S. cerevisiae

Mol Cell Biol, 1985 Jul, 5(7), 1743 - 9
Deletion analysis identifies a region, upstream of the ADH2 gene of Saccharomyces cerevisiae, which is required for ADR1-mediated derepression; Beier DR et al.; Deletion analysis was used to identify sequences upstream of the ADH2 gene of Saccharomyces cerevisiae that are required for its regulation . 5' and 3' internal deletions of the ADH2 control region were created in vitro, and the fragments were ligated adjacent to the ADH1 promoter and structural gene . Hybrid genes with 3' deletions extending from -119 to -216 (the start site of ADH2 transcription is designated +1) were fully repressed and derepressed to high levels . Hybrid genes with 3' deletions extending from -119 to -257 were repressed but failed to significantly derepress . Hybrid genes lacking the -216 to -257 region also failed to respond to ADR1-5c, a mutant allele of the unlinked regulatory gene ADR1, which confers constitutive expression on ADH2 . This implies that the region between these deletion endpoints, which includes a 22-base-pair sequence of dyad symmetry, is required for efficient derepression of an adjacent promoter . Internal deletions extending in the 3' direction from position -1141 confirmed these results . Deletion mutants lacking the region -1141 to -259 were normally regulated, whereas deletions extending from -1141 to -115 were not derepressible . These results support the hypotheses that the ADH2 promoter may normally be in an inactive conformation in the yeast chromosome and that derepression of ADH2 requires positive activation mediated through an upstream activation sequence located between 216 and 257 base pairs 5' to the start site of ADH2 transcription . No evidence for a DNA sequence mediating repression was obtained.

Biochimie, 1985 Jul-Aug, 67(7-8), 717 - 23
Synthetic oligodeoxyribonucleotides as tools in molecular genetics: the characterization of the CYC1 (iso-1-cytochrome c encoding) locus of Saccharomyces cerevisiae; Smith M; The use of synthetic oligodeoxyribonucleotides as tools for the isolation, characterization and mutagenesis of eukaryote genes has played a major role in the molecular definition of the CYC1 locus of Saccharomyces cerevisiae, which is the structural gene for the apoprotein of iso-1-cytochrome c . Thus, the possibility of using a synthetic oligodeoxyribonucleotide as a probe to identify and monitor the isolation of a specific gene was first established by model studies which defined the melting temperatures (TmS) for duplexes of oligonucleotides of different lengths and base compositions . This led to the isolation of the CYC1 locus using a synthetic 13 nucleotide probe . A more convenient strategy for determination of DNA sequences by the Sanger method was provided by using synthetic oligodeoxyribonucleotides as primers with denatured double-strand plasmid DNA as template . By this means, the sequence of the CYC1 locus was determined by "walking" along the gene without isolating restriction fragments of the DNA or separating DNA strands . Synthetic oligodeoxyribonucleotides, used as primers for reverse transcriptase with mRNA as template, were also used to precisely define the 5'- and 3'-ends of the iso-1-cytochrome c mRNA . Yet another application of synthetic oligodeoxyribonucleotides is their use as specific mutagens after in vitro incorporation into double-stranded DNA . In the case of iso-1-cytochrome c, this mutagenic strategy is being used to define the role of conserved amino-acids in cytochrome function.(ABSTRACT TRUNCATED AT 250 WORDS)

Can J Microbiol, 1985 Jul, 31(7), 654 - 6
Comparison of the mitochondrial endonucleases from Neurospora crassa and Saccharomyces cerevisiae; von Tigerstrom RG et al.; The endonucleases from Neurospora crassa and Saccharomyces cerevisiae are not closely related antigenically . They also differ with respect to their activity at pH 8, their degree of hydrophobicity, and their sensitivity to elevated temperatures . However, the two nucleases have similar specific activities, are inhibited by EDTA, and have nearly identical substrate specificities . Since the enzymes also have the same mode of action and intracellular location, these similarities may indicate that they have the same physiological role despite their structural differences.

Biochemistry, 1985 Jun 18, 24(13), 3332 - 7
Structure-activity relationships in the dodecapeptide alpha-factor of Saccharomyces cerevisiae: position 6 analogues are poor inducers of agglutinability; Baffi RA et al.; Five des-Trp1,Cha3,X6 analogues of alpha-factor, where X = Ala, Val, Ile, Nle, or D-Leu and X = Leu in the natural alpha-factor sequence, were prepared by solution-phase techniques utilizing isobutyl chloroformate or 1-hydroxybenzotriazole accelerated active esters as the coupling agents . Purification to 98% or greater homogeneity was accomplished by high-performance liquid chromatography on a reversed-phase muBondapak C18 column with methanol/water/trifluoroacetic acid as the mobile phase . Three of the synthesized analogues (X6 = Val, Ile, Nle) induced morphogenesis and increased agglutinability in a cells . These substitutions demonstrate that a gamma-branched side chain at position 6 is not essential for biological activity . All of the active analogues induced morphogenesis at lower concentrations than they induced enhanced agglutinability . These results and other structure-activity relationships {Baffi, R . A., Shenbagamurthi, P., Terrance, K., Becker, J . M., Naider, F., & Lipke, P . (1984) J . Bacteriol . 158, 1152-1156} indicate that the agglutination and morphological responses to alpha-factor can be varied independently . Replacement of Leu6 with Ala or D-Leu resulted in inactive analogues that were not antagonistic for alpha-factor activity . Cell-mediated hydrolysis experiments indicated that the biological activities of the alpha-factor analogues are independent of their rates of degradation . All position 6 analogues were hydrolyzed more slowly than the parent compound, suggesting that the enzyme which degrades alpha-factor is highly specific for the native structure.

Nucleic Acids Res, 1985 Jun 11, 13(11), 3791 - 804
Size and position of intervening sequences are critical for the splicing efficiency of pre-mRNA in the yeast Saccharomyces cerevisiae; Klinz FJ et al.; The size of the 309 bp actin gene intron of the yeast Saccharomyces cerevisiae was enlarged by inserting DNA fragments of different lengths and sequence . Enlarging the intron above 551 bp, the largest known yeast intron, led to a decrease in splicing efficiency . The effect on transcript splicing was dependent on the length of the inserted fragments rather than their sequence . It was also observed that insertion of the actin gene intron into different regions of the normally unsplit yeast YP2 gene, significantly influenced the efficiency of splicing of the resulting transcripts . The splicing efficiency of splicing of with the increase of the distance between the mRNA cap site and the intervening sequence.

J Biol Chem, 1985 Jun 10, 260(11), 6960 - 5
A comparative study of the characteristics of eIF-2 and eIF-2-ancillary factor activities from yeast Saccharomyces cerevisiae and rabbit reticulocytes; Ahmad MF et al.; The characteristics of yeast eukaryotic initiation factor 2 (eIF-2) and Co-eIF-2A have been studied and compared with those of the corresponding factors from rabbit reticulocytes . 1) Unlike eIF-2r, purified eIF-2y did not contain bound GDP . 2) Purified eIF-2y preparation contained GTPase activity and dephosphorylated GTP to GDP . 3) An anti-eIF-2r preparation which predominantly precipitated the gamma-subunit (Mr 54,000) of eIF-2r also precipitated the larger subunit (Mr 54,000) of eIF-2y . 4) Unlike eIF-2r, ternary complex formation by eIF-2y was not inhibited by Mg2+ . 5) Both Co-eIF-2A20y and Co-eIF-2r significantly enhanced Met-tRNAf binding to eIF-2y and, again, Mg2+ did not have any effect on this stimulated Met-tRNAf binding to eIF-2y . 6) Both Co-eIF-2A20y and Co-eIF-2r were similarly effective in stimulating Met-tRNAf binding to eIF-2r in the absence of Mg2+ . However, in the presence of Mg2+, Co-eIF-2A20y was significantly less effective than Co-eIF-2r as Co-eIF-2A20y did not promote displacement of GDP from eIF-2r X GDP . 7) eIF-2y bound {3H}GDP and this binding was significantly enhanced in the presence of Mg2+ . Also, {3H}GDP in the preformed eIF-2y X {3H}GDP complex was rapidly exchanged with exogenously added unlabeled GDP in the presence of Mg2+ . Co-eIF-2A20y had no effect on GDP binding to eIF-2y nor on GDP exchange reactions . 8) Reticulocyte heme-regulated protein synthesis inhibitor, which phosphorylated almost completely (in excess of 80%) the alpha-subunit (Mr 38,000) of eIF-2r, also phosphorylated similarly the smaller subunit (Mr 36,000) of eIF-2y . However, such phosphorylation had no significant effect on ternary complex formation, GDP binding, and GDP exchange reactions.

J Biol Chem, 1985 Jun 10, 260(11), 6955 - 9
Purification and properties of eukaryotic initiation factor 2 and its ancillary protein factor (Co-eIF-2A) from yeast Saccharomyces cerevisiae; Ahmad MF et al.; Two peptide chain initiation factor activities, eIF-2y and Co-eIF-2A20y, were purified from the high speed supernatant fraction of the yeast Saccharomyces cerevisiae and their properties were studied . 1) In sodium dodecyl sulfate-polyacrylamide gels, purified eIF-2y showed two major polypeptide bands corresponding to molecular weights of 54,000 and 36,000 . The Mr 54,000 band was significantly more intense than the Mr 36,000 band, indicating the possible presence of two polypeptides of equal molecular weight in this band . The molecular weight of eIF-2y, determined using a density gradient centrifugation method, was approximately 140,000 . 2) In sodium dodecyl sulfate-polyacrylamide gel, purified Co-eIF-2A20y showed a single polypeptide band corresponding to a molecular weight of 20,000 . A similar molecular weight for Co-eIF-2A20y was also found using a density gradient centrifugation method . 3) In partial reactions, eIF-2y bound Met-tRNAf in the presence of Mg2+ . The reaction required GTP . Co-eIF-2A20y stimulated Met-tRNAf binding to eIF-2y (2-3-fold) and also rendered the complex stable to 3 X 10(-5) M aurintricarboxylic acid . 4) This Co-eIF-2A20y activity was heat-labile and N-ethylmaleimide-insensitive . 5) Antibodies were prepared by injecting rabbits with homogeneous Co-eIF-2A20y . Such anti-Co-eIF-2A20y inhibited (60%) protein synthesis in a yeast cell-free protein synthesizing system and completely blocked Co-eIF-2A20y stimulation of Met-tRNAf . 40 S initiation complex formation . Protein synthesis inhibition by anti-Co-eIF-2A20y was almost completely reversed by preincubation of the antibodies specifically with homogeneous Co-eIF-2A20y.

Eur J Biochem, 1985 Jun 3, 149(2), 401 - 4
Mutations affecting the enzymes involved in the utilization of 4-aminobutyric acid as nitrogen source by the yeast Saccharomyces cerevisiae; Ramos F et al.; We present genetic evidence for the enzymes 4-aminobutyrate: 2-oxoglutarate aminotransferase (EC 2.6.1.19) and succinate-semialdehyde dehydrogenase {NAD(P)+} (EC 1.2.1.16) constituting the functional pathway for the utilization of 4-aminobutyric acid as a nitrogen source by Saccharomyces cerevisiae . We show that the pathway is induced by 4-aminobutyric acid and that the presence of the pathway enzymes probably requires the integrity of a positive control element.

Genetika, 1985 Jun, 21(6), 914 - 8
{Molecular nature of the direct mutations induced by 4-nitro-quinoline-N-oxide and 3-methyl-4-nitroquinoline-N-oxide in the ADE2 gene of Saccharomyces cerevisiae}; Kasinova GV et al.; The lethal and mutagenic effects and the nature of forward mutations in ADE2 gene induced by highly carcinogenic agent 4-nitroquinoline-N-oxide (4NQO) and its noncarcinogenic analogue 3-methyl-4-nitroquinoline-N-oxide (3M4NQO) have been examined in Saccharomyces cerevisiae . It is shown that 3M4NQO is more toxic than 4NQO . Both are very efficient mutagens: the mutagenic efficiency for ADE1 and ADE2 genes was 7.9 X 10(-5) for 4NQO and 10.5 X 10(-5) for 3M4NQO . The base pair substitutions are the main type of induced mutations in ADE2 gene (95 and 89% for 4NQO and 3M4NQO, respectively); among these 40% transversions for 4NQO and 63% for 3M4NQO, GC----AT transitions-32 and 31% for 4NQO and 3M4NQO, respectively, AT----GC transitions-23 and 22% for 4NQO and 3M4NQO, respectively . The results obtained indicate that 4NQO and 3M4NQO induce the same spectrum of mutations in ADE2 gene and that both mutagens are nonspecific in yeast cells.

Mol Cell Biol, 1985 Jun, 5(6), 1522 - 4
DNase I-hypersensitive sites in the galactose gene cluster of Saccharomyces cerevisiae; Proffitt JH; Five DNase I-hypersensitive regions were associated with the Saccharomyces cerevisiae galactose gene cluster during both galactose induction and glucose repression of transcription . Four hypersensitive regions were located in areas flanking the GAL cluster genes, and one site occurred within GAL10 . A DNase I-hypersensitive region located between the 5' ends of divergently transcribed GAL10 and GAL1 contained sequences essential for the transcription of both genes.

Mol Cell Biol, 1985 Jun, 5(6), 1512 - 21
Saccharomyces cerevisiae coordinates accumulation of yeast ribosomal proteins by modulating mRNA splicing, translational initiation, and protein turnover; Warner JR et al.; The rate of accumulation of each ribosomal protein is carefully regulated by the yeast cell to provide the equimolar ratio necessary for the assembly of the ribosome . The mechanisms responsible for this regulation have been examined by introducing into the yeast cell extra copies of seven individual ribosomal protein genes carried on autonomously replicating plasmids . In each case studied the plasmid-borne gene was transcribed to the same degree as the genomic gene . Nevertheless, the cell maintained a balanced accumulation of ribosomal proteins, using a variety of methods other than transcription . (i) Several ribosomal proteins were synthesized in substantial excess . However, the excess ribosomal protein was rapidly degraded . (ii) The excess mRNA for two of the ribosomal protein genes was translated inefficiently . We provide evidence that this was due to inefficient initiation of translation . (iii) The transcripts derived from two of the ribosomal protein genes were spliced inefficiently, leading to an accumulation of precursor RNA . We present a model which proposes the autogenous regulation of mRNA splicing as a eucaryotic parallel of the autogenous regulation of mRNA translation in procaryotes . Finally, the accumulation of each ribosomal protein was regulated independently . In no instance did the presence of excess copies of the gene for one ribosomal protein affect the synthesis of another ribosomal protein.

Proc Natl Acad Sci U S A, 1985 Jun, 82(11), 3785 - 9
RAS2 of Saccharomyces cerevisiae is required for gluconeogenic growth and proper response to nutrient limitation; Tatchell K et al.; Saccharomyces cerevisiae contains two genes with remarkable homology to members of the ras oncogene family . These two genes, RAS1 and RAS2, constitute an essential gene family since spores with disruptions of both genes fail to grow . We report here that strains containing RAS2 disruptions have three distinct phenotypes . First, they fail to grow efficiently on nonfermentable carbon sources . Second, they hyperaccumulate the storage carbohydrates glycogen and trehalose . Third, diploid cells homozygous for the RAS2 disruptions sporulate on rich media . Extragenic suppressors have been isolated that suppress the gluconeogenic defect . These suppressors fall into at least three complementation groups, mutations in two of which bypass the normal requirement of RAS for cell viability, allowing cells containing neither RAS gene to grow . The phenotype of the RAS2 mutant and extragenic suppressors implicate RAS with some function in the normal response to nutrient limitation.

Mutat Res, 1985 Jun-Jul, 150(1-2), 217 - 24
Modulation in cytochrome P-420 and P-450 content in Saccharomyces cerevisiae according to physiological conditions and genetic background; von Borstel RC et al.; The diploid strain D5 of Saccharomyces cerevisiae, relative to other strains of yeast, has a large amount of cytochrome P-450 present during the logarithmic phase of growth and a low amount of cytochrome P-420 . As the stationary phase of growth is approached, an increasing intensity of absorbance is observed at 420 nm . If the cells are suspended in buffer during mid-logarithmic growth, the absorbance at 450 nm disappears and absorbance at 420 nm is increased after the cells have been held in buffer for 24 h . At late logarithmic growth, the absorbance at 450 nm is still retained after the cells have been held in buffer for 24 h . Within 44 h of the time of harvest, the absorbance at 450 nm disappears completely and the absorbance at 420 nm is intense . Cytoplasmic petite variants of strain D5 have less of both cytochromes P-450 and P-420 than does the grande D5 strain; the absorbances at 450 and 420 nm are retained up to 96 h when the cells are held in buffer . Haploid spores of strain D5 exhibit absorbances at 450 and 420 nm during the logarithmic phase of growth, and these absorbances are retained after the cells are held in buffer for 24 h . An hypothesis is proposed which states that cytochrome P-450 is the membrane-bound form and cytochrome P-420 is free in the cytosol; the cytochromes interconvert and are active in either state until the associated enzymes disassociate.

DNA, 1985 Jun, 4(3), 203 - 10
Expression of rat liver cytochrome P-450MC cDNA in Saccharomyces cerevisiae; Oeda K et al.; Rat liver cytochrome P-450MC cDNA was inserted between the ADH1 promoter and terminator regions of the yeast expression vector pAAH5 . On introduction of the resulting recombinant plasmid pAMC1, Saccharomyces cerevisiae cells synthesized up to 8 X 10(5) molecules per cell of the cytochrome P-450MC protein, most of which was localized in yeast microsomes . Approximately half of the synthesized cytochrome contained heme in the enzyme molecule . These formed a functional electron-transport chain in the microsomes which exhibited aryl hydrocarbon hydroxylase activity toward benzo{a}pyrene.

J Gen Microbiol, 1985 Jun, 131 ( Pt 6), 1425 - 32
Regulation by ammonium of glutamate dehydrogenase (NADP+) from Saccharomyces cerevisiae; Bogonez E et al.; The activity of glutamate dehydrogenase (NADP+) (EC 1.4.1.4; NADP-GDH) of Saccharomyces cerevisiae is decreased under conditions in which intracellular ammonia concentrations increases . A high internal ammonia concentration can be obtained (a) by increasing the ammonium sulphate concentration in the culture medium, and (b) by growing the yeast either in acetate + ammonia media, where the pH of the medium rises during growth, or in heavily buffered glucose + ammonia media at pH 7.5 . Under these conditions cellular oxoglutarate concentrations do not vary and changes in NADP-GDH activity appear to provide a constant rate of oxoglutarate utilization . The following results suggest that the decrease in NADP-GDH activity in ammonia-accumulating yeast cells is brought about by repression of synthesis: (i) after a shift to high ammonium sulphate concentrations, the number of units of activity per cell decreased as the inverse of cell doubling; and (ii) the rate of degradation of labelled NADP-GDH was essentially the same in ammonia-accumulating yeast cells and in controls, whereas the synthesis constant was much lower in the ammonia-accumulating cells than in the controls.

J Cell Biol, 1985 Jun, 100(6), 1854 - 62
Outer plaque assembly and spore encapsulation are defective during sporulation of adenylate cyclase-deficient mutants of Saccharomyces cerevisiae; Uno I et al.; Sporulation in diploid cells homozygous for the cyr1-2 mutation of the yeast Saccharomyces cerevisiae was examined . This mutation causes a defect in adenylate cyclase and temperature-sensitive arrest in the G1 phase of the mitotic cell cycle . The cyr1-2/cyr1-2 diploid cells were able to initiate meiotic divisions, but produced predominantly two-spored asci at the restrictive temperature . Temperature-sensitive period for production of two-spored asci was approximately 12 h after the transfer of cells to the sporulation medium . The levels of cAMP increased during this period in the wild type and cyr1-2/cyr1-2 diploid cells incubated at the permissive temperature, but remained at an extremely low level in the cyr1-2/cyr1-2 diploid cells incubated at the restrictive temperature . Dyad analysis of the cyr1-2 strain indicated that meiotic products were randomly included into ascospores . Fluorescent microscopy of the cyr1-2/cyr1-2 diploid cells incubated at the restrictive temperature revealed that individual haploid nuclei were enclosed in each of the two spores after meiosis . About half of the cyr1-2/cyr1-2 diploid cells entered normal meiosis 1 producing two normal spindle pole bodies with inner and outer plaques, and the other half entered abnormal meiosis 1 producing one normal spindle pole body and one defective spindle pole body without out plaque . At meiosis II, some cells contained a pair of normal spindle pole bodies and other cells contained pairs of normal and abnormal spindle pole bodies.

Biochim Biophys Acta, 1985 May 30, 845(2), 151 - 7
The effect of chemical modification of Saccharomyces cerevisiae on electrophoretic mobility, cell-wall structure and amphotericin B uptake; Ellul H et al.; Saccharomyces cerevisiae NCYC 239 suspended in solutions of NaCl showed two distinct plateaus in plots of electrophoretic mobility vs . pH, corresponding to pKa values of approx . 2 and 5 . This is in contrast to cells suspended in buffer where only a single pKa (4) can be determined . Modification of cells with KI/I2 or nitrous acid led to altered electrophoretic mobility, indicating the presence of sulphydryl and amino groups, respectively, in the yeast cell surface, whereas uranyl nitrate modification had little effect, suggesting phosphate groups to be absent . Electron micrographs showed visible effects of KI/I2 and nitrous acid modification on cell membrane structure, and in these modified cells amphotericin B uptake was rapid . It is suggested that diffusion through the cell wall is the rate-limiting step for amphotericin B uptake . An activation energy of 20 kJ X mol-1 was determined for uptake of amphotericin B by unmodified cells.

J Biol Chem, 1985 May 25, 260(10), 5871 - 4
Nuclear mutants of Saccharomyces cerevisiae with altered subunits 4, 5, and 6 of cytochrome oxidase; Koerner TJ et al.; A collection of pet mutants of Saccharomyces cerevisiae has been screened for lesions in cytochrome oxidase . Three different complementation groups have been identified to consist of strains with altered forms of subunits 4, 5, or 6 that are known to be encoded by nuclear genes . The mutant proteins cross-react with antiserum to the holoenzyme or to the individual subunits but exhibit either an increase or decrease in size . In each instance the mutation imparts a respiratory deficient phenotype which is due to reduced levels of cytochrome oxidase activity in the mitochondria . These results indicate that each of the three proteins is required either for the catalytic activity or for the assembly of functional cytochrome oxidase.

Arch Biochem Biophys, 1985 May 15, 239(1), 184 - 90
Partial purification and properties of two histone acetyltransferases from the yeast, Saccharomyces cerevisiae; Lopez-Rodas G et al.; Two histone acetyltransferases, A and B, have been extracted and partially purified from yeast cells . The purification scheme included ammonium sulfate precipitation, and chromatography on DEAE-Sepharose and Sephadex G-200 . The basic properties of both enzymes closely correspond to those of acetyltransferase A and B found in higher eucaryotes . Yeast enzyme A elutes from DEAE-Sepharose prior to acetyltransferase B, and it is activated by low concentrations of DNA and strongly inhibited by p-chloromercuribenzoate (PCMB) . Enzyme B is inhibited by DNA over the entire range of concentrations tested and it is less sensitive to PCMB than enzyme A . When assayed with yeast whole histones, enzyme B shows a marked specificity toward histone H4, although H3 and H2B are also accepted as substrates . Enzyme A preferentially catalyzes the acetylation of yeast H2B and H3, with the other two core histones being acetylated to a much lesser extent.

J Biol Chem, 1985 May 10, 260(9), 5596 - 602
Attempted expression of a human initiator tRNA gene in Saccharomyces cerevisiae; Drabkin HJ et al.; In attempts to overproduce the wild type and, eventually, mutant human initiator methionine tRNAs for use in structure-function relationship studies, we have investigated the expression of the wild type human initiator tRNA gene in the yeast Saccharomyces cerevisiae, both in vitro and in vivo . We find that the yeast extract, while capable of accurately transcribing several yeast tRNA genes, does not transcribe the human initiator tRNA gene . In addition, when the human initiator tRNA gene is introduced into yeast as part of a 2 mu vector, no expression of the human tRNA gene was detected . A yeast alanine tRNA gene similarly introduced into yeast is expressed efficiently . The block in expression of the human tRNA gene is at the level of transcription and not processing . The yeast cell-free extract can accurately process precursors of the same human initiator tRNA made in a HeLa cell-free extract . Surprisingly, although the human tRNA gene has essentially the same intragenic control elements as the yeast initiator tRNA gene, the human tRNA gene competes extremely poorly for transcription factors in yeast extracts . In the course of screening a yeast DNA bank for initiator tRNA clones we have isolated and sequenced three yeast tRNA genes corresponding to glycine, alanine, and aspartic acid tRNAs . The sequence of glycine tRNA gene differs from the published tRNA sequence in having an additional nucleotide in the variable loop . The alanine tRNA gene codes for a new tRNA . All three genes are transcriptionally active in yeast extracts.

J Mol Biol, 1985 May 5, 183(1), 31 - 42
Partial suppression of an ochre mutation in Saccharomyces cerevisiae by multicopy plasmids containing a normal yeast tRNAGln gene; Pure GA et al.; We screened a yeast genomic library for recombinant DNA plasmids that complemented the ultraviolet (u.v.) sensitivity of a strain of Saccharomyces cerevisiae designated rad4-3 that is defective in excision repair of DNA . A multicopy plasmid (pNF4000) with a 9.4 X 10(3) base-pair yeast DNA insert partially complemented the u.v . sensitivity of rad4-3, but not of two other rad4 allelic mutants (rad4-2 and rad4-4), or of other u.v.-sensitive rad mutants . The yeast insert was analyzed by restriction mapping, DNA-DNA hybridization, DNA-tRNA hybridization and DNA sequencing . This analysis revealed the presence of a normal tRNAGln gene, a yeast sigma element situated 5' to the transfer RNA gene, a Ty element and a solo delta element . Deletion analysis of pNF4000 showed that the tRNAGln gene is required for partial complementation of the u.v . sensitivity of rad4-3 . Furthermore, a multicopy plasmid containing a tRNAGln gene derived from a different region of the yeast genome also partially complemented the u.v . sensitivity of rad4-3 . The rad4-3 mutation is suppressed following transformation with a plasmid containing the known ochre suppressor SUP11-o, indicating that it is an ochre mutation . We therefore conclude that when expressed in sufficient quantity, normal tRNAGln (which usually decodes the sense codon CAA) can weakly suppress the nonsense ochre codon UAA, and suggest that this represents an example of wobble occurring at the first rather than at the third position of the codon.

J Bacteriol, 1985 May, 162(2), 579 - 83
alpha-Aminoadipate as a primary nitrogen source for Saccharomyces cerevisiae mutants; Zaret KS et al.; In contrast to wild-type strains of the yeast Saccharomyces cerevisiae, lys2 and lys5 mutants are able to utilize alpha-aminoadipate as a primary source of nitrogen . Chattoo et al . (B . B . Chattoo, F . Sherman, D . A . Azubalis, T . A . Fjellstedt, D . Mehnert, and M . Ogur, Genetics 93:51-65, 1979) relied on this difference in the effective utilization of alpha-aminoadipate to develop a procedure for directly selecting lys2 and lys5 mutants . In this study we used a range of mutant strains and various media to determine why normal strains are unable to utilize alpha-aminoadipate as a nitrogen source . Our results demonstrate that the anabolism of high levels of alpha-aminoadipate through the biosynthetic pathway of lysine results in the accumulation of a toxic intermediate and, furthermore, that lys2 and lys5 mutants contain blocks leading to the formation of this intermediate.

Radiat Res, 1985 May, 102(2), 254 - 63
An attempt to stimulate mitosis in Saccharomyces cerevisiae with the ultraviolet luminescence from exponential phase cultures of this yeast; Quickenden TI et al.; Neither cell division nor growth of Saccharomyces cerevisiae were stimulated by the ultraviolet luminescence produced by adjacent exponential phase cultures of the yeast . The study included experiments in which the inocula (density = 5 X 10(7) cells cm-3) were irradiated and in which lag phase cultures (densities = 1 X 10(6) or 5 X 10(6) cells cm-3) were irradiated for 30 min with the yeast luminescence . These results do not support the claims of earlier workers that dividing cells can stimulate mitosis in optically coupled cultures by the so-called "mitogenetic effect."

