acta, 1988 Aug 10, 955(3), 346 - 51
Synergistic binding of glucose and aluminium ATP to hexokinase from Saccharomyces cerevisiae; Woolfitt AR et al.; The binding of glucose, AlATP and AlADP to the monomeric and dimeric forms of the native yeast hexokinase PII isoenzyme and to the proteolytically modified SII monomeric form was monitored at pH 6.7 by the concomitant quenching of intrinsic protein fluorescence . No fluorescence changes were observed when free enzyme was mixed with AlATP at concentrations up to 7500 microM . In the presence of saturating concentrations of glucose, the maximal quenching of fluorescence induced by AlATP was between 1.5 and 3.5% depending on species, and the average value of {L}0.5, the concentration of ligand at half-saturation, over all monomeric species was 0.9 +/- 0.4 microM . The presence of saturating concentrations of AlATP diminished {L}0.5 for glucose binding by between 260- and 670-fold for hexokinase PII and SII monomers, respectively (dependent on the ionic strength), and by almost 4000-fold for PII dimer . The data demonstrate extremely strong synergistic interactions in the binding of glucose and AlATP to yeast hexokinase, arising as a consequence of conformational changes in the free enzyme induced by glucose and in enzyme-glucose complex induced by AlATP . The synergistic interactions of glucose and AlATP are related to their kinetic synergism and to the ability of AlATP to act as a powerful inhibitor of the hexokinase reaction.

J Mol Biol, 1988 Aug 5, 202(3), 667 - 72
Testing for intron function in the essential Saccharomyces cerevisiae tRNA(SerUCG) gene; Ho CK et al.; The gene sup61+, which codes for the essential Saccharomyces cerevisiae tRNA(SerUCG), is the only single-copy tRNA gene in this organism know to contain an intron . To assess the role of this intron in tRNA gene expression, an intron-deleted sup61+ gene was constructed in vitro and introduced into the yeast genome . Isogenic intron- and intron+ strains were found to be indistinguishable by criteria that include growth rates, ability to undergo meiosis, levels of mature tRNA(SerUCG) transcribed in vivo, and the suppressor efficiency of amber- and ochre-specific alleles of this gene.

J Biol Chem, 1988 Aug 5, 263(22), 10836 - 42
The STE2 gene product is the ligand-binding component of the alpha-factor receptor of Saccharomyces cerevisiae; Blumer KJ et al.; The STE2 gene of Saccharomyces cerevisiae encodes a 431-residue protein containing seven hydrophobic segments that is thought to be an essential component of the cell-surface receptor for alpha-factor in MATa haploids . Methods were devised to prepare membrane fractions from MATa cells that retained high levels of alpha-factor binding activity, consistent with the view that the alpha-factor receptor resides in the plasma membrane . To demonstrate that the membrane constituent responsible for alpha-factor binding was the STE2 polypeptide, specific antibodies were generated and used to identify STE2-related polypeptides by radiolabeling, immunoprecipitation, and polyacrylamide gel electrophoresis . Under conditions of complete solubilization, the major form of the STE2 gene product detected was a glycoprotein with an apparent molecular weight of 49,000 . Affinity labeling of yeast membrane preparations by chemical cross-linking to 35S-alpha-factor indicated that a molecule of 49,000 molecular weight was the major alpha-factor-binding species . This alpha-factor-binding species was shown to be the product of the STE2 gene in three ways . First, MATa haploids carrying the STE2 gene on a multicopy plasmid overproduced alpha-factor binding activity about 15-fold . Second, MATa cells completely lacking a STE2 gene showed only nonspecific binding of alpha-factor (equivalent to the level displayed by MAT alpha haploids) and possessed no species that could be cross-linked to 35S-alpha-factor . Third, MATa cells expressing a truncated but functional STE2 gene (in which the COOH-terminal 135-hydrophilic residues were deleted) produced a protein detected by cross-linking to 35S-alpha-factor of apparent molecular weight 33,000, close to the size expected for the predicted abbreviated STE2 polypeptide . These findings demonstrate unequivocally that the STE2 gene product is the membrane component responsible for the ligand recognition function of the yeast alpha-factor receptor.

Genetics, 1988 Aug, 119(4), 797 - 804
Suppressors of a gpa1 mutation cause sterility in Saccharomyces cerevisiae; Miyajima I et al.; The Saccharomyces cerevisiae GPA1 gene encodes a protein highly homologous to the alpha subunit of mammalian G proteins and is essential for haploid cell growth . We have selected 77 mutants able to suppress the lethality resulting from disruption of GPA1 (gpa1::HIS3) . Two strains bearing either of two recessive mutations, sgp1 and sgp2, in combination with the disruption mutation, showed a cell type nonspecific sterile phenotype, yet expressed the major alpha-factor gene (MF alpha 1) as judged by the ability to express a MF alpha 1-lacZ fusion gene . The sgp1 mutation was closely linked to gpa1::HIS3 and probably occurred at the GPA1 locus . The sgp2 mutation was not linked to GPA1 and was different from the previously identified cell type nonspecific sterile mutations (ste4, ste5, ste7, ste11 and ste12) . sgp2 GPA1 cells showed a fertile phenotype, indicating that the mating defect caused by sgp2 is associated with the loss of GPA1 function . While expression of a FUS1-lacZ fusion gene was induced in wild-type cells by the addition of alpha-factor, mutants bearing sgp1 or sgp2 as well as gpa1::HIS3 constitutively expressed FUS1-lacZ . These observations suggest that GPA1 (SGP1) and SGP2 are involved in mating factor-mediated signal transduction, which causes both cell cycle arrest in the late G1 phase and induction of genes necessary for mating such as FUS1.

Biochem Genet, 1988 Aug, 26(7-8), 503 - 10
Indication for deletion of two introns in the oxi-3 gene of a respiratory-competent Saccharomyces cerevisiae strain; Mink M; Physical mapping of the mitochondrial DNA of the wild-type Saccharomyces cerevisiae strain RXII revealed that most of the restriction sites as well as the location of the apocytochrome b gene were identical in comparison with the known maps of the mitochondrial genome in other Saccharomyces cerevisiae strains . In the middle of the SalI linearized map of the RXII mitochondrial DNA, a deletion was detected which resulted in the loss of two EcoRI and one BamHI restriction sites . The corresponding region, however, exists in most other laboratory strains of Saccharomyces mapped so far . This region overlaps the introns aI2 and aI3 surrounding exon A3 sequences of the subunit 1 of the cytochrome oxidase gene . The nucleotide sequence of the subunit 1 gene showed that the BamHI site was located close to the aI3-A4 intron-exon junction and the distal EcoRI site close to the aI2-A2 boundary . I therefore conclude that these two introns are deleted in the mitochondrial genome of strain RXII . The exon A3 must have been conserved since this strain was respiratory competent . This result, while being a good example of the morphological diversity of a genome with the same function, may contribute to an understanding of the role of introns in the mitochondrial split genes in yeast.

Mol Cell Biol, 1988 Aug, 8(8), 3556 - 9
CDC33 encodes mRNA cap-binding protein eIF-4E of Saccharomyces cerevisiae; Brenner C et al.; The bcy1 mutation makes the cdc33 start mutant arrest at random points in the cell cycle instead of only at G1 . We cloned and sequenced CDC33 . This coding sequence is identical to that of the gene encoding the Saccharomyces cerevisiae 24-kilodalton mRNA cap-binding protein, eIF-4E.

Mol Gen Genet, 1988 Aug, 213(2-3), 421 - 4
Repair of double-strand breaks in plasmid DNA in the yeast Saccharomyces cerevisiae; Perera JR et al.; We studied the repair of double-strand breaks (DSB) in plasmid DNA introduced into haploid cells of the yeast Saccharomyces cerevisiae . The efficiency of repair was estimated from the frequency of transformation of the cells by an autonomously replicated linearized plasmid . The frequency of "lithium" transformation of Rad+ cells was increased greatly (by 1 order of magnitude and more) compared with that for circular DNA if the plasmid was initially linearized at the XhoI site within the LYS2 gene . This effect is due to recombinational repair of the plasmid DNA . Mutations rad52, rad53, rad54 and rad57 suppress the repair of DSB in plasmid DNA . The kinetics of DSB repair in plasmid DNA are biphasic: the first phase is completed within 1 h and the second within 14-18 h of incubating cells on selective medium.

J Biochem (Tokyo), 1988 Aug, 104(2), 192 - 5
A study on the identities of the three species of chromatin-associated proteinases in a mutant of Saccharomyces cerevisiae which lacks four major vacuolar proteinases; Motizuki M et al.; Using mutant strain ABYS1 of Saccharomyces cerevisiae lacking four main vacuolar proteinases, proteinase A, proteinase B, carboxypeptidase Y, and carboxypeptidase S, we examined the identities of chromatin-associated proteinases, ruling out possible contamination of the chromatin fraction by them . The chromatin of strain ABYS1 showed three peaks of proteolytic activity at pH 4, 7, and 11, and these activities were found to be derived from three species of proteinases, the aspartic, serine neutral, and serine alkaline ones . As these chromatin-associated proteinases of strain ABYS1 were identical in both quality and quantity to those of wild-type strain of yeast, we suggest that the yeast chromatin contains three species of specific proteinases as essential components.

Eur J Cell Biol, 1988 Aug, 46(3), 554 - 63
Nucleolar and nuclear envelope proteins of the yeast Saccharomyces cerevisiae; Hurt EC et al.; We have developed a fast and reliable purification protocol to obtain yeast nuclei in intact and pure form and in a reasonable yield . The purified nuclei appear homogeneous at the light and electron microscopic level, are highly enriched in the nuclear marker histone H2B and devoid of mitochondrial, vacuolar and cytosolic marker proteins . On sodium dodecyl sulfate (SDS)-polyacrylamide gels, the nuclear fraction contains unique proteins which distinguishes them from the major yeast subcellular fractions . Yeast nuclei were separated by detergent/salt extraction into soluble, insoluble and membrane fractions . Antibodies raised against subnuclear fractions lead to the identification of an integral nuclear membrane protein and a high-abundance 38-kDa protein which is located in the yeast nucleolus.

Proc Natl Acad Sci U S A, 1988 Aug, 85(16), 6007 - 11
Host function of MAK16: G1 arrest by a mak16 mutant of Saccharomyces cerevisiae; Wickner RB; The MAK16 gene was first defined as a gene whose mutation resulted in loss of M1 double-stranded RNA virus-like particles . The mak16-1 mutation also produces temperature-sensitive cell growth . We report here that mak16-1 cells arrest at the nonpermissive temperature in G1 phase, such that they are mating competent . We sequenced the MAK16 gene and found an open reading frame of 306 amino acids encoding a predicted protein of Mr 35,694 . Two typical nuclear localization signal sequences were found . MAK16-LacZ fusion proteins that include one of these putative signals entered the nucleus, while unfused beta-galactosidase did not, as judged by subcellular fractionation experiments . In the C-terminal third of the MAK16 open reading frame is an acidic region in which 25 of 41 residues are either glutamate or aspartate . This region contains potential phosphorylation sites for "casein kinases," protein kinases specific for serine or threonine residues in an acidic environment.

Genetics, 1988 Aug, 119(4), 779 - 90
Genetic control of intrachromosomal recombination in Saccharomyces cerevisiae . I . Isolation and genetic characterization of hyper-recombination mutations; Aguilera A et al.; Eight complementation groups have been defined for recessive mutations conferring an increased mitotic intrachromosomal recombination phenotype (hpr genes) in Saccharomyces cerevisiae . Some of the mutations preferentially increase intrachromosomal gene conversion (hpr4, hpr5 and hpr8) between repeated sequences, some increase loss of a marker between duplicated genes (hpr1 and hpr6), and some increase both types of events (hpr2, hpr3 and hpr7) . New alleles of the CDC2 and CDC17 genes were recovered among these mutants . The mutants were also characterized for sensitivity to DNA damaging agents and for mutator activity . Among the more interesting mutants are hpr5, which shows a biased gene conversion in a leu2-112::URA3::leu2-k duplication; and hpr1, which has a much weaker effect on interchromosomal mitotic recombination than on intrachromosomal mitotic recombination . These analyses suggest that gene conversion and reciprocal exchange can be separated mutationally . Further studies are required to show whether different recombination pathways or different outcomes of the same recombination pathway are controlled by the genes identified in this study.

Mol Gen Genet, 1988 Aug, 213(2-3), 400 - 8
Rad3 protein of Saccharomyces cerevisiae: overexpression and preliminary characterization using specific antibodies; Naumovski L et al.; The cloned RAD3 gene of Saccharomyces cerevisiae was tailored into expression vectors for overexpression of Rad3 protein in Escherichia coli and in yeast . In both organisms the overexpressed protein is detected as a species of molecular weight ca . 90 kDa, the size expected from the sequence of the cloned gene . The protein overexpressed in E . coli is largely insoluble; however the insoluble fraction was used to generate affinity-purified polyclonal antisera which proved to be powerful reagents for the initial characterization of Rad3 protein expressed in yeast . These studies showed that: (1) when overexpressed in yeast most of the Rad3 protein is detected in the soluble fraction of cell extracts; (2) endogenous Rad3 protein is untransformed cells is also ca . 90 kDa in size and is located in the cell nucleus; (3) Rad3/beta-galactosidase fusion protein partially purified on an affinity matrix is associated with DNA-dependent ATPase activity that is inhibited in the presence of anti-Rad3 antibodies, suggesting that Rad3 protein is an ATPase; and (4) Rad3 antibodies cross-react with two electrophoretically distinguishable polypeptides present in the nuclear fraction of human cells, and with a single polypeptide in extracts of Drosophila cells.

Mol Cell Biol, 1988 Aug, 8(8), 3150 - 9
Genetic analysis of small nuclear RNAs in Saccharomyces cerevisiae: viable sextuple mutant; Parker R et al.; Saccharomyces cerevisiae contains at least 24 distinct small nuclear RNAs (snRNAs), several of which are known to be essential for viability and to participate in the splicing of pre-mRNAs; the RNAs in this subset contain binding sites for the Sm antigen, a hallmark of metazoan snRNAs involved in mRNA processing . In contrast, we showed previously that the single-copy genes for three other snRNAs (snR3, snR4, and snR10) are not required for viability, although cells lacking snR10 are growth impaired at low temperature . None of these RNAs associates with the Sm antigen . To assess this apparent correlation, we cloned and sequenced the genes encoding three additional non-Sm snRNAs . Comparison of these genes with nine additional yeast snRNA genes revealed a highly conserved TATA box located 92 +/- 8 nucleotides 5' of the transcriptional start site . By using the technique of gene replacement with null alleles, each of these three single copy genes was shown to be completely dispensable . We constructed multiple mutants to test the hypothesis that, individually, each of these snRNAs is nonessential because the snRNAs play functionally overlapping roles . A mutant lacking five snRNAs (snR3, snR4, snR5, snR8, snR9) was indistinguishable from the wild type, and growth of the sextuple mutant was no more impaired than that in strains lacking only snR10 . This widespread dispensability of snRNAs was completely unexpected and forces us to reconsider the possible roles of these ubiquitous RNAs.

Mol Cell Biol, 1988 Aug, 8(8), 3094 - 103
A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth; Shih CK et al.; The antipsychotic drug trifluoperazine has been long considered a calmodulin inhibitor from in vitro studies but may function in vivo as a more general inhibitor by disturbing ion fluxes and altering the membrane potential . Resistance to trifluoperazine can arise in Saccharomyces cerevisiae cells by alterations in at least three distinct genetic loci . One locus, defined by a spontaneous dominant trifluoperazine resistance mutation (TFP1-408), was isolated and sequenced . The sequence of the TFP1-408 gene revealed a large open reading frame coding for a large protein of 1,031 amino acids with predicted hydrophobic transmembrane domains . A search of existing amino acid sequences revealed a significant homology with F0F1 ATP synthase . Mutant TFP1-408 cells did not grow efficiently in the presence of 50 mM CaCl2, whereas wild-type cells did . Wild-type cells became resistant to trifluoperazine in the presence of 50 mM CaCl2 or 50 mM MgCl2 . Mutant cells showed a higher rate of calcium transport relative to wild-type cells . These data suggest that the TFP1 gene product codes for a transmembrane ATPase-like enzyme possibly involved in Ca2+ transport or in generating a transmembrane ion gradient between two cellular compartments.

