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