Mol Cell Biol, 1985 May, 5(5), 907 - 15
Characterization of an essential Saccharomyces cerevisiae gene related to RNA processing: cloning of RNA1 and generation of a new allele with a novel phenotype; Atkinson NS et al.; The RNA1 gene product is believed to be involved in RNA metabolism due to the phenotype of a single conditionally lethal, temperature-sensitive allele, rna1-1 . We cloned the RNA1 gene and determined that it produces a 1,400-nucleotide polyadenylated transcript . On a multicopy plasmid, the mutant rna1-1 allele partially complements the rna1-1 temperature-sensitive growth defect . This suggests that the temperature-sensitive nature of the rna1-1 allele results from the synthesis of a product with lowered activity or stability at elevated temperatures or from a decrease in synthesis of the rna1-1 product at the restrictive temperature . A chromosomal disruption of RNA1 behaves as a recessive lethal mutation . Haploids bearing the disruption were isolated by sporulating a diploid heterozygous for the disrupted allele and the rna1-1 allele and possessing an episomal copy of the RNA1 gene . Analysis of the rescued haploids bearing the chromosomal disruption indicated that the recessive lethal phenotype of the RNA1 disruption is not merely due to a block in spore germination . Unexpectedly, diploids heterozygous for the disruption and the rna1-1 alleles become aneuploid for chromosome XIII at a frequency of 2 to 5% . It appears that the disrupted RNA1 allele on a multicopy plasmid also promotes aneuploidy for chromosome XIII . Promotion of aneuploidy seems to be a phenotype of this particular allele of RNA1.

Mutat Res, 1985 May, 149(3), 359 - 64
Mutagenic effect and mutation spectrum induced by 3H decay in the 8th position of DNA purine bases in Saccharomyces cerevisiae; Korolev VG et al.; Lethal and mutagenic effects and the mutation spectrum induced by 3H decay in the 8th position of adenine and guanine in yeast DNA have been studied . For haploid cells labelled with {8-3H}deoxyadenosinemonophosphate (8-3H-A) and {8-3H}deoxyguanosinemonophosphate (8-3H-G), the lethal efficiencies were determined as (3.0 +/- 0.8) X 10(-3) decay-1 and (3.8 +/- 0.6) X 10(-3) decay-1, respectively, and the mutagenic efficiencies as (5.7 +/- 1.1) X 10(-8) decay-1 and (8.7 +/- 1.4) X 10(-8) decay-1, respectively . The lethal effect of {8-3H}purines may be explained as being due to internal beta-irradiation . In contrast, the local effect of 3H-transmutation was twice as mutagenic as beta-irradiation when the induction of forward gene mutations was examined . Within the spectrum of mutations induced by 8-3H-G, a preference for GC----AT transitions was observed.

Mutat Res, 1985 May, 149(3), 339 - 51
Acetone, methyl ethyl ketone, ethyl acetate, acetonitrile and other polar aprotic solvents are strong inducers of aneuploidy in Saccharomyces cerevisiae; Zimmermann FK et al.; A diploid yeast strain D61.M was used to study induction of mitotic chromosomal malsegregation, mitotic recombination and point mutation . Several ketones (including acetone and methyl ethyl ketone) and some organic acid esters (including the methyl, ethyl and 2-methoxyethyl esters of acetic acid) and acetonitrile strongly induced aneuploidy but not recombination or point mutation . Only diethyl ketone induced low levels of recombination and point mutation in addition to aneuploidy . Related compounds were weak inducers of aneuploidy: methyl n-propyl ketone, the methyl esters of propionic and butyric acid, acetic acid esters of n- and iso-propanol and ethyl propionate . No mutagenicity was found with n-butyl and isoamyl acetate, ethyl formate, acetyl acetone (2,5-dipentanone) and dioxane . Methyl isopropyl ketone induced only some recombination and point mutation but no aneuploidy . Efficient induction was only observed with a treatment protocol in which growing cells were exposed to the chemicals during a growth period of 4 h at 28 degrees C followed by incubation in ice for more than 90 min, usually overnight for 16-17 h . Aneuploid cells could be detected in such cultures during a subsequent incubation at growth temperature if the chemical was still present . Detailed analysis showed that there was a high incidence of multiple events of chromosomal malsegregation . It is proposed that the mutagenic agents act directly on tubulin during growth so that labile microtubules are formed which dissociate in the cold . When cells are brought back to temperatures above the level critical for reassembly of tubulin and allowed to grow, faulty microtubules are formed.

J Bacteriol, 1985 May, 162(2), 763 - 7
Formation of septum-like structures at locations remote from the budding sites in cytokinesis-defective mutants of Saccharomyces cerevisiae; Slater ML et al.; Cell wall structures that partition membrane-bound portions of cytoplasm were formed at sites along the peripheral wall when a cytokinesis-defective cell division cycle mutant (cdc3) of Saccharomyces cerevisiae was grown at a restrictive temperature . The appearance of these structures, as observed in electron micrographs, was similar to that of normal septa . Aberrant septa were also detected in cytokinesis mutants harboring mutations cdc10, cdc11, and cdc12, after growth at 37 degrees C . Formation of the abnormal septa was abolished by the introduction, in a cdc3-containing strain, of additional cell cycle mutations that precluded events leading to cytokinesis and cell division . These results showed that septum formation can occur in the absence of cytokinesis . Formation of the abnormal structures was controlled by the same sequences of cell cycle events as formation of normal septa but was not subject to the spatial controls that ensure association of the septum with the budding site.

Biochim Biophys Acta, 1985 Apr 25, 834(2), 249 - 55
Purification of a phospholipase B inhibitor from Saccharomyces cerevisiae; Witt W et al.; The phospholipase B activity of plasma membrane vesicles from Saccharomyces cerevisiae is inhibited by the 100 000 X g supernatant of mechanically disrupted yeast cells . A 1850-fold purification of the inhibitor activity was achieved by gel filtration, ion exchange chromatography with DEAE-cellulose and hydrophobic interaction chromatography with Octyl-Sepharose . SDS-polyacrylamide gel electrophoresis of the purified inhibitor revealed two main bands with an apparent Mr of 60 000 and 26 500 . The phospholipase B activity was strongly reduced but not completely blocked by this preparation, while the lysophospholipase and transacylase reactions, which are catalyzed by the same membrane-bound enzymes (Witt, W . et al . (1984) Biochim . Biophys . Acta 795, 108-116), were not affected.

Eur J Biochem, 1985 Apr 15, 148(2), 405 - 6
Abnormal amino acid metabolism in mutants of Saccharomyces cerevisiae affected in the initiation of sporulation; Dickinson JR et al.; Amino acid metabolism was examined during sporulation in a wild-type strain of Saccharomyces cerevisiae and in a mutant homozygous for the spd1 mutation (derepression of sporulation) . In the wild-type initiation of sporulation involves a rise in intracellular glutamate . In the derepressed mutant there was no further increase of intracellular glutamate: glutamate was already present at a greater concentration than in the wild-type . These elevated glutamate levels are apparently due to reduction in the activity of 2-oxoglutarate dehydrogenase . One consequence of the elevated glutamate levels in diploids homozygous for the spd1 mutation is the production of considerable amounts of glycyl-L-proline, glutathione and saccharopine.

Nucleic Acids Res, 1985 Apr 11, 13(7), 2357 - 72
The nucleotide sequence of the RAD3 gene of Saccharomyces cerevisiae: a potential adenine nucleotide binding amino acid sequence and a nonessential acidic carboxyl terminal region; Reynolds P et al.; The RAD3 gene of Saccharomyces cerevisiae is required for excision of pyrimidine dimers and is essential for viability . We present the nucleotide sequence of the RAD3 protein coding region and its flanking regions, and the deduced primary structure of the RAD3 protein . In addition, we have mapped the 5' end of RAD3 mRNA . The predicted RAD3 protein contains 778 amino acids with a calculated molecular weight of 89,779 . A segment of the RAD3 protein shares homology with several adenine nucleotide binding proteins, suggesting that RAD3 protein may react with ATP . The twenty carboxyl terminal amino acids of RAD3 protein are predominantly acidic; however, deletion of this acidic region has no obvious effect on viability or DNA repair.

J Biol Chem, 1985 Apr 10, 260(7), 4531 - 40
Characterization of factors and DNA sequences required for accurate transcription of the Saccharomyces cerevisiae 5 S RNA gene; Taylor MJ et al.; Cell-free extracts prepared from yeast cells have previously been shown to direct selective and accurate in vitro transcription of tRNA and 5 S RNA genes . We have further analyzed the transcription factors and DNA sequences required for in vitro transcription of the yeast 5 S RNA gene . Fractionation of a yeast extract has identified a 5 S RNA gene-specific factor required, in addition to the two previously described tRNA factors (Klekamp, M . S., and Weil, P . A . (1982) J . Biol . Chem . 257, 8432-8441), for accurate transcription of the 5 S RNA gene by RNA polymerase III . Transcription of variant 5 S RNA genes has indicated that a region of the gene extending from residue +57 to residue +99 is essential for directing specific initiation of transcription . Although the 5' flanking and initial coding sequence is not absolutely required for transcription of the gene, some of the variant genes which have substitutions in this region are less actively transcribed than the wild-type gene . Transcription initiates on some of the variant genes at a position equivalent to +1 in the substituted sequence, while on other variant genes transcription initiates further upstream.

Appl Environ Microbiol, 1985 Apr, 49(4), 863 - 6
Image analysis method for the rapid counting of Saccharomyces cerevisiae cells; Costello PJ et al.; An image analysis system which incorporates a microscope, video camera, monitor, and Apple computer and which uses image area to count Saccharomyces cerevisiae cells is described and evaluated . Yeast cell suspensions of densities of up to 100 X 10(6) cells per ml can be counted when viewed in the counting chamber of a hemacytometer . The yeast image area measured depends upon the light intensity used to illuminate the yeast cells, the sharpness of the image focused on the monitor, and the grey level selected when scanning the digitized image on the monitor of the Apple computer, all of which can be controlled . The image area also depends upon the yeast strain and medium in which the culture is grown, but it is not affected by the concentration of sugar or ethanol in which the yeast cells are suspended . Yeast growth measured by image analysis can be calibrated to give results similar to those obtained with hemacytometer counting . Yeast cells can be counted in the presence of high cell densities of bacteria by adjusting the grey level at which the digitized image is scanned.

Mol Cell Biol, 1985 Apr, 5(4), 816 - 22
Isolation of the SUP45 omnipotent suppressor gene of Saccharomyces cerevisiae and characterization of its gene product; Himmelfarb HJ et al.; The Saccharomyces cerevisiae SUP45+ gene has been isolated from a genomic clone library by genetic complementation of paromomycin sensitivity, which is a property of a mutant strain carrying the sup45-2 allele . This plasmid complements all phenotypes associated with the sup45-2 mutation, including nonsense suppression, temperature sensitivity, osmotic sensitivity, and paromomycin sensitivity . Genetic mapping with a URA3+-marked derivative of the complementing plasmid that was integrated into the chromosome by homologous recombination demonstrated that the complementing fragment contained the SUP45+ gene and not an unlinked suppressor . The SUP45+ gene is present as a single copy in the haploid genome and is essential for viability . In vitro translation of the hybrid-selected SUP45+ transcript yielded a protein of Mr = 54,000, which is larger than any known ribosomal protein . RNA blot hybridization analysis showed that the steady-state level of the SUP45+ transcript is less than 10% of that for ribosomal protein L3 or rp59 transcripts . When yeast cells are subjected to a mild heat shock, the synthesis rate of the SUP45+ transcript was transiently reduced, approximately in parallel with ribosomal protein transcripts . Our data suggest that the SUP45+ gene does not encode a ribosomal protein . We speculate that it codes for a translation-related function whose precise nature is not yet known.

Mol Cell Biol, 1985 Apr, 5(4), 787 - 96
Alpha-factor structural gene mutations in Saccharomyces cerevisiae: effects on alpha-factor production and mating; Kurjan J; The role of alpha-factor structural genes MF alpha 1 and MF alpha 2 in alpha-factor production and mating has been investigated by the construction of mf alpha 1 and mf alpha 2 mutations that totally eliminate gene function . An mf alpha 1 mutant in which the entire coding region is deleted shows a considerable decrease in alpha-factor production and a 75% decrease in mating . Mutations in mf alpha 2 have little or no effect on alpha-factor production or mating . The mf alpha 1 mf alpha 2 double mutants are completely defective in mating and alpha-factor production . These results indicate that at least one alpha-factor structural gene product is required for mating in MAT alpha cells, that MF alpha 1 is responsible for the majority of alpha-factor production, and that MF alpha 1 and MF alpha 2 are the only active alpha-factor genes.

Mol Cell Biol, 1985 Apr, 5(4), 610 - 8
Expression of the Escherichia coli dam methylase in Saccharomyces cerevisiae: effect of in vivo adenine methylation on genetic recombination and mutation; Hoekstra MF et al.; The Escherichia coli DNA adenine methylase (dam) gene has been introduced into Saccharomyces cerevisiae on a yeast-E . coli shuttle vector . Sau3AI, MboI, and DpnI restriction enzyme digests and Southern hybridization analysis indicated that the dam gene is expressed in yeast cells and methylates GATC sequences . Analysis of digests of total genomic DNA indicated that some GATC sites are not sensitive to methylation . The failure to methylate may reflect an inaccessibility to the methylase due to chromosome structure . The effects of this in vivo methylation on the processes of recombination and mutation in mitotic cells were determined . A small but definite general increase was found in the frequency of mitotic recombination . A similar increase was observed for reversion of some auxotrophic markers; other markers demonstrated a small decrease in mutation frequency . The effects on mutation appear to be locus (or allele) specific . Recombination in meiotic cells was measured and was not detectably altered by the presence of 6-methyladenine in GATC sequences.

Proc Natl Acad Sci U S A, 1985 Apr, 82(7), 2106 - 10
Construction of telocentric chromosomes in Saccharomyces cerevisiae; Surosky RT et al.; We describe a simple method for the construction of large chromosomal deletions in yeast . Diploid yeast cells were transformed with DNA fragments that replace large regions of the chromosomes by homologous recombination . Using this method, we have constructed a telocentric chromosome III in which approximately equal to 100 kilobases (kb) of DNA has been removed from the left arm of the chromosome, so that the centromere is 12 kb from the left telomere . This telocentric chromosome is mitotically stable . Its rate of loss in a diploid strain is 2.5-7.4 X 10(-4) per cell division compared to a rate of loss of 0.36-1.8 X 10(-4) per cell division for a normal chromosome III . It also segregates 2+:2- with fidelity during meiosis . The construction of systematic deletions in a chromosome should be useful in determining the essential features for proper chromosomal segregation and replication.

Proc Natl Acad Sci U S A, 1985 Apr, 82(7), 2019 - 23
Influenza viral (A/WSN/33) hemagglutinin is expressed and glycosylated in the yeast Saccharomyces cerevisiae; Jabbar MA et al.; Recombinant plasmids were constructed in which genes coding for either the entire or the signal-minus (amino acid residues 2-17 deleted) hemagglutinin (HA) of WSN influenza virus were placed under the control of the alcohol dehydrogenase I gene promoter of Saccharomyces cerevisiae . Both recombinant plasmids were shown to direct the synthesis of HA-specific polypeptides that were detected by immunoprecipitation with antiviral antibodies . The complete HA produced in yeast had an approximate Mr of 70,000 and was glycosylated, as determined by the endoglycosidase H sensitivity, and was bound to membrane . Therefore, the complete HA polypeptide possessing the signal sequence probably traversed the yeast secretory pathways . Signal-minus HA, on the other hand, had a lower molecular weight and was nonglycosylated . The specific binding of yeast HA with antiviral antibodies could be competitively inhibited by influenza viral HA, demonstrating that the HA produced in yeast contained antigenic determinants of the native viral HA.

J Biol Chem, 1985 Mar 25, 260(6), 3542 - 7
Phosphorolytic cleavage of diadenosine 5',5'''-P1,P4-tetraphosphate . Properties of homogeneous diadenosine 5',5'''-P1,P4-tetraphosphate alpha, beta-phosphorylase from Saccharomyces cerevisiae; Guranowski A et al.; Novel enzymatic activity which splits diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) phosphorolytically has been found in extracts from Saccharomyces cerevisiae . One of the two alpha,beta-anhydride bonds between Ap4A phosphate residues undergoes phosphorolysis, and ATP (pppA) plus ADP (ppA) are the products of the reaction according to the equation: AppppA + P*i----pppA + p*pA The reaction is dependent on the presence of divalent metal ions; Mn2+ or Mg2+ sustain the greatest rates of reaction . Among analogues of the Ap4A substrate, Ap5A and Gp4G, but not p4A and Ap3A, are substrates, and corresponding products are p4A plus ADP, and GTP plus GDP, the phosphate being incorporated into the nucleoside 5'-diphosphates . In the reactions, phosphate can be substituted with arsenate . Arsenolysis of Ap4A, Ap5A, or Gp4G leads to ATP plus AMP, p4A plus AMP, and GTP plus GMP, respectively . The name diadenosine tetraphosphate alpha,beta-phosphorylase (ADP-forming) is proposed for the new enzyme . The phosphorylase has been purified to apparent homogeneity and behaves as a single polypeptide chain of Mr = 40,000 . Optimum activity of the enzyme is at pH 8.0 and the sulfhydryl groups are essential for catalysis . At saturating Ap4A, the rate constant for the reaction is 36 s-1 and the Km value for Ap4A is 60 microM (37 degrees C, 50 mM Hepes/KOH (pH 8.2), 500 microM MnCl2, 10 mM K2HPO4, 1 mM 2-mercaptoethanol, and 2% glycerol) . The Km values for phosphate and arsenate are 1 and 3 mM, respectively.

J Biol Chem, 1985 Mar 10, 260(5), 3108 - 15
Transfer RNA splicing in Saccharomyces cerevisiae . Secondary and tertiary structures of the substrates; Lee MC et al.; Secondary and tertiary structures of four yeast tRNA precursors that contain introns have been elucidated using limited digestion with a variety of single-strand- and double-strand-specific nucleases . The pre-tRNAs, representing the variety of intron sizes and potential structures, were: pre-tRNALeuCAA, pre-tRNALeuUAG, pre-tRNAIleUAU, and pre-tRNAPro-4UGG . Conventional tRNA cloverleaf structure is maintained in these precursors except that the anticodon loop is interrupted by the intron . The intron contains a sequence which is complementary to a portion of the anticodon loop and allows the formation of a double helix often extending the anticodon stem . The 5' and 3' splicing cleavage sites are located at either end of this helix and are single-stranded . The intron is the most sensitive region to nuclease cleavage, suggesting that it is on the surface of the molecule and available for interaction with the splicing endonuclease . Absence of Mg2+ or spermidine renders the dihydrouridine and T psi C loops of these precursors highly sensitive to nuclease digestion . These ionic effects mimic those observed for tRNAPhe and suggest that the tRNA portion of these precursors has native tRNA structure . We propose consensus secondary and tertiary structures which may be of significance to eventual understanding of the mechanism of yeast tRNA splicing.

J Biol Chem, 1985 Mar 10, 260(5), 3084 - 9
Elongation factor 1 alpha from Saccharomyces cerevisiae . Rapid large-scale purification and molecular characterization; Thiele D et al.; Cytoplasmic elongation factor 1 alpha (EF-1 alpha) was purified to homogeneity from the yeast Saccharomyces cerevisiae using a large-scale procedure . The three steps of purification used were batch adsorption on phosphocellulose, phosphocellulose chromatography and, as the last step, GDP-Sepharose or Biorex column chromatography . The protein is very basic (pI = 9.2) and has an apparent molecular mass of 49 kDa, as determined by polyacrylamide gel electrophoresis using denaturing conditions . It is one of the most abundant proteins in yeast (about 5% of total soluble protein), as shown by two-dimensional gel electrophoresis and by immunological titration . A strong immunological and structural homology was found between yeast EF-1 alpha and elongation factors from other sources . Common immunological features were found between yeast and wheat germ EF-1 alpha . Tryptic hydrolysis of yeast EF-1 alpha in the presence of 25% glycerol generated a large trypsin-resistant polypeptide (Mr = 43,000) which had the same NH2-terminal sequence as the proteolyzed product from rabbit reticulocyte, Artemia salina EF-1 alpha and Escherichia coli EF-Tu . Completed DNA sequence determination of one structural gene for yeast EF-1 alpha confirmed a remarkable conservation of several protein sequence domains in yeast and animal EF-1 alpha (Cottrelle, P., Thiele, D., Price, V., Memet, S., Micouin, J.Y., Marck, C., Buhler, J.M . Sentenac, A., and Fromageot, P . (1985) J . Biol . Chem . 260, 3090-3096).

Genetics, 1985 Mar, 109(3), 481 - 92
Mutations leading to expression of the cryptic HMRa locus in the yeast Saccharomyces cerevisiae; Kassir Y et al.; Mutations leading to expression of the silent HMRa information in Saccharomyces cerevisiae result in sporulation proficiency in mata1/MAT alpha diploids . An example of such a mutation is sir5-2, a recessive mutation in the gene SIR5 . As expected, haploids carrying the sir5-2 mutation are nonmaters due to the simultaneous expression of HMRa and HML alpha, resulting in the nonmating phenotype of an a/alpha diploid . However, sir5-2/sir5-2 mata1/MAT alpha diploids mate as alpha yet are capable of sporulation . The sir5-2 mutation is unlinked to sir1-1, yet the two mutations do not complement each other: mata1/MAT alpha sir5-2/SIR5 SIR1/sir1-1 diploids are capable of sporulation . In this case, recessive mutations in two unlinked genes form a mutant phenotype, in spite of the presence of the normal wild-type alleles . The PAS1-1 mutation, Provider of a Sporulation function, is a dominant mutation tightly linked to HMRa . PAS1-1 does not affect the mating ability of a strain, yet it allows diploids lacking a functional MATa locus to sporulate . It is proposed that PAS1-1 leads to partial expression of the otherwise cryptic a1 information at HMRa.

Arch Biochem Biophys, 1985 Mar, 237(2), 292 - 9
Extraction of proteins from Saccharomyces cerevisiae ribosomes under nondenaturing conditions; Lee JC et al.; The differential sensitivity of ribosomal proteins to removal by salts has been studied . Proteins were extracted from the large and small subunits of cytoplasmic ribosomes from Saccharomyces cerevisiae by washing the individual subunits with a series of solutions containing increasing concentrations of NH4Cl (0.74-3.56 M) for a defined time (20 min) at 0 degrees C . The molar ratio of magnesium to ammonium ions of 1:40 was maintained to protect the ribosomal subparticles from complete disassembly . Proteins extracted under each salt condition were analyzed for composition by two-dimensional polyacrylamide gel electrophoresis . The relative quantity of each protein was determined . Most proteins were not removed from the ribosomal particle completely by any one condition, but were preferentially enriched in a single fraction . Whereas most proteins could be solubilized, several proteins remained predominantly or exclusively with the final core particle . The kinetics of protein release from both subunits at a single NH4Cl concentration (0.74 M) were also studied . Release of protein was time dependent, i.e., longer extraction generally removed more of the same proteins . However, prolonged treatment (240 min) of subunits, even at the same salt concentration, resulted in removal of additional species of proteins in varying amounts . Among the ribosomal RNA species, only the 5 S RNA species was released from the ribosomal particles upon treatment.

Eur J Biochem, 1985 Mar 1, 147(2), 273 - 9
Intracellular maturation and secretion of acid phosphatase of Saccharomyces cerevisiae; Schonholzer F et al.; To elucidate intracellular maturation and secretion of acid phosphatase of Saccharomyces cerevisiae we prepared a monoclonal antibody that recognizes specifically the protein moiety of this cell surface glycoprotein . With this antibody membranes and soluble fractions of wild-type cells, grown in low-phosphate medium in the presence and absence of tunicamycin, were examined by the immunoblot technique . Similarly, secretory mutants, blocked at distinct steps in the secretory pathway at the restrictive temperature as well as a strain harboring several copies of the structural gene PHO5 for repressible acid phosphatase, were analyzed . The data suggest the following sequence of events in acid phosphatase maturation and secretion: three unglycosylated precursors with molecular masses of 60 kDa, 58 kDa and 56 kDa are synthesized into membranes of the endoplasmic reticulum, where these are core glycosylated in a membrane-bound form . They appear on sodium dodecyl sulfate gels as bands with molecular masses of 76 kDa and 80 kDa . Owing to a rate-limiting maturation step, occurring after core glycosylation, they can accumulate in a membrane-bound form . At the Golgi apparatus outer carbohydrate chains are attached to the core and the enzyme appears in a soluble form, indicating a release of acid phosphatase from the membrane between the endoplasmic reticulum and the Golgi . Pulse-chase experiments suggest that the time for acid phosphatase synthesis and its transport to the Golgi is about 5 min.

J Bacteriol, 1985 Mar, 161(3), 831 - 5
Rapid method for isolation and screening of cytochrome c oxidase-deficient mutants of Saccharomyces cerevisiae; McEwen JE et al.; We describe here a new method for the specific isolation of cytochrome c oxidase-deficient mutants of Saccharomyces cerevisiae . One unique feature of the method is the use of tetramethyl-p-phenylenediamine as a cytochrome c oxidase activity stain for yeast colonies . The staining of yeast colonies by tetramethyl-p-phenylenediamine is dependent upon a functional cytochrome c oxidase and is unaffected by other lesions in respiration . Since the tetramethyl-p-phenylenediamine colony staining reaction is rapid and simple, it greatly facilitates both the identification and characterization of cytochrome c oxidase-deficient mutants . Another feature of the method, which is made possible by the tetramethyl-p-phenylenediamine colony stain, is the use of an op1 parent strain for the isolation of nuclear pet or mitochondrial mit mutants in specific protein-coding genes . A parent strain that carries this marker selects against rho0 or rho- classes of pleiotropic respiratory-deficient mutants, since these are lethal in op1 strains . We have used this method to isolate 123 independently derived cytochrome c oxidase-deficient pet mutants and 300 independently derived mit mutants.

J Biol Chem, 1985 Feb 25, 260(4), 2253 - 7
Characterization of a specific alpha-mannosidase involved in oligosaccharide processing in Saccharomyces cerevisiae; Jelinek-Kelly S et al.; Fractionation of a crude extract from Saccharomyces cerevisiae X-2180 on Sepharose 6B in the presence of 0.5% Triton X-100 resolves two enzyme fractions containing alpha-mannosidase activity . Fraction I which is excluded from the gel contains alpha-mannosidase activity toward both p-nitrophenyl-alpha-D-mannopyranoside and Man9GlcNAc oligosaccharide as substrates, whereas Fraction II which is included in the gel contains only oligosaccharide alpha-mannosidase activity . The latter enzyme is very specific and removes a single mannose residue from Man9GlcNAc, whereas the alpha-mannosidase activity of Fraction I removes several mannose residues from Man9GlcNAc oligosaccharide . High resolution 1H NMR analysis of the Man8GlcNAc formed from Man9GlcNAc in the presence of the alpha-mannosidase of Fraction II showed only a single isomer with the following structure: (see formula; see text) This specific enzyme is most probably involved in processing of oligosaccharide during biosynthesis of mannoproteins . The mannose analog of 1-deoxynojirimycin (50-500 microM), dideoxy-1,5-imino-D-mannitol, inhibits the oligosaccharide alpha-mannosidase activities of Fractions I and II to about the same extent, but has no effect on the nonspecific alpha-mannosidase which acts on p-nitrophenyl-alpha-D-mannopyranoside.

Nucleic Acids Res, 1985 Feb 25, 13(4), 1327 - 39
Biogenesis of mitochondria: DNA sequence analysis of mit- mutations in the mitochondrial oli1 gene coding for mitochondrial ATPase subunit 9 in Saccharomyces cerevisiae; Ooi BG et al.; The nucleotide sequence of the yeast mitochondrial olil gene has been obtained in a series of mit- mutants with mutations in this gene, which codes for subunit 9 of of the mitochondrial ATPase complex . Subunit 9 is the proteolipid, 76 amino acids in length, necessary for the proton translocation function of the membrane Fo-sector . These mutants were classified on the basis of their rescue by a petite strain shown here to retain the entire wild-type olil gene . The mutation in one mit- strain removes a positively charged residue (Arg39----Met) which is likely to be located in a segment of subunit 9 that protrudes from the inner mitochondrial membrane . In a second mit- mutant, a negatively charged residue replaces a conserved glycine residue (Gly18----Asp) in a glycine-rich segment of the protein that is most likely embedded within the membrane . Other mit- mutations result in frameshifts with predicted products 7, 65 and 68 amino acid residues long . In each mit- mutant, there is the loss of one or more of the amino acid residues that are highly conserved among diverse species . The location and nature of specific changes pinpoint amino acid residues in subunit 9 essential to the activity of the mitochondrial ATPase complex.