Mol Cell Biol, 1988 Aug, 8(8), 3282 - 90
Characterization of an SNR gene locus in Saccharomyces cerevisiae that specifies both dispensible and essential small nuclear RNAs; Zagorski J et al.; A genetic locus is described that specifies two Saccharomyces cerevisiae small nuclear RNAs (snRNAs) . The genes specifying the two snRNAs are separated by only 67 base pairs and are transcribed in the same direction . The product RNAs contain 128 and 190 nucleotides and are designated snR128 and snR190, respectively . These RNAs resemble snRNAs of other eucaryotes in nuclear localization and possession of a 5' trimethylguanosine cap . Neither snRNA is related in sequence to previously described vertebrate or yeast snRNAs . Both RNAs exhibit properties consistent with nucleolar organization and hydrogen bonding to pre-rRNA species, suggesting possible roles in ribosome biogenesis . The snR128 species cosediments with deproteinized 27S pre-rRNA, whereas snR190 is associated with a 20S intermediate . Gene disruption in vitro followed by replacement of the chromosomal alleles reveals that SNR128 is essential, whereas SNR190 is not.

J Bacteriol, 1988 Aug, 170(8), 3778 - 81
Identification of mitochondrial and microsomal phosphatidylserine synthase in Saccharomyces cerevisiae as the gene product of the CHO1 structural gene; Kohlwein SD et al.; In Saccharomyces cerevisiae, the membrane-associated enzyme phosphatidylserine synthase (EC 2.7.8.8) is present in the mitochondria and the endoplasmic reticulum . The enzyme from both membrane fractions reacted with antiserum raised against a hybrid protein expressed from a TRPE-CHO1 fusion gene in Escherichia coli and was absent in a cho1 null mutant, strongly suggesting that both the mitochondrial and microsomal forms of phosphatidylserine synthase are the products of the CHO1 gene . The highest degree of purification of enzymatically active protein was 380- and 420-fold from the mitochondrial and the microsomal compartments, respectively . In both cases, the enzymatically active and immunoreactive material comigrated with a protein band of 30,000 apparent molecular weight . In the absence of protease inhibitors during the preparation of membranes, the enzyme underwent degradation to an enzymatically active protein of 23,000 apparent molecular weight.

Gene, 1988 Jul 30, 67(2), 223 - 8
A 28-bp segment of the Saccharomyces cerevisiae PHO5 upstream activator sequence confers phosphate control to the CYC1-lacZ gene fusion; Sengstag C et al.; Two regions within the Saccharomyces cerevisiae PHO5 upstream activator sequence (UAS) are involved in phosphate dependent transcription activation {Rudolph and Hinnen, Proc . Natl . Acad . Sci . USA 84 (1987) 1340-1344} . In experiments carried out in vivo we showed that one of these can compensate for the CYC1 UAS and expresses the heterologous CYC1-lacZ gene in response to phosphate starvation . A 28-bp segment is very efficient in gene activation, and a 19-bp subsegment that corresponds to the UASp consensus sequence brings about a weak but still detectable activation . As was observed with other UAS, gene activation is obtained with either orientation of the element, and tandem copies yield double lacZ activity compared to a single copy . No gene activation is observed in a pho4 and in a pho2 mutant . Absence of PHO2 reduces the basal expression of CYC1.

Gene, 1988 Jul 30, 67(2), 259 - 69
Retrovirus-like vectors for Saccharomyces cerevisiae: integration of foreign genes controlled by efficient promoters into yeast chromosomal DNA; Jacobs E et al.; Using modified Saccharomyces cerevisiae Ty1 elements located on a 2 mu plasmid, reverse-transcriptase-mediated transposition into yeast chromosomes of expression cassettes containing a foreign gene can be induced . These expression cassettes consist of the yeast ARG3 and CUP1 promoter sequences fused to the Escherichia coli galK structural gene . Expression cassettes as large as 2 kb can be inserted into Ty elements and transposed efficiently to various sites in the yeast genome . A third yeast promoter (from the yeast CAR1 gene) seems to be unsuitable for use in the expression cassette . This may be because it does not allow the transcription run-through necessary for Ty1 transposition . Ways of improving this vector system are discussed, as are its advantages over episomal vector systems.

Gene, 1988 Jul 30, 67(2), 229 - 45
Characterization of purified hepatitis B surface antigen containing pre-S(2) epitopes expressed in Saccharomyces cerevisiae; Langley KE et al.; The cloning and expression of the hepatitis B middle-protein surface antigen gene in the yeast Saccharomyces cerevisiae is described . A generalized expression vector carrying the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter was used . Expressed material, in the form of supramolecular particles, was purified and characterized . Severe proteolysis within the pre-S(2) region was observed for material expressed in a wild-type yeast host . This proteolysis was substantially reduced by utilization of a protease-deficient host . Immunoblotting of sodium dodecyl sulfate-polyacrylamide gels with several antibodies of differing specificity was performed to characterize the various protein species present . All species were analyzed by N-terminal sequencing after electroelution from gels . Carbohydrate staining of gels and glycosidase treatments of the purified antigen material indicated that full-length antigen was present in both glycosylated and unglycosylated forms . Glycosylation appeared to be of both asparagine-linked and threonine/serine-linked types . Site-directed mutagenesis was used to convert two arginine residues in the pre-S(2) region of the antigen to glutamine residues . The changes abolished reactivity with one polyclonal and two monoclonal antibodies specific for epitopes within the pre-S(2) region.

Nucleic Acids Res, 1988 Jul 25, 16(14B), 6737 - 52
Identification of a protein factor binding to the 5'-flanking region of a tRNA gene and being involved in modulation of tRNA gene transcription in vivo in Saccharomyces cerevisiae; Marschalek R et al.; Control mechanisms of tRNA gene transcription were studied in vivo in Saccharomyces cerevisiae . In order to be able to monitor in vivo transcription products of an individual tRNA gene, a 'tester gene' was used which is readily transcribed in vivo in yeast but does not cross-hybridize with any cellular yeast tRNA . A series of insertion mutants were constructed, modifying thereby the immediate and further distant 5'-flanking region of the 'tester tRNA gene' . Small linker molecules of different length and different sequence were inserted at positions -3 and -56 on the non-coding strand . Resulting tRNA gene variants were transformed into yeast cells and in vivo synthesized products were monitored by primer extension analysis . From the experimental data we suggest that a few essential nucleotides within the flanking region are able to determine the in vivo transcription activity of the 'tester tRNA gene' . Our results are rationalized on a biochemical level by protein binding assays: At least one protein binds to the 5'-flanking region of the 'tester tRNA gene' and different protein complexes are sequestered on active or less active tRNA gene variants.

Nucleic Acids Res, 1988 Jul 25, 16(14A), 6411 - 26
Replication of single-stranded DNA templates by primase-polymerase complexes of the yeast, Saccharomyces cerevisiae; Biswas EE et al.; A partially purified primase-polymerase complex from the yeast, Saccharomyces cerevisiae, was capable of replicating a single stranded circular phage DNA into a replicative form with high efficiency . The primase-polymerase complex exhibited primase activity and polymerase activity on singly primed circular ssDNA as well as on gapped DNA . In addition, it was able to replicate an unprimed, single-stranded, circular phage DNA through a coupled primase-polymerase action . On Biogel A-O.5m filtration the primase-polymerase activities appeared in the void volume, demonstrating a mass of greater than 500 kilodaltons . Primase and various primase-polymerase complexes synthesized unique primers on single stranded DNA templates and the size distribution of primers was dependent on the structure of the DNA and the nature of the primase-polymerase assembly.

Nucleic Acids Res, 1988 Jul 25, 16(14A), 6373 - 84
Close association of a DNA replication origin and an ARS element on chromosome III of the yeast, Saccharomyces cerevisiae; Huberman JA et al.; Two dimensional gel electrophoretic techniques were used to locate all functional DNA replication origins in a 22.5 kb stretch of yeast chromosome III . Only one origin was detected, and that origin is located within several hundred bp of an ARS element.

Nucleic Acids Res, 1988 Jul 25, 16(14B), 7119 - 31
The Saccharomyces cerevisiae RAD18 gene encodes a protein that contains potential zinc finger domains for nucleic acid binding and a putative nucleotide binding sequence; Jones JS et al.; The RAD18 gene of Saccharomyces cerevisiae is required for postreplication repair of UV damaged DNA . We have isolated the RAD18 gene, determined its nucleotide sequence and examined if deletion mutations of this gene show different or more pronounced phenotypic effects than the previously described point mutations . The RAD18 gene open reading frame encodes a protein of 487 amino acids, with a calculated molecular weight of 55,512 . The RAD18 protein contains three potential zinc finger domains for nucleic acid binding, and a putative nucleotide binding sequence that is present in many proteins that bind and hydrolyze ATP . The DNA binding and nucleotide binding activities could enable the RAD18 protein to bind damaged sites in the template DNA with high affinity . Alternatively, or in addition, RAD18 protein may be a transcriptional regulator . The rad18 deletion mutation resembles the previously described point mutations in its effects on viability, DNA repair, UV mutagenesis, and sporulation.

Science, 1988 Jul 15, 241(4863), 317 - 22
The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae; Weinert TA et al.; Cell division is arrested in many organisms in response to DNA damage . Examinations of the genetic basis for this response in the yeast Saccharomyces cerevisiae indicate that the RAD9 gene product is essential for arrest of cell division induced by DNA damage . Wild-type haploid cells irradiated with x-rays either arrest or delay cell division in the G2 phase of the cell cycle . Irradiated G1 and M phase haploid cells arrest irreversibly in G2 and die, whereas irradiated G2 phase haploid cells delay in G2 for a time proportional to the extent of damage before resuming cell division . In contrast, irradiated rad9 cells in any phase of the cycle do not delay cell division in G2, but continue to divide for several generations and die . However, efficient DNA repair can occur in irradiated rad9 cells if irradiated cells are blocked for several hours in G2 by treatment with a microtubule poison . The RAD9-dependent response detects potentially lethal DNA damage and causes arrest of cells in G2 until such damage is repaired.

J Biol Chem, 1988 Jul 15, 263(20), 9674 - 81
Kinetic evidence for a critical rate of protein synthesis in the Saccharomyces cerevisiae yeast cell cycle; Moore SA; The kinetics of cell cycle initiation were measured at pH 2.7 for cells that had been arrested at the "start" step of cell division with the polypeptide pheromone alpha-factor . Cell cycle initiation was induced by the removal of alpha-factor . The rate at which cells completed start was identical to the rate of subsequent bud emergence . After short times of prearrest with alpha-factor (e.g . 5.2 h), the kinetics of bud emergence were biphasic, indicative of two subpopulations of cells that differed by greater than 10-fold in their rates of cell cycle initiation . The subpopulation that exhibited a slow rate of cell cycle initiation is comprised of cells that resided in G1 prior to start at the time of removal of alpha-factor, whereas the subpopulation that initiated the cell cycle rapidly is comprised of cells that had reached and become blocked at start . A critical concentration of cycloheximide was found to reintroduce slow budding cells into a population of 100% fast budding cells, suggesting that the two subpopulations differ with respect to attainment of a critical rate of protein synthesis that is necessary for the performance of start . Cycloheximide and an increase in the time of prearrest with alpha-factor had opposite effects on both the partitioning of cells between the two subpopulations and the net rate of protein synthesis per cell, consistent with this conclusion . Cell cycle initiation by the subpopulation of fast budding cells required protein synthesis even though the critical rate of protein synthesis had been achieved during arrest . It is concluded that alpha-factor inhibits the synthesis of and/or inactivates specific proteins that are required for the performance of start, but alpha-factor does not prevent attainment of the critical rate of protein synthesis.

J Biol Chem, 1988 Jul 5, 263(19), 9142 - 8
Purification and characterization of C1, the catalytic subunit of Saccharomyces cerevisiae cAMP-dependent protein kinase encoded by TPK1; Zoller MJ et al.; In the yeast Saccharomyces cerevisiae, three genes TPK1, TPK2, and TPK3 encode catalytic subunits of cAMP-dependent protein kinase . We have purified and characterized the catalytic subunit, C1, encoded by the TPK1 gene . In order to purify C1 completely free of C2 and C3, a strain was constructed that contained only the TPK1 gene and genetic disruptions of the other two TPK genes . The cellular level of C1 was increased by expressing the genes for C1 (TPK1) and yeast regulatory subunit (BCY1) on multiple copy plasmids within this strain . Purification was accomplished by a two-column procedure in which holoenzyme was chromatographed on Sephacryl-200, then bound to an anti-regulatory subunit immunoaffinity column . Pure C1 was released from the antibody column by addition of cAMP . The protein migrated on a sodium dodecyl sulfate-polyacrylamide gel with an Mr of 52,000 . Kinetic analysis showed that the apparent Km for ATP and Leu-Arg-Arg-Ala-Ser-Leu-Gly was 33 and 101 microM, respectively . The kcat was determined to be 640 min-1 . The protein weakly autophosphorylated, incorporating less than 0.1 mol of phosphate/mol of catalytic subunit . NH2-terminal sequencing revealed that the protein was blocked.

J Biol Chem, 1988 Jul 5, 263(19), 9094 - 101
Independent genes coding for three acidic proteins of the large ribosomal subunit from Saccharomyces cerevisiae; Remacha M et al.; The yeast ribosome contains three acidic proteins, L44, L44', and L45, closely related from a structural point of view, that seem to play a functional role similar to that of proteins L7 and L12 in the bacterial ribosome . By screening a cDNA bank in lambda gt11 with specific polyclonal and monoclonal antibodies, recombinant phages expressing each one of the acidic proteins have been cloned . A unique copy of each gene is detected using the phage cDNA inserts as probes in nitrocellulose blots of yeast DNA digested with different restriction enzymes . The inserts were subcloned in the plasmid pUC19, and their physical maps and nucleotide sequences were determined . By using the cDNA inserts as probes in genomic DNA banks, DNA fragments carrying the acidic protein genes have been cloned, characterized, and sequenced . The results conclusively show that the three yeast acidic proteins are coded by independent genes and are not the result of a post-translational modification of the product of a unique gene, as in bacteria . Like most ribosomal protein genes, the gene for protein L44' has an intron and two upstream stimulatory boxes (UASrpg) fitting closely to the consensus sequence . The genes coding for proteins L44 and L45 lack introns and seem also exceptional in other characteristics of their sequences . Proteins L44 and L45 have amino acid sequences with about 80% similarity . Protein L44' is only 63% similar to the other two polypeptides . The three proteins have highly conserved carboxyl termini comprising the last 30 amino acids, and the first 10 amino acids of L44 and L45 are identical . The results cast doubts about the possibility of a similar role for the different acidic ribosomal proteins.

Mutat Res, 1988 Jul-Aug, 200(1-2), 183 - 92
Genotoxicity of excess thymidylate in thymidylate low-requiring Saccharomyces cerevisiae is associated with changes in phosphate metabolism; Holderried J et al.; When dTMP in concentrations greater than 100 microM is offered to growing cells of thymidylate low-requiring yeast strains it is both mutagenic and toxic . At exposure concentrations greater than 1 mM dTMP interferes significantly with the low-affinity phosphate permease even in the presence of exogenous phosphate concentrations of 6 mM . Chemical analysis and 31P NMR spectroscopy reveal that excess dTMP disturbs phosphate metabolism in thymidylate low-requiring strains but not in the wild type . The most prominent changes in phosphorus-containing molecules are found in polyphosphates of which up to 20% are broken down within a 20-min time span with a concomitant increase in orthophosphate pools.

Proc Natl Acad Sci U S A, 1988 Jul, 85(13), 4789 - 93
The copper, zinc-superoxide dismutase gene of Saccharomyces cerevisiae: cloning, sequencing, and biological activity; Bermingham-McDonogh O et al.; The gene for copper, zinc-superoxide dismutase (Cu,Zn-SOD; EC 1.15.1.1) from the yeast Saccharomyces cerevisiae has been cloned, sequenced, and shown to have physiological activity . The gene was isolated from a lambda gt11 library by using a long, unique deoxyoligonucleotide probe . The probe sequence was deduced from the known amino acid sequence by using a computer-generated yeast codon preference table . The sequence of the coding and flanking regions is reported . The cloned gene was expressed and shown to be active in vivo . A 3.2-kilobase fragment containing the coding region and 160 upstream bases, subcloned in a yeast/Escherichia coli shuttle vector, was used to transform a yeast strain lacking Cu,Zn-SOD activity . The presence of the Cu,Zn-SOD gene-containing plasmid corrected the characteristic dioxygen sensitivity of this strain . Electrophoretic transfer blots with antibody to yeast Cu,Zn-SOD showed the presence of the protein in transformants and wild-type yeast but not in the mutant . The role of Cu,Zn-SOD in defense against dioxygen toxicity is discussed in the light of these findings.

Cytometry, 1988 Jul, 9(4), 394 - 404
A single-cell assay of beta-galactosidase activity in Saccharomyces cerevisiae; Wittrup KD et al.; A novel assay of single-cell exogenous beta-galactosidase activity in Saccharomyces cerevisiae has been developed . Intracellular fluorescence due to the hydrolysis of resorufin-beta-D-galactopyranoside attains a steady state between production of resorufin and its subsequent leakage from the cell . The cells are permeabilized with Triton X-100, and the assay is performed at 0 degrees C . These conditions were chosen to minimize intercellular fluorescence communication . Free resorufin in the extracellular space is bound by bovine serum albumin to prevent its uptake by cells . Two regimes of fluorescence accumulation are observed, one limited by the rate of diffusion of substrate into the cell, and one limited by the rate of enzymatic cleavage of the substrate . A quantitative correlation between fluorescence and beta-galactosidase activity is obtained under optimized assay conditions.

Genetika, 1988 Jul, 24(7), 1141 - 52
{Killer systems of Saccharomyces cerevisiae yeasts}; Nesterova GF; The killer systems of Saccharomyces cerevisiae are a peculiar group of cytoplasmic symbionts of primitive eukaryotes . The genetic material of these symbionts is double-stranded RNA . Their basic properties are linearity of genome, its fragmentation, resulting in two separately replicating major and minor segments, and the ability to control the synthesis of secretory proteins--mycocins which can kill the taxonomically related strains . Secretion of mycocins also confers immunity to their action . The strains containing killer symbionts are toxigenic and resistant to their own toxins, while those with no killer double-stranded RNA are sensitive to mycocins . The killer systems of Saccharomyces cerevisiae possess some properties relevant to viruses and evidently are evolved during the evolution of infectious viruses . Occurrence of such systems in monocellular eucaryotic organisms is an example of genome complication in the course of putting together the virus-like components . The peculiarities of replication and expression of killer systems and their utilization for the construction of vector molecules are discussed.

EMBO J, 1988 Jul, 7(7), 2233 - 40
A major 125-kd membrane glycoprotein of Saccharomyces cerevisiae is attached to the lipid bilayer through an inositol-containing phospholipid; Conzelmann A et al.; A number of plasma membrane glycoproteins of mammalian and protozoan origin are released from cells by phosphatidylinositol-specific phospholipase C . Some of these proteins have been shown to be attached to the lipid bilayer via a covalently linked, structurally complex glycophospholipid . Here we establish the existence of similarly linked glycoproteins in the yeast Saccharomyces cerevisiae . The most abundant of these is a tightly membrane-bound glycoprotein of 125 kd . The detergent-binding moiety of this protein can be removed by phosphatidylinositol-specific phospholipase C of bacterial origin or from Trypanosoma brucei . Metabolic labeling indicates that the protein contains covalently attached fatty acid and inositol . It also contains the cross-reacting determinant (CRD), an antigen found previously on the glycophospholipid anchor of protozoan and mammalian origin . Treatment of the protein with endoglycosidases F and H results in a 95-kd species . In the secretion mutant sec18, grown at 37 degrees C, the vesicular transport of glycoproteins is reversibly blocked between the rough endoplasmic reticulum and the Golgi apparatus . We find that sec18 cells, when grown at 37 degrees C, do add phospholipid anchors to newly synthesized glycoproteins . This indicates that these anchors are added in the rough endoplasmic reticulum.

EMBO J, 1988 Jul, 7(7), 2211 - 9
Effects of histone H4 depletion on the cell cycle and transcription of Saccharomyces cerevisiae; Kim UJ et al.; We have constructed a yeast strain (UKY403) in which the sole histone H4 gene is under control of the GAL1 promoter . This allows the activation of H4 mRNA synthesis on galactose and its repression on glucose . UKY403 cells, pre-synchronized in G1 with alpha-mating factor, have been used to show that glucose treatment results in the loss of approximately half the chromosomal nucleosomes . This depletion is only partially reversible when the H4 gene is reactivated on galactose . It was found that the resultant lethality manifests itself first in S phase, the period of nucleosome assembly, but leads to highly synchronous arrest in G2 and a virtually complete block in chromosomal segregation . Histone H4-depleted chromatin was analyzed for its efficiency as a template for all three RNA polymerases . Using pulse-labeling, we find no evidence for altered transcription by RNA polymerase I (25S, 18S and 5.8S rRNAs) or RNA polymerase III (5S rRNA, tRNAs) . Northern blot analysis was used to measure levels of RNA polymerase II transcripts . There was little effect on the activation or repression of the CUP1 chelatin gene . While there may be some decrease in the level of certain mRNAs (e.g . HIS4, ARG4) other message levels (HIS3, TRP1) show little change upon glucose repression . Therefore, nucleosome loss certainly does not have a general effect on transcription.

Mol Cell Biol, 1988 Jul, 8(7), 2980 - 3
A new RAS mutation that suppresses the CDC25 gene requirement for growth of Saccharomyces cerevisiae; Camonis JH et al.; In the yeast Saccharomyces cerevisiae, the activation of adenylate cyclase requires the products of the RAS genes and of CDC25 . We isolated several dominant extragenic suppressors of the yeast cdc25 mutation . They did not suppress a thermosensitive allele of the adenylate cyclase gene (CDC35) . One of these suppressors was a mutated RAS2 gene in which the transition C/G----T/A at position 455 resulted in replacement of threonine 152 by isoleucine in the protein . The same mutation in a v-Ha-ras gene reduces the affinity of p21 for guanine nucleotides (L.A . Feig, B . Pan, T.M . Roberts, and G.M . Cooper, Proc . Natl . Acad . Sci . USA 83:4607-4611, 1986) . These results support a model in which the CDC25 gene product is the GDP-GTP exchange factor regulating the activity of the RAS gene product.

Mol Cell Biol, 1988 Jul, 8(7), 2763 - 9
Bent DNA functions as a replication enhancer in Saccharomyces cerevisiae; Williams JS et al.; Previous studies have demonstrated that bent DNA is a conserved property of Saccharomyces cerevisiae autonomously replicating sequences (ARSs) . Here we showed that bending elements are contained within ARS subdomains identified by others as replication enhancers . To provide a direct test for the function of this unusual structure, we analyzed the ARS activity of plasmids that contained synthetic bent DNA substituted for the natural bending element in yeast ARS1 . The results demonstrated that deletion of the natural bending locus impaired ARS activity which was restored to a near wild-type level with synthetic bent DNA . Since the only obvious common features of the natural and synthetic bending elements are the sequence patterns that give rise to DNA bending, the results suggest that the bent structure per se is crucial for ARS function.

Mol Cell Biol, 1988 Jul, 8(7), 2745 - 52
ACE1 regulates expression of the Saccharomyces cerevisiae metallothionein gene; Thiele DJ; Copper resistance in Saccharomyces cerevisiae is mediated, in large part, by the CUP1 locus, which encodes a low-molecular-weight, cysteine-rich metal-binding protein . Expression of the CUP1 gene is regulated at the level of transcriptional induction in response to high environmental copper levels . This report describes the isolation of a yeast mutant, ace1-1, which is defective in the activation of CUP1 expression upon exposure to exogenous copper . The ace1-1 mutation is recessive and lies in a genetic element that encodes a trans-acting CUP1 regulatory factor . The wild-type ACE1 gene was isolated by in vivo complementation and restores copper inducibility of CUP1 expression and copper resistance to the otherwise copper-sensitive ace1-1 mutant . Linkage analysis and gene deletion experiments verified that this gene represents the authentic ACE1 locus . ACE1 maps to the left arm of chromosome VII, 9 centimorgans centromere distal to lys5 . The ACE1 gene appears to play a direct or indirect positive role in activation of CUP1 expression in response to elevated copper concentrations.

Mol Cell Biol, 1988 Jul, 8(7), 2690 - 7
LEU3 of Saccharomyces cerevisiae activates multiple genes for branched-chain amino acid biosynthesis by binding to a common decanucleotide core sequence; Friden P et al.; LEU3 of Saccharomyces cerevisiae encodes an 886-amino-acid polypeptide that regulates transcription of a group of genes involved in leucine biosynthesis and has been shown to bind specifically to a 114-base-pair DNA fragment of the LEU2 upstream region (P . Friden and P . Schimmel, Mol . Cell . Biol . 7:2707-2717, 1987) . We show here that, in addition to LEU2, LEU3 binds in vitro to sequences in the promoter regions of LEU1, LEU4, ILV2, and, by inference, ILV5 . The largely conserved decanucleotide core sequence shared by the binding sites in these genes is CCGGNNCCGG . Methylation interference footprinting experiments show that LEU3 makes symmetrical contacts with the conserved bases that lie in the major groove . Synthetic oligonucleotides (19 to 29 base pairs) which contain the core decanucleotide and flanking sequences of LEU1, LEU2, LEU4, and ILV2 have individually been placed upstream of a LEU3-insensitive test promoter . The expression of each construction is activated by LEU3, although the degree of activation varies considerably according to the specific oligonucleotide which is introduced . A promoter construction with substitutions in the core sequence remains LEU3 insensitive, however . One of the oligonucleotides (based on a LEU2 sequence) was also tested and shown to confer leucine-sensitive expression on the test promoter . The results demonstrate that only a short sequence element is necessary for LEU3-dependent promoter binding and activation and provide direct evidence for an expanded repertoire of genes that are activated by LEU3.

Genetics, 1988 Jul, 119(3), 499 - 506
Isolation and characterization of mutants which show an oversecretion phenotype in Saccharomyces cerevisiae; Sakai A et al.; We have isolated mutants responsible for an oversecretion phenotype in Saccharomyces cerevisiae, using a promoter of SUC2 and the gene coding for alpha-amylase from mouse as a marker of secretion . These mutations defined two complementation groups, designated as ose1 (over secretion) and rgr1 (resistant to glucose repression) . The ose1 mutant produced an oversecretion of amylase by 12- to 15-fold under derepressing conditions; however, the amylase mRNA was present at nearly the same amount as it was in the parent cells . No expression of the amylase gene was detected under repressing conditions . The rgr1 mutant oversecreted amylase by 11- to 13-fold under repressing conditions by 15- to 18-fold under derepressing conditions . The rgr1 mutant showed pleiotropic effects on the following cellular functions: (1) resistance to glucose repression, (2) temperature-sensitive lethality, (3) sporulation deficieny in homozygous diploid cells, and (4) abnormal cell morphology . The rgr1 mutation was not allelic with ssn6 and cyc9, and failed to suppress snf1.

Proc Natl Acad Sci U S A, 1988 Jul, 85(13), 4735 - 9
Chitin synthase 2 is essential for septum formation and cell division in Saccharomyces cerevisiae; Silverman SJ et al.; Previous work led to the puzzling conclusion that chitin synthase 1, the major chitin synthase activity in Saccharomyces cerevisiae, is not required for synthesis of the chitinous primary septum . The mechanism of in vivo synthesis of chitin has now been clarified by cloning the structural gene for the newly found chitin synthase 2, a relatively minor activity in yeast . Disruption of the chitin synthase 2 gene results in the loss of well-defined septa and in growth arrest, establishing that the gene product is essential for both septum formation and cell division.

Mol Cell Biol, 1988 Jul, 8(7), 2942 - 54
Meiotic recombination between repeated transposable elements in Saccharomyces cerevisiae; Kupiec M et al.; We have measured the frequency of meiotic recombination between marked Ty elements in the Saccharomyces cerevisiae genome . These recombination events were usually nonreciprocal (gene conversions) and sometimes involved nonhomologous chromosomes . The frequency of ectopic gene conversion among Ty elements appeared lower than expected on the basis of previous studies of recombination between artificially constructed repeats . The conversion events involved either a subset of the total Ty elements in the genome or the conversion tract was restricted to a small region of the Ty element . In addition, the observed conversion events were very infrequently associated with reciprocal exchange.

Genetics, 1988 Jul, 119(3), 527 - 34
Molecular genetics of serine and threonine catabolism in Saccharomyces cerevisiae; Petersen JG et al.; The catabolic L-serine (L-threonine) deaminase of Saccharomyces cerevisiae allows the yeast to grow on media with L-serine or L-threonine as sole nitrogen source . A mutant, cha1 (catabolism of hydroxyamino acids), lacking this enzyme activity has been isolated . We have cloned the CHA1 gene by complementation of a cha1 mutation . Northern analysis showed that CHA1 mRNA has a size of about 1200 ribonucleotides . CHA1 is probably the structural gene for the enzyme; it is an abundant RNA in cells grown with serine and threonine as nitrogen source, whereas it is not detected when cells are grown on ammonium or proline, i.e., the transcription of the CHA1 gene is induced by serine or threonine . Under induced growth conditions haploid ilv1 CHA1 strains do not require isoleucine, i.e., the catabolic deaminase is able to substitute for the biosynthetic threnonine deaminase encoded by the ILV1 gene . We have identified a nuclear, recessive mutation, sil1, that suppresses ilv1 mutations by increased transcription of the CHA1 gene under growth conditions leading to partial induction . The sil1 mutation could exert its effect by increasing the effective pools of the hydroxyamino acids . Alternatively SIL1 may encode a negatively acting regulatory protein for CHA1.

Genetics, 1988 Jul, 119(3), 517 - 26
Conditional mutants of RPC160, the gene encoding the largest subunit of RNA polymerase C in Saccharomyces cerevisiae; Gudenus R et al.; A 18.4-kb fragment of the yeast genome containing the gene of the largest subunit of RNA polymerase C (RPC160) was cloned by hybridization to a previously isolated fragment of that gene . RPC160 maps on chromosome XV, tightly linked but not allelic to the essential gene TSM8740 . Temperature sensitive (ts) mutant alleles were constructed by in vitro mutagenesis with NaHSO3 and substituted for the wild-type allele on the chromosome . Four of them were unambiguously identified as rpc160 mutants by failure to complement a fully defective mutation rpc160::URA3 . The faithful transcription of a yeast tRNA gene by mutant cell-free extracts is strongly reduced as compared to wild-type . In vivo, the rpc160 mutations specifically affect the synthesis of tRNA in a temperature sensitive way, with comparatively little effect on the synthesis of 5S rRNA and no effect on 5.8S rRNA . An unlinked mutation (pcil-3) suppresses the temperature sensitive phenotype of the rpc160-41 mutation.

J Cell Biol, 1988 Jul, 107(1), 9 - 15
Selective excision of the centromere chromatin complex from Saccharomyces cerevisiae; Kenna M et al.; We have taken advantage of the known structural parameters associated with centromere DNA in vivo to construct a CEN fragment that can be selectively excised from the chromatin DNA with restriction endonucleases . CEN3 DNA is organized in chromatin such that a 220-250-bp region encompassing the elements of centromere homology is resistant to nuclease digestion . Restriction enzyme linkers encoding the Bam HI-recognition site were ligated to a 289 base pair DNA segment that spans the 220-250-bp protected core (Bloom et al., 1984) . Replacement of this CEN3-Bam HI linker cassette into a chromosome or plasmid results in formation of a complete structural and functional centromeric unit . A centromere core complex that retains its protected chromatin conformation can be selectively excised from intact nuclei by restriction with the enzyme Bam HI . The centromeric protein-DNA complex is therefore not dependent upon the intact torsional constrains on linear chromosomes for its structural integrity . Isolation of this complex provides a novel approach to characterizing authentic centromeric proteins bound to DNA in their native state.