Eur J Biochem, 1985 Feb 15, 147(1), 191 - 6
Mitochondrial transcription and processing of transcripts during release from glucose repression in 'resting cells' of Saccharomyces cerevisiae; Zennaro E et al.; Mitochondrial transcription and processing of transcripts have been investigated at different stages of release from glucose repression in resting cells of Saccharomyces cerevisiae . Transcripts were identified by hybridization with nick-translated or terminally labelled gene-specific probes . This allowed the determination of the steady-state levels of individual transcripts in the mitochondrial RNA population . Results showed different gene-specific patterns of response to respiratory induction: no increase in the level of transcripts (oxi2); a rapid increase in the steady-state levels of all transcripts (cob); a very strong increase in the processing of the high-molecular-mass precursors (oxi3 and oli2); an increase in the level of stable circular transcripts (oxi3) . As a whole the results indicate specific and differentiated effects of release from glucose repression on the expression of the different mitochondrial genes and demonstrate the importance of processing events in mitochondrial regulation.

Mol Gen Mikrobiol Virusol, 1985 Feb, (2), 33 - 5
{Gene transfer using fusion of isolated cell nuclei with protoplasts of the yeast Saccharomyces cerevisiae}; Becher D et al.; Hybrid clones of Saccharomyces cerevisiae with different genotypes have been obtained by polyethyleneglycol induced fusion of isolated cellular nuclei with protoplasts . The genetic instability of complete nuclei after fusion results in formation of different genotypes.

Int J Pept Protein Res, 1985 Feb, 25(2), 187 - 96
Synthesis and biological activity of N epsilon-acyl derivatives of a Saccharomyces cerevisiae mating pheromone; Shenbagamurthi P et al.; We report a general method for acylation of the N epsilon-amino group of the lysyl residue in peptides . The procedure involves acylation using p-nitrophenyl esters and 1-hydroxybenzotriazole in organic solvents to yield a series of fatty acyl mating pheromones of Saccharomyces cerevisiae . The fatty acyl group does not influence coupling of peptide fragments . Biological activities of the synthesized alpha-factor mating pheromones derivatized with acetyl, butyryl, caprylyl and lauryl groups are nearly equivalent to the activity of unacylated alpha-factor . The N epsilon-stearyl-alpha-factor is biologically inactive . The procedures reported in this communication can be used to increase hydrophobicity of lysine-containing peptides when the lysyl group is not essential for activity.

Can J Microbiol, 1985 Feb, 31(2), 109 - 18
Ultrastructure of Saccharomyces cerevisiae strain AG1-7 and its responses to changes in environment; Willison JH et al.; Asynchronous populations of the budding yeast Saccharomyces cerevisiae strain AG1-7 were examined by freeze-fracture electron microscopy for ultrastructural changes occurring in response to changes in the environment, specifically the following: temperature (23 or 37 degrees C); cell density (exponential, early stationary, and stationary phases); various periods of nitrogen starvation at low cell density, and return of nitrogen-starved cells to nitrogen-replete medium . This information has been gathered in preparation for ultrastructural examination of comparable responses of temperature-sensitive cell-cycle mutants . The plasma membrane was found to be particularly responsive to changes in environment . A high proportion (75%) of cells in exponential phase populations at 37 degrees C displayed paracrystalline arrays of plasma membrane particles, whereas this proportion was much lower (20%) at 23 degrees C in the same medium; plasma membrane grooves were longer at 37 than at 23 degrees C . In budded cells, the mother cell displayed paracrystalline arrays more frequently than the bud . Entry of cells into stationary phase, either through permitting population growth or by limiting nitrogen supply, resulted in increases in numbers of paracrystalline arrays and grooves . Groove depth also increased . The paracrystalline-array and groove-density responses were independent, both during entry into stationary phase and during the subsequent lag phase . Unusual groove forms appeared during stationary phase in high cell density populations, but not in low cell density nitrogen-starved populations . "Aggregate" and "geometric" tonoplast forms, previously described in strain A364A when grown under some of the conditions used here, were not found in AG1-7 under any of the conditions used here . It was demonstrated that particle-free patches can arise rapidly on the tonoplast of AG1-7 in response to temperature change from 37 to 23 degrees C . During stationary phase, spherosomes (lipid droplets) increased in size, particularly in response to nitrogen depletion . After 72 h of nitrogen starvation, about 10% of cell volume consisted of spherosomes . Changes in vacuolar content and mitochondrial form were also noted during entry into stationary phase.

Genetics, 1985 Feb, 109(2), 303 - 32
Meiotic recombination between duplicated genetic elements in Saccharomyces cerevisiae; Jackson JA et al.; We have studied the meiotic recombination behavior of strains carrying two types of duplications of an 18.6-kilobase HIS4 Bam HI fragment . The first type is a direct duplication of the HIS4 Bam HI fragment in which the repeated sequences are separated by Escherichia coli plasmid sequences . The second type, a tandem duplication, has no sequences intervening between the repeated yeast DNA . The HIS4 genes in each region were marked genetically so that recombination events between the duplicated segments could be identified . Meiotic progeny of the strains carrying the duplication were analyzed genetically and biochemically to determine the types of recombination events that had occurred . Analysis of the direct vs . tandem duplication suggests that the E . coli plasmid sequences are recombinogenic in yeast when homozygous . In both types of duplications recombination between the duplicated HIS4 regions occurs at high frequency and involves predominantly interchromosomal reciprocal exchanges (equal and unequal crossovers) . The striking observation is that intrachromosomal reciprocal recombination is very rare in comparison with interchromosomal reciprocal recombination . However, intrachromosomal gene conversion occurs at about the same frequency as interchromosomal gene conversion . Reciprocal recombination events between regions on the same chromatid are the most infrequent exchanges . These data suggest that intrachromosomal reciprocal exchanges are suppressed.

J Bacteriol, 1985 Feb, 161(2), 778 - 80
Interaction of UAG suppressors and omnipotent suppressors in Saccharomyces cerevisiae; Song JM et al.; Haploids bearing the dominant UAG suppressor, SUP7-a, and various alleles of the omnipotent suppressor sup35 were examined . The presence of the UAG suppressor reduced the efficiency of some alleles of sup35, and caused other sup35 alleles to be lethal . A nonclassical interaction of the dominant suppressor tRNA and the ribosome is proposed to explain these observations.

J Bacteriol, 1985 Feb, 161(2), 769 - 71
Expression of a Saccharomyces cerevisiae photolyase gene in Escherichia coli; Sancar GB; A 3.3-kilobase PvuII fragment carrying the PHR1 gene of Saccharomyces cerevisiae has been cloned into an Escherichia coli expression vector and introduced into E . coli strains deficient in DNA photolyase . Complementation of the E . coli phr-1 mutation was observed, strongly suggesting that the yeast PHR1 gene encodes a DNA photolyase.

J Bacteriol, 1985 Feb, 161(2), 596 - 601
Alpha mating type-specific expression of mutations leading to constitutive agglutinability in Saccharomyces cerevisiae; Doi S et al.; Two mutants of Saccharomyces cerevisiae have been isolated and characterized . The mutants were constitutively agglutinable at 36 degrees C, the temperature at which wild-type cells agglutinate only after induction by mating pheromone . The mutant cells had other properties specific for the normal alpha cell type, i.e., conjugation with a cells, response to a mating pheromone, and production of alpha mating pheromone . The two mutations, cag1 and cag2, were recessive and expressed only in alpha cells . cag1 is linked very closely to the MAT locus, but cag2 is unlinked to the MAT locus . These cag mutations complemented ste3-1 . These results indicate that CAG genes are novel alpha-specific genes involved in the regulation of sex agglutinin synthesis.

Biochemistry, 1985 Jan 29, 24(3), 753 - 9
Saccharomyces cerevisiae structural cell wall mannoprotein; Frevert J et al.; A novel mannoprotein fraction with an average molecular weight of 180 000 has been isolated from Saccharomyces cerevisiae mnn9 mutant cell wall that was solubilized by beta-glucanase digestion . The same material could be extracted from purified wall fragments with 1% sodium dodecyl sulfate . The protein component, 12% by weight, is rich in proline, whereas the carbohydrate, mainly mannose, is about evenly distributed between asparagine and hydroxyamino acids . Endoglucosaminidase H digestion of the isolated mannoprotein reduced its average molecular weight to 150 000, but the mannoprotein, while still embedded in the cell wall, was inaccessible to the enzyme . Biosynthesis and translocation of the mannoprotein were investigated by following incorporation of {3H}proline into this fraction . In the presence of tunicamycin, both mnn9 and wild-type X2180 cells made a mannoprotein fraction with an average molecular weight of 140 000, whereas in the absence of the glycosylation inhibitor, the mnn9 mutant made material with a molecular weight of 180 000 and the mannoprotein made by wild-type cells was too large to penetrate the polyacrylamide gel . Although the cell wall mannoprotein was resistant to heat and proteolytic enzymes, attempts to isolate the carbohydrate-free component failed to yield any characteristic peptide material.

J Biol Chem, 1985 Jan 25, 260(2), 1271 - 9
Nucleolytic processing of a tRNAArg-tRNAAsp dimeric precursor by a homologous component from Saccharomyces cerevisiae; Engelke DR et al.; A subcellular extract from Saccharomyces cerevisiae has been used to transcribe cloned yeast tRNA genes in vitro and to process the primary transcripts at the 5' and 3' termini . Chromatographic fractionation of the extract has separated the transcription components from two distinct nucleolytic activities: an endonuclease that cleaves the precursors to produce mature 5' termini; and a 3'-5' exonuclease . These fractions have been used to elaborate a processing pathway for the dimeric primary transcript of the yeast tRNAArg-tRNAAsp gene pair . Under optimal conditions in vitro this gene is expressed at a rate of 200 transcripts/gene/hour, initiating at position -10 with respect to the mature 5' terminus of tRNAArg and terminating near position +160 . The primary transcripts are cleaved by an endonuclease to give tRNAAsp with a mature 5' terminus, and a pre-tRNAArg monomer with a 5' leader and 3' trailer sequences . A second endonuclease cleavage of pre-tRNAArg generates the mature 5' terminus of tRNAArg . The endonuclease cleavages are not ordered . Exonuclease activity(ies) remove the spacer sequences from the 5' mature tRNAArg, and trim the 3' trailer portion from tRNAAsp . Exonucleolytic removal of the 3' trailer does not require prior endonuclease action, but removal of the spacer sequences from pre-tRNAArg is incomplete without prior removal of the 5' leader sequences.

FEBS Lett, 1985 Jan 7, 179(2), 307 - 10
An antisuppressor mutant of Saccharomyces cerevisiae deficient in isopentenylated tRNA has reduced delta 2-isopentenylpyrophosphate: tRNA-delta 2-isopentenyl transferase activity; Laten HM et al.; We have previously reported the isolation and initial characterization of a mutation in Saccharomyces cerevisiae, designated mod5-1, that reduces the capacity of altered tyrosine tRNAs to suppress ochre nonsense mutations . The mutation results in the virtual elimination of the modified tRNA nucleoside, N6-delta 2-(isopentenyl) adenosine, normally found adjacent to the anticodons of certain tRNA species . We demonstrate here that MOD5 codes for delta 2-isopentenylpyrophosphate: tRNA-delta 2-isopentenyl transferase, or a protein that regulates its synthesis.

Eur J Biochem, 1985 Jan 2, 146(1), 95 - 100
Purification and characterization of the indole-3-glycerolphosphate synthase/anthranilate synthase complex of Saccharomyces cerevisiae; Prantl F et al.; The indole-3-glycerolphosphate synthase/anthranilate synthase complex from Saccharomyces cerevisiae was purified to apparent homogeneity . The native complex with Mr approximately equal to 130 000 consists of two different subunits, the TRP2 gene product with Mr = 64 000 and the TRP3 gene product with Mr = 58 000 . The larger polypeptide was identified as anthranilate synthase and is active in vitro with ammonia as cosubstrate without need of complex formation . The smaller polypeptide carries both glutamine amidotransferase activity and indole-3-glycerolphosphate synthase activity . Various steady-state kinetic parameters as well as the amino acid composition of the two polypeptides were determined.

Curr Genet, 1985, 10(1), 35 - 7
Suppression of temperature sensitive mutations in oncogene-related CDC genes in Saccharomyces cerevisiae by catabolite repression resistance and cytoplasmic petite mutations; Egilsson V et al.; The "start" cell division control genes CDC36 and CDC28 have been reported to contain a certain sequence homology to tissue oncogenes (ets and some protein kinase encoding oncogenes respectively) . Here we report that temperature sensitive mutations in these genes are suppressed in cytoplasmic "petite" mutants and catabolite repression resistant mutants.

Curr Genet, 1985, 10(1), 21 - 7
Maintenance and copy number control of ARS1 plasmids in Saccharomyces cerevisiae . Evidence of a mating type effect; Gullov K et al.; Following mating of a and alpha isogenic haploids we observe that the frequency of plasmid bearing cells, during selective growth, increases three fold . By examining the mitotic stability, the frequency of plasmid bearing cells during the cell cycle and the copy number of ARS1 plasmids in isogenic haploid and diploid cells, we show that the apparent stability of circular ARS1 plasmids in a/alpha cells is largely due to a diminished copy number in these cells . This observation is fully comprehensible with the model for plasmid segregation as presented by Murray and Szostak (1983) . In order to account for the differences in copy numbers, alpha and alpha/alpha isogenic strains were compared . Likewise a number of mating type nonspecific sterile mutants were compared with the parental Ste+ strain . It seems that a diminished copy number is established when the MATa1/MAT alpha 2 regulatory system (Klar et al . 1981) is switched on, since the effect is observed in Sir- strains only.

Curr Genet, 1985, 10(1), 15 - 20
A mutant of Saccharomyces cerevisiae with impaired maintenance of centromeric plasmids; Larionov VL et al.; A mutant with unstable maintenance of hybrid plasmids containing either one of the centromeric loci CEN3, CEN6, CEN11 and ars1 or the replicator of the 2 mu plasmid has been obtained . The frequency of loss of hybrid plasmids in the mutant was up to 3.10(-1) per one generation versus 10(-2) in the original strain . The unstable maintenance of minichromosomes in the mutant is controlled by a recessive nuclear gene, named SMC for stability of minichromosomes . Loss of some minichromosomes is connected with impairment of their segregation in cell division . In diploids homozygous for smc mitotic chromosomal segregation is not affected but sporulation is impaired . The question of adequacy of usage of minichromosomes for selection of mutants with impaired function of centromeric loci is discussed.

Microbiol Sci, 1985 Jan, 2(1), 10 - 3
Saccharomyces cerevisiae as a model eukaryote for studies on mitochondriogenesis; Trivedi A et al.; A great deal of progress has been made in elucidating the underlying mechanisms which control the interplay between the nuclear and mitochondrial genomes during biogenesis of mitochondria . The advantage of using the yeast Saccharomyces cerevisiae in these studies over other eukaryotic cells will be discussed.

Curr Genet, 1985, 9(7), 529 - 32
UV-inducible proteins in Saccharomyces cerevisiae; Rolfe M; Two UV-inducible proteins have been detected in the yeast, Saccharomyces cerevisiae . The proteins have molecular weights of 78,000 Daltons and 23,000 Daltons . This induction is specific for UV-irradiation as exposure to X-rays, mitomycin C and heat shock does not result in the synthesis of the proteins . The larger (78 kD) protein is induced in various rad strains and in a rho degree cir degree strain . Attempts are being made to isolate the genes coding for these inducible proteins.

Curr Genet, 1985, 9(5), 341 - 4
General and specific controls of lysine biosynthesis in Saccharomyces cerevisiae; Urrestarazu LA et al.; Six of the eight enzymes of the alpha-aminoadipate pathway for the biosynthesis of lysine in Saccharomyces cerevisiae were examined for repressibility to lysine and for susceptibility to the general control of amino acid biosynthesis . All of the enzymes exhibited a 2 to 4 fold lower level of specific activity in the wildtype strain X2180 when grown in lysine supplemented medium as compared to minimal medium . However, levels of only three of the enzymes, alpha-aminoadipate reductase, saccharopine reductase, and saccharopine dehydrogenase, were derepressed in the leaky lysine mutant 7305d and leaky arginine mutant 7853-6c when grown in minimal medium . These observations are characteristic of enzymes under general control of amino acid biosynthesis . The remaining three enzymes, homocitrate synthetase, homoaconitase and homoisocitrate dehydrogenase were repressed in 7305d cells grown in minimal or lysine supplemented medium.

Mol Gen Genet, 1985, 200(2), 291 - 4
Mutation affecting the specific regulatory control of lysine biosynthetic enzymes in Saccharomyces cerevisiae; Ramos F et al.; A Saccharomyces cerevisiae mutant which exhibits a considerably increased cellular lysine pool has been isolated and characterized . Assay of enzymes of the lysine and arginine pathways shows that the mutation harboured by this mutant alters the specific repression of lysine but does not influence the general control of amino acid biosynthesis . Because it is recessive to the wild-type allele and acts pleiotropically on the synthesis of several lysine pathway enzymes, this regulatory mutation has been denominated lys80-1 (or lysR--1) . It is believed to affect the synthesis or the structure of a factor which plays a negative role in the control of LYS gene expression.

Mol Gen Genet, 1985, 199(1), 59 - 63
Expression of the RAD1 and RAD3 genes of Saccharomyces cerevisiae is not affected by DNA damage or during the cell division cycle; Nagpal ML et al.; The RAD1 and RAD3 genes of Saccharomyces cerevisiae are required for excision repair of UV damaged DNA . In addition, the RAD3 gene is essential since rad3 deletions are recessive lethals . We have examined the induction of the RAD1 and RAD3 genes by DNA damage and during the cell division cycle . We have made fusions of the RAD1 and RAD3 genes with the Escherichia coli lacZ gene encoding beta-galactosidase . Beta-galactosidase activity was measured in a Rad+ yeast strain containing the RAD1-lacZ or the RAD3-lacZ fusion, either in a multicopy replicating plasmid or as a single copy integrant resulting from transformation with an integrating plasmid which transforms yeast by homologous recombination in the yeast genome . No induction of beta-galactosidase activity occurred after ultraviolet light (UV) or 4-nitroquinoline-1-oxide (NQO) treatment . Haploid cells of mating type a were synchronized by treatment with alpha factor and beta-galactosidase activity was determined during different cell cycle stages . No change in beta-galactosidase activity was observed in the strain containing the RAD1-lacZ or the RAD3-lacZ fusion integrated in the yeast genome.

Curr Genet, 1985, 10(3), 187 - 95
Saccharomyces cerevisiae strains sensitive to inorganic mercury . II . Effect of glucose; Sakamoto E et al.; Saccharomyces cerevisiae strains sensitive to inorganic mercury (Ono and Sakamoto 1985) did not grow well on the medium rich in glucose and poor in peptone . This growth inhibition, like growth inhibition caused by inorganic mercury, was relieved by exogenous tyrosine . Sugars such as fructose and mannose were as inhibitory as glucose, but glycerol was not at all . Galactose was inhibitory but not so much as glucose . A gal2 mutation (defective in galactose uptake) partly relieved growth inhibition caused by excess galactose . Moreover, it was found that some of revertants which gained ability to grow well in the presence of excess glucose were defective in the glucose uptake . From these observations, we conclude that growth inhibition of the inorganic mercury sensitive strains by excess sugar is a consequence of the catabolite regulation . In other words, the inorganic mercury sensitive strains are hyper-sensitive to the catabolite regulation due to the presence of the HGS2-1 allele.

Curr Genet, 1985, 9(7), 533 - 8
UV-inducible transcripts in Saccharomyces cerevisiae; Rolfe M; Differential colony hybridisation has been used to identify DNA sequences in Saccharomyces cerevisiae corresponding to RNA transcripts whose levels increase 5-10 fold following UV-irradiation . Four sequences have been identified, three of which share sequence homology and hybridize to the same set of genomic DNA fragments . The fourth sequence appears to be distinct, however each DNA sequence hybridizes to a similar sized RNA transcript which is approximately 4.0 kb long . The relationships between these DNA sequences and their potential protein products is discussed.

Curr Genet, 1985, 9(4), 253 - 7
Cloning and mapping of CDC40, a Saccharomyces cerevisiae gene with a role in DNA repair; Kassir Y et al.; The cdc40 mutation has been previously shown to be a heat-sensitive cell-division-cycle mutation . At the restrictive temperature, cdc40 cells arrest at the end of DNA replication, but retain sensitivity to hydroxyurea (Kassir and Simchen 1978) . The mutation has also been shown to affect commitment to meiotic recombination and its realization . Here we show that mutant cells are extremely sensitive to Methyl-Methane Sulfonate (MMS) when the treatment is carried out at restrictive temperature . Incubation at 37 degrees C prior to, or after MMS treatment at 23 degrees C, does not result in lower survival . It is concluded that the CDC40 gene product has a role in DNA repair, possibly holding together or protecting the DNA during the early stages of repair . The CDC40 gene was cloned on a 2.65 kb DNA fragment . A 2 mu plasmid carrying the gene was integrated and mapped to chromosome IV, between trp4 and ade8, by the method of marker loss . Conventional tetrad analysis has shown cdc40 to map 1.7 cM from trp4.

Basic Life Sci, 1985, 36, 231 - 42
Characterization of a tightly centromere-linked gene essential for meiosis in the yeast Saccharomyces cerevisiae; Yeh E et al.; The centromere region in the yeast Saccharomyces cerevisiae is characterized by short DNA fragments, less than 1,000 bp in length, that are capable of stabilizing entire chromosomes throughout mitotic and meiotic cell divisions . The CEN fragments are organized in a unique chromatin structure and are surrounded by ordered arrays of nucleosomal subunits . RNA transcripts are found 200-300 bp from the centromere, and lie within this ordered chromatin array . No transcripts have been detected through the centromere itself . We have examined the expression and cellular function of a tightly centromere-linked transcript on chromosome 11, (CEN11)L . The (CEN11)L transcript is present at constitutive levels throughout the mitotic and meiotic cell cycles . Disruption of the coding sequences in vivo has no effect on cell viability or mitotic growth, but the cells are unable to sporulate . Genetic complementation with known mutants in sporulation (spo10, spo13) has defined (CEN11)L as a new locus that appears to be required during both meiotic segregation divisions.

Folia Microbiol (Praha), 1985, 30(6), 501 - 5
Effect of clomiphene on the content of sterols and fatty acids in Saccharomyces cerevisiae; Rezanka T et al.; During cultivation of Saccharomyces cerevisiae clomiphene regulates both quantitative and qualitative production of sterols and fatty acids as identified by gas chromatography and mass spectrometry . The content of sterols decreases to 75%, the production of fatty acids is comparable with that in the control . The occurrence of sterols increases; sterols with methyl group in position 4, without double bond in position 22 and with double bond in position 24(25) or 24(28) predominate . Among fatty acids shorter saturated and monoene acids are primarily produced, 2-hydroxy acids practically disappeared.

Gene, 1985, 39(1), 95 - 101
Complete nucleotide sequence of the hexokinase PI gene (HXK1) of Saccharomyces cerevisiae; Kopetzki E et al.; The nucleotide sequence of the yeast glycolytic hexokinase isoenzyme PI-gene, HXK1, has been determined by sequencing the yeast DNA insert of the previously isolated plasmid HXK1 clone {Entian et al., Mol . Gen . Genet . 198 (1984) 50-54} . The structural gene sequence included 1452 bp coding for 484 amino acid (aa) residues corresponding to the Mr of 153 605 for the HXK1 monomer . Several initiation regions and termination points were located using nuclease S1 mapping . The HXK1 sequence was 76% homologous with that of HXK2, which is responsible for triggering glucose repression in yeasts . Since HXK1 is not involved in this regulatory system, the regulatory function of HXK2 must correspond to one or more of the differences between both isoenzymes . Most changes in the amino acid sequence were statistically distributed; however, four clustered regions with more than five altered aa residues were identified.

Gene, 1985, 38(1-3), 205 - 14
The histidine permease gene (HIP1) of Saccharomyces cerevisiae; Tanaka J et al.; The histidine-specific permease gene (HIP1) of Saccharomyces cerevisiae has been mapped, cloned, and sequenced . The HIP1 gene maps to the right arm of chromosome VII, approx . 11 cM distal to the ADE3 gene . The gene was isolated as an 8.6-kb BamHI-Sau3A fragment by complementation of the histidine-specific permease deficiency in recipient yeast cells . We sequenced a 2.4-kb subfragment of this BamHI-Sau3A fragment containing the HIP1 gene and identified a 1596-bp open reading frame (ORF) . We confirmed the assignment of the 1596-bp ORF as the HIP1 coding sequence by sequencing a hip1 nonsense mutation . Analysis of the amino acid (aa) sequence of the HIP1 gene reveals several hydrophobic stretches, but shows no obvious N-terminal signal peptide . We have constructed a deletion of the HIP1 gene in vitro and replaced the wild-type copy of the gene with this deletion . The hip1 deletion mutant can grow when it is supplemented with 30 mM histidine, 50 times the amount required for the growth of HIP1 cells . Revertants of this deletion mutant able to grow on a normal level of histidine arise by mutation in unlinked genes . Both these observations suggest that there are additional, low-affinity pathways for histidine uptake.

Mol Gen Genet, 1985, 201(1), 99 - 106
The use of plasmid DNA to probe DNA repair functions in the yeast Saccharomyces cerevisiae; White CI et al.; The survival of plasmid YRp12 treated in vitro with ultraviolet- or gamma-radiation, or with restriction endonucleases, has been used to investigate in vivo RAD gene activity in Saccharomyces cerevisiae . Yields of pyrimidine dimers or single and double strand breaks in plasmid DNA were assayed by physical methods . The biological effects of these damages were assayed by transformation of wild-type cells and rad mutants from each of the major groups of radiosensitive mutants . After UV-irradiation plasmid survival depended qualitatively on the same host functions that are needed for cellular survival . After gamma-irradiation no such correspondence was found . Apart from a RAD52-dependent stimulation of transformation efficiency at low doses, other host repair functions had little effect . Stimulation of transformation corresponded with the production of double- but not single-strand breaks in plasmid sequences homologous with the yeast genome and may be linked with a transient increase in mitotic stability . More generally these data also show that transformation events using the LiCl protocol may entail the uptake of a very low number of plasmid molecules per cell over a 10-fold range of DNA concentrations.

Mol Gen Genet, 1985, 201(1), 1 - 6
Partial deprivation of GTP initiates meiosis and sporulation in Saccharomyces cerevisiae; Varma A et al.; We have investigated the physiological conditions under which meiosis and the ensuing sporulation of Saccharomyces cerevisiae are initiated . Initiation of sporulation occurs in response to carbon, nitrogen, phosphorus, or sulfur deprivation, and also, when met auxotrophs are partially starved for methionine, but not after starvation of other amino acid auxotrophs . It also occurs after partial starvation of pur or gua auxotrophs for guanine but not after starvation of ura auxotrophs for uracil . Under all these sporulation conditions the concentrations of both guanine nucleotides (GTP) and S-adenosylmethionine (SAM) decrease whereas those of other nucleotides show no trend . We show that the decrease of guanine nucleotides is essential for the initiation of meiosis and sporulation: when a gua auxotroph, also lacking one of the two SAM synthetases, is starved for guanine but supplemented with 0.1 mM methionine, GTP decreases while SAM slightly increases and yet the cells sporulate.

Mol Gen Genet, 1985, 200(3), 458 - 62
An improved assay for DNA ligase reveals temperature-sensitive activity in cdc9 mutants of Saccharomyces cerevisiae; Barker DG et al.; A sensitive and quantitative assay for DNA ligase has been developed which is suitable for the analysis of crude cell extracts of yeast . The assay is sufficiently sensitive to detect the low levels of DNA ligase activity remaining in cdc9 mutants of Saccharomyces cerevisiae . Indeed, we have been able to show that this residual activity is temperature-sensitive, thus establishing finally that CDC9 is the structural gene for DNA ligase.

Mol Gen Genet, 1985, 200(1), 74 - 9
Isolation of the catalase A gene of Saccharomyces cerevisiae by complementation of the cta1 mutation; Cohen G et al.; As a first step in an analysis of the DNA regions involved in the control of the catalase A gene of Saccharomyces cerevisiae by glucose, heme, and oxygen this gene has been cloned . Catalase A-deficient mutants were obtained by UV mutagenesis of a ctt1 mutant strain specifically lacking catalase T . All the catalase A-deficient mutants obtained fall into one complementation group . The single recessive mutation causing specific lack of catalase A was designated cta1 . Several overlapping DNA fragments complementing the cta1 mutation were obtained by transforming ctt1 cta1 double mutants with a yeast gene library in vector YEp13 . Hybrid selection of RNA with the help of one of the cloned DNAs followed by in vitro translation of this RNA and identification of the protein synthesized with catalase A-specific antibodies showed that the catalase A structural gene has been cloned . A single copy of this gene is present in the yeast genome . Transcription of the catalase A gene cloned into vector YEp13 is repressed by glucose . The DNA isolated hybridizes to a 1.6 kb polyA+-RNA virtually absent from heme-deficient cells, presumably catalase A mRNA.