Gene, 1988 Jun 30, 66(2), 313 - 8
Construction of versatile Escherichia coli--yeast shuttle vectors for promoter testing in Saccharomyces cerevisiae; Zvonok NM et al.; The promoterless PHO5 gene of the yeast Saccharomyces cerevisiae, encoding the repressible acid phosphatase (AP) was utilized as a reporter gene for the construction of a novel vector system for selection and functional analysis of yeast promoters . The Escherichia coli-yeast shuttle plasmids, pZHB81 and pZHB82, contain different arrays of unique restriction sites located upstream of the PHO5 coding region . Yeast promoters could be screened from random DNA fragments (cloned in the upstream sites) for their ability to direct the expression of the PHO5 gene in transformed (AP-deficient) yeast host cells . AP-expressing transformants were selected directly on agar plates by using a routine colony staining method . Relative promoter strength was assessed by direct assay for AP activity in cell lysates.

Gene, 1988 Jun 30, 66(2), 205 - 13
Transcript characterisation, gene disruption and nucleotide sequence of the Saccharomyces cerevisiae WH12 gene; Kelly DE et al.; WH12 is a gene which plays a prominent role in regulating growth and proliferation in Saccharomyces cerevisiae . It is expressed as a 2.0-kb mRNA transcript . Disruption of this transcript in a WH12+ cell results in the mutant phenotype being displayed . The nucleotide sequence of the cloned gene has been determined and found to include a 487-codon long open reading frame coding for a 55.3-kDa protein . The protein showed no extensive homologies to any previously identified protein . The 5' and 3' noncoding regions contained many of the features found in other yeast genes.

Gene, 1988 Jun 30, 66(2), 223 - 34
Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae; Castanon MJ et al.; cDNA clones encoding human lysozyme were isolated from a human histiocytic cell line (U-937) and a human placenta cDNA library . The clones, ranging in size from 0.5 to 0.75 kb, were identified by direct hybridization with synthetic oligodeoxynucleotides . The nucleotide sequence coding for the entire protein was determined . The derived amino acid sequence has 100% homology with the published amino acid (aa) sequence; the leader sequence codes for 18 aa . Expression and secretion of human lysozyme in Saccharomyces cerevisiae was achieved by placing the cloned cDNA under the control of a yeast gene promoter (ADH1) and the alpha-factor peptide leader sequence.

J Biol Chem, 1988 Jun 25, 263(18), 8569 - 75
Identification of a novel stress-inducible glycoprotein in Saccharomyces cerevisiae . I . Preliminary characterization; Verma R et al.; We have identified a novel stress-inducible protein in Saccharomyces cerevisiae by pulse-labeling with {35S}methionine and two-dimensional gel analysis . The protein was characterized biochemically to gain further insight into mechanisms regulating the stress response . It has a Mr = 118,000 and exists in two forms of pI = 4.2 (p118A) and pI = 4.3 (p118B) . p118A and p118B are modified by N-glycosylation . Tunicamycin treatment revealed the presence of precursor proteins of Mr = 105,000, pI = 4.1 (p105A) and pI = 4.25 (p105B) . The synthesis of p118A and p118B was almost completely shut off in cycling cells and was increased 11-fold following a mild heat shock . Both forms of p118 decayed in a biphasic manner under induced conditions . A tight correlation was observed in the kinetics of thermotolerance induction and p118A synthesis . Other forms of stress such as sulfur starvation which lead to arrest in the unbudded phase also resulted in enhanced synthesis of both p118A and p118B . However, in cell division cycle mutants blocked at various stages at the restrictive temperature, p118A and p118B had different synthetic patterns . Taken together, these data imply a role for induced p118 in proliferation arrest in the unbudded state.

J Biol Chem, 1988 Jun 25, 263(18), 8576 - 82
Modulation of expression of the stress-inducible p118 of Saccharomyces cerevisiae by cAMP . II . A study of p118 expression in mutants of the cAMP cascade; Verma R et al.; In the preceding paper, we have identified a protein of Mr = 118,000 which is induced by stress conditions that lead to cessation of DNA synthesis and cell division (Verma, R., Iida, H., and Pardee, A.B . (1988) J . Biol . Chem . 263, 8569-8575) . In the current study, we have investigated the possible role this protein may play in cellular proliferation by studying p118 expression in mutants of the cAMP metabolic pathway . The cyr 1-2 mutant gene encodes a thermolabile adenylate cyclase whose activity is only 7% of wild type even at permissive temperatures (23 degrees C) . We have found that at 23 degrees C, the G1 period was 5-fold longer in cyr 1-2 than in CYR1+ cells and that p118 was constitutively expressed in these slow cycling mutants . Addition of 8-bromo-cAMP to cyr 1-2 mutants restored growth at both the restrictive and permissive temperatures and resulted in a shut-off in the synthesis of p118 . The effect of the analog on p118 expression was rapid, preceding the increase in cell number and percentage-budded cells . In contrast to wild type cells, p118 synthesis was not induced by sulfur starvation in RAS2val19 mutants possessing high levels of adenylate cyclase activity and bcy1 mutants defective in the regulatory subunit of cAMP-dependent protein kinase . A large body of evidence exists supporting a role of cAMP in positive control of cell proliferation . It is therefore possible that conditions which decrease cAMP arrest growth through a chain of events that include p118 induction.

Nucleic Acids Res, 1988 Jun 24, 16(12), 5587 - 601
Sequence and genetic analysis of a dispensible 189 nucleotide snRNA from Saccharomyces cerevisiae; Thompson JR et al.; The structure of a Saccharomyces cerevisiae gene that encodes a small nuclear RNA (snRNA) of 189 nucleotides is described . This gene, designated SNR189, is located 400 base pairs upstream of the CRY1 gene on yeast chromosome III . Gene replacement analysis revealed the SNR189 gene to be dispensable for growth under a variety of culture conditions . The snR189 sequence lacks homology with other sequenced yeast or metazoan snRNAs.

Nucleic Acids Res, 1988 Jun 24, 16(12), 5291 - 303
SnR30: a new, essential small nuclear RNA from Saccharomyces cerevisiae; Bally M et al.; The gene for a previously unidentified small nuclear RNA has been cloned from Saccharomyces cerevisiae and its nucleotide sequence has been determined . The RNA, snR30, was mapped to a unique coding sequence 605 nucleotides long . SnR30 appears to be one of the most abundant snRNAs of S, cerevisiae in that it can be resolved by ethidium bromide staining on one-dimensional denaturing gels of total yeast RNA . Like other snRNAs, snR30 is enriched in nuclei preparations and possesses a trimethyl guanosine cap structure at its 5' end . After substituting one allele of the wild type gene in a diploid strain for a deleted gene, after sporulation, haploid strains carrying the deletion were unable to grow, indicating that snR30 is required for an essential, but as yet, unknown function . The nucleotide sequence close to the initiation site of the SNR30 gene is similar to that of other yeast SNR genes whose transcripts are associated with pre-rRNA, suggesting that snR30 is related to this group of snRNAs.

J Biol Chem, 1988 Jun 15, 263(17), 8099 - 105
Exonuclease V from Saccharomyces cerevisiae . A 5'----3'-deoxyribonuclease that produces dinucleotides in a sequential fashion; Burgers PM et al.; A novel deoxyribonuclease, exonuclease V, has been purified approximately 30,000-fold from Saccharomyces cerevisiae . Exonuclease V is localized in the nucleus . The nuclease degrades single-stranded, but not double-stranded, DNA from the 5'-end . The products of exonuclease action are dinucleotides, except the 3'-terminal tri- and tetranucleotides which are not degraded . Studies with synthetic oligo- and polynucleotides with specified sequence elements showed that exonuclease V cleaves off dinucleotides as primary digestion products . Thus, the polymers (pT)9pA(pT)n and (pT)10pA(pT)n yielded pTpA and pApT as digestion products, respectively . Removal of the 5'-terminal phosphate from the DNA substrate results in reduced binding of the enzyme to the substrate . In addition, the initial hydrolytic cut by exonuclease V on the dephosphorylated substrate produces a mixture of dinucleoside monophosphates and trinucleoside diphosphates . The enzyme is processive in action.

Gene, 1988 Jun 15, 66(1), 45 - 54
Nucleotide sequence of the SUP2 (SUP35) gene of Saccharomyces cerevisiae; Kushnirov VV et al.; A nucleotide sequence of the yeast Saccharomyces cerevisiae omnipotent suppressor SUP2 (SUP35) gene is presented . The sequence contains a single open reading frame (ORF) of 2055 bp, which may encode a 76.5-kDa protein . A single transcript of 2.3 kb corresponding to a complete ORF is found . Analysis of codon bias suggests that the SUP2 gene is not highly expressed . The C-terminal part of the deduced amino acid sequence shows a high homology to yeast elongation factor EF-1 alpha, whereas the N-terminal part is unique for the SUP2 protein . The N terminus contains a number of short repeating elements and possesses an unusual amino acid composition . Analysis of the nucleotide and deduced amino acid sequences indicates that three additional proteins could possibly be expressed, two of which might be initiated on internal ATG codons and a third might be formed by alternative splicing . One of these proteins is supposed to be imported into mitochondria . Possible functions of the SUP2 gene product(s), especially its putative activity as a soluble factor controlling the fidelity of translation, are discussed.

J Biol Chem, 1988 Jun 5, 263(16), 7691 - 702
Purification and properties of the major nuclease from mitochondria of Saccharomyces cerevisiae; Dake E et al.; The vast majority of nuclease activity in yeast mitochondria is due to a single polypeptide with an apparent molecular weight of 38,000 . The enzyme is located in the mitochondrial inner membrane and requires non-ionic detergents for solubilization and activity . A combination of heparin-agarose and Cibacron blue-agarose chromatography was employed to purify the nuclease to approximately 90% homogeneity . The purified enzyme shows multiple activities: 1) RNase activity on single-stranded, but not double-stranded RNA, 2) endonuclease activity on single- and double-stranded DNA, and 3) a 5'-exonuclease activity on double-stranded DNA . Digestion products with DNA contain 5'-phosphorylated termini . Antibody raised against an analogous enzyme purified from Neurospora crassa (Chow, T . Y . K., and Fraser, M . (1983) J . Biol . Chem . 258, 12010-12018) inhibits and immunoprecipitates the yeast enzyme . This antibody inhibits 90-95% of all nuclease activity present in solubilized mitochondria, indicating that the purified nuclease accounts for the bulk of mitochondrial nucleolytic activity . Analysis of a mutant strain in which the gene for the nuclease has been disrupted supports this conclusion and shows that all detectable DNase activity and most nonspecific RNase activity in the mitochondria is due to this single enzyme.

Genetics, 1988 Jun, 119(2), 273 - 87
Meiotic disjunction of homologs in Saccharomyces cerevisiae is directed by pairing and recombination of the chromosome arms but not by pairing of the centromeres; Surosky RT et al.; We explored the behavior of meiotic chromosomes in Saccharomyces cerevisiae by examining the effects of chromosomal rearrangements on the pattern of disjunction and recombination of chromosome III during meiosis . The segregation of deletion chromosomes lacking part or all (telocentric) of one arm was analyzed in the presence of one or two copies of a normal chromosome III . In strains containing one normal and any one deletion chromosome, the two chromosomes disjoined in most meioses . In strains with one normal chromosome and both a left and right arm telocentric chromosome, the two telocentrics preferentially disjoined from the normal chromosome . Homology on one arm was sufficient to direct chromosome disjunction, and two chromosomes could be directed to disjoin from a third . In strains containing one deletion chromosome and two normal chromosomes, the two normal chromosomes preferentially disjoined, but in 4-7% of the tetrads the normal chromosomes cosegregated, disjoining from the deletion chromosome . Recombination between the two normal chromosomes or between the deletion chromosome and a normal chromosome increased the probability that these chromosomes would disjoin, although cosegregation of recombinants was observed . Finally, we observed that a derivative of chromosome III in which the centromeric region was deleted and CEN5 was integrated at another site on the chromosome disjoined from a normal chromosome III with fidelity . These studies demonstrate that it is not pairing of the centromeres, but pairing and recombination along the arms of the homologs, that directs meiotic chromosome segregation.

Genetics, 1988 Jun, 119(2), 249 - 60
Unlinked noncomplementation: isolation of new conditional-lethal mutations in each of the tubulin genes of Saccharomyces cerevisiae; Stearns T et al.; Mutations in genes of Saccharomyces cerevisiae that code for proteins that interact with beta-tubulin were sought by screening for unlinked mutations that fail to complement mutations in the single beta-tubulin-encoding gene (TUB2) . Among the first three noncomplementing mutations examined, two are linked to TUB2 while one is unlinked . The unlinked mutation was shown to be a conditional-lethal allele of the major alpha-tubulin-encoding gene (TUB1) and represents the first such mutation in that gene . The tub1-1 mutation itself causes a cold-sensitive cell-cycle arrest, and confers supersensitivity to the antimicrotubule drug benomyl . These phenotypes occur in the presence of a wild-type copy of the minor alpha-tubulin-encoding gene, TUB3; the combination of tub1-1 and a tub3 null mutation is inviable in haploids . Through further application of this method, new mutations in TUB2 and TUB3 were isolated as unlinked noncomplementers of tub1-1 . The noncomplementation between tub1 and tub2 mutations is gene specific and allele specific, suggesting that the phenotype is due to an interaction at the protein level . We conclude that isolation of unlinked noncomplementing mutations is likely to be a generally useful method for isolating mutations in interacting gene products.

Eur J Biochem, 1988 Jun 1, 174(2), 297 - 302
Study of the coinduction by fatty acids of catalase A and acyl-CoA oxidase in standard and mutant Saccharomyces cerevisiae strains; Skoneczny M et al.; Evidence is presented that Saccharomyces cerevisiae can metabolize fatty acids via the inducible peroxisomal beta-oxidation pathway even when these acids are not the sole carbon source . The fatty acids of chain length of C10-C18 induce acyl-CoA oxidase simultaneously with catalase A but have no effect on catalase T and acyl-CoA dehydrogenase . The coinduction of both acyl-CoA oxidase and catalase A is recorded in strains with both active catalase A and T or displaying only catalase A activity . In mutants lacking catalase A, the induction of acyl-CoA oxidase is observed without a concomitant increase in catalase activity . After centrifugation in a linear Ficoll gradient of the particulate fraction from the cells grown on ethanol and oleate the activity of acyl-CoA oxidase cosediments with catalase A . The relationship of catalase A to acyl-CoA oxidase is discussed.

Exp Cell Res, 1988 Jun, 176(2), 360 - 5
Localization of the regulatory subunit of cAMP-dependent protein kinase in Saccharomyces cerevisiae; Uno I et al.; The subcellular distribution of the regulatory subunit of cAMP-dependent protein kinase in Saccharomyces cerevisiae cells was determined by subcellular fractionation and indirect immunofluorescence microscopy using the bcy1 mutant deficient in the regulatory subunit as control . The regulatory subunit of cAMP-dependent protein kinase showing cAMP-binding activity was identified as a single protein of 50 kDa by photoaffinity labeling and immunoblotting . The regulatory subunit was concentrated in a nuclear fraction in addition to a cytoplasmic fraction . By comparison of the regulatory subunit distribution with the DNA localization, the area detected by the indirect immunofluorescence was identified as the nucleus.

Proc Natl Acad Sci U S A, 1988 Jun, 85(11), 3683 - 7
ATP-independent DNA strand transfer catalyzed by protein(s) from meiotic cells of the yeast Saccharomyces cerevisiae; Sugino A et al.; An activity that catalyzes the transfer of a strand from a duplex linear molecule of DNA to a complementary circular single strand can be detected in crude extracts from mitotic and meiotic cells of the yeast Saccharomyces cerevisiae by adding yeast single-stranded DNA binding proteins . This DNA strand-transfer activity increases greater than 15-fold during meiosis in MATa/MAT alpha diploids prior to the detection of a 100- to 1000-fold increase in homologous chromosomal recombination . No increase is observed in MATa/MATa or MAT alpha/MAT alpha cells, which do not undergo meiosis when shifted to meiotic medium, suggesting the activity is related to meiotic recombination . The activity is named strand-transfer protein alpha (STP alpha) and has been extensively purified from the meiotic cells (6 hr after exposure to sporulation medium) . The apparent molecular mass of STP alpha is 38 kDa under denaturing conditions . The DNA strand-transfer reaction catalyzed by STP alpha requires homologous single-stranded and double-stranded DNA and Mg2+ but no nucleotide cofactor . Yeast single-stranded DNA binding proteins stimulate the reaction at least 10-fold . Among the products analyzed by electron microscopy were typical strand-exchange structures.