J Basic Microbiol, 1985, 25(3), 161 - 74
Electron microscopic study of purified polysaccharide components glucans and mannan of the cell walls in the yeast Saccharomyces cerevisiae; Kopecka M; In this study electron-microscopic characteristics of platinum shadowed or negatively stained preparations of purified beta-(1----3)-D-glucan, beta-(1----6)-D-glucan, and mannan were examined . These polysaccharides were isolated from cell walls of the yeast Saccharomyces cerevisiae . While purified samples of beta-(1----6)-D-glucan and mannan proved to be amorphous in structure and homogenous in appearance, the purified beta-(1----3)-D-glucan, isolated and presented to us as alkali-insoluble yeast glucan A2, was not homogenous . It consisted of (i) fibrillar component, (ii) amorphous matrix, and (iii) chitin bud scars . The ultrastructure of beta-(1----3)-D-glucan present in the glucan A2 sample did not change after treatment with 0.5 M acetic acid at 75 degrees C for 2 hours . After treatment with 1 M NaOH for 3 days at 4 degrees C scar material was removed by centrifugation and after a subsequent acidification of supernatant with acetic acid both the microfibrillar and the amorphous components were still present . It was concluded that beta-(1----3)-D-glucan component consists of molecules probably differing in their physico-chemical properties such as D . P., the degree of branching, conformation, and that cannot be separated by the methods currently used for their isolation.

Gene, 1985, 34(1), 55 - 61
Molecular cloning of the RAD10 gene of Saccharomyces cerevisiae; Prakash L et al.; We have cloned the RAD10 gene of Saccharomyces cerevisiae and physically mapped it to a 1.0-kb DNA fragment . Strains containing disruptions of the RAD10 gene were found to show enhanced UV sensitivity compared with the previously characterized rad10-1 or rad10-2 mutants . The UV sensitivity of the disruption mutant is comparable to the highly UV sensitive rad1-19, rad2-delta, and rad3-2 mutants.

Antonie Van Leeuwenhoek, 1985, 51(1), 1 - 10
Parental age and the life-span of zygotes of Saccharomyces cerevisiae; Muller I; Isolated cells of Saccharomyces cerevisiae were mated by micromanipulation and the reproductive capacity of the resulting zygotes was determined . The mating frequency was dependent on the age of the parents: conjugations between young cells and cells which had completed more than two thirds of their life-span were very rare events . The life-span of a zygote was very similar to the life-span of its shorter-lived parent . If one of the parent cells had budded several times prior to fusion, the life-span of the zygote was reduced correspondingly, i.e . there was no 'rescue by hybridization.' In four crosses the distribution of buds on both of the parent cells was recorded . In three of these four crosses the buds were evenly distributed, and in one the alpha-parent had three times as many buds as the a-parent.

Microbios, 1985, 42(167), 37 - 44
Growth and adaptation of Saccharomyces cerevisiae at different cadmium concentrations; Minney SF et al.; The effect of 0, 5, 10 and 25 mg l-1 cadmium on the growth of Saccharomyces cerevisiae in defined medium has been investigated . It was found that the length of the lag phase increased with cadmium concentration and that metal uptake during the lag phase occurred only at a cadmium concentration of 25 mg l-1 . However, metal uptake occurred at all cadmium concentrations during the exponential phase . The yeast was gradually adapted to cadmium by a series of subcultures which resulted in a decrease in the length of the lag phase . Adaptation also caused a reduction in the cadmium uptake during the lag phase at 25 mg l-1 cadmium but did not affect uptake during the exponential phase at any concentration . A single passage through cadmium-free medium partially reversed the adaptation process . Sulphide production was enhanced significantly when the yeast was grown in the presence of increasing cadmium concentrations . However, at 5 mg l-1 cadmium, adapted cells produced less sulphide than unadapted cells, whilst at 10 and 25 mg l-1 cadmium the production of sulphide was similar for adapted and unadapted cells.

Microbios, 1985, 42(167), 17 - 25
Biochemical and genetic analysis of an acatalasic rho+ mutant of Saccharomyces cerevisiae; Maqueda M et al.; The previously described acatalasic rho+ strain R-6 has been studied in order to determine the type of mutation responsible for its inability to produce catalase . Induction conditions for catalase activity and temperature influence on the behaviour of this strain were assayed . Furthermore, haploid progeny arising from crosses between R-6 and other strains (rho+, rho- and rho0) was examined for catalase levels . From the data obtained a hypothetical interaction between nuclear and mitochondrial genomes in relation to catalase biosynthesis is discussed.

Mol Gen Genet, 1985, 199(1), 21 - 5
Cloning and mapping of the sporulation gene, spoT7, in Saccharomyces cerevisiae; Tanaka H et al.; In order to isolate a DNA fragment able to complement a sporulation-deficient mutation in Saccharomyces cerevisiae, a simple screening procedure was devised which was based on the difference in osmotic sensitivity between protoplasts and spores . A plasmid (pHT7) containing a 13 kb DNA insert that complemented the spoT7 mutation was isolated from a yeast genomic library prepared in the vector YEp13 . Gene spoT7 was linked to rna1 at 1.2 cM and to mak27 at 7.2 cM on the right arm of chromosome XIII . Mapping of the cloned gene following integration into the chromosome showed that the cloned gene was allelic to spoT7 and that a part of the RNA1 gene was also cloned into the same fragment . Gene spoT7 was localized on a 5 kb DNA fragment by further subcloning.

Mol Gen Genet, 1985, 199(1), 14 - 20
A mutant plasmid with increased stability of holding and polymerization in Saccharomyces cerevisiae; Harashima S et al.; A yeast mutant plasmid, pX, showing increased stability of holding in mitotic and meiotic cell division, was isolated from an unstable plasmid, YRp7, which consists of pBR322 and a 1.4 kilobase (kb) fragment of Saccharomyces cerevisiae carrying the TRP1 gene and the autonomously replicating sequence, ARS1 . The pX plasmid exists as circular molecules in a series of polymeric forms from the monomer to the 20-mer or more, consisting, except for the monomer, of even numbers of unit molecules in tandem arrangement . The pX monomer consists solely of a yeast DNA fragment of 849 base pairs (bp) containing the TRP1 and ARS1 sequences derived from the 1.4 kb yeast fragment of YRp7 . The copy number of pX was calculated to be 20 as monomer units per genome . The ARS1 region was delimited to 80 bp by this mutation and the region essential for the ARS function was discussed.

Gene, 1985, 33(2), 215 - 26
Factors affecting heterologous gene expression in Saccharomyces cerevisiae; Mellor J et al.; The 'promoter' fragment from the yeast phosphoglycerate kinase (PGK) gene has been used to direct the expression of human interferon-alpha-2 (IFN alpha 2) on a high-copy-number plasmid in yeast . The yields of IFN alpha 2 are only 1-3% of yeast total protein, whereas the maximum yield of PGK produced by the PGK gene on a high-copy-number plasmid is at least 50% . IFN alpha 2 is turned over more rapidly than PGK but in addition a major reason for the relatively low level of IFN alpha 2 is that IFN-specific RNA levels are much lower . This does not reflect differences in plasmid copy number or integrity, or differences in the 5' and 3' untranslated regions of the transcripts or DNA flanking regions . It appears that the presence of heterologous coding sequences, or the absence of specific yeast sequences causes a reduction in heterologous RNA levels in yeast.

Gene, 1985, 33(2), 159 - 68
Transcription of the human adenovirus E1a gene in Saccharomyces cerevisiae; Handa H et al.; The early region 1a (E1a) and its flanking sequences of human adenovirus type 5 (Ad5) have been cloned in the yeast-Escherichia coli shuttle vector YEp13 and transferred into the yeast Saccharomyces cerevisiae . The E1a-specific RNAs were produced in the transformed yeast cells . The 5' ends of these transcripts were capped but were lacking 10 to 45 nucleotides from the 5' end of the proper E1a mRNA . These transcripts terminated approx . 1000 nucleotides downstream from the proper 3' end . No splicing of the E1a-specific RNA could be detected in the yeast cells.

Basic Life Sci, 1985, 31, 425 - 34
Mutation induction by excess deoxyribonucleotides in Saccharomyces cerevisiae; Brendel M; Excess dTMP is toxic and mutagenic with exponentially growing dTMP efficient uptaking yeast strains 831 rho+ and 833 rho . The respiratory deficient strain 833 exhibits a tenfold sensitivity to the genotoxicity of excess dTMP . Mutant yield in the forward mutation system CAN1----can1 after dTMP excess is comparable to that found after irradiation with UV254nm . Excess dTMP is a poor mutagen in stationary phase cells of both strains . Mutagenicity of excess dTMP is not found in an ochre mutant allele (ade2-1) . Exposure of exponentially growing cells to other deoxyribonucleotides (dCMP, dAMP, and dGMP) reveal these nucleotides to have mutagenic potential as well.

Zentralbl Mikrobiol, 1985, 140(1), 3 - 11
{Effect of cadmium, zinc, lead and mercury on enzyme activity in Saccharomyces cerevisiae}; Grafl HJ et al.; The in vivo influence of cadmium, zinc, lead, and mercury on the activities of intracellular enzymes in Saccharomyces cerevisiae has been studied . Cadmium and lead cause a significant increase of the enzyme activities . Zinc and mercury do not affect the enzyme activities but they intensify considerably the effects of cadmium and lead . A reduction of enzyme activities is found only rarely . Interactions between the heavy metals tested can lead to synergism or to antagonism or rather to an addition of the effects . The findings suggest that under in vivo conditions heavy metals show only an indirect influence on intracellular enzymes.

Chromosoma, 1985, 91(2), 113 - 20
Synaptonemal complexes of normal and mutant yeast chromosomes (Saccharomyces cerevisiae); Moens PB et al.; Synaptonemal complex (SC) analysis of six laboratory yeast strains showed the SC karyotypes to be repeatable within strains . Chromosomal differences were found between strains . In five of the strains, two SCs insert into the nucleolus . This represents a single bivalent with a nucleolar organizer in a medial position as is suggested by genetic data or two bivalents each with a terminal nucleolar organizer . In the first interpretation, n = 16; in the second, n = 17 . Strain Tris has a single nucleolar SC and n = 17 . In strains DCx374, DCx416 and x8366a the genetically determined rearrangements of linkage group III could not be identified . Presumably the short SC (0.33 micron) associated with linkage group III cannot accommodate an inversion loop or a translocation configuration . The strains however were found to harbour a reciprocal translocation involving the nucleolar chromosome . Trisomy for one of the longer chromosomes was observed in Tris and spo10 . It is concluded that rearrangements of the medium and long but not short yeast chromosomes can be detected cytologically . Measurements of nuclear volumes show SC length to vary with artifactually induced swelling of the nucleus . Linear regression of SC length over nuclear radius indicates that actual SC length is only about one-half the observed length . As a result the DNA packing per SC unit length is higher than previously estimated.

Anal Biochem, 1985 Jan, 144(1), 179 - 85
Purification of phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae and its use for bicarbonate assay; Tortora P et al.; Electrophoretically homogeneous phosphoenolpyruvate carboxykinase (EC 4.1.1.49) from Saccharomyces cerevisiae was obtained in high yields by means of a two-step purification procedure consisting of ion-exchange chromatography and affinity chromatography on adenosine 5'-monophosphate-Sepharose 4B . In the latter step the binding of the enzyme to the resin specifically required the presence of Mn2+ . The enzyme was eluted when Mn2+ was removed by addition of ethylenediaminetetraacetate to the elution buffer . Homogeneity, molecular weight, and subunit composition of phosphoenolpyruvate carboxykinase were checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration . A factor which caused an underestimation of the enzyme activity in crude extracts was identified as adenylate kinase . Finally, a method is proposed for the enzymatic assay of bicarbonate using a purified phosphoenolpyruvate carboxykinase preparation.

Mol Cell Biol, 1985 Jan, 5(1), 226 - 35
Regulation of CDC9, the Saccharomyces cerevisiae gene that encodes DNA ligase; Peterson TA et al.; We have cloned CDC9, the structural gene for Saccharomyces cerevisiae DNA ligase, and investigated its transcriptional regulation both as a function of cell cycle stage and after UV irradiation . The steady-state level of DNA ligase mRNA increases at least fourfold in late G1, after the completion of start but before S phase . This high level of CDC9 mRNA then decays with an apparent half-life of ca . 20 min and remains at a low basal level throughout the rest of the cell cycle . The accumulation of CDC9 mRNA in late G1 is dependent upon the completion of start but not the CDC7 and CDC8 functions . Exposure of cells to UV light elicits an eightfold increase in DNA ligase mRNA levels.

Mol Cell Biol, 1985 Jan, 5(1), 17 - 26
RAD3 gene of Saccharomyces cerevisiae: nucleotide sequence of wild-type and mutant alleles, transcript mapping, and aspects of gene regulation; Naumovski L et al.; We determined the complete nucleotide sequence of the RAD3 gene of Saccharomyces cerevisiae . The coding region of the gene contained 2,334 base pairs that could encode a protein with a calculated molecular weight of 89,796 . Analysis of RAD3 mRNA by Northern blots and by S1 nuclease mapping indicated that the transcript was approximately 2.5 kilobases and did not contain intervening sequences . Fusions between the RAD3 gene and the lac'Z gene of Escherichia coli were constructed and used to demonstrate that the RAD3 gene was not inducible by DNA damage caused by UV radiation or 4-nitroquinoline-1-oxide . Two UV-sensitive chromosomal mutant alleles of RAD3, rad3-1 and rad3-2, were rescued by gap repair of a centromeric plasmid, and their sequences were determined . The rad3-1 mutation changed a glutamic acid to lysine, and the rad3-2 mutation changed a glycine to arginine . Previous studies have shown that disruption of the RAD3 gene results in loss of an essential function and is associated with inviability of haploid cells . In the present experiments, plasmids carrying the rad3-1 and rad3-2 mutations were introduced into haploid cells containing a disrupted RAD3 gene . These plasmids expressed the essential function of RAD3 but not its DNA repair function . A 74-base-pair deletion at the 3' end of the RAD3 coding region or a fusion of this deletion to the E . coli lac'Z gene did not affect either function of RAD3.

Farmaco {Sci}, 1985 Jan, 40(1), 3 - 13
Photobiological properties of furothiocoumarins in Saccharomyces cerevisiae; Juttermann R et al.; Nine furothiocoumarins corresponding to psoralen, 8-methylpsoralen, 3-carbethoxy-8-methylpsoralen, 8-methoxypsoralen, pseudopsoralen, angelicin, isopseudopsoralen, allopsoralen and pseudoisopsoralen were synthesized by treating furocoumarins with phosphorus pentasulfide . Photobiological studies on haploid yeast cells (Saccharomyces cerevisiae) revealed that the furothiocoumarins exert some photoactivity on cell survival and the induction of mitochondrial damage . In most cases, the furothiocoumarins were less active than their furocoumarinic counterparts and exhibited a preference for a monofunctional type of action . A certain photochemotherapeutic activity can be suggested.

Radiat Environ Biophys, 1985, 24(1), 1 - 7
Influence of different inhibitors on the activity of the RAD54 dependent step of DNA repair in Saccharomyces cerevisiae; Siede W et al.; The recombinagenic pathway of DNA repair in yeast was characterized by the effect of different inhibitors on the temperature-dependent survival after gamma-irradiation in haploid cells of the thermoconditional mutant rad54-3 . Blocking protein synthesis with cycloheximide in replicating cells caused partial inhibition of the RAD54 dependent function but some repair activity remained detectable . This indicates that gamma-rays can induce RAD54 activity above some constitutive level of function . Inhibition of DNA replication by hydroxyurea efficiently blocked the RAD54 dependent function in stationary-phase cells but not in logarithmic-phase cells . In logarithmic-phase cells, we found a strong inhibitory effect of caffeine on the RAD54 mediated repair process.

Proc Natl Acad Sci U S A, 1985 Jan, 82(1), 168 - 72
RAD6 gene of Saccharomyces cerevisiae encodes a protein containing a tract of 13 consecutive aspartates; Reynolds P et al.; The RAD6 gene of Saccharomyces cerevisiae is required for postreplication repair of UV-damaged DNA, for induced mutagenesis, and for sporulation . We have mapped the transcripts and determined the nucleotide sequence of the cloned RAD6 gene . The RAD6 gene encodes two transcripts of 0.98 and 0.86 kilobases which differ only in their 3' termini . The transcribed region contains an open reading frame of 516 nucleotides . The rad6-1 and rad6-3 mutant alleles, which we have cloned and sequenced, introduce amber and ochre nonsense mutations, respectively, into the open reading frame, proving that it encodes the RAD6 protein . The RAD6 protein predicted by the nucleotide sequence is 172 amino acids long, has a molecular weight of 19,704, and contains 23.3% acidic and 11.6% basic residues . Its most striking feature is the highly acidic carboxyl terminus: 20 of the 23 terminal amino acids are acidic, including 13 consecutive aspartates . RAD6 protein thus resembles high mobility group proteins HMG-1 and HMG-2, which each contain a carboxyl-proximal tract of acidic amino acids.

Mutat Res, 1985 Jan-Feb, 148(1-2), 47 - 57
Lethal and mutagenic effects photoinduced in haploid yeast (Saccharomyces cerevisiae) by two new monofunctional pyridopsoralens compared to 3-carbethoxypsoralen and 8-methoxypsoralen; Averbeck D et al.; The photobiological effects of two monofunctional pyridopsoralens (PPs), pyrido{3,4-c}psoralen and pyrido{3,4-c}-7-methylpsoralen were studied and compared to those of 3-carbethoxypsoralen (3-CPs) and 8-methoxypsoralen (8-MOP) in a haploid wild-type strain of yeast (Saccharomyces cerevisiae) . The capacity of PPs to photoinduce lethal effects in the presence of 365-nm radiation was not only higher than that of the monofunctional compound 3-CPs, but also higher than that of the bifunctional compound 8-MOP . This activity was apparently independent of oxygen, and it was found that it was probably due to the induction of monoadducts in DNA . A high effectiveness of PPs on the induction of cytoplasmic 'petite' mutations was observed suggesting a high photoaffinity towards mitochondrial DNA . In contrast to 8-MOP, the strong cell killing activity of PPs was not accompanied by a strong inducing effect on nuclear mutations (HIS+ reversions or canR forward mutations) . For these endpoints, PPs were less effective per unit dose of 365-nm radiation and also less efficient per viable cell than 8-MOP . From this, it appears that the lesions photoinduced by the former compounds show a more lethal than (nuclear) mutagenic potential . Furthermore, the fact that PPs were even less mutagenic (nuclear) per viable cell than the monofunctional compound 3-CPs suggests that the activity of these agents may differ in frequency and nature of lesions induced . The photobiological activity of PPs in haploid yeast appears to be in line with the recent proposition for their use in photochemotherapy.

J Bacteriol, 1985 Jan, 161(1), 385 - 92
Protein synthesis during transition and stationary phases under glucose limitation in Saccharomyces cerevisiae; Boucherie H; Metabolic changes have been investigated during continuous growth of yeast cells inoculated in glucose-containing medium until the cells entered the stationary phase in response to glucose exhaustion . Well in advance of glucose exhaustion, a transition phase was observed, characterized by a decrease in the growth rate and a progressive reduction of protein and RNA accumulation . Two-dimensional gel analysis of the proteins synthesized during this stage showed that the pattern of proteins remained similar to that of log-phase cells . When the cells entered the stationary phase, protein accumulation was 10% of that in log-phase cells, and incorporation of labeled RNA precursor was undetectable . Analysis of protein synthesis gave evidence that the synthesis of 95% of the proteins present in log-phase cells was arrested in stationary-phase cells . Among the 20 proteins whose synthesis continues throughout the stationary phase were identified actin, aldehyde dehydrogenase, enolase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and five heat shock proteins . In addition, the synthesis of six new proteins was observed . The occurrence of these new proteins in stationary-phase cells is presumed to result from the release of carbon catabolite repression due to glucose exhaustion.

Genetics, 1985 Jan, 109(1), 21 - 35
Genetic analysis of mutants of Saccharomyces cerevisiae resistant to the herbicide sulfometuron methyl; Falco SC et al.; Sulfometuron methyl (SM), a potent new sulfonylurea herbicide, inhibits growth of the yeast Saccharomyces cerevisiae on minimal media . Sixty-six spontaneous mutants resistant to SM were isolated . All of the resistance mutations segregate 2:2 in tetrads; 51 of the mutations are dominant, five are semidominant and ten are recessive . The mutations occur in three linkage groups, designated SMR1, smr2 and smr3 . Several lines of evidence demonstrate that the SMR1 mutations (47 dominant and four semidominant) are alleles of ILV2 which encodes acetolactate synthase (ALS), the target of SM . First, SMR1 mutations result in the production of ALS enzyme activity with increased resistance to SM . Second, molecular cloning of the ILV2 gene permitted the isolation of mutations in the cloned gene which result in the production of SM-resistant ALS . Finally, SMR1 mutations map at the ILV2 locus . The smr2 mutations (four recessive, two dominant and one semidominant) map at the pdr 1 (pleiotropic drug resistance) locus and show cross-resistance to other inhibitors, typical of mutations at this locus . The smr3 mutations (six recessive and two dominant) define a new gene which maps approximately midway between ADE2 and HIS3 on the right arm of chromosome XV.

Gene, 1985, 40(2-3), 285 - 90
One strand of ars189 from the maxicircle of Crithidia fasciculata transforms Saccharomyces cerevisiae more efficiently than its complementary strand as a single stranded DNA; Kim R et al.; An autonomously replicating element (ars189) has been isolated from the maxicircle DNA of an insect trypanosomatid Crithidia fasciculata . This 189-bp fragment contains two copies of the yeast consensus ARS sequence of (A/T)TTTATPuTTT(T/A), has an A + T composition of 79.4%, and shows a large asymmetry in the distribution of adenine and thymine residues between the two strands . The complementary strands of ars189 have been cloned into an M13 vector containing the URA3 gene of Saccharomyces cerevisiae . When these circular single-stranded (ss) DNAs were used to transform yeast spheroplasts, the M13 chimeric DNA carrying the strand of ars189 rich in adenine generated approximately four times more yeast Ura + transformants than the construct containing the thymine-rich strand . In contrast, both strands of yeast ARS1 cloned into an M13 vector transformed yeast at an equivalent level . The conversion of ARS-containing ss DNAs to duplex forms in vivo and their subsequent autonomous replication have been verified by Southern hybridization analysis of extracts from yeast transformants.

Gene, 1985, 36(3), 349 - 55
Homology between the photoreactivation genes of Saccharomyces cerevisiae and Escherichia coli; Yasui A et al.; A cloned fragment of Saccharomyces cerevisiae chromosomal DNA carrying the photoreactivation gene (PHR) has been sequenced . The fragment contains a 1695-bp intronless open reading frame (ORF) coding for a polypeptide of 564 amino acids (aa) . The phr gene of Escherichia coli was also sequenced, and the sequence is in agreement with the published data . The yeast PHR gene has a G + C content of 36.2%, whereas 53.7% was found for the E . coli gene . Despite the difference in G + C content there is a 35% homology between the deduced aa sequences . This homology suggests that both genes have originated from a common ancestral gene.

Gene, 1985, 36(3), 225 - 34
The RAD2 gene of Saccharomyces cerevisiae: nucleotide sequence and transcript mapping; Nicolet CM et al.; We have determined the nucleotide (nt) sequence of a segment of the yeast chromosome carrying the RAD2 gene . The coding region consists of 2925 bp and could encode a protein of 975 amino acids with a calculated Mr of 111 100 . A major transcriptional start point was mapped to a position of approx . 22 bp upstream from the first ATG codon . A number of minor transcriptional start points were also identified in this region, all of them 5' to the putative translational start codon . We noted a number of consensus nt sequences in the 5' and 3' non-coding regions of the RAD2, RAD1 and RAD3 genes of Saccharomyces cerevisiae . In addition, three regions of amino acid sequence homology in the putative RAD1 and RAD2 polypeptides were observed.

Mol Gen Genet, 1985, 200(3), 407 - 14
The expression of the MET25 gene of Saccharomyces cerevisiae is regulated transcriptionally; Sangsoda S et al.; The MET25 gene of Saccharomyces cerevisiae was cloned by functional complementation after transformation of a yeast met25 mutant . Subcloning of the DNA fragment bearing MET25 located the gene on a 2.3 kb region . The gene was formally identified by integration at the chromosomal MET25 locus . The cloned MET25 gene was used as a probe to measure the MET25 messenger RNA in a wild-type strain grown under conditions which promoted or failed to promote repression of MET25 expression . It was found that, under repression conditions, MET25 messenger RNA was reduced tenfold when compared with non-repression conditions . This suggests that the expression of MET25 is regulated transcriptionally . The direction of transcription, the size of the transcript and the position of the transcribed part of the gene were determined . Deletion mapping of the regulatory region was carried out . Deleted plasmids were introduced back into yeast cells and tested for their ability to complement met25 mutations and to promote regulation of expression of the MET25 gene by exogenous methionine . By this method the regulatory region was found to be confined to a 130 bp region.

Mol Gen Genet, 1985, 199(3), 396 - 400
Expression of an Escherichia coli phr gene in the yeast Saccharomyces cerevisiae; Langeveld SA et al.; A 2 kb DNA fragment, containing the photoreactivation gene phr1 from Escherichia coli, was inserted at the BamH1 site in the tet gene of the yeast--E . coli shuttle vector pJDB207 . Photoreactivation--deficient Saccharomyces cerevisiae cells transformed with this plasmid showed photoreactivation of killing after UV irradiation of the cells, while extracts of transformed cells exhibited photoreactivating activity in vitro . Far more photoreactivating enzyme molecules were found when the gene was inserted in the plasmid in the opposite orientation to the tet gene as compared with a plasmid carrying the inserted gene in the same orientation . Photoreactivating enzyme encoded by the E . coli phr1 gene and produced in transformed yeast cells has characteristics of the E . coli photoreactivating enzyme (flavoprotein) as judged from the influence of ionic strength on photoreactivating activity.

Gene, 1985, 34(2-3), 269 - 81
Transcriptional regulation of the MET3 gene of Saccharomyces cerevisiae; Cherest H et al.; The MET3 gene, coding for ATP sulfurylase (ATPS), an enzyme implicated in methionine biosynthesis in Saccharomyces cerevisiae, was cloned by functional complementation, after transformation, of a yeast met3 mutant strain . The cloned MET3 gene was used as a probe to measure the specific MET3 messenger RNA in a wild-type strain grown under conditions which promote or fail to promote repression of ATPS synthesis . It was found that the level of MET3 messenger RNA is reduced ten-fold when the strain is grown under conditions where ATPS synthesis is repressed, suggesting that the MET3 expression is regulated transcriptionally . The direction of transcription and the size of the transcript have been determined.

Biochimie, 1985 Jan, 67(1), 35 - 43
{Control of the cell division cycle and sporulation in Saccharomyces cerevisiae by the cyclic AMP system}; Jacquet M et al.; This paper reviews recent data on the adenylate cyclase system of the yeast Saccharomyces cerevisiae . Since the discovery of yeast adenylate cyclase mutants and the possibility of molecular biological analysis, adenylate cyclase and the subsequent steps in the cAMP cascade have become subject of intense investigation . CYR1, the structural gene for the adenylate cyclase catalytic subunit is necessary for cell division and in diploid cells is involved in the choice between sporulation and cell division . The cell division cycle in yeast is initiated by a step called START, which has been defined by mutations causing an arrest of the cells in an unbudded state . One class of mutation causes the cell to arrest at the same stage of the cell division cycle as the pheromone implicated in conjugation . A second class causes cells to cease growth in a different manner, but one which is similar to the arrest brought about by nutient deprivation . The adenylate cyclase gene belongs to the second class and has been identified as CDC35 . Two genes of the first class have been cloned and sequenced . CDC28 codes for a kinase which has homology with the src proto-oncogene family . CDC36 is partly homologous with the oncogene ets . Two genes related to the ras oncogene family have also been implicated in the control of START . START can be dissociated in two subsequent phases, the first being controlled by the AMPc system and the second including proto-oncogenes . A model in which cAMP is a positive indicator of available nutrients such as nitrogen has been constructed.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibiot Med Biotekhnol, 1985 Jan, 30(1), 19 - 21
{Natural interferon inducers: double-stranded RNA of killer plasmids of Saccharomyces cerevisiae}; Duzhak AB et al.; Methods for isolation and purification of yeast double spiral RNA (dsRNA) are described . The most simple method includes reprecipitation of dsRNA in solutions of lithium chloride and its interphase distribution in the system of chloroform: isoamilic alcohol: water . Preparations with the content (w/w) of dsRNA from 30 to 90 per cent were obtained . They possess a high interferon-inducing activity and an antiviral effect on the experimental infection caused by the virus of mouse encephalomyocarditis.

Mol Cell Biol, 1985 Jan, 5(1), 248 - 52
Transcriptional and post-transcriptional control of PHO8 expression by PHO regulatory genes in Saccharomyces cerevisiae; Kaneko Y et al.; A DNA fragment bearing the PHO8 gene, which encodes repressible alkaline phosphatase of Saccharomyces cerevisiae, was cloned . Northern hybridizations with the PHO8 DNA as probe indicated that the PHO8 transcript is 1.8 kilobases in length and is more abundant in cells grown in low-phosphate medium than in high-phosphate medium . The pho9 mutant, whose phenotype is defective in the activity of repressible alkaline phosphatase, produced as much of the PHO8 transcript as did the PHO9+ cells . Hence, the PHO9 product should act at the post-transcriptional level . The pho4 mutant could not derepress the PHO8 transcript, whereas the pho80 mutant could, irrespective of the amount of Pi in the medium, as has been suggested by genetic study.

Curr Genet, 1985, 10(3), 163 - 9
Sporulation-regulated genes of Saccharomyces cerevisiae; Holaway BL et al.; We have characterized 46 hybrid phage which hybridize preferentially to mRNA from sporulating cells . Cross-hybridization experiments demonstrate that 27 distinct SPR (Sporulation regulated) sequences are represented among these phage . The SPR genes can be grouped into three classes: early, middle, and late . The early class shows an accumulation of transcripts soon after transfer to sporulation medium and continues to accumulate RNA throughout sporulation . Transcripts of the middle class increase in level at about the time of DNA synthesis, rise rapidly in abundance until meiosis II, then accumulate more slowly for at least the next 3 h . Late gene transcripts begin to accumulate at about the time of meiosis I, increase 10- to 20-fold in the next 2 h, then remain constant in late sporulating cells.

Int J Biochem, 1985, 17(7), 775 - 80
Properties of ATPase activity in intact vegetative cells and sporulating cells of yeast Saccharomyces cerevisiae; Ota A; The properties of ATPase activity were examined in the intact cells of yeast . The activity was stimulated by Mg2+, Mn2+ and Co2+ . The activity was inhibited by NaN3 and by high concentrations of NaF, NaVO3 and PCMB . Optimal pH for the activity was approximately 8 . The maximum value of the activity was obtained in the cells at the early stationary phase and it decreased in 3 hr after transfer to sporulation medium.

Curr Genet, 1985, 9(6), 435 - 9
Primary structure of a gene for subunit V of the cytochrome c oxidase from Saccharomyces cerevisiae; Seraphin B et al.; We have isolated a gene coding for cytochrome c oxidase subunit V by genetic complementation in yeast . This protein is made as a 153 amino acid long precursor; its amino-terminal extension of 20 amino acids contains four basic residues and no acidic one, a feature common to most pre-sequences of imported mitochondrial proteins.

Curr Genet, 1985, 9(4), 259 - 62
The DEL1 mutator gene in Saccharomyces cerevisiae does not act in trans; Picologlou S et al.; DEL1 strains of the yeast Saccharomyces cerevisiae exhibit a high rate of deletions of the three linked genes, CYC1, OSM1, and RAD7 . Classical genetic methods showed that DEL1 segregated as a single Mendelian gene closely linked to CYC1 . In addition, genetic evidence suggested that DEL1 was both cis- and trans-dominant (Liebman et al . 1979) . Molecular analysis of deletions isolated from a haploid DEL1 strain established that deletion formation was mediated by recombination between yeast transposable elements, Ty's (Liebman et al . 1981) . We now report the molecular characterization of deletions isolated from diploids in the trans configuration . This analysis reveals that these deletions probably arose in a two-step process involving mitotic recombination followed by Ty-mediated deletion formation in cis.

Acta Microbiol Pol, 1985, 34(3-4), 231 - 41
Catalase T deficient mutants of Saccharomyces cerevisiae; Traczyk A et al.; A method for the isolation of catalase T deficient mutants of Saccharomyces cerevisiae is described . Ten mutants lacking catalase T and belonging to 5 complementation groups were isolated . CTT1 locus was identified as the structural gene for catalase T . It is under the control of CTT2, CTT3 and CTT4 loci.

Gene, 1985, 36(1-2), 1 - 13
A new putative gene in the mitochondrial genome of Saccharomyces cerevisiae; Colin Y et al.; The 2200-bp ori2-ori7 region of the mitochondrial (mt) genome of Saccharomyces cerevisiae has been sequenced on the genome of a petite, b7, excised at those ori sequences from wild-type strain B . The region contains an open reading frame, ORF5, which is transcribed into a 900-nucleotide (nt) RNA in both the parental wild-type strain and its derived petite, b7 . This RNA uses as a template the strand used by most mt transcripts . Its start point is located 337 nt upstream of ORF5; and a messenger termination site has been found 900 nt downstream of the initiation site . These data suggest that ORF5 is a new mitochondrial gene . The G + C content of ORF5 is only 15.7%; 90% of the G + C base pairs of ORF5 are comprised in a palindromic G + C cluster similar to that present in the varl gene . The coding capacity of ORF5 is 46 amino acids (aa), mainly represented by methionine, phenylalanine, arginine, valine, asparagine, isoleucine and tyrosine . The aa composition and the codon usage of ORF5 are reminiscent of those of varl and of other intergenic ORFs.

J Biol Chem, 1984 Dec 25, 259(24), 15401 - 7
Isolation and sequence of the structural gene for cytochrome c oxidase subunit VI from Saccharomyces cerevisiae; Wright RM et al.; Using synthetic oligodeoxyribonucleic acid probes we have identified and isolated COX6, the structural gene for subunit VI of cytochrome c oxidase from Saccharomyces cerevisiae . The nucleotide sequence of COX6 predicts an amino acid sequence, for the mature subunit VI polypeptide, which is in perfect agreement with that determined previously . The nucleotide sequence of COX6 also predicts that subunit VI is derived from a precursor with a highly basic 40-amino acid NH2-terminal presequence . This precursor has been observed after in vitro translations programmed by yeast poly(A+)RNA . Northern blot analysis of poly(A+) RNA from strain D273-10B reveals that COX6 is homologous to three RNAs of 1800, 900, and 700 bases in length . By means of Southern blot analysis, the cloned gene was shown to be co-linear with yeast chromosomal DNA and to exist in a single copy in the yeast genome . An additional open reading frame, consisting of 82 codons, terminates 22 codons upstream from COX6 . It is "in frame" with the COX6 coding region.

Nucleic Acids Res, 1984 Dec 21, 12(24), 9367 - 82
Transfer RNA splicing in Saccharomyces cerevisiae: defining the substrates; Ogden RC et al.; The primary sequences of all the tRNA precursors which contain intervening sequences and which accumulate in the Saccharomyces cerevisiae rnal mutant are presented . A combination of DNA and RNA sequence analysis has led to elucidation of the primary sequence of four hitherto uncharacterized precursors . The location of the intervening sequence has in all cases been unambiguously determined by analysis of the intermediates in the splicing reaction . Secondary structures based upon the tRNA cloverleaf are shown for all the tRNA precursors and discussed with respect to common recognition by the yeast splicing endonuclease.

Nucleic Acids Res, 1984 Dec 11, 12(23), 8951 - 70
Homologous versus heterologous gene expression in the yeast, Saccharomyces cerevisiae; Chen CY et al.; DNA sequences normally flanking the highly expressed yeast 3-phosphoglycerate kinase (PGK) gene have been placed adjacent to heterologous mammalian genes on high copy number plasmid vectors and used for expression experiments in yeast . For many genes thus far expressed with this system, expression has been 15-50 times lower than the expression of the natural homologous PGK gene on the same plasmid . We have extensively investigated this dramatic difference and have found that in most cases it is directly proportional to the steady-state levels of mRNAs . We demonstrate this phenomenon and suggest possible causes for this effect on mRNA levels.

J Biol Chem, 1984 Dec 10, 259(23), 14406 - 12
Extensive purification and characterization of chromatin-bound histone acetyltransferase from Saccharomyces cerevisiae; Travis GH et al.; A strong correlation has been established between reversible acetylation of histones and transcriptional activation of chromatin . However, the function of histone acetylation remains unknown . We have approached this question by purifying histone acetyltransferase 15,000-fold from yeast and characterizing it enzymatically . Biochemical properties, including the pH and temperature optima and the Michaelis-Menten constants for both acetyl coenzyme A and histones, are similar to those reported for histone acetyltransferases from higher eukaryotes . Yeast histone acetyltransferase has a native molecular weight of 110,000 as determined by gel filtration and is tightly bound to chromatin . It displays high-substrate specificity for histones . It acetylates all four core histones in the order: H4 greater than H2B greater than H2A . 10-fold higher histone acetyltransferase activity is observed for free histones when compared to yeast polynucleosomes as a substrate.

J Biol Chem, 1984 Dec 10, 259(23), 14347 - 9
Identification of an altered elongation factor in temperature-sensitive mutant ts 7'-14 of Saccharomyces cerevisiae; Herrera F et al.; Postpolysomal extracts from wild-type (wt A364A) and temperature-sensitive (ts 7'-14) yeast cells were preincubated for short periods of time at the nonpermissive temperature (37-41 degrees C) prior to incubations for protein synthesis at 20 degrees C . Whereas wt A364A extracts were relatively unaffected by preincubation at the elevated temperature, mutant extracts lost their ability to translate exogenous natural mRNA and poly(U) . Phe-tRNA synthetase and ribosomes from ts 7'-14 cells were not inactivated by preincubation at 37-41 degrees C, but a cytosolic component required for chain elongation, as measured by poly(U) translation, was extensively inactivated . The three elongation factors (EF-1, EF-2, and EF-3) required for chain elongation in yeast were resolved chromatographically . Only one factor, EF-3, was able to restore the poly(U)-translational activity of mutant extracts inactivated at the elevated temperature . Heat-inactivated yeast cytosols, which did not support protein synthesis with yeast ribosomes, were perfectly able to translate poly(U) with rat liver ribosomes, which require only EF-1 and EF-2 . These and other experiments indicated that the genetically altered component in 7'-14 mutant cells is EF-3.

J Biol Chem, 1984 Dec 10, 259(23), 14966 - 72
Isolation of chitin synthetase from Saccharomyces cerevisiae . Purification of an enzyme by entrapment in the reaction product; Kang MS et al.; Chitin synthetase, in the zymogen form, was extracted with digitonin from a particulate fraction from Saccharomyces cerevisiae and converted into active form by treatment with immobilized trypsin . When the activated enzyme was incubated with UDP-GlcNAc and other components of an assay mixture, a chitin precipitate formed, trapping a large portion of the synthetase . The enzyme was easily extracted frm the chitin gel with a recovery of approximately 50% and an enrichment of approximately 100-fold . Further purification was obtained by repeating the chitin step . After polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the purified synthetase showed a major band corresponding to Mr 63,000, a weaker band at Mr 74,000, and some other minor bands . Under nondenaturing conditions, an Mr of 570,000 was calculated for the enzyme from Stokes radius and sedimentation coefficient determinations . After electrophoresis in a nondenaturing gel and incubation with the components of the standard assay, chitin was formed and precipitated in the gel, yielding an opaque band . Soluble oligosaccharides were not precursors for insoluble chitin, suggesting that synthesis of chitin chains takes place by a processive mechanism . N-Acetylglucosamine stimulated the purified synthetase only slightly and did not participate as a primer in the reaction . The same chain length, somewhat more than 100 units of GlcNAc, was determined in samples of chitin that had been synthesized either in vivo, or with a membrane preparation or with purified synthetase . These results suggest that chitin synthetase itself is capable both of initiating chitin chains without a primer and of determining their length.

J Biol Chem, 1984 Dec 10, 259(23), 14394 - 8
Characterization of Saccharomyces cerevisiae thymidylate kinase, the CDC8 gene product . General properties, kinetic analysis, and subcellular localization; Jong AY et al.; Thymidylate kinase is the product of the CDC8 gene of Saccharomyces cerevisiae (Jong, A.Y.S., Kuo, C.-L., and Campbell, J.L . (1984) J . Biol . Chem . 259, 11052-11059) . In this communication we report the catalytic properties of the enzyme . The enzyme catalyzes the phosphorylation of deoxythymidine monophosphate to form deoxythymidine diphosphate in the presence of phosphate donor . ATP and dATP are the most efficient phosphate donors . In addition to dTMP, the yeast enzyme can use dUMP and 5-iodo-dUMP as phosphate acceptors . Kinetic analysis gives a Km of 0.5 mM for dTMP and 2 mM for dUMP . dTMP has a 7-fold greater rate constant than dUMP . Thymidylate kinase requires a divalent cation and is active over the entire range of pH from 6 to 9 . The relative inhibitory effects of related compounds on yeast thymidylate kinase activity are in the order of dTDP greater than thymidine greater than 5-iodo-dUMP greater than ADP greater than or equal to dADP greater than dUMP greater than dTTP greater than dUDP, if dTMP is used as the phosphate acceptor . dTDP is a competitive inhibitor, with a Ki of 0.62 mM . Subcellular fractionation indicates that thymidylate kinase is found in the combined nuclear and cytoplasmic fraction but not in the mitochondria.

Gene, 1984 Dec, 32(1-2), 75 - 82
Molecular cloning of the GAL80 gene from Saccharomyces cerevisiae and characterization of a gal80 deletion; Yocum RR et al.; An integrated GAL1-lacZ fusion provided a useful phenotypic marker for the gal80- regulatory mutation in Saccharomyces cerevisiae . On minimal glucose plates containing a beta-galactosidase indicator, a GAL80 strain containing the fusion gave white colonies, whereas a gal80- strain gave blue colonies . This color difference was used to isolate the GAL80 gene from a plasmid bank by complementation of the gal80- mutant . The putative GAL80 gene was located on a 2.6-kb HindIII-SalI fragment and has been subcloned into an integrating vector . Genetic analysis showed that the clone integrated at the GAL80 locus . A deletion that covered the entire GAL80 region was constructed in vitro and transplaced into the yeast genome to give an isogenic pair of GAL80 and gal80 deletion strains . Glucose repression of a GAL1-lacZ fusion was normal in the gal80 deletion strain, implying that the GAL80 gene product is not involved in glucose repression.

Mol Cell Biol, 1984 Dec, 4(12), 2811 - 7
DNA polymerases, deoxyribonucleases, and recombination during meiosis in Saccharomyces cerevisiae; Resnick MA et al.; We utilized strains of Saccharomyces cerevisiae that exhibit high efficiency of synchrony of meiosis to examine several aspects of meiosis including sporulation, recombination, DNA synthesis, DNA polymerase I and II, and Mg2+-dependent alkaline DNases . The kinetics of commitment to intragenic recombination and sporulation are similar . The synthesis of DNA, as measured directly with diphenylamine, appears to precede the commitment to recombination . Both DNA polymerase I and II activities and total DNA-synthesizing activity in crude extracts increase two- to threefold before the beginning of meiotic DNA synthesis . Increases of 10- to 20-fold over mitotic levels are found for Mg2+-dependent alkaline DNase activity in crude extracts before and during the commitment to meiotic intragenic recombination . Of particular interest is the comparable increase in a nuclease under the control of the RAD52 gene; this enzyme has been identified by the use of antibody raised against a similar enzyme from Neurospora crassa . Since the RAD52 gene is essential for meiotic recombination, the nuclease is implicated in the high levels of recombination observed during meiosis . The effects observed in this report are meiosis specific since they are not observed in an alpha alpha strain.

Mol Cell Biol, 1984 Dec, 4(12), 2750 - 7
Upstream region required for regulated expression of the glucose-repressible SUC2 gene of Saccharomyces cerevisiae; Sarokin L et al.; The SUC2 gene produces two mRNAs with different 5' ends that encode two forms of invertase . The 1.9-kilobase mRNA encoding secreted invertase is regulated by glucose repression (carbon catabolite repression), and the 1.8-kilobase mRNA encoding intracellular invertase is produced constitutively at low levels . To identify 5' noncoding sequences essential for regulated expression of SUC2, we constructed in vitro a series of deletions and inserted them into the yeast genome at the chromosomal SUC2 locus . Analysis of the effects of each deletion on SUC2 gene expression identified an upstream region required for derepression of secreted invertase synthesis . The 3' boundary of this region is near -418 . The 5' boundary does not appear to be sharply defined, but lies ca . 100 base pairs upstream . A deletion extending from -418 to -140 allowed high-level derepression, indicating that no essential sequences lie between the upstream region and the TATA box at -133 and that the upstream region can be moved 279 base pairs closer to the transcriptional start site . Interactions between the deletions and several unlinked mutations affecting the regulation of SUC2 gene expression were examined . Sequences between -1,900 and -86 are dispensable for expression of the 1.8-kilobase mRNA.

Mutat Res, 1984 Dec, 129(3), 327 - 35
The effect of newly induced mutations on the fitness of genotypes and populations of yeast (Saccharomyces cerevisiae); Orthen E et al.; This paper analyses the fate of artificially induced mutations and their importance to the fitness of populations of the yeast, Saccharomyces cerevisiae, an increasingly important model organism in population genetics . Diploid strains, treated with UV and EMS, were cultured asexually for approximately 540 generations and under conditions where the asexual growth was interrupted by a sexual phase . Growth rates of 100 randomly sampled diploid clones were estimated at the beginning and at the end of the experiment . After the induction of sporulation the growth rates of 100 randomly sampled spores were measured . UV and EMS treatment decreases the average growth rate of the clones significantly but increases the variability in comparison to the untreated control . After selection over approximately 540 generations, variability in growth rates was reduced to that of the untreated control . No increase in mean population fitness was observed . However, the results show that after selection there still exists a large amount of hidden genetic variability in the populations which is revealed when the clones are cultivated in environments other than those in which selection took place . A sexual phase increased the reduction of the induced variability.

Genetics, 1984 Dec, 108(4), 827 - 31
Location of the genes that control induction of the allantoin-degrading enzymes in Saccharomyces cerevisiae; Turoscy V et al.; In an effort to understand the regulation of allantoin degradation in Saccharomyces cerevisiae, we isolated two classes of mutants, each defective in the induction process associated with production of the pathway enzymes . Mutation at one locus (DAL80) results in constitutive expression of the genes involved in allantoin catabolism . Mutation at the second locus (DAL-81) results in the loss of ability to induce these enzymes . This report describes genetic data indicating that the DAL80 and DAL81 loci are situated approximately 13 cM from the centromere on the right arm of chromosome XI and 9 cM proximal to the DAL1 locus on chromosome IX, respectively.

J Bacteriol, 1984 Dec, 160(3), 895 - 902
DNA repair in Saccharomyces cerevisiae: purification and characterization of apurinic endonucleases; Armel PR et al.; Five chromatographically distinct apurinic endonucleases (D1, D2, D3, D4, and E) were purified from Saccharomyces cerevisiae 234, 122, 1,000, 4,550, and 5,490-fold, respectively . All appeared to be class II apurinic endonucleases and were not contaminated with exonuclease or nonspecific endonuclease activities under the reaction conditions used . All had similar pH optima, but endonucleases D4 and E showed higher salt requirements and endonuclease D4 had a lower Mg2+ requirement for optimal activity than the other endonucleases . Endonuclease D4 also nicked OsO4-treated DNA . The molecular weights of the apurinic endonucleases as determined by glycerol gradient sedimentation analysis were 37,000, 49,000, and 10,000, for endonucleases E, D4, and D2, respectively . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of samples of radioiodinated endonuclease E showed the presence of two proteins.

J Bacteriol, 1984 Dec, 160(3), 874 - 8
Saccharomyces cerevisiae does not accumulate ethanol against a concentration gradient; Guijarro JM et al.; It has been reported that yeast cells accumulate ethanol against a concentration gradient . We initiated a study of the mechanism involved in this phenomenon . However, we found that this accumulation does not occur and that ethanol permeates the yeast cell plasma membrane by simple diffusion . The following evidence supports this conclusion . (i) Uptake and outflow of ethanol in yeast cells followed first-order kinetics and were insensitive to the presence of structural analogs of ethanol, to drastic pH changes, and to the action of reagents of amino and thiol groups . These results strongly suggest that ethanol permeates the yeast cell plasma membrane without involvement of any carrier . (ii) The outflow rate of ethanol seems greater than the ability of this organism to produce ethanol, indicating that intracellular accumulation of ethanol is not possible . (iii) The intracellular concentration of ethanol found was similar to the concentration in culture media in all tested conditions . With the available information, it is difficult to ascertain the reasons for the discrepancy between our results and those previously reported by other authors . However, the inadequacy of the cell-sampling procedure and of the chromatographic conditions used by those authors suggests that the discrepancy may be due to artifacts in the measurements of ethanol.

J Bacteriol, 1984 Dec, 160(3), 1196 - 8
Mating ability during chemically induced G1 arrest of cells of the yeast Saccharomyces cerevisiae; Bedard DP et al.; Diploid formation by haploid cells of Saccharomyces cerevisiae was tested during and after treatment with chemical agents which bring about arrest at the cell cycle regulatory step "start." All compounds, except sinefungin, allowed efficient mating . During sinefungin treatment, zygote formation, but not karyogamy, was affected.

Mol Cell Biol, 1984 Dec, 4(12), 2767 - 73
Three regulatory systems control production of glutamine synthetase in Saccharomyces cerevisiae; Mitchell AP et al.; Production of glutamine synthetase in Saccharomyces cerevisiae is controlled by three regulatory systems . One system responds to glutamine levels and depends on the positively acting GLN3 product . This system mediates derepression of glutamine synthetase in response to pyrimidine limitation as well, but genetic evidence argues that this is an indirect effect of depletion of the glutamine pool . The second system is general amino acid control, which couples derepression of a variety of biosynthetic enzymes to starvation for many single amino acids . This system operates through the positive regulatory element GCN4 . Expression of histidinol dehydrogenase, which is under general control, is not stimulated by glutamine limitation . A third system responds to purine limitation . No specific regulatory element has been identified, but depression of glutamine synthetase is observed during purine starvation in gln3 gcn4 double mutants . This demonstrates that a separate purine regulatory element must exist . Pulse-labeling and immunoprecipitation experiments indicate that all three systems control glutamine synthetase at the level of subunit synthesis.

Mol Cell Biol, 1984 Dec, 4(12), 2758 - 66
Regulation of glutamine-repressible gene products by the GLN3 function in Saccharomyces cerevisiae; Mitchell AP et al.; Mutants of the yeast Saccharomyces cerevisiae have been isolated which fail to derepress glutamine synthetase upon glutamine limitation . The mutations define a single nuclear gene, GLN3, which is located on chromosome 5 near HOM3 and HIS1 and is unlinked to the structural gene for glutamine synthetase, GLN1 . The three gln3 mutations are recessive, and one is amber suppressible, indicating that the GLN3 product is a positive regulator of glutamine synthetase expression . Four polypeptides, in addition to the glutamine synthetase subunit are synthesized at elevated rates when GLN3+ cultures are shifted from glutamine to glutamate media as determined by pulse-labeling and one- and two-dimensional gel electrophoresis . The response of all four proteins is blocked by gln3 mutations . In addition, the elevated NAD-dependent glutamate dehydrogenase activity normally found in glutamate-grown cells is not found in gln3 mutants . Glutamine limitation of gln1 structural mutants has the opposite effect, causing elevated levels of NAD-dependent glutamate dehydrogenase even in the presence of ammonia . We suggest that there is a regulatory circuit that responds to glutamine availability through the GLN3 product.

Gene, 1984 Dec, 32(3), 263 - 74
Expression of heterologous genes in Saccharomyces cerevisiae from vectors utilizing the glyceraldehyde-3-phosphate dehydrogenase gene promoter; Bitter GA et al.; The promoter region from the cloned glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Saccharomyces cerevisiae (Musti et al., 1983) has been characterized . A 653-bp TaqI restriction fragment with a 3' border 24 bp upstream from the ATG initiation codon was isolated and demonstrated to contain all sequences necessary for promoter function in vivo . This DNA segment was converted to a portable promoter by cloning it into M13mp9, and the entire nucleotide sequence of the portable promoter was determined . Two generalized yeast expression vectors have been constructed utilizing the GPD portable promoter . The expression vectors include the yeast 2 mu origin of replication and amplification functions, such that the plasmids are maintained at high copy number in ciro yeast hosts . These vectors direct synthesis of a consensus alpha-interferon (IFN-alpha Con1) as 1% of total cell protein . Hepatitis B surface antigen (HBsAg) was also expressed from these vectors . The 5' end of the HBsAg gene was replaced with a synthetic DNA segment which restored the deleted GPD untranslated leader and utilized optimal yeast codons for the first 30 amino acids . The partially synthetic gene resulted in a 10- to 15-fold increased expression level from GPD vectors yielding HBsAg polypeptide as 2-4% of total cell protein.

Mol Cell Biol, 1984 Dec, 4(12), 2837 - 42
Primary structure of the nuclear PUT2 gene involved in the mitochondrial pathway for proline utilization in Saccharomyces cerevisiae; Krzywicki KA et al.; The PUT2 gene, believed to encode delta 1-pyrroline-5-carboxylate dehydrogenase, has been completely sequenced . The DNA contains an open reading frame of 1,725 base pairs encoding a protein of 575 amino acids . Transcript mapping with both S1 nuclease and primer extension methods revealed numerous initiation sites of RNA synthesis 50 to 80 base pairs downstream from several TATA boxes . The deduced amino acid sequence of delta 1-pyrroline-5-carboxylate dehydrogenase contains a highly basic amino terminus that may serve as the signal sequence that targets this protein to the mitochondrion.

Mol Cell Biol, 1984 Dec, 4(12), 2735 - 44
Primary structure of the RAD52 gene in Saccharomyces cerevisiae; Adzuma K et al.; The RAD52 gene of Saccharomyces cerevisiae, which is involved in genetic recombination and DNA repair, was cloned by transformation of rad52-1 mutant cells to methyl methanesulfonate resistance with BamHI fragments of Rad+ genomic DNA inserted into the Escherichia coli-S . cerevisiae shuttle vector YRp7 . A plasmid carrying a 2.0-kilobase BamHI fragment was found to partially complement methyl methanesulfonate sensitivity of the rad52-1 mutant . By using this fragment as a hybridization probe, a plasmid that fully complemented the methyl methanesulfonate sensitivity of the mutant was isolated, which carries a 3.3-kilobase SalI fragment containing most of the 2.0-kilobase BamHI fragment . Analysis of the nucleotide sequence of the SalI fragment revealed the presence of a large open reading frame of 1,512 nucleotides . The rad52-1 mutant DNA has a single-base change in this reading frame, which leads to an amino acid substitution . Analysis of mRNA synthesized in yeast by the S1 mapping technique disclosed possible transcription initiation and termination points of the RAD52 gene and suggested formation of the gene product without splicing of the transcript.

EMBO J, 1984 Dec 1, 3(12), 2825 - 30
Cloning of the RNA2 gene of Saccharomyces cerevisiae; Lee MG et al.; The RNA2 gene of Saccharomyces cerevisiae, which has been implicated in splicing the transcripts of nuclear protein coding genes, has been cloned by complementation of the temperature-sensitive growth defect of an rna2-1 mutant strain . The cloned sequence also suppresses the accumulation of unspliced precursor transcripts of the actin gene in an rna2-1 mutant . The gene has been localised to a 3.2-kb DNA restriction fragment and the corresponding low abundance 2.8-kb transcript identified and the 5' ends mapped . Evidence that this cloned sequence represents the RNA2 gene includes homologous integration at the rna2-1 locus and disruption of the RNA2 gene by insertional inactivation . The disrupted allele confers a recessive lethal phenotype, indicating an essential function for the RNA2 gene product.

Biochem J, 1984 Dec 1, 224(2), 497 - 503
Transcription of a yeast ribosomal RNA minigene in Saccharomyces cerevisiae; Quincey RV et al.; A transcription system using intact yeast has been developed for investigating which sequences are implicated in the initiation of transcription of yeast rRNA genes . The system employs an rRNA minigene that consists of the initiation and termination sites for rRNA biosynthesis separated by approx . 700 base pairs of vector DNA in the Escherichia coli-yeast shuttle vector, pJDB207 . Two recombinants containing this minigene were constructed; one retained all of the nontranscribed spacer DNA upstream from the initiation site, the other retained 208 base pairs of this DNA . Transcripts of this structurally unique minigene in RNA from yeast transformed with these recombinants were readily detected by nuclease S1 mapping . These transcripts were initiated at the site used by the host rRNA genes, were approx . 3-fold more abundant in the recombinant retaining all of the nontranscribed spacer and were less abundant when the yeast was not growing.