J Bacteriol, 1988 Jun, 170(6), 2870 - 2
Saccharomyces cerevisiae acquires resistance to 2-deoxyglucose at a very high frequency; Heredia MF et al.; We have found that Saccharomyces cerevisiae acquires spontaneously increasing resistance to 2-deoxyglucose at a very high frequency . This finding allows the easy isolation of different types of resistant strains of interest for metabolic studies with 2-deoxyhexoses . On the other hand, it sounds a note of caution in the widespread use of 2-deoxyglucose as a selective agent for the isolation of yeast mutants with impaired hexose transport or phosphorylation systems.

J Bacteriol, 1988 Jun, 170(6), 2654 - 8
Derepression of high-affinity glucose uptake requires a functional secretory system in Saccharomyces cerevisiae; Bisson LF; The expression of high-affinity glucose uptake in Saccharomyces cerevisiae strains carrying conditional mutations conferring a block of secretion and cell surface growth (sec) revealed a requirement for a functional secretory pathway for derepression of carrier activity . Thus, in strains carrying the sec1-1, sec4-2, sec7-1, sec14-3, or sec17-1 mutation, no high-affinity carrier activity was expressed after a shift to derepressing glucose concentrations at the nonpermissive temperature . In the case of sec18-1, however, derepression of carrier activity did occur at both the permissive and nonpermissive temperature, but not to the same extent as found in the wild-type strain, suggesting that SEC18 function may not be essential for expression of carrier activity . In sec1-1, accumulation of high-affinity carrier activity (or a component thereof) in presecretory vesicles during incubation at the nonpermissive temperature was demonstrated . The presence of a high glucose concentration in the medium did not affect transfer of that accumulated carrier function to the cell surface . Carrier function did not accumulate in strains carrying the other sec mutations . Analysis of the stability of high-affinity carrier activity at 37 degrees C demonstrated rapid and unexpected loss of carrier activity not affected by the presence of glucose in the medium . Thus, blockage of cell surface growth seems to affect turnover rates of hexose carrier activities.

J Biochem (Tokyo), 1988 Jun, 103(6), 1004 - 10
Primary structure of Saccharomyces cerevisiae NADPH-cytochrome P450 reductase deduced from nucleotide sequence of its cloned gene; Yabusaki Y et al.; We isolated cDNA (pgCYR, about 2.1 kb) and genomic DNA (pgGYR, about 4 kb) clones coding for NADPH-cytochrome P450 reductase by immunoscreening of yeast Saccharomyces cerevisiae cDNA and genomic DNA libraries in phage lambda gt11 . The clones were sequenced and found to encode a protein of 691 amino acid residues with a calculated molecular weight of 76,737 daltons . The amino-terminal sequence (excluding the initial methionine residue) deduced therefrom was in agreement with the protein sequence of the yeast reductase . In addition, the deduced sequence included the partial amino acid sequence determined with the papain-solubilized reductase . The total amino acid sequence of the yeast reductase showed 33-34% similarity with those of the rat, rabbit, pig, and trout reductases . In spite of low similarity in the total amino acid sequences, the possible functional domains related to binding of FAD, FMN, and NADPH were well conserved among all five species compared.

Mol Cell Biol, 1988 Jun, 8(6), 2484 - 93
Mutations in a gene encoding the alpha subunit of a Saccharomyces cerevisiae G protein indicate a role in mating pheromone signaling; Jahng KY et al.; Mutations which allowed conjugation by Saccharomyces cerevisiae cells lacking a mating pheromone receptor gene were selected . One of the genes defined by such mutations was isolated from a yeast genomic library by complementation of a temperature-sensitive mutation and is identical to the gene GPA1 (also known as SCG1), recently shown to be highly homologous to genes encoding the alpha subunits of mammalian G proteins . Physiological analysis of temperature-sensitive gpa1 mutations suggests that the encoded G protein is involved in signaling in response to mating pheromones . Mutational disruption of G-protein activity causes cell-cycle arrest in G1, deposition of mating-specific cell surface agglutinins, and induction of pheromone-specific mRNAs, all of which are responses to pheromone in wild-type cells . In addition, mutants can conjugate without the benefit of mating pheromone or pheromone receptor . A model is presented where the activated G protein has a negative impact on a constitutive signal which normally keeps the pheromone response repressed.

J Bacteriol, 1988 Jun, 170(6), 2687 - 91
Mutants of Saccharomyces cerevisiae with defective vacuolar function; Kitamoto K et al.; Mutants of the yeast Saccharomyces cerevisiae that have a small vacuolar lysine pool were isolated and characterized . Mutant KL97 (lys1 slp1-1) and strain KL197-1A (slp1-1), a prototrophic derivative of KL97, did not grow well in synthetic medium supplemented with 10 mM lysine . Genetic studies indicated that the slp1-1 mutation (for small lysine pool) is recessive and is due to a single chromosomal mutation . Mutant KL97 shows the following pleiotropic defects in vacuolar functions . (i) It has small vacuolar pools for lysine, arginine, and histidine . (ii) Its growth is sensitive to lysine, histidine, Ca2+, heavy metal ions, and antibiotics . (iii) It has many small vesicles but no large central vacuole . (iv) It has a normal amount of the vacuolar membrane marker alpha-mannosidase but shows reduced activities of the vacuole sap markers proteinase A, proteinase B, and carboxypeptidase Y.

J Bacteriol, 1988 Jun, 170(6), 2683 - 6
Dynamic aspects of vacuolar and cytosolic amino acid pools of Saccharomyces cerevisiae; Kitamoto K et al.; By using the Cu2+ method (Y . Ohsumi, K . Kitamoto, and Y . Anraku, J . Bacteriol . 170:2676-2682, 1988) for differential extraction of the vacuolar and cytosolic amino acid pools from yeast cells, the amino acid compositions of the two pools extracted from Saccharomyces cerevisiae cells, grown in synthetic medium supplemented with various amino acids, were determined . Histidine and lysine in the medium expanded the vacuolar pool extremely . Glutamate also accumulated in the cells, but mainly in the cytosol . The composition of amino acids in the cytosolic pool was fairly constant, in contrast to that in the vacuolar pool . Cells grown in synthetic medium supplemented with 10 mM arginine accumulated arginine in the vacuoles at a concentration of about 430 mM . This large arginine pool was metabolically active and was effectively utilized during nitrogen starvation . Arginine efflux from the vacuoles was coupled with K+ influx, with an arginine/K+ exchange ratio of 1, as judged by the initial rate . The vacuolar arginine pool was exchangeable with lysine added to the medium and was decreased by treatment of the cells with the mating pheromone, alpha-factor.

Microbiologia, 1988 Jun, 4(2), 97 - 105
Cellular location of asparaginase activity in Saccharomyces cerevisiae and regulation of this activity by nitrogen compounds; Mormeneo S et al.; Three asparaginase activities have been detected in Saccharomyces cerevisiae . One is found outside the permeability barrier; a second one is found inside and is soluble in the cell, and the third one is localized in a system of membrane particles . Synthesis of the membrane and external asparaginases require "de novo" synthesis of RNA and protein . The synthesis of exocellular asparaginase is inhibited by several nitrogen compounds (catabolite repression) . This inhibition might take place at the transcriptional level . Moreover, this isoenzyme is reversibly inactivated by its natural substrates (catabolite inhibition by substrates) . The half life of external asparaginase mRNA was calculated by two independent methods and values of 7.5 and 9.5 min were found.

J Gen Microbiol, 1988 Jun, 134 ( Pt 6), 1661 - 6
Water stress plating hypersensitivity of yeasts: protective role of trehalose in Saccharomyces cerevisiae; Mackenzie KF et al.; Water stress plating hypersensitivity was studied in two strains of Saccharomyces cerevisiae, one of them being a mutant incapable of accumulating trehalose to significant levels . The wild-type strain was grown in a defined medium with glucose, maltose or ethanol as carbon/energy source . In each case plating hypersensitivity was demonstrated and resistance to the stress developed in the second half of the exponential growth phase . Development of resistance was accompanied by accumulation of trehalose and was apparently unrelated to glycerol content which, under these conditions, was always low . A qualitatively similar trend was observed in the mutant grown on glucose but trehalose levels remained low and recovery of stress resistance was only slight . Dinitrophenol induced trehalose breakdown in resting yeast and simultaneously induced the onset of plating hypersensitivity . A negative correlation was demonstrated between trehalose content and 'plating discrepancy' (log colony count on 'normal' agar-log colony count on stressing agar) for both strains under all experimental conditions . The correlation held for trehalose contents up to about 50 mg (g dry yeast)-1, above which the yeasts were apparently fully resistant . Trehalose was evidently a more effective compatible solute, per mole, than glycerol.

Biosci Rep, 1988 Jun, 8(3), 287 - 91
Immunological identification of two lamina-like proteins in Saccharomyces cerevisiae; Galcheva-Gargova Z et al.; Proteins from Saccharomyces cerevisiae were tested for their crossreactivity with antibodies raised against nuclear lamina proteins of Ehrlich Ascites Tumour cells . The results of the immunoblotting experiments and ELISA suggest the existence of at least two proteins (65 and 59 kDa) which are immunologically related to the nuclear lamina proteins of higher eukaryotes.

Yeast, 1988 Jun, 4(2), 93 - 106
Pleiotropic mutations in Saccharomyces cerevisiae affecting sterol uptake and metabolism; Lewis TL et al.; Sterol uptake control mutants (upc-) have been isolated via ethylmethanesulfonate mutagenesis from wild-type Saccharomyces cerevisiae . These mutants are heme and sterol competent but possess the ability to accumulate exogenous sterol(s) under aerobic conditions . Previous studies demonstrate sterol uptake only in a hem-, erg- background; however, the Upc- strains described here are Hem+ and do not require exogenous sterol for growth . We were unable to obtain viable hem+, erg-, upc+ recombinants; such combinations appear to be lethal . Isolates of Upc mutants demonstrated different levels of sterol uptake, and sterol analysis revealed a broad phenotypic range with regard to amounts and accumulation of ergosterol and non-ergosterol sterols . Assays of acyl CoA: ergosterol acyltransferase and sterol ester hydrolase showed no apparent difference in activity between Upc mutants and the wild type.

J Med Vet Mycol, 1988 Jun, 26(3), 153 - 62
Isolation and analysis of ketoconazole resistant mutants of Saccharomyces cerevisiae; Watson PF et al.; Nine mutants of Saccharomyces cerevisiae which are resistant to ketoconazole, have been isolated and characterized . In each case the mutation is nuclear in origin and allelic to a previously described mutation, erg3, which gives rise to a block in the delta 5-6 desaturation step of ergosterol biosynthesis . The significance of this second site mutation to the point of inhibitory action of ketoconazole, that is the P-450-mediated C-14 demethylation of lanosterol, is discussed.

Genetika, 1988 Jun, 24(6), 993 - 7
{Genetic control of plasmid recombination in the cotransformation of Saccharomyces cerevisiae: the role of RAD52 and FLP genes}; Kozhina TN et al.; In our earlier works we observed high frequency of recombination between two chimeric plasmids of different types, when they were introduced into yeast cells via cotransformation . Incapability of one of these plasmids to replicate autonomously in yeast cell is the necessary condition for such recombination . The high efficiency of this process point to the differences between interplasmid recombination and other types of yeast recombination . In this work, we studied the participation of two genes in the control of interplasmid exchanges . These are RAD52 responsible for normal processes of meiotic and mitotic recombination and highly specific gene FLP located on 2 mkm DNA which specifies site-specific recombination in the region of inverted sequences of this plasmid . The mutation rad52 in the recipient strain was shown to sharply decrease the efficiency of recombination between integrative and episome plasmids during cotransformation . The absence of FLP gene in the recipient strain (cirO) has no influence on this process.

EMBO J, 1988 Jun, 7(6), 1805 - 13
Multiple regulatory mechanisms control the expression of the RAS1 and RAS2 genes of Saccharomyces cerevisiae; Breviario D et al.; Expression of the RAS1 and RAS2 genes of Saccharomyces cerevisiae has been examined at the transcriptional and translational levels . When dextrose is the carbon source, the steady-state amount of RAS1 mRNA and the rate of RAS1 protein synthesis are reduced in parallel as cells approach the mid-exponential phase of growth . RAS1 mRNA levels and protein synthesis are very low at all stages of growth when ethanol rather than dextrose is provided as the sole carbon source . The rate of RAS2 protein synthesis is regulated differently . In cells cultured on dextrose, it is lowest in the early exponential phase, increases approximately 10-fold and remains nearly constant as cells approach stationary phase . By contrast, RAS2 mRNA is found at uniformly high levels at all phases of exponential growth, suggesting that the translational efficiency of RAS2 mRNA is repressed during the early exponential phase . This repression is not observed when ethanol is the sole carbon source . Nutrient starvation, resulting in G1 arrest and sporulation in diploids, leads to greatly decreased amounts of RAS2 mRNA, accomplished in part by selective repression of RAS2 transcripts with particular 5' ends . However, this reduction in RAS2 mRNA levels has little effect on the rate of RAS2 protein synthesis, suggesting that the translational efficiency of RAS2 mRNA is stimulated by nutrient starvation . The combination of transcriptional and translational controls which regulate yeast RAS gene expression seems to ensure that one or the other RAS proteins will be produced over a wide range of physiological states.

Mol Gen Genet, 1988 Jun, 212(3), 495 - 504
A consensus transcription termination sequence in the promoter region is necessary for efficient gene expression of the TRP1 gene of Saccharomyces cerevisiae; Braus G et al.; The TRP1 gene of Saccharomyces cerevisiae is the only TRP gene which is not derepressible by the general control regulatory system . In the TRP1 promoter transcription starts at five initiation sites, organized in two clusters . The two transcripts of the first, more upstream cluster include a long leader sequence of approximately 200 bp . A transcriptional terminator element located in the 5' region of the TRP1 gene is essential for accurate gene expression . In partial TRP1 promoters lacking the terminator, like the original EcoRI TRP1 fragment used in numerous vectors, plasmid-encoded transcription is initiated predominantly in adjacent vector regions, resulting mainly in large, poorly translated transcripts . This poor translation is not due to mRNA instability . The effect can be suppressed by introducing artificial transcription barriers between vector sequences and the truncated EcoRI TRP1 fragment.

Mol Gen Genet, 1988 Jun, 212(3), 459 - 65
Genetic mapping of the Saccharomyces cerevisiae DNA polymerase I gene and characterization of a pol1 temperature-sensitive mutant altered in DNA primase-polymerase complex stability; Lucchini G et al.; The cloned DNA polymerase I gene has been used to map the POL1 locus on the left arm of chromosome XIV, between MET4 and TOP2 . Temperature-sensitive mutants in POL1 have been obtained by in vitro mutagenesis of the cloned gene and in vivo replacement of the wild-type allele with the mutated copy . Physiological and biochemical characterization of one temperature-sensitive mutant (pol1-1) shows that cells shifted to the non-permissive temperature can complete one round of cell division and DNA replication before they arrest . Analysis of DNA polymerase I in crude extracts and in partially purified preparations indicates that the pol1-1 mutation results in a conformational change and affects the stability of the DNA primase-polymerase complex.

Genes Dev, 1988 Jun, 2(6), 664 - 76
Ribosomal protein synthesis is not regulated at the translational level in Saccharomyces cerevisiae: balanced accumulation of ribosomal proteins L16 and rp59 is mediated by turnover of excess protein; Tsay YF et al.; We have investigated the mechanisms whereby equimolar quantities of ribosomal proteins accumulate and assemble into ribosomes of the yeast Saccharomyces cerevisiae . Extra copies of the cry1 or RPL16 genes encoding ribosomal proteins rp59 or L16 were introduced into yeast by transformation . Excess cry1 or RPL16 mRNA accumulated in polyribosomes in these cells and was translated at wild-type rates into rp59 or L16 proteins . These excess proteins were degraded until their levels reached those of other ribosomal proteins . Identical results were obtained when the transcription of RPL16A was rapidly induced using GAL1-RPL16A promoter fusions, including a construct in which the entire RPL16A 5'-noncoding region was replaced with the GAL1 leader sequence . Our results indicate that posttranscriptional expression of the cry1 and RPL16 genes is regulated by turnover of excess proteins rather than autogenous regulation of mRNA splicing or translation . The turnover of excess rp59 or L16 is not affected directly by mutations that inactivate vacuolar hydrolases.