Proc Natl Acad Sci U S A, 1984 Dec, 81(24), 7860 - 4
Upstream activation sites of the CYC1 gene of Saccharomyces cerevisiae are active when inverted but not when placed downstream of the "TATA box"; Guarente L et al.; The ability of the upstream activation sites (UASs) of the yeast CYC1 gene to function when inverted or when positioned downstream of the "TATA box" is investigated . Inversion of a 130-base-pair DNA fragment bearing the UASs leaves the activity of the sites almost completely intact . In contrast, positioning the sites downstream of the TATA box or in the intron of a CYC1-ribosomal protein 51-lacZ tribrid gene almost totally abolishes their activity . In the latter construct, the separation between the UASs and TATA box is roughly equivalent to that between the elements in the intact CYC1 promoter region . The UASs are shown not to interrupt transcription of splicing in this construct since a GAL10 UAS positioned upstream of the TATA box gives rise to galactose-inducible expression of the tribrid gene . The inability of the UASs to function in the intron is partly due to sequences between the intron and the TATA box that block the activation signal . However, a large component of the inactivity of the sites in the intron appears to be their downstream location . This result is discussed in light of possible mechanisms of upstream activation in yeast.

J Bacteriol, 1984 Dec, 160(3), 1093 - 100
Isolation and characterization of the RNA2+, RNA4+, and RNA11+ genes of Saccharomyces cerevisiae; Soltyk A et al.; We used genetic complementation to isolate DNA fragments that encode the Saccharomyces cerevisiae genes RNA2+, RNA4+, and RNA11+ and to localize the genes on the cloned DNA fragments . RNA blot-hybridization analyses coupled with genetic analyses indicated the RNA2+ is coded by a 3.0-kilobase (kb) transcript, RNA4+ is coded by a 1.6-kb transcript, and RNA11+ is coded by a 1.3-kb or a 1.7-kb transcript or both; none of the cloned genes contains detectable introns . All three genes were transcribed into messages of very low abundance (approximately 20 times lower than a ribosomal protein message) . DNA blot-hybridization revealed that all cloned genes are represented only once in the yeast chromosome . mRNA for RNA2+ and RNA4+ is produced in approximate proportion to gene dosage, whereas RNA11+ transcription appears to be not nearly so dependent on gene dosage . On a medium-copy plasmid (5 to 10 copies per cell), each cloned gene complemented mutations only in its own gene, indicating that each gene encodes a unique function . Genetic analysis by integrative transformation indicated that we cloned the RNA2+, RNA4+, and RNA11+ structural genes and not second-site suppressors.

J Bacteriol, 1984 Dec, 160(3), 1078 - 87
Nucleotide sequence of the Saccharomyces cerevisiae arginase gene (CAR1) and its transcription under various physiological conditions; Sumrada RA et al.; We have determined the nucleotide sequence of the yeast CAR1 gene along with its 5' and 3' flanking sequences . The structural portion of this gene encodes a protein of 333 amino acid residues with a calculated Mr value of 35,616 . The transcripts produced from this gene are modestly heterogeneous at their 5' and 3' termini . Most 5' termini map to a position 42 to 49 base pairs upstream from the ATG at the beginning of the open reading frame . The 3' termini map to a position 108 to 127 base pairs downstream of the amber codon which terminates the open reading frame . A variety of potentially significant sequences were identified in the regions flanking the structural portion of the gene.

J Biol Chem, 1984 Nov 25, 259(22), 13923 - 9
Messenger RNA guanylyltransferase from Saccharomyces cerevisiae . I . Purification and subunit structure; Itoh N et al.; GTP:mRNA guanylyltransferase, an enzyme that catalyzes the transfer of the GMP moiety from GTP to the 5' end of the RNA to form a cap structure (G(5')pppN-), has been purified to an apparent homogeneity from Saccharomyces cerevisiae . The mRNA 5'-triphosphatase activity hydrolyzing the gamma-phosphoryl group from pppN-RNA was co-purified with mRNA guanylyltransferase activity through column chromatographies on CM-Sephadex and poly(U)-Sepharose, and centrifugation through glycerol gradients, suggesting that these two activities are physically associated . An 820,w value of 7.3, and Mr = 140,000 were estimated from the sedimentation behavior in glycerol gradients . Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two major polypeptides, Mr = 45,000 (alpha) and 39,000 (beta), were detected with the purified enzyme preparation . Their molar ratios were close to unity when estimated by the relative density of silver staining . These results suggest that the yeast mRNA-capping enzyme is an oligomeric protein which may consist of two alpha and two beta chains (alpha 2 beta 2).

J Biol Chem, 1984 Nov 25, 259(22), 13930 - 6
Messenger RNA guanlyltransferase from Saccharomyces cerevisiae . II . Catalytic properties; Itoh N et al.; Highly purified mRNA-capping enzyme from Saccharomyces cerevisiae catalyzes (a) removal of the gamma-phosphoryl group from the 5'-end of the newly formed mRNA and (b) guanylylation of the resulting diphosphoryl end . Characteristics of the two reactions catalyzed by this enzyme are studied . Guanylyltransferase is most active at pH 7.0 in the presence of 3 mM Mg2+, and utilizes GTP as a guanylyl donor with an apparent Km of 5 microM, and ppGCC (A2, U2, G)n as a guanylyl acceptor with two Km values of 0.5 and 4 microM . It catalyzes GTP-PPi exchange in the absence of the acceptor RNA, and forms a covalent enzyme-GMP intermediate having Mr = 45,000 in sodium dodecyl sulfate gel electrophoresis . RNAs with 5'-diphosphoryl as well as 5'-triphosphoryl ends are capped, while mononucleotides such as GDP and ppGp are inert . Since guanylyltransferase can utilize ppGpC and ppGpCpC as acceptors, the presence of at least one phosphodiester bond seems to be sufficient for the acceptor activity . However, oligonucleotides of longer chain length are preferred . RNA 5'-triphosphatase associated with the purified enzyme requires Mg2+ and exhibits a broad pH optimum from 6.5 to 8.5, and an apparent Km value for pppA-terminated poly(A) is 1.4 microM . The enzyme is specific for the gamma-phosphoryl group at the 5'-terminus of RNA and does not hydrolyze ATP . It can hydrolyze the gamma-phosphoryl group of pppGp, but the RNA substrates with longer chain length are preferred.

Arch Biochem Biophys, 1984 Nov 15, 235(1), 159 - 66
Effect of exogenous addition of hemin on the biogenesis of mitochondrial membranes during glucose repression in Saccharomyces cerevisiae; Gopalan G et al.; Exogenous addition of hemin alleviated glucose repression by promoting mitochondrial membrane functions . It prevented the release of mitochondrial proteins into the cytosol by decreasing the activities of phospholipase C and phospholipase D . It restored oligomycin sensitivity of mitochondrial ATPase associated with repressed mitochondria . It enhanced cardiolipin content twofold, and decreased the sterol:phospholipid ratio . Studies on the amino acid incorporation into isolated mitochondria showed that hemin can also promote biogenesis of the organelle by stimulating amino acid incorporation into mitochondrial proteins, and this stimulation appeared to be mediated through some cytosolic factor(s).

Nucleic Acids Res, 1984 Nov 12, 12(21), 7975 - 85
Yeast may not contain histone H1: the only known 'histone H1-like' protein in Saccharomyces cerevisiae is a mitochondrial protein; Certa U et al.; It is likely that histone H1 is involved in the condensation of chromatin in eukaryotes . However, both the presence of histone H1 in yeast and the extent of yeast chromatin condensation are controversial . A 20 kD protein copurifies with yeast chromatin and was shown by other investigators to have characteristics of histone H1 protein . In an attempt to obtain a positive identification of the 20 kD protein, we purified the protein to homogeneity and raised antibodies against it . We show here by immunofluorescence that the 20 kD protein does not localize to the nucleus but to cytoplasmic particles resembling mitochondria . Furthermore, we show by Western-blot analysis that anti-20 kD protein antibodies react to protein isolated from purified mitochondria . Finally, we present evidence based on size, charge, amino acid composition and immunological cross reactivity to suggest that the yeast 20 kD protein is likely to be the mitochondrial DNA-binding HM protein . This leaves no candidate for histone H1 in yeast.

Nucleic Acids Res, 1984 Nov 12, 12(21), 8001 - 16
Tandemly arranged variant 5S ribosomal RNA genes in the yeast Saccharomyces cerevisiae; McMahon ME et al.; Most of the ribosomal RNA genes of the yeast Saccharomyces cerevisiae are about 9 kilobases (kb) in size and encode both the 35S rRNA (processed to produce the 25S, 18S, and 5.8S species) and 5S rRNA . These genes are arranged in a single tandem array of 100 repeats . Below, we present evidence that at the centromere-distal end of this array is a tandem arrangement of a different type of rRNA gene . Each of these repeats is 3.6 kb in length and encodes a single 5S rRNA . The coding sequence of this gene is different from that of the "normal" 5S gene in three positions located at the 3' end of the gene.

J Biol Chem, 1984 Nov 10, 259(21), 13273 - 81
Metabolism of added orthovanadate to vanadyl and high-molecular-weight vanadates by Saccharomyces cerevisiae; Willsky GR et al.; The effect of vanadium oxides on living systems may involve the in vivo conversion of vanadate and vanadyl ions . The addition of 5 mM orthovanadate (VO4(3-), V(V)), a known inhibitor of the (Na,K)-ATPase, to yeast cells stopped growth . In contrast, the addition of 5 mM vanadyl (VO2+, V(IV) stimulated growth . Orthovanadate addition to whole cells is known to stimulate various cellular processes . In yeast, both ions inhibited the plasma membrane Mg2+ ATPase and were transported into the cell as demonstrated with {48V}VO4(3-) and VO2+ . ESR spectroscopy has been used to measure the cell-associated paramagnetic vandyl ion, while 51V NMR has detected cell-associated diamagnetic vanadium (e.g . V(V)) . Cells were exposed to both toxic (5 mM) and nontoxic (1 mM) concentrations of vanadate in the culture medium . ESR showed that under both conditions, vanadate became cell associated and was converted to vanadyl which then accumulated in the cell culture medium . 51V NMR studies showed the accumulation of new cell-associated vanadium resonances identified as dimeric vanadate and decavanadate in cells exposed to toxic amounts of medium vanadate (5 mM) . These vanadate compounds did not accumulate in cells exposed to 1 mM vanadate . These studies confirm that the inhibitory form of vanadium usually observed in in vitro experiments is vanadate, in one or more of its hydrated forms . These data also support the hypothesis that the stimulatory form of vanadium usually observed in whole cell experiments is the vanadyl ion or one or more of its liganded derivatives.

Mol Cell Biol, 1984 Nov, 4(11), 2356 - 63
Specific transcripts are elevated in Saccharomyces cerevisiae in response to DNA damage; McClanahan T et al.; Differential hybridization has been used to identify genes in Saccharomyces cerevisiae displaying increased transcript levels after treatment of cells with UV irradiation or with the mutagen/carcinogen 4-nitroquinoline-1-oxide (NQO) . We describe the isolation and characterization of four DNA damage responsive genes obtained from screening ca . 9,000 yeast genomic clones . Two of these clones, lambda 78A and pBR178C, contain repetitive elements in the yeast genome as shown by Southern hybridization analysis . Although the genomic hybridization pattern is distinct for each of these two clones, both of these sequences hybridize to large polyadenylated transcripts ca . 5 kilobases in length . Two other DNA damage responsive sequences, pBRA2 and pBR3016B, are single-copy genes and hybridize to 0.5- and 3.2-kilobase transcripts, respectively . Kinetic analysis of the 0.5-kilobase transcript homologous to pBRA2 indicates that the level of this RNA increases more than 15-fold within 20 min after exposure to 4-nitroquinoline-1-oxide . Moreover, the level of this transcript is significantly elevated in cells containing the rad52-1 mutation which are deficient in DNA strand break repair and gene conversion . These results provide some of the first evidence that DNA damage stimulates transcription of specific genes in eucaryotic cells.

Mol Cell Biol, 1984 Nov, 4(11), 2347 - 55
Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae; Emr SD et al.; The yeast SUC2 gene codes for the secreted enzyme invertase . A series of 16 different-sized gene fusions have been constructed between this yeast gene and the Escherichia coli lacZ gene, which codes for the cytoplasmic enzyme beta-galactosidase . Various amounts of SUC2 NH2-terminal coding sequence have been fused in frame to a constant COOH-terminal coding segment of the lacZ gene, resulting in the synthesis of hybrid invertase-beta-galactosidase proteins in Saccharomyces cerevisiae . The hybrid proteins exhibit beta-galactosidase activity, and they are recognized specifically by antisera directed against either invertase or beta-galactosidase . Expression of beta-galactosidase activity is regulated in a manner similar to that observed for invertase activity expressed from a wild-type SUC2 gene: repressed in high-glucose medium and derepressed in low-glucose medium . Unlike wild-type invertase, however, the invertase-beta-galactosidase hybrid proteins are not secreted . Rather, they appear to remain trapped at a very early stage of secretory protein transit: insertion into the endoplasmic reticulum (ER) . The hybrid proteins appear only to have undergone core glycosylation, an ER process, and do not receive the additional glycosyl modifications that take place in the Golgi complex . Even those hybrid proteins containing only a short segment of invertase sequences at the NH2 terminus are glycosylated, suggesting that no extensive folding of the invertase polypeptide is required before initiation of transmembrane transfer . beta-Galactosidase activity expressed by the SUC2-lacZ gene fusions cofractionates on Percoll density gradients with ER marker enzymes and not with other organelles . In addition, the hybrid proteins are not accessible to cell-surface labeling by 125I . Accumulation of the invertase-beta-galactosidase hybrid proteins within the ER does not appear to confer a growth-defective phenotype to yeast cells . In this location, however, the hybrid proteins and the beta-galactosidase activity they exhibit could provide a useful biochemical tag for yeast ER membranes.

Gene, 1984 Nov, 31(1-3), 257 - 61
Nucleotide sequence of the 3' terminal region of the LEU2 gene from Saccharomyces cerevisiae; Froman BE et al.; The 3' terminal region from the LEU2 gene of yeast has been sequenced . Two open reading frames (ORF) have been identified, one of which constitutes the 3' terminus of beta-isopropylmalate dehydrogenase, the product of the LEU2 gene . A noncoding spacer region located between the ORFs contains two consensus-type transcriptional terminators . The terminator-like sequences are oriented in opposing directions on opposite DNA strands . The noncoding spacer region may represent a single terminator for the LEU2 gene or two separate terminators involved in blocking convergent transcription from an as yet unidentified yeast gene.

Mutat Res, 1984 Nov-Dec, 132(5-6), 161 - 9
Analysis of replication of DEB-alkylated DNA in yeast: bypass replication in a rad3 mutant of Saccharomyces cerevisiae; Lecka-Czernik B et al.; We presented indirect evidence that in an excision-deficient rad3 mutant of yeast exposed to diepoxybutane (DEB), DNA synthesis continued past the damaged sites . This bypass replication was confined to the first post-treatment round of replication and was followed by inhibition of DNA synthesis . Analyses by alkaline sucrose gradient sedimentation and by alkaline elution from filters revealed that in mutant cells the first post-treatment round of replication proceeded at a similar rate to that in untreated cells and was not accompanied by strand scission of template DNA . The post-treatment synthesis was presumably of an error-prone type, as the frequency of reversion to ade2-1 prototrophy was increased . In contrast, in the isogenic wild-type strain, the post-treatment incorporation of radioactivity into DNA was slightly reduced and newly replicated DNA fragments were of lower molecular weight than in control cells . There was also some strain scission in template DNA, presumably resulting from excision-repair.

Mol Cell Biol, 1984 Nov, 4(11), 2529 - 31
Cell cycle phases in the unequal mother/daughter cell cycles of Saccharomyces cerevisiae; Brewer BJ et al.; During cell division in the yeast Saccharomyces cerevisiae mother cells produce buds (daughter cells) which are smaller and have longer cell cycles . We performed experiments to compare the lengths of cell cycle phases in mothers and daughters . As anticipated from earlier indirect observations, the longer cell cycle time of daughter cells is accounted for by a longer G1 interval . The S-phase and the G2-phase are of the same duration in mother and daughter cells . An analysis of five isogenic strains shows that cell cycle phase lengths are independent of cell ploidy and mating type.

Mol Cell Biol, 1984 Nov, 4(11), 2509 - 17
Rearrangements of highly polymorphic regions near telomeres of Saccharomyces cerevisiae; Horowitz H et al.; We have examined the mitotic and meiotic properties of telomeric regions in various laboratory strains of yeast . Using a sequence (Y probe) derived from a cloned yeast telomere (J . Szostak and E . Blackburn, Cell 29:245-255, 1982), we found that various strains of Saccharomyces cerevisiae show extensive polymorphisms of restriction endonuclease fragment length . Some of the variation in the lengths of telomeric fragments appears to be under the control of a small number of genes . When DNA from various strains was digested with endonuclease KpnI, nearly all of the fragments homologous to the Y probe were found to be of different size . The pattern of fragments in different strains was extremely variable, with a greater degree of polymorphism than that observed for fragments containing the mobile TY1 element . Tetrad analysis of haploid meiotic segregants from diploids heterozygous for many different Y-homologous KpnI fragments revealed that most of them exhibited Mendelian (2:0) segregation . However, only a small proportion of these fragments displayed the obligate 2:2 parental segregation expected of simple allelic variants at the same chromosome end . From the segregations of these fragments, we concluded that some yeast telomeres lack a Y-homologous sequence and that the chromosome arms containing a Y-homologous sequence are different among various yeast strains . Regions near yeast telomeres frequently undergo rearrangement . Among eight tetrads from three different diploids, we have found three novel Y-homologous restriction fragments that appear to have arisen during meiosis . In all three cases, the appearance of a new fragment was accompanied by the loss of another band . In one of these cases, the rearrangement leading to a novel fragment arose in an isogenic diploid, in which both homologous chromosomes should have been identical . Among these same tetrads we also found examples of apparent mitotic gene conversions and mitotic recombination involving telemetric regions.

Mol Cell Biol, 1984 Nov, 4(11), 2479 - 85
The INO2 and INO4 loci of Saccharomyces cerevisiae are pleiotropic regulatory genes; Loewy BS et al.; We isolated a mutant of Saccharomyces cerevisiae defective in the formation of phosphatidylcholine via methylation of phosphatidylethanolamine . The mutant synthesized phosphatidylcholine at a reduced rate and accumulated increased amounts of methylated phospholipid intermediates . It was also found to be auxotrophic for inositol and allelic to an existing series of ino4 mutants . The ino2 and ino4 mutants, originally isolated on the basis of an inositol requirement, are unable to derepress the cytoplasmic enzyme inositol-1-phosphate synthase (myo-inositol-1-phosphate synthase; EC 5.5.1.4) . The INO4 and INO2 genes were, thus, previously identified as regulatory genes whose wild-type product is required for expression of the INO1 gene product inositol-1-phosphate synthase (T . Donahue and S . Henry, J . Biol . Chem . 256:7077-7085, 1981) . In addition to the identification of a new ino4-allele, further characterization of the existing series of ino4 and ino2 mutants, reported here, demonstrated that they all have a reduced capacity to convert phosphatidylethanolamine to phosphatidylcholine . The pleiotropic phenotype of the ino2 and ino4 mutants described in this paper suggests that the INO2 and INO4 loci are involved in the regulation of phospholipid methylation in the membrane as well as inositol biosynthesis in the cytoplasm.

Genetika, 1984 Nov, 20(11), 1792 - 7
{Nuclear mutants of Saccharomyces cerevisiae K2 yeasts with decreased killer activity}; Filatov AA et al.; Recessive mutations in two chromosomal unlinked genes kir1 and kir2 of Saccharomyces cerevisiae K2 result in weak killer activity or in complete loss of killer capacity . Kir1 is located on chromosome 7 and is linked to ade7 and ski6 . The kir1 and kir2 mutants reveal no alteration of cell membrane . They normally excrete acid phosphatase and have a normal level of mating and sporulation . The analysis of the plasmid nucleic acid in two strains containing the mutant alleles kir1-12 and kir2-23 shows the increased content of L double-stranded DNA, the content of M double-stranded RNA being increased.

Proc Natl Acad Sci U S A, 1984 Nov, 81(21), 6594 - 8
Expression of human alpha 1-antitrypsin cDNA in the yeast Saccharomyces cerevisiae; Cabezon T et al.; Nucleotide sequences coding either for the precursor or the mature form of human alpha 1-antitrypsin have been placed under the control of the yeast ARG3 expression signals . Recombinant plasmids pRIT10782 and pRIT10787 express the precursor or the mature alpha 1-antitrypsin species, respectively, in two different yeast strains, with yields ranging between 0.3 and 1% of total soluble proteins . The alpha 1-antitrypsin synthesized in yeasts was specifically recognized by polyclonal and monoclonal antibodies raised against human alpha 1-antitrypsin . In addition, it was shown to be biologically active in its mature form only, with optimal activity in a peptidase-deficient yeast strain.

Biosci Rep, 1984 Nov, 4(11), 963 - 72
Expression of a Drosophila heat-shock gene in cells of the yeast Saccharomyces cerevisiae; Nicholson RC et al.; A 3.52-kilobase (kb) segment of Drosophila melanogaster DNA carrying the 2.15-kb transcribed sequence for the 70 000-dalton heat-shock protein (hsp70) and 1.14-kb of the 5' flanking sequence was inserted into an autonomously replicating chimeric plasmid and used to transform the yeast Saccharomyces cerevisiae . The Drosophila gene is efficiently transcribed in the transformed cells, yielding a transcript which is 21 nucleotides shorter than the normal Drosophila mRNA at the 5' end . Significant increases in the amount of Drosophila-specific RNA occur when the transformed cells are subjected to heat shock, indicating that the Drosophila gene is inducible in the yeast cells.

Mol Cell Biol, 1984 Nov, 4(11), 2420 - 7
Negative regulation of STE6 gene expression by the alpha 2 product of Saccharomyces cerevisiae; Wilson KL et al.; The alpha 2 product of the alpha mating type locus of Saccharomyces cerevisiae is proposed to be a negative regulator of a set of dispersed genes concerned with specialized properties of a cells . This set of genes includes those, termed a-specific STE genes (STE2, STE6, and STE14), which are required for mating by a cells but not by alpha cells . We cloned the STE6 gene to determine whether its expression is limited to a cells and, if so, whether its expression is inhibited in alpha cells by the alpha 2 product . Expression of STE6 was assayed in two ways: by blot hybridization, RNA and by beta-galactosidase activity in strains carrying a STE6-lacZ hybrid gene . We found that STE6 expression was limited to a cells and was negatively regulated by the alpha 2 product . STE6 RNA was not detectable in strains containing the wild-type alpha 2 gene product . Expression of STE6 was at least 150-fold lower in alpha cells than in a cells, based on beta-galactosidase activities in a and alpha cells carrying the STE6-lacZ gene . These results confirmed that the alpha 2 product is a negative regulator of gene expression and showed that it acts at the level of RNA production . We also examined the phenotype of a mutant carrying an insertion mutation of the STE6 gene, the ste6::lacZ allele . In addition, an a-specific defect in mating, this mutant was greatly reduced (but not completely deficient) in a-factor production . Other phenotypes characteristic of a cells--Barrier activity, agglutination, and response to alpha-factor--were normal . STE6 thus appears to be necessary for biosynthesis of a-factor.

Mutat Res, 1984 Nov-Dec, 141(3-4), 161 - 4
Drug synergism or antagonism in the induction of diploid meiotic products in Saccharomyces cerevisiae; Sora S et al.; The induction of yeast diploid meiotic products by treatment with bleomycin and mitomycin C is reduced when sporulating cells are treated in combination with propranolol, and increased when they are treated in combination with caffeine . We show that bleomycin and mitomycin C act by blocking the second meiotic division . The frequency of this event appears to be directly related to the intracellular cAMP concentration which is known to be influenced in opposite ways by caffeine and propranolol.

Mol Cell Biol, 1984 Nov, 4(11), 2396 - 405
Isolation and characterization of the RNA2, RNA3, and RNA11 genes of Saccharomyces cerevisiae; Last RL et al.; Temperature-sensitive mutations in the genes RNA2 through RNA11 cause accumulation of intervening sequence containing precursor mRNAs in Saccharomyces cerevisiae . Three different plasmids have been isolated which complement both the temperature-sensitive lethality and precursor mRNA accumulation when introduced into rna2, rna3, and rna11 mutant strains . The yeast sequences on these plasmids have been shown by Southern transfer hybridization and genetic mapping to be derived from the RNA2, RNA3, and RNA11 genomic loci . Part of the RNA2 gene is homologous to more than one region of the yeast genome, whereas the RNA3 and RNA11 genes are single copy . RNAs homologous to these loci have been identified by RNA transfer hybridization, and the specific RNAs which are associated with the Rna+ phenotype have been mapped . This was done by a combination of transcript mapping, subcloning, and in vitro mutagenesis . The transcripts are found to be enriched in polyadenylated RNA and are of very low abundance (0.01-0.001% polyadenylated RNA).

Biochim Biophys Acta, 1984 Oct 24, 796(1), 102 - 9
Partial purification and properties of phosphatidate phosphatase in Saccharomyces cerevisiae; Hosaka K et al.; Using an aqueous dispersion of {32P}phosphatidate as substrate we detected phosphatidate phosphatase (EC 3.1.3.4) activity in a cell-free extract of the yeast, Saccharomyces cerevisiae . The activity was found in both the membrane and the soluble fractions . The enzyme was purified from the soluble fraction about 600-fold . The purification procedure involved (NH4)2SO4 fractionation, poly(ethylene glycol) 6000 fractionation and column chromatography on DEAE-Sepharose, Sephadex G-100 and Blue-Sepharose . The purified enzyme almost absolutely required Mg2+ for activity . The molecular weight of the enzyme was estimated by analytical gel filtration on Sephadex G-100 to be approx . 75000 . The enzyme was highly specific for phosphatidate . The apparent Km for phosphatidate was approx . 0.05 mM . The optimum pH was between 7.0 and 8.0.

J Biol Chem, 1984 Oct 10, 259(19), 12054 - 62
Biochemical and physiological aspects of glutamine synthetase inactivation in Saccharomyces cerevisiae; Mitchell AP et al.; Saccharomyces cerevisiae glutamine synthetase is inactivated in vivo by the addition of glutamine or ammonia . Inactivation is characterized by a specific loss of synthetase activity; transferase activity remains stable . Several physiological perturbations cause inactivation, such as carbon starvation or limitation for a required amino acid, which could cause a buildup of glutamine . The kinetics of reappearance of synthetase activity after inactivation suggest that the process is reversible in vivo . No change in the native size of the enzyme was associated with inactivation but there appears to be a change in the immunological properties of the enzyme subunit.

J Gen Microbiol, 1984 Oct, 130 ( Pt 10), 2629 - 37
Adenine phosphoribosyltransferase mutants in Saccharomyces cerevisiae; Woods RA et al.; Mutants of Saccharomyces cerevisiae deficient in adenine phosphoribosyltransferase (A-PRT, EC 2,4,2,7) have been isolated following selection for resistance to 8-azaadenine in a prototrophic strain carrying the ade4-su allele of the gene coding for amidophosphoribosyltransferase (EC 2,4,2,14) . The mutants were recessive and defined a single gene, apt1 . They did not excrete purine when combined with ade4+ . The mutants appeared to retain some A-PRT activity in crude extracts, and strains of the genotype ade2 apt1 responded to both adenine and hypoxanthine . Mutants deficient in adenine aminohydrolase (EC 3,5,4,2) activity, aah1, and hypoxanthine:guanine phosphoribosyltransferase (EC 2,4,2,8) activity, hpt1, were used to synthesize the genotypes apt1 hpt1 aah+ and apt1 hpt+ aah1 . The absence of A-PRT activity in strains with these genotypes confirmed the hypothesis that the residual A-PRT activity of apt1 mutants was due to adenine aminohydrolase and hypoxanthine:guanine phosphoribosyltransferase acting in concert.

Gene, 1984 Oct, 30(1-3), 121 - 8
Isolation and characterization of the RAD2 gene of Saccharomyces cerevisiae; Higgins DR et al.; We have cloned the RAD2 gene of Saccharomyces cerevisiae and used it to determine the size and direction of its transcript and to make rad2 deletion mutants . The RAD2 gene encodes a 3.3-kb transcript and the direction of transcription is leftwards, from EcoRI towards Bg/II . Deletions of the RAD2 gene have no effect on viability of vegetative cells or spores, or on sporulation.

Mol Cell Biol, 1984 Oct, 4(10), 2062 - 71
Expression of the Saccharomyces cerevisiae GAL7 gene on autonomous plasmids; Baker SM et al.; We used a combination of cloned DNA fragments encoding the GAL7 gene, yeast plasmid vectors, and chromosomal gal7 deletions to characterize the in vivo transcription of the GAL7 gene on autonomously replicating plasmids . Our results demonstrated that a plasmid-borne 3.1-kilobase DNA fragment containing the GAL7 gene provides sufficient information to mimic the regulated expression of the chromosomal location . Normal expression of GAL7 could occur in the absence of DNA encoding the functional genes of the GAL cluster region and was not altered when the gene was adjacent to other plasmid elements such as autonomously replicating sequences or centromeres . The chromosomal and single-copy centromeric plasmid locations of GAL7 were indistinguishable in their response to growth conditions (induction by galactose, repression by glucose) and positive and negative regulatory factors (GAL4 and GAL80) . Increasing the gene dosage to more than 200 copies per cell resulted in constitutive expression of the GAL7 mRNA; fully induced mRNA levels were increased more than 10-fold at these high gene dosages . When cells were shifted from noninducing to inducing conditions, the initial time of appearance and the rate of accumulation of GAL7 mRNA were altered in cell populations containing multiple GAL7 genes . The induction kinetics and final accumulation of the chromosomal GAL10 mRNA were also affected by the presence of multiple copies of the GAL7 gene; these results are consistent with a model involving limiting amounts of regulatory factors.

Mol Cell Biol, 1984 Oct, 4(10), 2052 - 61
Codon recognition during frameshift suppression in Saccharomyces cerevisiae; Gaber RF et al.; A genetic approach has been used to establish the molecular basis of 4-base codon recognition by frameshift suppressor tRNA containing an extra nucleotide in the anticodon . We have isolated all possible base substitution mutations at the position 4 (N) in the 3'-CCCN-5' anticodon of a Saccharomyces cerevisiae frameshift suppressor glycine tRNA encoded by the SUF16 gene . Base substitutions at +1 frameshift sites in the his4 gene have also been obtained such that all possible 4-base 5'-GGGN-3' codons have been identified . By testing for suppression in different strains that collectively represent all 16 possible combinations of position 4 nucleotides, we show that frameshift suppression does not require position 4 base pairing . Nonetheless, position 4 interactions influence the efficiency of suppression . Our results suggest a model in which 4-base translocation of mRNA on the ribosome is directed primarily by the number of nucleotides in the anticodon loop, whereas the resulting efficiency of suppression is dependent on the nature of position 4 nucleotides.

Mol Cell Biol, 1984 Oct, 4(10), 1985 - 98
Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae; Yocum RR et al.; We present the DNA sequence of a 914-base pair fragment from Saccharomyces cerevisiae that contains the GAL1-GAL10 divergent promoter, 140 base pairs of GAL10 coding sequence, and 87 base pairs of GAL1 coding sequence . From this fragment, we constructed four pairs of GAL1-lacZ and GAL10-lacZ fusions on various types of yeast plasmid vectors . On each type of vector, the fused genes were induced by galactose and repressed by glucose . The response of a GAL1-lacZ fusion to gal4 and gal80 regulatory mutations was similar to the response of intact chromosomal GAL1 and GAL10 genes . A set of deletions that removed various portions of the GAL10 regulatory sequences from a GAL10-CYC1-lacZ fusion was constructed in vitro . These deletions defined a relatively guanine-cytosine-rich region of 45 base pairs that contained sequences necessary for full-strength galactose induction and an adjacent guanine-cytosine rich 55 base pairs that contained sequences sufficient for weak induction.

J Cell Biol, 1984 Oct, 99(4 Pt 1), 1441 - 50
A heat shock-resistant mutant of Saccharomyces cerevisiae shows constitutive synthesis of two heat shock proteins and altered growth; Iida H et al.; A heat shock-resistant mutant of the budding yeast Saccharomyces cerevisiae was isolated at the mutation frequency of 10(-7) from a culture treated with ethyl methane sulfonate . Cells of the mutant are approximately 1,000-fold more resistant to lethal heat shock than those of the parental strain . Tetrad analysis indicates that phenotypes revealed by this mutant segregated together in the ratio 2+:2- from heterozygotes constructed with the wild-type strain of the opposite mating type, and are, therefore, attributed to a single nuclear mutation . The mutated gene in the mutant was herein designated hsr1 (heat shock response) . The hsr1 allele is recessive to the HSR1+ allele of the wild-type strain . Exponentially growing cells of hsr1 mutant were found to constitutively synthesize six proteins that are not synthesized or are synthesized at reduced rates in HSR1+ cells unless appropriately induced . These proteins include one hsp/G0-protein (hsp48A), one hsp (hsp48B), and two G0-proteins (p73, p56) . Heterozygous diploid (hsr1/HSR1+) cells do not synthesize the proteins constitutively induced in hsr1 cells, which suggests that the product of the HSR1 gene might negatively regulate the synthesis of these proteins . The hsr1 mutation also led to altered growth of the mutant cells . The mutation elongated the duration of G1 period in the cell cycle and affected both growth arrest by sulfur starvation and growth recovery from it . We discuss the problem of which protein(s) among those constitutively expressed in growing cells of the hsr1 mutant is responsible for heat shock resistance and alterations in the growth control.

J Bacteriol, 1984 Oct, 160(1), 80 - 6
Secretion can proceed uncoupled from net plasma membrane expansion in inositol-starved Saccharomyces cerevisiae; Atkinson KD et al.; Secretion of acid phosphatase and invertase was examined in an inositol-requiring ino1 mutant of the yeast Saccharomyces cerevisiae . Inositol starvation is known to block plasma membrane expansion, presumably due to restricted membrane phospholipid synthesis . If membrane expansion and extracellular protein secretion are accomplished by the same intracellular transport process, one would expect secretion to fail coordinately with cessation of plasma membrane growth in inositol-starved cells . In glucose-grown, inositol-starved cells, plasma membrane expansion and acid phosphatase secretion stopped coordinately, and intracellular acid phosphatase accumulated . In sucrose-grown, inositol-starved cells, plasma membrane growth halted, but secretion of both acid phosphatase and invertase continued until the onset of inositol-less death . Although glucose-grown and sucrose-grown cells differ in their ability to secrete when deprived of inositol, they exhibited the same disturbances in phospholipid synthesis . Phosphatidylinositol synthesis failed, and its precursors phosphatidic acid and CDP-diglyceride accumulated equally in both cultures . Sucrose-grown yeast cells appear to accomplish normal levels of extracellular protein secretion by an inositol-independent mechanism . In glucose-grown yeasts, both plasma membrane expansion and secretion are inositol dependent.

J Bacteriol, 1984 Oct, 160(1), 222 - 6
Saccharomyces cerevisiae aldolase mutants; Lobo Z; Six mutants lacking the glycolytic enzyme fructose 1,6-bisphosphate aldolase have been isolated in the yeast Saccharomyces cerevisiae by inositol starvation . The mutants grown on gluconeogenic substrates, such as glycerol or alcohol, and show growth inhibition by glucose and related sugars . The mutations are recessive, segregate as one gene in crosses, and fall in a single complementation group . All of the mutants synthesize an antigen cross-reacting to the antibody raised against yeast aldolase . The aldolase activity in various mutant alleles measured as fructose 1,6-bisphosphate cleavage is between 1 to 2% and as condensation of triose phosphates to fructose 1,6-bisphosphate is 2 to 5% that of the wild-type . The mutants accumulate fructose 1,6-bisphosphate from glucose during glycolysis and dihydroxyacetone phosphate during gluconeogenesis . This suggests that the aldolase activity is absent in vivo.

Mol Cell Biol, 1984 Oct, 4(10), 2161 - 9
Molecular cloning and nucleotide sequence analysis of the Saccharomyces cerevisiae RAD1 gene; Yang E et al.; We have screened a yeast genomic library for complementation of the UV sensitivity of mutants defective in the RAD1 gene and isolated a plasmid designated pNF1000 with an 8.9-kilobase insert . This multicopy plasmid quantitatively complemented the UV sensitivity of two rad1 mutants tested but did not affect the UV resistance of other rad mutants . The location of the UV resistance function in pNF1000 was determined by deletion analysis, and an internal fragment of the putative RAD1 gene was integrated into the genome of a RAD1 strain . Genetic analysis of several integrants showed that integration occurred at the chromosomal RAD1 site, demonstrating that the internal fragment was derived from the RAD1 gene . A 3.88-kilobase region of pNF1000 was sequenced and showed the presence of a small open reading frame 243 nucleotides long that is apparently unrelated to RAD1, as well as a 2,916-nucleotide larger open reading frame presumed to encode RAD1 protein . Depending on which of two possible ATG codons initiates translation, the size of the RAD1 protein is calculated at 110 or 97 kilodaltons.

Mol Cell Biol, 1984 Oct, 4(10), 2023 - 30
A positive regulatory site and a negative regulatory site control the expression of the Saccharomyces cerevisiae CYC7 gene; Wright CF et al.; A series of BAL31 deletions were constructed in vitro in the upstream region of the Saccharomyces cerevisiae CYC7 gene, encoding the iso-2-cytochrome c protein . These deletions identified two sites which play a role in governing the expression of this gene . A positive site, the deletion of which led to decreased CYC7 expression, lay ca . 240 base pairs 5' to the translational initiation codon (-240) . A negative site, the deletion of which led to greatly increased levels of CYC7 expression, lay at ca . -300 bp . Deletion of both these sites resulted in low wild-type-like expression of the gene . Therefore, these two sites appear to act antagonistically to give the low wild-type levels of CYC7 expression . Within the region defined as containing the positive site, there is a sequence which bears some homology to the upstream activation sites in the regulated gene, CYC1, encoding the iso-1-cytochrome c protein.

J Biol Chem, 1984 Sep 25, 259(18), 11509 - 11
An arginine/histidine exchange transport system in vacuolar-membrane vesicles of Saccharomyces cerevisiae; Sato T et al.; High-affinity arginine-uptake activity in vacuolar-membrane vesicles from the yeast Saccharomyces cerevisiae was detected in the presence of ATP and histidine . Histidine increased the initial rate of uptake and the steady level of accumulation of arginine . When the vesicles were preloaded with histidine in the absence of ATP and diluted, transient uptake of arginine took place with concomitant downhill efflux of histidine from the vesicles . Kinetic examination indicated the existence of a unique transport system which catalyzed exchange of 1 mol of arginine outside with 1 mol of histidine inside the vacuolar membrane . The Ktentry for arginine and the Ktexit for histidine were determined to be 0.1 and 1.1 mM, respectively.

J Biol Chem, 1984 Sep 25, 259(18), 11505 - 8
Substrate specificities of active transport systems for amino acids in vacuolar-membrane vesicles of Saccharomyces cerevisiae . Evidence of seven independent proton/amino acid antiport systems; Sato T et al.; The substrate specificities of the amino acid transport systems of vacuoles of the yeast, Saccharomyces cerevisiae, were investigated using purified vacuolar-membrane vesicles (Ohsumi, Y., and Anraku, Y . (1981) J . Biol . Chem . 256, 2079-2082) . Ten amino acids: arginine, lysine, histidine, phenylalanine, tryptophan, tyrosine, glutamine, asparagine, isoleucine, and leucine, were taken up actively into the vesicles . Kinetic studies indicated the presence of seven independent H+/amino acid antiport systems with narrow substrate specificity, which were all driven by a proton motive force established by ATP hydrolysis . The Kt and Vmax values, and the specific inhibitors for the arginine, arginine-lysine, histidine, phenylalanine-tryptophan, tyrosine, glutamine-asparagine, and isoleucine-leucine transport systems were determined.

Biochim Biophys Acta, 1984 Sep 14, 805(1), 59 - 71
Subcellular fractionation of actively growing protoplasts of Saccharomyces cerevisiae; Martinez JP et al.; Cell homogenates obtained from partially regenerated Saccharomyces cerevisiae protoplasts were fractionated by a procedure using a combination of continuous and discontinuous sucrose gradients, under experimental conditions that minimize possible artifacts due to centrifugation and resuspension . At least five different membranous organelle fractions (plasma membrane, mitochondria, rough endoplasmic reticulum, smooth endoplasmic reticulum-like structures and small-sized particulated structures) were isolated . Subcellular fractions were characterized by assaying established marker enzymes . Radioactive labelled {(U-3H}uracil) ribosomes were also used as a further characterization criterion of the rough endoplasmic reticulum . Comparative SDS-polyacrylamide gel electrophoresis of the protein constituents of the isolated membrane-bound organelles suggest that the polypeptide pattern could also be used as an additional marker for some of these structures . Finally, subcellular distribution of chitin synthase was determined using this fractionation procedure, and two partially zymogenic enzyme pools (one inside the cell associated to particles which sediments at high speed, and the second one associated to the plasma membrane) were found.

J Biol Chem, 1984 Sep 10, 259(17), 10857 - 62
Phosphatidylserine synthesis in Saccharomyces cerevisiae . Purification and characterization of membrane-associated phosphatidylserine synthase; Bae-Lee MS et al.; Membrane-associated phosphatidylserine synthase (CDP-diacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) was purified from the microsomal fraction of Saccharomyces cerevisiae strains S288C and VAL2C(YEpCHO1) . VAL2C(YEpCHO1) contains a hybrid plasmid bearing the structural gene for phosphatidylserine synthase and overproduces the enzyme 6-7 fold (Letts, V . A., Klig, L . S., Bae-Lee, M., Carman, G . M., and Henry, S . A . (1983) Proc . Natl . Acad . Sci . U . S . A . 80, 7279-7283) compared to wild-type S288C . The purification procedure included Triton X-100 extraction of the microsomal membranes, CDP-diacylglycerol-Sepharose affinity chromatography, and DE-53 chromatography . The procedure yielded a preparation from each strain containing a major peptide band (Mr = 23,000) upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Phosphatidylserine synthase was dependent on manganese and Triton X-100 for maximum activity at pH 8.0 . The apparent Km values for serine and CDP-diacylglycerol were 0.58 mM and 60 microM, respectively . Thioreactive agents inhibited enzyme activity . The enzyme was thermally labile above 40 degrees C . Results of isotopic exchange reactions between substrates and products suggest that the enzyme catalyzes a sequential Bi Bi reaction.

Eur J Biochem, 1984 Sep 3, 143(2), 251 - 6
Molecular cloning of the gene encoding CDPdiacylglycerol-inositol 3-phosphatidyl transferase in Saccharomyces cerevisiae; Nikawa J et al.; The structure gene encoding CDPdiacylglycerol-inositol 3-phosphatidyltransferase was isolated by complementation in a Saccharomyces cerevisiae mutant after transformation with a comprehensive gene library containing Sau3AI partial restriction fragments of wild-type yeast DNA inserted into the YEp13 shuttle vector . Introduction of the cloned plasmid into the mutant restored both growth of cells and CDPdiacylglycerol-inositol 3-phosphatidyltransferase activity . A subcloning study indicated that the complementing gene was contained within a 1700-base-pair fragment of DNA . Expression of the cloned sequence was independent of its orientation in the vector . CDPdiacylglycerol-inositol 3-phosphatidyltransferase activity in the transformant was eight fold higher than that in the wild-type strain . Although the mutant enzyme had a greatly increased Km for myo-inositol, the enzyme in the transformant had an apparent Km for myo-inositol equal to that of the wild-type enzyme, indicating the presence of the wild-type structure gene on the recombinant plasmid . The elevated level of enzyme activity in the transformant did not lead to an increase in the content of phosphatidylinositol . In contrast, supplementation of the culture medium with increasing concentrations of myo-inositol resulted in an increase in the phosphatidylinositol content . Thus, the availability of myo-inositol is a critical regulatory factor in yeast phosphatidylinositol synthesis.

Mol Cell Biol, 1984 Sep, 4(9), 1853 - 63
High-resolution mapping of DNase I-hypersensitive sites of Drosophila heat shock genes in Drosophila melanogaster and Saccharomyces cerevisiae; Costlow N et al.; High-resolution analysis of the chromatin structure of the promoter regions of five Drosophila heat shock genes showed a similar location for the hypersensitive sequences relative to the start of transcription . For each of the five genes examined--those coding for hsp27, hsp26, hsp23, hsp70, and hsp83--the DNase I-hypersensitive sites in Drosophila melanogaster nuclei mapped to two regions upstream of the coding region . These sites occurred on the average, 115 and 17 base pairs upstream from the start of transcription of the five heat shock genes examined . This latter site corresponded to sequences at or near the TATA consensus sequence . Sites even further upstream of the hsp27, hsp26, and hsp83 genes were also evident . Additionally, for the two genes examined--hsp70 and hsp83--the DNase I-hypersensitive sites were preserved, at least within this level of resolution (+/- 10 base pairs), when the Drosophila genes were integrated into the Saccharomyces cerevisiae genome . This result indicates that the signals responsible for generating these hypersensitive sites are inherent in the DNA sequences and, in this case, are not highly species specific.

Mol Cell Biol, 1984 Sep, 4(9), 1747 - 53
Double-stranded RNAs that encode killer toxins in Saccharomyces cerevisiae: unstable size of M double-stranded RNA and inhibition of M2 replication by M1; Sommer SS et al.; The sizes of M1 and M2 (but not L) change rapidly with growth, varying by perhaps as much as 33% . Size variation is seen within 76 generations . In addition, the exclusion of M2 by M1 or L-A-E {( EXL}) is mediated by inhibition of replication or segregation, not by enhanced degradation of preexisting molecules.

Mol Cell Biol, 1984 Sep, 4(9), 1682 - 8
Cytoplasmic and secreted Saccharomyces cerevisiae invertase mRNAs encoded by one gene can be differentially or coordinately regulated; Perlman D et al.; A single structural gene, SUC2, encodes both secreted and cytoplasmic invertase in Saccharomyces cerevisiae . It is known that the unprocessed polypeptides which differ by a secretion signal sequence are encoded by separate mRNAs . This unusual transcriptional organization raises the question as to the degree to which the transcripts can be independently regulated . To define a system for studying this problem, we examined invertase transcription after various physiological perturbations of cells: rapid catabolite derepression, heat shock, and cell cycle arrest . With each treatment, fluctuations in mRNA levels for both cytoplasmic and secreted invertase were observed . We concluded that (i) catabolite-derepressed synthesis of the mRNAs occurs rapidly after a drop in glucose, is a sustained response, and does not require de novo protein synthesis; (ii) heat shock transcription of both invertase mRNAs is, in contrast, a brief and transient response requiring de novo protein synthesis; and (iii) alpha-mating hormone treatment (G1 phase arrest and release) results in regular and coordinated synthesis of both mRNAs midway between rounds of histone mRNA synthesis . We propose that invertase mRNA regulation involves constitutively synthesized transcriptional factors (observed during catabolite derepression) and transient factors (observed during heat shock and possibly during synchronous growth) . Moreover, the mRNA levels for secreted and cytoplasmic invertase can be independently regulated.

J Bacteriol, 1984 Sep, 159(3), 1013 - 7
Expression of kinase-dependent glucose uptake in Saccharomyces cerevisiae; Bisson LF et al.; There are both low- and high-affinity mechanisms for uptake of glucose in Saccharomyces cerevisiae; high-affinity uptake somehow depends on the presence of hexose kinases (L . F . Bisson and D . G . Fraenkel, Proc . Natl . Acad . Sci . U.S.A . 80:1730-1734, 1983; L . F . Bisson and D . G . Fraenkel, J . Bacteriol . 155:995-1000, 1983) . We report here on the effect of culture conditions on the level of high-affinity uptake . The high-affinity component was low during growth in high concentrations of glucose (100 mM), increased as glucose was exhausted from the medium, and decreased again during prolonged incubation in the stationary phase . The higher level of uptake was found in growth on low concentrations of glucose (0.5 mM) and in growth on normal concentrations of galactose, lactate plus glycerol, or ethanol . These results suggest that some component of high-affinity uptake is repressible by glucose . A shift from medium with 100 mM glucose to medium with 5 mM glucose resulted in up to a 10-fold increase in the level of high-affinity uptake within 90 min; the increase did not occur in the presence of cycloheximide or 2,4-dinitrophenol or in buffer alone with low glucose, suggesting that protein synthesis or energy metabolism (or both) was required . Reimposition of the high glucose concentration caused loss of high-affinity uptake, a process not prevented by cycloheximide . The use of hexokinase single-gene mutants showed that the derepression of high-affinity uptake was not clearly correlated with changes in levels of the kinases themselves . These results place the phenomenon of high- and low-affinity uptake in a physiological context, in that high-affinity uptake seems to be expressed best in conditions where it might be needed . Apparent similarities between glucose uptake in yeast and animal cells are noted.

Biochem J, 1984 Sep 1, 222(2), 293 - 8
Transport of maltose in Saccharomyces cerevisiae . Effect of pH and potassium ions; Loureiro-Dias MC et al.; The transport of maltose in Saccharomyces cerevisiae has been generally accepted as a H+-sugar symport, with a stoichiometrical ratio of 1:1 . A simultaneous exit of K+ from the cells with the initial uptake of maltose has been reported previously . By using a K+-selective electrode and radioactive maltose, we were able to measure the exit of 1 mol of K+/mol of maltose taken up by the cells in the first 10-15 s . This stoichiometrical ratio is pH-independent . So, uptake of protons in a non-buffered cell suspension or exit of K+ in a buffered one can be used to measure initial rates of maltose uptake . We have used a K+ electrode and a pH electrode to study the effect of external pH and K+ respectively on the kinetic parameters of maltose transport . The following results were obtained: the apparent half-saturation constant for maltose (Km) increased from 5.2 mM at pH 5.8 to 38.0 mM at pH 7.8; the same increase in pH halved the apparent maximum uptake rate (Vmax); K+ had an inhibitory effect, decreasing Vmax . and increasing Km at pH values above 5; K+ had a stimulating effect at pH values below or equal to 4 . Under physiological conditions, i.e . lower pH outside, neutral pH inside and much higher {K+} inside the cell, and assuming symmetry of the system, a higher affinity for maltose is to be expected in the outer face of the plasma membrane . This behaviour of the system could explain, by itself, the maintenance of the high concentration of free maltose inside the cell (necessary because of the low affinity of the maltase), without significant back transport to the outside.

Radiat Res, 1984 Sep, 99(3), 582 - 90
The effect of cycloheximide on repair in a temperature conditional radiation-sensitive mutant of Saccharomyces cerevisiae; Budd M et al.; Previous results {M . Budd and R . K . Mortimer, Mutat . Res . 103, 19-24 (1982)} have shown that rad54-3 strains are temperature conditional for double-strand break repair . At the temperature where survival is high, 23 degrees C, rad54-3 strains are able to repair X-ray-induced double-strand breaks, while at the temperature where survival is low, 36 degrees C, these strains are unable to repair rad54-3 strains provide a system to study the effects of drugs that block protein synthesis such as cycloheximide on repair of X-ray damage . Repair of X-ray damage is studied by irradiating rad54-3 cells, incubating them at the permissive temperature, 23 degrees C, for 5 hr, shifting the cells to the restrictive temperature, 36 degrees C, and assaying for colony-forming ability . Comparing the survival of these cells with those which had been continuously incubated at the restrictive temperature after irradiation shows the extent of repair . Addition of cycloheximide at the time of irradiation causes an inhibition of repair . If cycloheximide is added a short time after irradiation, an enhanced recovery is observed compared with the addition of the drug at the time of irradiation . One explanation for the enhanced recovery is an increased synthesis of repair enzymes after irradiation.

J Bacteriol, 1984 Sep, 159(3), 1018 - 26
Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity; Zlotnik H et al.; A beta-glucanase (Z-glucanase) from Zymolyase was freed from a protease (Z-protease) by affinity chromatography on alpha 2-macroglobulin-Sepharose columns and used to solubilize proteins from isolated cell walls of Saccharomyces cerevisiae . The cell wall proteins were labeled with 125I and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography . The bulk of the labeled material had very low mobility . Its mannoprotein nature was demonstrated by precipitation with specific antibodies and by conversion to a band with an average molecular weight of 94,000 after incubation with endo-beta-N-acetylglucosaminidase . The intact mannoproteins were hydrolyzed by Z-protease, but were resistant to the enzyme when the carbohydrate was first removed by endo-beta-N-acetylglucosaminidase . In intact cells, lysis of cell walls by Z-glucanase required a previous incubation with z-protease, which led to solubilization of most of the 125I-labeled proteins . Other proteases that did not attack the cell wall mannoproteins were unable to substitute for Z-protease . The specific effect of Z-protease is consistent with the notion that mannoproteins form a surface layer of the cell wall that penetrates the wall to some depth and shields glucans from attack by Z-glucanase . Mannoproteins, however, do not appear to cover the inner face of the cell wall, because isolated cell walls, in contrast to intact cells, were completely solubilized by Z-glucanase in the absence of protease . The function of mannoproteins in determining cell wall porosity was highlighted by the finding that horseradish peroxidase (Mr, 40,000) causes lysis of cells that had been treated with Z-protease . Depletion of mannoproteins by Z-protease also resulted in the disappearance of a darkly stained surface layer of the cell wall, as observed by electron microscopy . Other agents that facilitate cell lysis by Z-glucanase, such as 2-mercaptoethanol, digitonin, and high concentrations of salts, caused little or no solubilization of mannoprotein . We assume that they perturb and loosen the structure of the mannoprotein network, thereby increasing its porosity . The implications of our results for the construction of the yeast cell wall and the anchoring of mannoprotein to the cell are discussed.

Mol Cell Biol, 1984 Sep, 4(9), 1871 - 9
Two genes for ribosomal protein 51 of Saccharomyces cerevisiae complement and contribute to the ribosomes; Abovich N et al.; We cloned and sequenced the second gene coding for yeast ribosomal protein 51 (RP51B) . When the DNA sequence of this gene was compared with the DNA sequence of RP51A (J.L . Teem and M . Rosbash, Proc . Natl . Acad . Sci . U.S.A . 80:4403--4407, 1983), the following conclusions emerged: both genes code for a protein of 135 amino acids; both open reading frames are interrupted by a single intron which occurs directly after the initiating methionine; the open reading frames are 96% homologous and code for the same protein with the exception of the carboxy-terminal amino acid; DNA sequence homology outside of the coding region is extremely limited . The cloned genes, in combination with the one-step gene disruption techniques of Rothstein (R . J . Rothstein, Methods Enzymol . 101:202-211, 1983), were used to generate haploid strains containing mutations in the RP51A or RP51B genes or in both . Strains missing a normal RP51A gene grew poorly (180-min generation time versus 130 min for the wild type), whereas strains carrying a mutant RP51B were relatively normal . Strains carrying mutations in the two genes grew extremely poorly (6 to 9 h), which led us to conclude that RP51A and RP51B were both expressed . The results of Northern blot and primer extension experiments indicate that strains with a wild-type copy of the RP51B gene and a mutant (or deleted) RP51A gene grow slowly because of an insufficient amount of RP51 mRNA . The growth defect was completely rescued with additional copies of RP51B . The data suggest that RP51A contributes more RP51 mRNA (and more RP51 protein) than does RP51B and that intergenic dosage compensation, sufficient to rescue the growth defect of strains missing a wild-type RP51A gene, does not take place.

Mol Cell Biol, 1984 Sep, 4(9), 1864 - 70
Cloning and mapping of Saccharomyces cerevisiae photoreactivation gene PHR1; Schild D et al.; The yeast Saccharomyces cerevisiae, like most organisms, is able to directly repair pyrimidine dimers by using a photoreactivating enzyme and visible light . Cells carrying the phr1 mutation were shown previously to be unable to photoreactivate dimers, but neither the map position nor the primary gene product of the PHR1 gene has been determined . We have cloned this gene and determined its map position . A plasmid containing a 6.4-kilobase yeast DNA insert has been isolated and shown to restore photoreactivation in a phr1 strain . A 3.1-kilobase subclone has also been shown to complement phr1 . The original plasmid was targeted to integrate into chromosomal DNA at a site homologous to the insert by cutting within the insert . Two of these integrants have been mapped on the right arm of chromosome XV; the integrants have been further mapped at ca . 13 centimorgans from prt1 . It has also been independently determined that phr1 maps at this location . Thus, we have determined the map position of PHR1 and also have shown that the plasmid contains PHR1 rather than a suppressor of the phr1 mutation.