Appl Environ Microbiol, 1988 Jun, 54(6), 1420 - 5
The mannoprotein of Saccharomyces cerevisiae is an effective bioemulsifier; Cameron DR et al.; The mannoprotein which is a major component of the cell wall of Saccharomyces cerevisiae is an effective bioemulsifier . Mannoprotein emulsifier was extracted in a high yield from whole cells of fresh bakers' yeast by two methods, by autoclaving in neutral citrate buffer and by digestion with Zymolase (Miles Laboratories; Toronto, Ontario, Canada), a beta-1,3-glucanase . Heat-extracted emulsifier was purified by ultrafiltration and contained approximately 44% carbohydrate (mannose) and 17% protein . Treatment of the emulsifier with protease eliminated emulsification . Kerosene-in-water emulsions were stabilized over a broad range of conditions, from pH 2 to 11, with up to 5% sodium chloride or up to 50% ethanol in the aqueous phase . In the presence of a low concentration of various solutes, emulsions were stable to three cycles of freezing and thawing . An emulsifying agent was extracted from each species or strain of yeast tested, including 13 species of genera other than Saccharomyces . Spent yeast from the manufacture of beer and wine was demonstrated to be a possible source for the large-scale production of this bioemulsifier.

Mol Cell Biol, 1988 Jun, 8(6), 2523 - 35
Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae; Hegemann JH et al.; Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII) . We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system . The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential . Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold . Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)) . These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.

DNA, 1988 Jun, 7(5), 355 - 60
Efficient secretion and processing of heterologous proteins in Saccharomyces cerevisiae is mediated solely by the pre-segment of alpha-factor precursor; Ernst JF; A novel processing site was identified in fusions of the alpha-factor precursor of Saccharomyces cerevisiae following its 19 amino-terminal residues (pre-segment) . Fusions of the pre-segment to heterologous proteins, including aminoglycoside phosphotransferase (APH) and human granulocyte-macrophage colony stimulating factor (hGM-CSF), were as efficiently secreted and processed as corresponding pre-pro fusions . Pre- and pre-pro fusions to hGM-CSF were identically N- and O-glycosylated . While pre-pro fusions to interleukin-1 beta were not cleaved, pre-fusions were correctly processed during secretion . The high secretion efficiency of pre-fusions suggests that the pro-segment of the alpha-factor precursor is not required for efficient secretion and processing of protein fusions.

J Biochem (Tokyo), 1988 Jun, 103(6), 954 - 61
Construction of a human cytochrome c gene and its functional expression in Saccharomyces cerevisiae; Tanaka Y et al.; The nucleotide sequences of a partial cDNA and three pseudogenes of human cytochrome c were determined . The complete nucleotide sequences which encode human cytochrome c were constructed on the basis of one of the pseudogenes by in vitro mutagenesis . The constructed human cytochrome c was functionally expressed in Saccharomyces cerevisiae . The recombinant human cytochrome c was purified and characterized.

Mol Cell Biol, 1988 Jun, 8(6), 2572 - 80
Transposable element-mediated enhancement of gene expression in Saccharomyces cerevisiae involves sequence-specific binding of a trans-acting factor; Goel A et al.; In our studies on the regulation of adjacent-gene expression by Ty sequences, we demonstrated that a single-base-pair change (T-A----C-G) in the epsilon sequence of Ty917-derived elements is primarily responsible for enhancement of beta-galactosidase expression from lacZ fusion plasmids . Using an electrophoretic gel mobility assay, we showed that the same base pair transition is required for binding of a trans-acting factor, TyBF, from crude cell extracts in vitro . We identified the site of TyBF binding and determined the guanine nucleotide contact sites required for TyBF interaction . We propose that TyBF binding to cis-acting Ty2 sequences activates adjacent-gene transcription.

Mol Cell Biol, 1988 Jun, 8(6), 2342 - 9
Physical monitoring of mating type switching in Saccharomyces cerevisiae; Connolly B et al.; The kinetics of mating type switching in Saccharomyces cerevisiae can be followed at the DNA level by using a galactose-inducible HO (GAL-HO) gene to initiate the event in synchronously growing cells . From the time that HO endonuclease cleaves MAT a until the detection of MAT alpha DNA took 60 min . When unbudded G1-phase cells were induced, switched to the opposite mating type in "pairs." In the presence of the DNA synthesis inhibitor hydroxyurea, HO-induced cleavage occurred but cells failed to complete switching . In these blocked cells, the HO-cut ends of MATa remained stable for at least 3 h . Upon removal of hydroxyurea, the cells completed the switch in approximately 1 h . The same kinetics of MAT switching were also seen in asynchronous cultures and when synchronously growing cells were induced at different times of the cell cycle . Thus, the only restriction that confined normal homothallic switching to the G1 phase of the cell cycle was the expression of HO endonuclease . Further evidence that galactose-induced cells can switch in the G2 phase of the cell cycle was the observation that these cells did not always switch in pairs . This suggests that two chromatids, both cleaved with HO endonuclease, can interact independently with the donors HML alpha and HMRa.

Mol Cell Biol, 1988 Jun, 8(6), 2275 - 9
Oxygen-dependent upstream activation sites of Saccharomyces cerevisiae cytochrome c genes are related forms of the same sequence; Cerdan ME et al.; In Saccharomyces cerevisiae, the two genes, CYC1 and CYC7, that encode the isoforms of cytochrome c are expressed at different levels . Oxygen regulation is mediated by the expression of the CYP1 gene, and the CYP1 protein interacts with both CYC1 upstream activation sequence 1 (UAS1) and CYC7 UASo . In this study, the homology between the CYP1-binding sites of both genes was investigated . The most noticeable difference between the CYC1 and CYC7 UASs is the presence of GC base pairs at the same positions in a repeated sequence in CYC7 compared with CG base pairs in CYC1 . Directed mutagenesis changing these GC residues to CG residues in CYC7 led to CYC1-like expression of CYC7 both in a CYP1 wild-type strain and in a strain carrying the semidominant mutation CYP1-16 which reverses the oxygen-dependent expression of the two genes . Our results strongly support the hypothesis that the CYP1-binding sites in CYC1 and CYC7 are related forms of the same sequence and that the CYP1-16 protein has altered specificity for the variant forms of the consensus sequences in both genes.

Eur J Biochem, 1988 Jun 1, 174(2), 399 - 404
In vivo and in vitro evidence for a proton leakage through the inner mitochondrial membrane in a mutant of Saccharomyces cerevisiae; Manon S et al.; Mutants of Saccharomyces cerevisiae were isolated which supported three mutations: two unlinked chromosomic mutations conferring thermosensitivity and cold sensitivity respectively, and a mitochondrial mutation conferring paromomycin sensitivity . When studied on isolated mitochondria, these mutants exhibited low phosphorylation efficiency and great proton permeability of their inner mitochondrial membrane . Experiments were carried out on whole cells: determination of growth rates, cellular yields and cellular respiration, either in the presence of triethyltin, an ATP synthase inhibitor, or in the presence of uncoupler, demonstrating that the proton leakage is actually a physiological phenomenon linked to the cold-sensitive phenotype . Experiments performed on isolated mitochondria confirmed the existence of such a proton leakage.

Philos Trans R Soc Lond B Biol Sci, 1988 May 31, 319(1193), 97 - 105
Translational regulation of mitochondrial gene expression by nuclear genes of Saccharomyces cerevisiae; Fox TD et al.; We describe several yeast nuclear mutations that specifically block expression of the mitochondrial genes encoding cytochrome c oxidase subunits II (COXII) and III (COXIII) . These recessive mutations define positive regulators of mitochondrial gene expression that act at the level of translation . Mutations in the nuclear gene PET111 completely block accumulation of COXII, but the COXII mRNA is present in mutant cells at a level approximately one-third of that of the wild type . Mitochondrial suppressors of pet111 mutations correspond to deletions in mtDNA that result in fusions between the coxII structural gene and other mitochondrial genes . The chimeric mRNAs encoded by these fusions are translated in pet111 mutants; this translation leads to accumulation of functional COXII . The PET111 protein probably acts directly on coxII translation, because it is located in mitochondria . Translation of the mitochondrially coded mRNA for COXIII requires the action of at least three nuclear genes, PET494, PET54 and a newly discovered gene, provisionally termed PET55 . Both the PET494 and PET54 proteins are located in mitochondria and therefore probably act directly on the mitochondrial translation system . Mutations in all three genes are suppressed in strains that contain chimeric coxIII mRNAs with the 5'-untranslated leaders of other mitochondrial transcripts fused to the coxIII coding sequence . The products of all three nuclear genes may form a complex and carry out a single function.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1988 May 30, 65(2), 203 - 17
Synthesis of functional mouse cytochromes P-450 P1 and chimeric P-450 P3-1 in the yeast Saccharomyces cerevisiae; Cullin C et al.; Mouse liver cytochrome P-450 P1 was produced in the yeast Saccharomyces cerevisiae transformed by various expression vectors . The relative efficiency of the phosphoglycerate kinase and GAL10-CYC1 promoters to direct the P-450 P1 mRNA synthesis was determined . The level of protein synthesis was found to be dependent on the amount of the 5'-noncoding sequence of the original cDNA removed during the construction . Yeast-synthesised P-450 P1 was found to be integrated into the microsomal membrane in a fully functional form, as judged by Western blotting, optical spectra and enzymatic activities . The amount of P-450 reached up to 0.6% of the microsomal protein level . A nucleotide sequence coding for a chimeric enzyme in which 40 N-terminal codons of P-450 P1 were replaced by 36 N-terminal codons of P-450 P3 was constructed and expressed in yeast . The resulting protein retained full P-450 P1 activity and was produced with a similar efficiency suggesting that the P-450 N-terminal sequence is not involved in structures critical for the substrate specificities of the P1 isoenzyme.

J Mol Biol, 1988 May 20, 201(2), 247 - 60
Intra-chromosomal gene conversion induced by a DNA double-strand break in Saccharomyces cerevisiae; Ray A et al.; We have stimulated mitotic and meiotic gene conversion between non-tandem direct repeats of ADE4 by a defined double-strand break imparted in vivo to one of two copies of the gene . The experimental design permitted us to distinguish unambiguously between reciprocal intra-chromosomal crossing over and non-reciprocal break-join events that could accompany the induced conversions . We observed that (1) less than 10% of the induced conversion events are accompanied by intra-chromosomal crossing over in both mitosis and meiosis; (2) non-reciprocal break-join is not stimulated by the double-strand breaks; (3) a double-strand break in meiosis is repaired off intra-chromosomal homology (if available) with approximately sevenfold preference over repair off the homologous chromosome . Our observations, analyzed in the light of previous investigations of spontaneous inter and intra-chromosomal crossing over and gene conversion, lead to the view that chromosomal configuration constrains intra-chromosomal crossing over accompanying conversion between closely spaced repeated genes during resolution of the conversion intermediate.

Biochem Biophys Res Commun, 1988 May 16, 152(3), 962 - 7
Role of disulfide bonds in folding and secretion of human lysozyme in Saccharomyces cerevisiae; Taniyama Y et al.; We examined folding and secretion of human lysozyme using four mutants each lacking two cysteines expressed in a yeast secretion system . Our results have revealed that the formation of the disulfide bond Cys6/Cys128 in human lysozyme is a prerequisite for correct folding in vivo in yeast . Substitution of Ala for Cys77 and Cys95 gave eight-fold greater secretion of a molecule with almost the same specific activity as that of the native enzyme . Substitutions of the other cysteines gave molecules that were secreted at a lower rate and had lower specific activities than the native enzyme . These are the first findings that the individual disulfide bonds of human lysozyme have different functions in folding and secretion in vivo.

J Biol Chem, 1988 May 15, 263(14), 6908 - 15
PHO5-LACZ hybrid proteins block translocation of native acid phosphatase in Saccharomyces cerevisiae; Wolfe PB; A set of protein hybrids composed of variable portions of the amino-terminal residues of the yeast phosphate-repressible acid phosphatase (product of PHO5) and an active fragment of bacterial beta-galactosidase has been constructed . When these PHO5-LACZ hybrids are expressed in a yeast strain carrying an intact chromosomal PHO5 gene, they show a size-dependent interference with the secretion of native acid phosphatase . Hybrid proteins containing approximately 50 residues of acid phosphatase do not affect secretion of native acid phosphatase . Hybrids containing greater than 200 residues of acid phosphatase reduce the amount of secreted acid phosphatase more than by 50% . The interference with secretion is specific for acid phosphatase . The hybrids do not affect secretion of invertase, and do not confer a growth-deficient phenotype on yeast . Both the hybrid proteins and acid phosphatase accumulate in non-glycosylated, membrane-bound forms which are sensitive to proteolysis from the cytoplasmic side of the membrane . The hybrids and accumulated acid phosphatase co-migrate on Percoll density gradients with markers of the endoplasmic reticulum, but not with markers of the Golgi or secretory vesicles . These results suggest that PHO5-LACZ hybrid proteins specifically block secretion of native acid phosphatase by interfering with enzyme after targeting but before translocation across the endoplasmic reticulum.

J Biol Chem, 1988 May 15, 263(14), 6783 - 90
Mitochondrial import of the ADP/ATP carrier protein in Saccharomyces cerevisiae . Sequences required for receptor binding and membrane translocation; Smagula C et al.; The ADP/ATP carrier of yeast (309 amino acids) is an abundant transmembrane protein of the mitochondrial inner membrane whose import involves well-defined steps (Pfanner, N., and Neupert, W . (1987) J . Biol . Chem . 262, 7528-7536) . Analysis of the in vitro import of gene fusion products containing ADP/ATP carrier (AAC) sequences at the amino terminus and mouse dihydrofolate reductase (DHFR) at the carboxyl terminus indicates that the first 72 amino acids of the soluble carrier protein, a hydrophilic region of the protein, are not by themselves sufficient for initial binding to the AAC receptor on the mitochondrial surface . However, an AAC-DHFR gene fusion containing the first 111 residues of the ADP/ATP carrier protein exhibited binding to mitochondria at low temperature (2 degrees C) and internalization at 25 degrees C to a mitochondrial space protected from proteinase K in the same manner as the wild-type ADP/ATP carrier protein . The AAC-DHFR protein, in contrast to the wild-type AAC protein imported into mitochondria under optimal conditions, remained extractable at alkaline pH and appeared to be blocked at an intermediate step in the AAC import pathway . Based on its extraction properties, this AAC-DHFR hybrid is proposed to be associated with a proteinaceous component of the import apparatus within mitochondria . These data indicate that the import determinants for the AAC protein are not located at its extreme amino terminus and that protein determinants distal to the first 111 residues of the carrier may be necessary to move the protein beyond the alkali-extractable step in the biogenesis of a functional AAC protein.

Nucleic Acids Res, 1988 May 11, 16(9), 3845 - 61
Characterization of products derived from self-splicing of intron aI5 alpha which is located in the mitochondrial COX I gene of Saccharomyces cerevisiae; Winter AJ et al.; We have characterized the in vitro self-splicing of intron aI5 alpha containing precursor RNA from the yeast mitochondrial gene coding for cytochrome oxidase subunit I . This intron follows the rules for group I self-splicing introns and all the characteristic products have been identified . In addition we have detected abnormal RNA products with features that indicate that the self-splicing behaviour of this intron is more complex . Two intron circles are formed by use of a major and minor intron-internal site for circle closure . A cryptic 5'-splice site located in the 3' exon results in guanosine nucleotide mediated opening at a position 30 nt downstream of the normal 3' splice site . The reactions can all be explained on the basis of the "splice guide" model proposed by Davies et al (1982 Nature 300 719-724) . Although the sequence motifs at cyclization and splice sites occur more often in this intron, only some of them are allowed to interact with the internal guide sequence, suggesting that both primary structure and spatial folding of the RNA are involved in formation of productive reaction sites.

J Mol Biol, 1988 May 5, 201(1), 69 - 80
Effect of DNA structure and nucleotide sequence on Holliday junction resolution by a Saccharomyces cerevisiae endonuclease; Evans DH et al.; Previous studies have demonstrated that mitotic Saccharomyces cerevisiae cells contain an endonuclease that cleaves Holliday junctions . In this paper, the cleavage of a number of model branched substrates has been characterized in detail . Three-armed Y-branched molecules were not substrates for the enzyme . Holliday junction substrates constructed from wild-type lambda att sites were resolved in a concerted reaction by paired single-strand breaks that contained 5'-phosphate and 3'-hydroxyl groups and were often symmetrically related . Holliday junctions were also constructed using DNAs derived from lambda safG and safT mutants to alter the nucleotide sequence immediately flanking the cross-strand exchange . These one to six base-pair changes in nucleotide sequence were observed to have dramatic effects on both the directionality and rate of resolution . More than 90% of wild-type junctions were cleaved in only one direction, while Holliday junctions composed of safT DNA were cleaved equally in both possible directions . Hybrid junctions composed of half wild-type DNA and half safG DNA were cleaved in the same orientation as the wild-type junction but at one-seventh of the rate, while junctions constructed completely from safG DNA were not cleaved at all . The cleavage sites were mapped at the nucleotide level and the locations of the paired nicks made by the endonuclease were also found to be affected by the sequence of the substrates and in such a way as to account for the directionality of cleavage . These results have important consequences for the interpretation of genetic experiments, since they provide biochemical evidence that some of the non-random nature of genetic recombination might be due to non-randomly distributed resolution processes.

J Biol Chem, 1988 May 5, 263(13), 6058 - 62
Yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase . Properties of phospho and dephospho forms and of two mutants in which serine 11 has been changed by site-directed mutagenesis; Marcus F et al.; The properties of dephospho- and phosphofructose-1,6-bisphosphatase from the yeast Saccharomyces cerevisiae and of two mutant enzymes in which the phosphorylatable Ser11 had been changed by site-directed mutagenesis (Ser----Ala and Ser----Asp) were studied to clarify the role of cyclic AMP-dependent phosphorylation of yeast fructose-1,6-bisphosphatase . The mutant enzymes and wild type Ser11 fructose-1,6-bisphosphatase were overexpressed and purified to homogeneity . Phosphofructose-1,6-bisphosphatase was prepared by in vitro phosphorylation . The comparison of the properties of the above enzymes demonstrated that all four had similar maximum activity . However, the phosphoenzyme was about 3-fold more sensitive to AMP and fructose 2,6-bisphosphate inhibition than the dephosphoenzyme, suggesting that regulation operates in vivo by this mechanism, leading to decreased enzyme activity . The purified mutant enzymes Ala11 and Asp11 exhibited properties closely similar to those of dephospho- and phosphofructose-1,6-bisphosphatase, respectively . These results indicate that the functional group at residue 11 is an important factor in the regulation of fructose-1,6-bisphosphatase activity and that Ser(P) can be functionally substituted by Asp in this enzyme.

Mol Cell Biol, 1988 May, 8(5), 2257 - 60
Distribution of telomere-associated sequences on natural chromosomes in Saccharomyces cerevisiae; Zakian VA et al.; Pulsed-field gel electrophoresis was used to examine the distribution of telomere-associated sequences on individual chromosomes in four strains of Saccharomyces cerevisiae . The pattern of X and Y' distribution was different for each strain . At least one chromosome in each strain lacked Y', and in some strains, chromosome I, the smallest yeast chromosome, lacked detectable amounts of both X and Y'.

Mol Cell Biol, 1988 May, 8(5), 2184 - 94
Transcription interferes with elements important for chromosome maintenance in Saccharomyces cerevisiae; Snyder M et al.; Transcription directed into a Saccharomyces cerevisiae autonomously replicating sequence (ARS) causes high-frequency loss of minichromosomes . Conditionally stable artificial yeast chromosomes were constructed that contain an inducible GAL promoter upstream of ARS1 . Under growth conditions in which the promoter was inactive, these chromosomes were mitotically stable; however, when the GAL promoter was induced, the chromosomes became extremely unstable as a result of transcriptional impairment of ARS function . This interference by the GAL promoter occurred only in cis but can occur from either side of ARS1 . Transcriptional interference of ARS function can be monitored readily by using a visual colony-color assay (P . Hieter, C . Mann, M . Snyder, and R.W . Davis, Cell 40:381-392, 1985), which was further developed as a sensitive in vivo assay for sequences which rescue ARS from transcription . DNA fragments from the 3' ends of genes, inserted downstream of the GAL promoter, protected ARS function from transcriptional interference . This assay is expected to be independent of both RNA transcript stability and processing . Philippsen et al . have shown that transcription into a yeast centromere inhibits CEN function in vivo (L . Panzeri, I . Groth-Clausen, J . Shepard, A . Stotz, and P . Philippsen, Chromosomes Today 8:46-58, 1984) . We identified two 200- to 300-base-pair DNA fragments flanking CEN4 that rescued ARS1 from transcription . Both of these fragments protected ARS from transcription when inserted in either orientation . The 3' ends of stable transcripts are encoded by fragments that protected the ARS from transcription, suggesting that the protection was achieved by transcription termination . It is suggested that protection of elements important for the replication and segregation of eucaryotic chromosomes from transcription is necessary for their proper function in vivo.

Mol Cell Biol, 1988 May, 8(5), 2132 - 9
Transcriptional-translational regulatory circuit in Saccharomyces cerevisiae which involves the GCN4 transcriptional activator and the GCN2 protein kinase; Roussou I et al.; GCN4 protein mediates the transcriptional activation of amino acid biosynthetic genes in Saccharomyces cerevisiae by specifically binding to DNA sequences in their 5'-regulatory regions . GCN4 expression is regulated at the level of translation, with translational derepression occurring under conditions of amino acid starvation . The product of the GCN2 gene is essential for translational derepression of GCN4 . Sequence analysis of the GCN2 gene reveals that the GCN2 protein has a domain highly homologous to the catalytic domain of all known protein kinases . Furthermore, gcn2 strains are deficient in a protein kinase activity corresponding to a protein with the calculated molecular weight deduced from the GCN2 open reading frame . Therefore it is likely that GCN2 encodes a protein kinase, which may be directly involved in translational regulation of the GCN4 mRNA . Transcription of the GCN2 gene is increased when cells are cultured in amino acid starvation medium . This transcriptional activation is mediated by the GCN4 protein, which binds to the promoter region of the GCN2 gene . Thus, this system is modulated by a transcriptional-translational regulatory circuit, which is activated by amino acid starvation . Activation is not the result of a simple quantitative increase of either one of the identified components of the circuit.

Genes Dev, 1988 May, 2(5), 517 - 27
SCH9, a gene of Saccharomyces cerevisiae that encodes a protein distinct from, but functionally and structurally related to, cAMP-dependent protein kinase catalytic subunits; Toda T et al.; A new gene, SCH9, was isolated from Saccharomyces cerevisiae by its ability to complement a cdc25ts mutation . Sequence analysis indicates that it encodes a 90,000-dalton protein with a carboxy-terminal domain homologous to yeast and mammalian cAMP-dependent protein kinase catalytic subunits . In addition to suppressing loss of CDC25 function, multicopy plasmids containing SCH9 suppress the growth defects of strains lacking the RAS genes, the CYR1 gene, which encodes adenylyl cyclase, and the TPK genes, which encode the cAMP-dependent protein kinase catalytic subunits . Cells lacking SCH9 grow slowly and have a prolonged G1 phase of the cell cycle . This defect is suppressed by activation of the cAMP effector pathway . We propose that SCH9 encodes a protein kinase that is part of a growth control pathway which is at least partially redundant with the cAMP pathway.

Can J Microbiol, 1988 May, 34(5), 680 - 5
Effect of valine and the herbicide sulfometuron methyl on acetolactate synthase activity in nuclear and plasmid-borne sulphometuron methyl resistant Saccharomyces cerevisiae strains; Maiti SN et al.; Acetolactate synthase (ALS) specific activity was evaluated in isogenic lines of Saccharomyces cerevisiae carrying the wild-type ILV2 gene or mutations in this gene for resistance to the herbicide sulfometuron methyl (SM) . Statistical comparisons were made between two nuclear alleles and among five alleles borne on a YE chimaeric plasmid transformed into a strain carrying a 1.5-kilobase deletion in the nuclear ILV2 gene . Decreased ALS activity of plasmid-borne SM-resistant mutations was shown not to be caused by copy number effects . ALS-specific activity in strains carrying the wild-type ILV2 allele exhibited strong feedback inhibition by valine and was sensitive to SM . All nuclear and plasmid-borne SM-resistance alleles resulted in ALS-specific activity highly resistant to SM and resistant to valine feedback inhibition.

Mikrobiologiia, 1988 May-Jun, 57(3), 491 - 3
{Distribution of trehalose between the cells and the rehydration medium in dehydrated Saccharomyces cerevisiae}; Zikmanis PB et al.; The work was concerned with studying the balance of trehalose distribution between the rehydration medium and Saccharomyces cerevisiae cells grown in a chemically defined medium and dehydrated using the convective technique . A direct linear correlation between the viability of populations and the overall residual trehalose content in the cells and in the medium after the rehydration of dry yeast cells was shown to be most important . An inverse correlation was established between the viability of yeast cells and the amount of trehalose mobilised by the cells in the process of rehydration.

J Biochem (Tokyo), 1988 May, 103(5), 858 - 62
Expression of a rat liver microsomal cytochrome P-450 catalyzing testosterone 16 alpha-hydroxylation in Saccharomyces cerevisiae: vitamin D3 25-hydroxylase and testosterone 16 alpha-hydroxylase are distinct forms of cytochrome P-450; Hayashi S et al.; Rat cytochrome P-450(M-1) cDNA was expressed in Saccharomyces cerevisiae TD1 cells by using a yeast-Escherichia coli shuttle vector consisting of P-450(M-1) cDNA, yeast alcohol dehydrogenase promoter and yeast cytochrome c terminator . The yeast cells synthesized up to 2 X 10(5) molecules of P-450(M-1) per cell . The microsomal fraction prepared from the transformed cells contained 0.1 nmol of cytochrome P-450 per mg of protein . The expressed cytochrome P-450 catalyzed 16 alpha- and 2 alpha-hydroxylations of testosterone in accordance with the catalytic activity of P-450(M-1), but did not hydroxylate vitamin D3 or 1 alpha-hydroxycholecalciferol at the 25 position . The expressed cytochrome P-450 also catalyzed the oxidation of several drugs and did not show 25-hydroxylation activity toward 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol . However, it cross-reacted with the polyclonal and monoclonal antibodies elicited against purified P-450cc25 which catalyzed the 25-hydroxylation of vitamin D3 . These results indicated that P-450(M-1) cDNA coded the 2 alpha- and 16 alpha-hydroxylase of testosterone, and that these two positions of testosterone are hydroxylated by a single form of cytochrome P-450 . Vitamin D3 25-hydroxylase and testosterone 16 alpha- and 2 alpha-hydroxylase are different gene products, although these two hydroxylase activities are immunochemically indistinguishable.

Genetika, 1988 May, 24(5), 773 - 80
{Cloning of the gene of Saccharomyces cerevisiae yeasts that determines resistance to the toxic action of cadmium ions}; Ianushka AP et al.; A gene conferring resistance to cadmium in Saccharomyces cerevisiae was isolated from a yeast gene library created on the basis of the pL3 vector . The phenotype of resistance is only expressed in the yeast cells with cloned DNA inserted into a multicopy plasmid . Integration of the plasmid into chromosome or introduction of the centromeric region into the plasmid decreases the level of cadmium resistance . The cloned Sau3A I fragment of the yeast chromosome is 3.5 kbp in size . Restriction analysis and subcloning experiments showed the gene to be located within 1.6 kbp of the XhoI-Sau3A I fragment of DNA . Instability was observed in the vicinity of the XhoI-Sau3A I fragment of the yeast DNA in Escherichia coli.

Biochem J, 1988 May 1, 251(3), 857 - 64
Isolation and expression of a pea vicilin cDNA in the yeast Saccharomyces cerevisiae; Watson MD et al.; A cDNA clone containing the complete coding sequence for vicilin from pea (Pisum sativum L.) was isolated . It specifies a 50,000-Mr protein that in pea is neither post-translationally processed nor glycosylated . The cDNA clone was expressed in yeast from a 2 micron plasmid by using the yeast phosphoglycerate kinase promoter and initiator codon . The resultant fusion protein, which contains the first 16 amino acid residues of phosphoglycerate kinase in addition to the vicilin sequence, was purified and subsequently characterized . It has slightly slower mobility on SDS/polyacrylamide-gel electrophoresis than standard pea vicilin and forms a mixture of multimers, some of which resemble the native protein.

EMBO J, 1988 May, 7(5), 1483 - 8
Purification and characterization of the inducible a agglutinin of Saccharomyces cerevisiae; Watzele M et al.; A cell surface glycoprotein induced by the mating pheromone alpha factor in Saccharomyces cerevisiae a cells has been purified to homogeneity . At 4 x 10(-9) M it strongly inhibits mating-type-specific agglutination between a and alpha cells . The protein is solely O-glycosylated . It consists of 29% carbohydrate and its apparent molecular mass is 22 kd on SDS gels . After HF treatment it behaves like a protein of 13 kd; therefore its true molecular mass probably is close to 18 kd . Mild periodate treatment destroys the biological activity of the purified protein . The protein contains one cysteine, no arginine, and 27% of the amino acids are serine and threonine residues, two thirds of which are glycosylated . With a polyclonal antibody the glycoprotein can already be detected at the cell surface 15 min after pheromone addition . The inducible antigen is not expressed in a specific phase of the cell cycle; it first appears exclusively on the growing bud . Mother cells express the antigen on their surface only after the daughter cells have separated; it is then localized at the tip of the pear-shaped 'shmoo' . Using the secretory ts-mutant sec 18 is shown that a mannosylated precursor of a agglutinin accumulates at the endoplasmic reticulum.

Nucleic Acids Res, 1988 Apr 25, 16(8), 3297 - 312
Sequence and expression of NUC1, the gene encoding the mitochondrial nuclease in Saccharomyces cerevisiae; Vincent RD et al.; The DNA sequence and studies on the expression of the NUC1 gene from Saccharomyces cerevisiae are presented . The NUC1 locus is located in the distal portion of the left arm of Chromosome X and encodes the major nuclease found in mitochondria . The inferred amino acid sequence of NUC1 predicts that the nuclease is basic, rich in prolines, of average hydrophobicity, and has a molecular weight for the primary translation product of 37,209 daltons . NUC1 is very poorly expressed, consistent with the codon usage bias determined from the DNA sequence and our previous determination of the number of enzyme molecules per cell . Mapping of the 5' terminus of the NUC1 mRNA reveals that the mRNA has a long 400 base untranslated leader in which are found three open reading frames, each initiated by an AUG . The possibility that these upstream open reading frames contribute to the poor expression of the NUC1 gene is discussed.

Gene, 1988 Apr 15, 64(1), 87 - 96
Construction of plasmids which lead to overproduction of yeast PHR1 photolyase in Saccharomyces cerevisiae and Escherichia coli; Sancar GB et al.; The PHR1 gene of Saccharomyces cerevisiae encodes a DNA photolyase which is normally present in fewer than 300 copies per cell . We have constructed plasmids in which PHR1 expression in yeast and Escherichia coli is under the control of strong, inducible promoters thereby leading to the regulated overproduction of biologically active photolyase . Under inducing conditions, E . coli cells carrying the tac-PHR1 plasmid pCB1241 accumulate up to 8% of total cellular protein as yeast photolyase; similarly, the GAL10-PHR1 fusion plasmid pGBS107 directs the synthesis of at least 1800-2400 molecules of photolyase per log-phase yeast cell . In both plasmids translation begins at the first ATG in the PHR1 open reading frame (ORF) . Constructs in which translation initiates at the second or third ATG fail to complement yeast and E . coli phr1 mutations, indicating that the first ATG in the PHR1 ORF is the translational start site in vivo and that all or part of the N-terminal 78 amino acids are required for activity.

J Biol Chem, 1988 Apr 15, 263(11), 5158 - 63
Solubilization and purification of alpha-mannosidase, a marker enzyme of vacuolar membranes in Saccharomyces cerevisiae; Yoshihisa T et al.; Yeast alpha-mannosidase, a marker enzyme of vacuolar membranes, was solubilized and purified from commercial bakers' yeast . The alpha-mannosidase was solubilized efficiently with 10 mM Na2CO3 . A high pH (greater than 8.5) and a sufficient amount of a detergent such as 0.2% (w/v) Triton X-100 were required to keep the enzyme in a soluble state . This suggested that the enzyme is either a peripheral membrane protein or an ecto-type integral membrane protein . After 4,300-fold purification by conventional chromatography, the alpha-mannosidase gave a single band on nondenaturing polyacrylamide gel electrophoresis, but could be fractionated into active isoforms, which consisted of 107-, 73-, and 31-kDa polypeptides, with a Mono Q anion exchange fast protein liquid chromatography system . Apparent molecular weight of the native enzyme was determined as 560,000 . It suggested that the composition of isoforms will be described as (107 kDa)n (73 kDa)6-n (31 kDa)6-n, where n is 0-6 . The 107- and 73-kDa polypeptides were purified further under denaturing conditions . One-dimensional peptide map analysis and immunological analysis of these polypeptides indicated that they are closely related proteins . Immunoblotting of crude cell lysates revealed that the 107-kDa polypeptide appeared first, and then the 73-kDa polypeptide appeared along growth phase . It suggested that proteolytic conversion of the 107-kDa polypeptide occurs to form the 73- and 31-kDa polypeptides and leads to formation of isoforms of the enzyme.

FEBS Lett, 1988 Apr 11, 231(1), 253 - 8
Iron storage in Saccharomyces cerevisiae; Raguzzi F et al.; A ferritin-like molecule was purified from iron-loaded cells of Saccharomyces cerevisiae, but its iron content was very low and was not representative of the cellular iron content . A study of the intracellular distribution of iron has shown that the vacuoles are involved in the storage of iron in the yeast cell . Moreover, it seems that this vacuolar iron can be further utilised by the cells for iron-requiring processes such as mitochondriogenesis.

Biochemistry, 1988 Apr 5, 27(7), 2288 - 94
Base cleavage specificity of angiogenin with Saccharomyces cerevisiae and Escherichia coli 5S RNAs; Rybak SM et al.; The base cleavage specificity of angiogenin toward naturally occurring polyribonucleotides has been determined by using rapid RNA sequencing technology . With 5S RNAs from Saccharomyces cerevisiae and Escherichia coli, angiogenin cleaves phosphodiester bonds exclusively at cytidylic or uridylic residues, preferably when the pyrimidines are followed by adenine . However, not all of the existent pyrimidine bonds in the 5S RNAs are cleaved, likely owing to elements of structure in the substrate . Despite the high degree of sequence homology between angiogenin and ribonuclease A (RNase A), which includes all three catalytic as well as substrate binding residues, the cleavage patterns with natural RNAs are unique to each enzyme . Angiogenin significantly hydrolyzes certain bonds that are not appreciably attacked by RNase A and vice versa . The different cleavage specificities of angiogenin and RNase A may account for the fact that the former is angiogenic while the latter is not.

Eur J Biochem, 1988 Apr 5, 173(1), 131 - 7
Reprogrammed expression of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae . Expression in vitro from a chemically synthesized gene and import into isolated mitochondria; Farrell LB et al.; A synthetic gene has been designed and constructed by total chemical synthesis as a first step in the functional relocation from the mitochondrion to the nucleus of a gene encoding subunit 9 of the yeast mitochondrial ATPase complex . This gene (NAP9) incorporates codons frequently used in nuclear genes of Saccharomyces cerevisiae and additionally includes a series of unique restriction enzyme cleavage sites to facilitate future systematic manipulations of the gene and its protein product . Following the expression of the NAP9 gene by transcription and translation in vitro, a radiolabelled protein was produced which displayed a gel electrophoretic mobility and solubility in chloroform/methanol characteristic of the authentic subunit 9 proteolipid encoded in vivo by the mitochondrial oli1 gene . In order to achieve import into mitochondria of yeast subunit 9, a fusion was made between the NAP9 gene and DNA encoding the cleavable presequence of the nuclearly encoded precursor to subunit 9 from Neurospora crassa . Following expression in vitro, the resultant fusion protein was imported and appropriately processed by isolated yeast mitochondria . The import of yeast subunit 9 was less efficient than that observed in parallel import experiments with yeast subunit 8 attached to the same presequence or with the naturally occurring intact N . crassa subunit 9 precursor . Yeast subunit 9 lacking a leader sequence is not imported into mitochondria but, unlike subunit 8, it does not embed itself into the outer membrane, in spite of its highly hydrophobic character.

Eur J Cancer Clin Oncol, 1988 Apr, 24(4), 591 - 6
'Petite' mutagenesis by anticancer drugs in Saccharomyces cerevisiae; Ferguson LR et al.; The mitochondria of cancer cells are potential targets for chemotherapy . Drugs which primarily affect mitochondrial DNA can be screened using a 'petite' mutagenesis assay in Saccharomyces cerevisiae . We have used this approach to estimate the antimitochondrial effects of a range of current clinical and experimental antitumour drugs with varying modes of action . Of agents currently in the clinic, the antimetabolites 5-fluorouracil and methotrexate were extremely effective in inducing this respiratory defect, providing cells were growing during treatment . Adriamycin, BCNU, bleomycin, methyl CCNU, cis-platinum, chlorambucil, daunomycin, nitracine, nitrogen mustard and hycanthone were also weakly effective 'petite' mutagens, in either growing or non-growing conditions . None of the currently used agents but some experimental drugs induced high numbers of 'petite' mutants during growing or non-growing conditions . To date, where such agents have been tested clinically, they have proved either ineffective or very toxic . It is possible that antimitochondrial effects on non-proliferating cellular tissues such as the heart might cause unacceptable toxicity and preclude the clinical use of such agents . For those agents effective against proliferating cells, the mitochondria could be an important target for chemotherapy in some cell types . This type of drug appears relatively uncommon in the clinic at present . The 'petite' mutagenesis assay could be more widely used as a screen to optimize this property in development of analogues of current clinical agents, or in developing new types of anticancer drug.

Genetics, 1988 Apr, 118(4), 609 - 17
Mutations affecting the tRNA-splicing endonuclease activity of Saccharomyces cerevisiae; Winey M et al.; Two unlinked mutations that alter the enzyme activity of tRNA-splicing endonuclease have been identified in yeast . The sen1-1 mutation, which maps on chromosome 12, causes temperature-sensitive growth, reduced in vitro endonuclease activity, and in vivo accumulation of unspliced pre-tRNAs . The sen2-1 mutation does not confer a detectable growth defect, but causes a temperature-dependent reduction of in vitro endonuclease activity . Pre-tRNAs do not accumulate in sen2-1 strains . The in vitro enzyme activities of sen1-1 and sen2-1 complement in extracts from a heterozygous diploid, but fail to complement in mixed extracts from separate sen1-1 and sen2-1 haploid strains . These results suggest a direct role for SEN gene products in the enzymatic removal of introns from tRNA that is distinct from the role of other products known to affect tRNA splicing.

Genetics, 1988 Apr, 118(4), 601 - 7
Nonsense mutations in essential genes of Saccharomyces cerevisiae; Riles L et al.; A new method for isolating nonsense mutations in essential yeast genes has been used to develop a collection of 115 ochre mutations that define 94 complementation groups . The mutants are isolated in a genetic background that includes an ochre suppressor on a metastable plasmid and a suppressible colony-color marker on a chromosome . When the parental strain is plated on a rich medium, the colonies display a pattern of red, plasmid-free sectors on a white background . Mutants containing an ochre mutation in any essential yeast gene give rise to nonsectoring, white colonies, since cell growth is dependent on the presence of the plasmid-borne suppressor . Analysis of the data suggests that mutations are being recovered from a pool of approximately 250 genes.

J Cell Biol, 1988 Apr, 106(4), 1075 - 81
Prepro-carboxypeptidase Y and a truncated form of pre-invertase, but not full-length pre-invertase, can be posttranslationally translocated across microsomal vesicle membranes from Saccharomyces cerevisiae; Hansen W et al.; We have determined that prepro-carboxypeptidase Y and a truncated form of pre-invertase can be translocated across the yeast microsomal membrane post-translationally in a homologous in vitro system . The yeast secretory protein prepro-alpha-factor which was previously shown to be an efficient posttranslational translocation substrate is therefore not unique in this regard, but rather the yeast ER protein translocation machinery is generally capable of accepting substrates from a ribosome-free, soluble pool . However, within our detection limits, full-length pre-invertase could not be translocated posttranslationally, but was translocated co-translationally . This indicates that not every fully synthesized pre-protein can use this pathway, presumably because normal or aberrant folding characteristics can interfere with translocation competence.

Arch Biochem Biophys, 1988 Apr, 262(1), 171 - 80
In situ behavior of the pyrimidine pathway enzymes in Saccharomyces cerevisiae . 3 . Catalytic and regulatory properties of carbamylphosphate synthetase: channeling of carbamylphosphate to aspartate transcarbamylase; Belkaid M et al.; The present work reports direct evidence for the channeling of carbamylphosphate from carbamylphosphate synthetase to aspartate transcarbamylase in the multifunctional protein that catalyzes the two first reactions of the pyrimidine pathway in Saccharomyces cerevisiae . This phenomenon is almost certainly related to the previously reported observation that the apparent in situ catalytic mechanism of aspartate transcarbamylase is altered by the association of this enzyme to carbamylphosphate synthetase . As a prerequisite of this investigation, the in situ catalytic and regulatory properties of carbamylphosphate synthetase were studied in the permeabilized cells of a strain that contains the wild-type multifunctional protein but is devoid of the carbamylphosphate synthetase specific for the arginine pathway.

J Gen Microbiol, 1988 Apr, 134 ( Pt 4), 1063 - 9
Low concentrations of trifluoperazine arrest the cell division cycle of Saccharomyces cerevisiae at two specific stages; Eilam Y et al.; Low concentrations of trifluoperazine (TFP) reversibly inhibited vegetative growth of Saccharomyces cerevisiae . The cell cycle was analysed by flow cytometry using haploid a cells synchronized by alpha-factor arrest and several temperature-sensitive cell division cycle mutants (cdc) . Cells were pulse-labelled with fluorescein-labelled concanavalin A (ConA-FITC) to determine cell division or stained with propidium iodide to determine the stage of cell cycle arrest by TFP . Cell growth was estimated from the changes in the relative intensity of scattered light, and budding was determined microscopically . When TFP was added before Start on release from alpha-factor arrest, after release of cdc28-arrested cells, and at transition from stationary phase to vegetative growth, cell growth, budding and DNA synthesis were inhibited . When TFP was added after execution of spindle pole body duplication, cell growth, bud emergence and DNA synthesis were not inhibited but cell division was inhibited and the cells arrested with buds at G2M Using cdc mutants, the second stage of arrest by TFP was determined to be just before medial-nuclear division.

Microbiologica, 1988 Apr, 11(2), 143 - 50
A synthetic medium for Saccharomyces cerevisiae; Vidotto V et al.; Growth of Saccharomyces cerevisiae strain Castelli 20 has been observed in a minimal synthetic medium using different glucose concentrations and without biotin, during the first 30 hrs of its development at 28 degrees C . The yeast's growth was observed spectrophotometrically at 675 nm reading the Optical Density every hour . The minimal medium of Sheperd et al . (1980), with glucose 15 g/L and biotin was modified: the vitamin was eliminated and the concentration of glucose was gradually reduced down to 0.5 g/L . At 5 g/L of glucose concentration and without vitamins the strain grew very well . From our results up to 30 hrs biotin has no influence on the yeast's growth . The medium would be useful to study Saccharomyces cerevisiae physiology during the early period of its development.

J Bacteriol, 1988 Apr, 170(4), 1945 - 9
Effect of calcofluor white on chitin synthases from Saccharomyces cerevisiae; Roncero C et al.; The growths of Saccharomyces cerevisiae wild-type strain and another strain containing a disrupted structural gene for chitin synthase (chs1::URA3), defective in chitin synthase 1 (Chs1) but showing a new chitin synthase activity (Chs2), were affected by Calcofluor . To be effective, the interaction of Calcofluor with growing cells had to occur at around pH 6 . Treatment of growing cells from these strains with the fluorochrome led to an increase in the total levels of Chs1 and Chs2 activities measured on permeabilized cells . During treatment, basal levels (activities expressed in the absence of exogenous proteolytic activation) of Chs1 and Chs2 increased nine- and fourfold, respectively, through a mechanism dependent on protein synthesis, since the effect was abolished by cycloheximide . During alpha-factor treatment, both Chs1 and Chs2 levels increased; however, as opposed to what occurred during the mitotic cell cycle, there was no further increase in Chs1 or Chs2 activities by Calcofluor treatment.

Curr Genet, 1988 Apr, 13(4), 291 - 7
Plasmid associations with residual nuclear structures in Saccharomyces cerevisiae; Conrad MN et al.; Acentric yeast plasmids are mitotically unstable, apparently because they cannot freely diffuse after replicating and therefore are not included in the daughter nucleus . This behavior could result if plasmids remain attached to structural elements of the nucleus after replicating . Since DNA replication is believed to take place on the nuclear matrix, we tested whether there was a correlation between the mitotic stability of a given plasmid and the extent to which it was found associated with residual nuclear structures . Residual nuclei were prepared from yeast nuclei by extraction with either high salt, 2 M NaCl, or low salt, 10 mM lithium diiodosalicylate (LIS) . Hybridization analysis was used to estimate the fraction of plasmid molecules remaining after nuclei were extracted . We examined the extent of matrix association of three ARS1 plasmids, Trp1-RI circle (1.45 kb), YRp7 (5.7 kb) and p lambda BAT (45.1 kb) with mitotic loss rates ranging from 3-25% . In addition we examined the matrix binding of the endogenous 2 micron plasmid and the 2 micron-derived YEp13 which is relatively stable in the presence of 2 micron and less stable in cir degree strains . Among the ARS1 plasmids we observed a negative correlation between stability and matrix association, consistent with models in which binding to the nuclear matrix prevents passive segregation of ARS1 plasmid molecules . No such correlation was observed among the 2 micron plasmids . Among all plasmids examined there is a positive correlation between size and matrix association.

Mol Cell Biol, 1988 Apr, 8(4), 1432 - 42
The Saccharomyces cerevisiae genome contains functional and nonfunctional copies of transposon Ty1; Boeke JD et al.; Saccharomyces cerevisiae Ty elements are transposons closely related to retroviruses . The DNA sequence of a functional Ty element (TyH3) is presented . The long terminal repeat sequences are different, suggesting that TyH3 is a recombinant Ty element . A chromosomal Ty element near the LYS2 gene, Ty173, was found to be nonfunctional, even though it has no detectable insertions or deletions . The defect in Ty173 transposition is caused by a missense mutation giving rise to a Leu-to-Ile substitution in the TYB (pol) open reading frame . Several chromosomal Ty elements carry this lesion in their DNA, indicating that nonfunctional Ty elements are common in the yeast genome.

J Cell Biochem, 1988 Apr, 36(4), 323 - 7
ADP-ATP carrier of Saccharomyces cerevisiae contains a mitochondrial import signal between amino acids 72 and 111; Smagula CS et al.; The ADP-ATP carrier (also referred to as the adenine nucleotide translocator) of Saccharomyces cerevisiae is encoded by a nuclear gene, translated in the cytosol, and imported into the mitochondrial inner membrane . In order to study the determinants of mitochondrial import, a series of fusion proteins, consisting of the first 21, 72, and 111 amino acids of the ADP-ATP carrier, joined to mouse dihydrofolate reductase were generated . Dihydrofate reductase is a cytoslic protein that does not bind mitochondria . The reticulocyte lysate reaction containing the 35S-methionine-labeled protein was incubated with mitochondria in a buffer containing 3% BSA . Following incubation for import, the reactions were treated with 1 mM PMSF or 25 micrograms/ml proteinase K; mitochondria were reisolated and analyzed by gel electrophoresis . The 21 and 72 amino acid hybrid proteins showed a low level of binding to mitochondria: the bound form was entirely protease accessible . The 111 amino acid hybrid protein was imported to a protease-protected location within mitochondria . It is concluded that the first 72 amino acids of the ADP-ATP carrier do not suffice to import the protein into mitochondria and that the region between amino acids 72 and 111, a region that contains a transmembrane-spanning domain, constitutes at least part of the mitochondrial import signal.

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