Mol Cell Biol, 1984 Sep, 4(9), 1725 - 9
Micronuclear DNA of Oxytricha nova contains sequences with autonomously replicating activity in Saccharomyces cerevisiae; Colombo MM et al.; Oxytricha nova is a hypotrichous ciliate with micronuclei and macronuclei . Micronuclei, which contain large, chromosomal-sized DNA, are genetically inert but undergo meiosis and exchange during cell mating . Macronuclei, which contain only small, gene-sized DNA molecules, provide all of the nuclear RNA needed to run the cell . After cell mating the macronucleus is derived from a micronucleus, a derivation that includes excision of the genes from chromosomes and elimination of the remaining DNA . The eliminated DNA includes all of the repetitious sequences and approximately 95% of the unique sequences . We cloned large restriction fragments from the micronucleus that confer replication ability on a replication-deficient plasmid in Saccharomyces cerevisiae . Sequences that confer replication ability are called autonomously replicating sequences . The frequency and effectiveness of autonomously replicating sequences in micronuclear DNA are similar to those reported for DNAs of other organisms introduced into yeast cells . Of the 12 micronuclear fragments with autonomously replicating sequence activity, 9 also showed homology to macronuclear DNA, indicating that they contain a macronuclear gene sequence . We conclude from this that autonomously replicating sequence activity is nonrandomly distributed throughout micronuclear DNA and is preferentially associated with those regions of micronuclear DNA that contain genes.

J Bacteriol, 1984 Sep, 159(3), 837 - 42
Role of inositol-containing sphingolipids in Saccharomyces cerevisiae during inositol starvation; Hanson BA; The in vitro lipid requirements of UDP-N-acetylglucosamine-dolichol phosphate N-acetylglucosamine-1-phosphotransferase for the inositol-containing sphingolipids from Saccharomyces cerevisiae were characterized in terms of concentration and specificity . The effects of combinations of lipids, especially phosphatidylinositol and the inositol-containing sphingolipids, were also tested on the transferase . Phosphatidylinositol and phosphatidylglycerol stimulated the enzyme 3.3- and 2.8-fold, respectively . The inositol-containing sphingolipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine did not stimulate the activity of the transferase . Phosphatidylcholine and phosphatidylethanolamine in combination with phosphatidylinositol had no effect on the transferase activity; however, the inositol-containing sphingolipids markedly inhibited the stimulation of the transferase by phosphatidylinositol . This inhibition by the sphingolipids was prevented if phosphatidylcholine, in addition to the other lipids, was present in the assay mixture . In addition, changes due to inositol starvation in the in vivo membrane lipid environment, i.e., phosphatidylinositol and the inositol-containing sphingolipids, were analyzed to determine whether they corresponded to the observed in vitro effects . Three hours after the beginning of inositol starvation, there were 9- and 14-fold reductions in the accumulation of phosphatidylinositol in membrane fractions IIA (vesicles) and IV (endoplasmic reticulum), respectively, although there was only a 6-fold reduction in membrane fraction I (plasma membrane) . The accumulation of {14C}inositol into inositol-containing sphingolipids also reflected the differences in the cellular location of membranes.

J Bacteriol, 1984 Sep, 159(3), 1065 - 7
Deletion of plasmid sequences during Saccharomyces cerevisiae transformation; Clancy S et al.; Saccharomyces cerevisiae was transformed with DNA by the lithium acetate method . Mutation of nonselected markers on the transforming vector was observed at a frequency several orders of magnitude higher than spontaneous mutation frequencies . These mutations were shown to be deletions . Linearization of the vector before transformation stimulated deletion formation.

Genetics, 1984 Sep, 108(1), 53 - 66
Regulation of repressible acid phosphatase by cyclic AMP in Saccharomyces cerevisiae; Matsumoto K et al.; One of the cyr 1 mutants (cyr 1-2) in yeast produced low levels of adenylate cyclase and cyclic AMP at 25 degrees and was unable to derepress acid phosphatase . Addition of cyclic AMP to the cyr1-2 cultures elevated the level of repressible acid phosphatase activity . The bcy1 mutation, which suppresses the cyr1-2 mutation by allowing activity of a cyclic AMP-independent protein kinase, also allows acid phosphatase synthesis without restoring adenylate cyclase activity . The CYR3 mutant had structurally altered cyclic AMP-dependent protein kinase and was unable to derepress acid phosphatase . The cyr1 locus was different from pho2, pho4 and pho81, which were known to regulate acid phosphatase synthesis . Mutants carrying cyr1-2 and pho80, PHO81c, PHO82 or pho85 mutations, which confer constitutive synthesis of repressible acid phosphatase, produced acid phosphatase . The cyr1-2 mutant produced significantly low levels of invertase and alpha-D-glucosidase . These results indicated that cyclic AMP-dependent protein kinase exerts its function in the synthesis of repressible acid phosphatase and other enzymes.

Proc Natl Acad Sci U S A, 1984 Sep, 81(17), 5330 - 4
Secretion of foreign proteins from Saccharomyces cerevisiae directed by alpha-factor gene fusions; Bitter GA et al.; Fusions between the cloned yeast alpha-factor structural gene and chemically synthesized DNA segments encoding human protein analogs have been constructed . The gene fusions encode hybrid proteins that include the first 89 amino acids of the native alpha-factor precursor fused to either a small (beta-endorphin, 31 amino acids) or large (alpha-interferon, 166 amino acids) foreign protein . Proteolytic cleavage sites involved in alpha-factor maturation from the native precursor immediately precede the foreign peptide in the hybrid protein . The alpha-factor promoter was utilized to express the gene fusions in Saccharomyces cerevisiae and resulted in the efficient secretion of the foreign proteins into the culture medium . The processing of the hybrid proteins has been characterized by amino acid sequence analysis of the secreted proteins . The proteolytic cleavages involved in the maturation of alpha-factor peptides from the native precursor also occur accurately in the hybrid protein . In addition, cleavages occurred on the carboxyl side of two lysines within the beta-endorphin peptide . Internal cleavages in the interferon protein were also detected . However, in this case, the cleavages occurred at a very low frequency such that greater than 95% of the secreted interferon remained intact.

J Biol Chem, 1984 Aug 25, 259(16), 10422 - 9
The purification and characterization of DNA topoisomerases I and II of the yeast Saccharomyces cerevisiae; Goto T et al.; The ATP-independent type I and the ATP-dependent type II DNA topoisomerase of the yeast Saccharomyces cerevisiae have been purified to near homogeneity, and the purification procedures are reported . Both purified topoisomerases are single subunit enzymes with monomer weights of Mr = 90,000 and 150,000 for the type I and type II enzyme, respectively . Sedimentation and gel filtration data suggest that the type I enzyme is monomeric and the type II enzyme is dimeric . Similar to other purified eukaryotic topoisomerases, the yeast type I enzyme does not require a divalent cation for activity, but is stimulated 10-20-fold in the presence of 7-10 mM Mg(II) or Ca(II) . Mn(II) is about 25% as efficient as Mg(II) in this stimulation but Co(II) is inhibitory . The yeast type II topoisomerase has an absolute requirement for a divalent cation: Mg(II) is the most effective, whereas Mn(II), Ca(II), or Co(II) supports the reaction to a lesser extent . The type II enzyme also requires ATP or dATP; the nonhydrolyzable ATP analogues adenylyl imidodiphosphate and adenylyl (beta,gamma-methylene)diphosphonate are potent inhibitors . Both yeast topoisomerases are completely inhibited by N-ethylmaleimide at 0.5 mM . In addition, the type II enzyme, but not the type I enzyme, is inhibited to various extents by coumermycin, ethidium, and berenil . Both topoisomerases are nuclear enzymes; no topoisomerase specific to mitochondria has been detected.

Nucleic Acids Res, 1984 Aug 24, 12(16), 6397 - 414
Analysis of full-length cDNA clones carrying GAL1 of Saccharomyces cerevisiae: a model system for cDNA expression; Miyajima A et al.; A cDNA cloning vector that allows expression in Saccharomyces cerevisiae has been developed using the plasmid primer approach described by Okayama and Berg {Mol . Cell . Biol . 2:161-170(1982)} . The vector contains ARS1 and TRP1 for plasmid maintenance in yeast and the ADC1 or GAL1 promoter and the TRP5 terminator for expression of the cloned cDNA . Using this system, several recombinants with nearly full-length GAL1 cDNA inserts in a cDNA library made with galactose-induced yeast mRNA were identified . By measurement of galactokinase mRNA and its protein, the expression of GAL1 cDNA was shown to be under the control of the promoter placed upstream of the cDNA insert . Nucleotide sequence analysis revealed that the 3'-ends of the GAL1 cDNA inserts were not unique, indicating that polyA tails were added to GAL1 transcripts at multiple sites in the GAL1 gene . Genetic complementation of appropriate yeast mutants permitted the isolation of clones containing the coding sequences for GAL1, HIS3, and LEU2 from the same cDNA library.

FEBS Lett, 1984 Aug 20, 174(1), 47 - 9
An edeine resistant mRNA-dependent protein synthesis system from a Saccharomyces cerevisiae mutant; Herrera F et al.; A cell-free protein synthesizing system from a mutant of Saccharomyces cerevisiae translated exogenous mRNA in the presence of 2 microM edeine, while a similar system from wild-type strain was completely inhibited by the drug . The mutant ribosomes showed an affinity for {125I}edeine comparable to the wild-type ribosomes, thereby suggesting that these macromolecules alone were not responsible for the edeine-resistant capacity of the mutant.

Biochim Biophys Acta, 1984 Aug 15, 795(1), 117 - 24
Secretion of phospholipase B from Saccharomyces cerevisiae; Witt W et al.; Phospholipase B and lysophospholipase activity is secreted from yeast cells (Saccharomyces cerevisiae) growing aerobically in batch cultures during the exponential phase . A glycoprotein with both activities running on SDS-polyacrylamide slab gels as a broad band between 200 000 and 280 000 Da was purified about 2500-fold by gel filtration, chromatofocusing and hydrophobic interaction chromatography with octyl-Sepharose . The secreted phospholipase has a slightly higher carbohydrate content of 41 mumol/mg protein compared to a form of the enzyme associated to the plasma membrane described in the previous communication (Witt, W., Schweingruber, M.E . and Mertsching, A . (1984) Biochim . Biophys . Acta 795, 108-116) and exerts very similar enzymatic properties . Fatty acids are set free from lysophosphatidylcholine with a 68-fold higher rate than from phosphatidylcholine with a concomitant generation of the corresponding diacyl compound . pH optima of 3.0 and 3.5 were determined with phosphatidylcholine and lysophosphatidylcholine, respectively . During the enzymatic degradation of the cell wall, high amounts of phospholipase activity were released, indicating that the enzyme is present in the periplasmatic space or associated to cell wall components.

Biochim Biophys Acta, 1984 Aug 15, 795(1), 108 - 16
Phospholipase B from the plasma membrane of Saccharomyces cerevisiae . Separation of two forms with different carbohydrate content; Witt W et al.; Two forms of phospholipase B could be solubilized from the plasma membrane of Saccharomyces cerevisiae, separated by gel filtration with Sephacryl S-300 and identified by SDS-polyacrylamide gel electrophoresis as glycoproteins of the apparent molecular weights of about 220 000 (phospholipase B1) and 145 000 (phospholipase B2) . The enzymes are very similar in respect to their catalytic properties . Both forms converted lysophosphatidylcholine to diacylphosphatidylcholine and unesterified fatty acids . The carbohydrate content of the glycoproteins could be reduced by treatment with endoglycosidase H and HF . By incubation of phospholipase B1 and phospholipase B2 with endoglycosidase H from Streptomyces griseus, one main protein with an apparent Mr of 67 000 and the same residual carbohydrate content was obtained . Treatment with HF reduced phospholipase B1 and phospholipase B2 to proteins with an apparent Mr of 52 000 and 67 000, respectively . These results could indicate that the two forms are similar in respect to their protein moieties . An antiserum raised in mice against phospholipase B2 showed no crossreactivity with phospholipase B1 as detected by immunoblot analysis . The reactivity of phospholipase B2 was diminished or abolished by progressive removal of carbohydrate . These results were taken as indications for differences in the carbohydrate component of the two enzyme forms.

Mol Cell Biol, 1984 Aug, 4(8), 1454 - 9
Differential regulation of the 70K heat shock gene and related genes in Saccharomyces cerevisiae; Ellwood MS et al.; Saccharomyces cerevisiae contains a family of genes related to Hsp70, the major heat shock gene of Drosophila melanogaster . The transcription of three of these genes, which show no conservation of sequences 5' to the protein-coding region, was analyzed . The 5' flanking regions from the three genes were fused to the Escherichia coli beta-galactosidase structural gene and introduced into yeasts on multicopy plasmids, putting the beta-galactosidase production under yeast promoter control . Analysis of beta-galactosidase mRNA and protein production in these transformed strains revealed that transcription from the three promoters is differentially regulated . The number of transcripts from one promoter is vastly increased for a brief period after heat shock, whereas mRNA from another declines . Transcripts from a third gene are slightly enhanced upon heat shock; however, multiple 5' ends of the mRNA are found, and a minor species increases in amount after heat shock . Transcription of these promoters in their native state on the chromosome appears to be modulated in the same manner.

Mol Cell Biol, 1984 Aug, 4(8), 1618 - 26
Conservative replication of double-stranded RNA in Saccharomyces cerevisiae by displacement of progeny single strands; Sclafani RA et al.; Saccharomyces cerevisiae contains two double-stranded RNA (dsRNA) molecules, L and M, encapsulated in virus-like particles . After cells are transferred from dense (13C 15N) to light (12C 14N) medium, only two density classes of dsRNA are found, fully light (LL) and fully dense (HH) . Cells contain single-stranded copies of both dsRNAs and, at least for L dsRNA, greater than 99% of these single strands are the positive protein-encoding strand . Single-stranded copies of L and M dsRNA accumulate rapidly in cells arrested in the G1 phase . These results parallel previous observations on L dsRNA synthesis and are consistent with a role of the positive single strands as intermediates in dsRNA replication . We propose that new positive strands are displaced from parental molecules and subsequently copied to produce the completely new duplexes.

Arch Microbiol, 1984 Aug, 138(4), 310 - 4
Alpha-mating-type-specific suppression and a-mating-type-specific enhancement by tunicamycin of sexual agglutinability in the yeast Saccharomyces cerevisiae; Hasegawa S et al.; Effects of tunicamycin (TM) on the sexual agglutinability and zygote formation of Saccharomyces cerevisiae were studied using the two kinds of haploid strains, inducible and constitutive for sexual agglutinability . Induction of sexual agglutinability by opposite mating type sex pheromone of inducible strains was inhibited by TM in alpha mating type but not in a mating type . The recovery by temperature-shift-down from the temperature-suppressed sexual agglutinability of constitutive strains was enhanced by TM in a mating type but rather inhibited in alpha mating type . Pretreatment with TM of constitutive strains enhanced sexual agglutinability in a mating type but not in alpha mating type . The above-mentioned a-mating-type-specific agglutinability-enhancing actions of TM were discussed in relation to the action mechanism of alpha pheromone which induces or enhances the sexual agglutinability of a cells . Zygote formation was inhibited by TM in both constitutive and inducible strains at concentrations which showed only partially inhibitory effect on sexual agglutinability.

Eur J Biochem, 1984 Aug 1, 142(3), 551 - 7
Isolation and properties of 5-aminolevulinate synthase from the yeast Saccharomyces cerevisiae; Volland C et al.; 5-Aminolevulinate synthase from yeast mitochondria has been purified to homogeneity for the first time . By using affinity chromatography on agarose-hexane-CoA, gel filtration and DEAE-Sepharose chromatography, the enzyme was purified about 7000-fold with an overall yield of 40% . The specific activity of the final preparation was 39000 nmol of 5-aminolevulinate h-1 mg-1 of protein at 30 degrees C . As judged by gel filtration, polyacrylamide gradient gel and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the enzyme appeared to be composed of two identical subunits of a relative molecular mass of 53000 . Electrophoresis of sodium-dodecyl-sulfate-solubilized yeast homogenate followed by immune replica analysis showed that the value of 53000 is the Mr of a non-degraded form . The purified enzyme had an isoelectric point of 5.3 and a pH optimum of 7.4 . Pyridoxal 5'-phosphate has been shown to be an essential cofactor . The enzyme activity was sensitive to thiol blocking reagents . Hemin, but not heme, inhibited the activity of the purified enzyme.

J Bacteriol, 1984 Aug, 159(2), 797 - 9
Flocculation of Saccharomyces cerevisiae tup1 mutants; Lipke PN et al.; Strains of Saccharomyces cerevisiae carrying a mutation in the TUP1 locus exhibited calcium-dependent flocculation . The flocculation had none of the characteristics of sexual agglutination . The flocculation differed from that exhibited by a FLO1 strain in the effect of pH on cation dependence and sensitivity to chemical inactivation.

Mol Cell Biol, 1984 Aug, 4(8), 1521 - 7
Disruption of regulatory gene GAL80 in Saccharomyces cerevisiae: effects on carbon-controlled regulation of the galactose/melibiose pathway genes; Torchia TE et al.; In Saccharomyces cerevisiae, the transcriptional expression of the galactose-melibiose catabolic pathway genes is under the control of at least three regulatory genes, GAL4, GAL80, and GAL3 . We have isolated the GAL80 gene and have studied the effect of a null mutation on the carbon-controlled regulation of the MEL1 and GAL cluster genes . The null mutation was achieved in vivo by replacing the chromosomal wild-type GAL80 allele with an in vitro-created GAL80 deletion-disruption mutation . Enzyme activities and RNA levels for the GAL cluster and MEL1 genes were constitutively expressed in the null mutant strain grown on glycerol-lactate and were higher than in the isogenic wild-type yeast strain when compared after growth on galactose . Carbon catabolite repression of the GAL cluster and MEL1 genes, which occurs at the level of transcription, is retained in the null mutant . Deletion of the GAL80 gene in a gal4 cell does not restore GAL cluster and MEL1 gene expression . The data demonstrate that (i) the GAL80 protein is a purely negative regulator, (ii) the GAL80 protein does not mediate carbon catabolite repression, and (iii) the GAL4 protein is not simply an antagonizer of GAL80-mediated repression.

Mol Cell Biol, 1984 Aug, 4(8), 1440 - 8
Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae; Johnston M et al.; The GAL1 and GAL10 genes of Saccharomyces cerevisiae are divergently transcribed, with 606 base pairs of DNA separating their transcription initiation sites . These two genes are stringently coregulated: their expression is induced ca . 1,000-fold in cells growing on galactose and is repressed by growth on glucose . The nucleotide sequence of the region of DNA between these genes and the precise sites of transcription initiation are presented here . The most notable feature of the nucleotide sequence of this region is a 108-base-pair guanine-plus-cytosine-rich stretch of DNA located approximately in the middle of the region between GAL1 and GAL10 . Analysis of the effects of mutations that alter the region between these two genes, constructed in vitro or selected in vivo, suggest that these guanine-plus-cytosine-rich sequences are required for the expression of both genes . The region of DNA between GAL1 and GAL10 is sufficient for regulation of expression of these genes: fusion of the region to the yeast HIS3 gene places HIS3 under GAL control.

Proc Natl Acad Sci U S A, 1984 Aug, 81(15), 4642 - 6
Alpha-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae; Brake AJ et al.; Saccharomyces cerevisiae cells were transformed with plasmids containing hybrid genes in which the sequence encoding mature human epidermal growth factor was joined to sequences encoding the leader region (preprosegment) of the precursor of the yeast mating pheromone alpha-factor . These cells accurately process the hybrid protein and efficiently secrete authentic biologically active human epidermal growth factor into the medium.

J Biol Chem, 1984 Jul 25, 259(14), 9332 - 8
Mitochondrial gene expression in saccharomyces cerevisiae . II . Fidelity of translation in isolated mitochondria from wild type and respiratory-deficient mutant cells; McKee EE et al.; The fidelity of mitochondrial translation has been examined in isolated yeast mitochondria incubated in an optimized protein-synthesizing medium (McKee, E . E., and Poyton, R . O., (1984) J . Biol . Chem . 259, 9320-9331) . These studies have revealed: that isolated mitochondria synthesize bona fide mitochondrial gene products which are identical in kind and relative amounts to those synthesized in vivo; that mitochondria isolated from both mitochondrial mit- mutants and nuclear Pet mutants, which retain the capacity for mitochondrial protein synthesis, produce a mutant pattern of mitochondrial gene products which is similar to that produced in vivo; and that isolated mitochondria synthesize up to 7% of their protein mass in vitro at a rate of about one polypeptide bond/polypeptide chain/s . These studies also reveal that isolated wild type yeast mitochondria are competent in all steps in mitochondrial translation, including initiation . Using pactamycin as a specific inhibitor of translational initiation we have demonstrated that polypeptide chain initiation continues throughout a 60-min incubation period . By using this in vitro system to calculate the stoichiometry of synthesis of the major proteins coded by yeast mitochondrial DNA we have found that the var1 polypeptide is synthesized at a level which is significantly lower than all other mitochondrial gene products and that cytochrome c oxidase subunits I, II, and III and ATPase subunit 9 are synthesized in nearly equimolar amounts . These results suggest that the synthesis of these four gene products is controlled coordinately.

J Biol Chem, 1984 Jul 25, 259(14), 9320 - 31
Mitochondrial gene expression in saccharomyces cerevisiae . I . Optimal conditions for protein synthesis in isolated mitochondria; McKee EE et al.; An in vitro mitochondrial protein-synthesizing system, which makes use of intact yeast mitochondria, has been developed in order to study mitochondrial gene expression and its control by nuclear-coded proteins . Studies with this system have revealed that: isolated mitochondria synthesize polypeptide gene products which can be radiolabeled to high specific radioactivities when incubated in a "protein-synthesizing medium" that has been optimized with respect to each of its components; two energy-generating systems, endogenous oxidative phosphorylation and an exogenous ATP-regenerating system, support the highest level of protein synthesis; and the omission of an oxidizable substrate results in the synthesis of two new polypeptides (19.5 and 18 kDa) and a decrease in the amounts of cytochrome c oxidase subunits I and II which are synthesized . They have also revealed that added adenine and guanine nucleotides increase the overall level of protein synthesis and that the added guanine nucleotides facilitate polypeptide chain elongation . Although isolated mitochondria which have been optimized for protein synthesis synthesize normal gene products (McKee, E . E., McEwen, J . E., and Poyton, R . O., (1984) J . Biol . Chem . 259, 9332-9338) they still respond to an added dialyzed S-100 fraction from yeast cells by increasing their level of protein synthesis . This stimulation is observed in the presence of optimal concentrations of GTP, making it unlikely that guanyl nucleotides or enzymes which synthesize them are the sole stimulatory factors present in cellular cytosolic fractions, as suggested by Ohashi and Schatz (Ohashi, A., and Schatz, G . (1980) J . Biol . Chem . 255, 7740-7745).

FEBS Lett, 1984 Jul 23, 173(1), 199 - 203
Localization of the thermosensitive X-prolyl dipeptidyl aminopeptidase in the vacuolar membrane of Saccharomyces cerevisiae; Bordallo C et al.; Most of the X-prolyl dipeptidyl aminopeptidase activity of Saccharomyces cerevisiae was found to be associated with purified vacuolar membranes (specific activity approx . 75-times higher than in the protoplast lysate) . The tonoplast-bound enzyme is thermosensitive . Another heat-resistant enzyme was found in the protoplast lysate . The tonoplast-bound thermosensitive enzyme shows an apparent Km of 0.06 mM against L-alanyl-L-prolyl-p-nitroanilide while the heat-resistant enzyme shows an apparent Km of 0.4 mM against the same substrate.

Biochimie, 1984 Jul-Aug, 66(7-8), 583 - 8
Influence of oxygen on the microsomal electron transport system in Saccharomyces cerevisiae; Bertrand JC et al.; The influence of oxygen on the level of microsomal electron transport chain components has been studied during the growth of Saccharomyces cerevisiae . Enzyme activities and cytochrome content were assayed in microsomal fractions prepared from a protoplast lysate free from mitochondrial contamination . It was found that the cytochrome P-450 and cytochrome b5 content, to get her with the NADPH-cytochrome (P-450)-reductase and NADH-cytochrome (b5)-reductase activities, were increased in the cells as the pO2 of the medium was decreasing . At the same time an increase in the membrane surface of the endoplasmic reticulum can be observed.

Carbohydr Res, 1984 Jul 1, 129, 87 - 97
Synthesis and mass spectra of the partially methylated and partially ethylated anhydro-D-mannitol acetates derived by reductive cleavage of permethylated and perethylated Saccharomyces cerevisiae D-mannans; Bowie JU et al.; Reductive cleavage of per-O-ethylated or per-O-methylated Saccharomyces cerevisiae D-mannans and subsequent acetylation had previously been shown to produce the expected derivatives of 1,5-anhydro-D-mannitol . Described herein is the independent synthesis of each of these derivatives, namely, 1,5-anhydro-2,3,4,6-tetra-O-methyl-, -2,3,4,6-tetra-O-ethyl-, -2-O-acetyl-3,4,6-tri-O-methyl-, -2-O-acetyl-3,4,6-tri-O-ethyl-, -3-O-acetyl-2,4,6-tri-O-methyl-, -3-O-acetyl-2,4,6-tri-O-ethyl-, -6-O-acetyl-2,3,4-tri-O-methyl-, -6-O-acetyl-2,3,4-tri-O-ethyl-, -2,6-di-O-acetyl-3,4-di-O-methyl-, -2,6-di-O-acetyl-3,4-di-O-ethyl-, -3,6-di-O-acetyl-2,4-di-O-methyl-, and -3,6-di-O-acetyl-2,4-di-O-ethyl-D-mannitol . The 1H-n.m.r . spectra, chemical-ionization (NH3) mass spectra, and electron-impact mass spectra for all of these derivatives are tabulated.

Mutat Res, 1984 Jul, 137(1), 47 - 9
Induction of gene conversion and reverse mutation by manganese sulphate and nickel sulphate in Saccharomyces cerevisiae; Singh I; Manganese sulphate and nickel sulphate were tested for the induction of gene conversion at the trp5 locus and reverse mutation at the ilv1 locus in D7 strain of Saccharomyces cerevisiae . Nickel sulphate was more toxic than manganese sulphate but did not induce any reverse mutation and gave a weak positive response for conversion . Manganese sulphate was also only slightly mutagenic and convertogenic.

Exp Cell Res, 1984 Jul, 153(1), 259 - 65
Meiotic karyotype of the yeast Saccharomyces cerevisiae; Kuroiwa T et al.; A cytogenetic study of the meiotic chromosomes of the budding yeast Saccharomyces cerevisiae was undertaken by high resolution epifluorescence microscopy . Condensation of chromatin into separate chromosomes takes place during prophase I . At metaphase I, there are 16 separate and distinct bivalents which are roughly classified into three groups by morphological differences and DNA content.

Mol Cell Biol, 1984 Jul, 4(7), 1238 - 45
Regulation of basal and induced levels of the MEL1 transcript in Saccharomyces cerevisiae; Post-Beittenmiller MA et al.; The MEL1 gene in Saccharomyces cerevisiae is required for the production of alpha-galactosidase and for the catabolism of melibiose . Production of alpha-galactosidase is induced by galactose or melibiose and repressed by glucose . Inducibility is controlled by the positive and negative regulatory proteins GAL4 and GAL80, respectively . We have cloned the MEL1 gene to study its transcriptional expression and regulation . Evidence is presented that the MEL1 gene encodes alpha-galactosidase and that mel0 is a naturally occurring allele which lacks the alpha-galactosidase-coding sequences . RNAs prepared from wild-type cells and from cells carrying either the noninducible gal4-2 or GAL80S-100 allele grown on three different carbon sources were examined by Northern hybridization analyses . In wild-type cells under noninducing conditions, such as growth on glycerol-lactic acid, the MEL1 transcript was detected at a basal level which was 1 to 2% of the fully induced level . The basal level of expression was diminished in cells carrying the gal4-2 mutant allele but not in cells carrying the GAL80S-100 allele . The basal and induced RNA levels are repressed by glucose . Size determinations of the MEL1 transcripts detected in glycerol-lactic acid- and galactose-grown cells provided no evidence for two distinct transcripts.

Mol Cell Biol, 1984 Jul, 4(7), 1326 - 33
Positive regulatory interactions of the HIS4 gene of Saccharomyces cerevisiae; Lucchini G et al.; The role of cis- and trans-acting elements in the expression of HIS4 has been examined by using HIS4-lacZ fusions in which lacZ expression is dependent upon the HIS4 5' noncoding region . The cis-acting sequences involved in regulation were defined by studying the effects of the wild-type and various deletions and their revertants on regulation via the general control of amino acid biosynthesis . The role of trans-acting genes was analyzed by studying the regulation of the HIS4-lacZ fusions in strains carrying mutations in the GCN (AAS) or GCD (TRA) genes and in strains carrying the GCN genes on high-copy-number plasmids . These studies have led to the following conclusions . (i) HIS4 is positively regulated by the general control . (ii) At least one copy of the 5'TGACTC3' repeat at -136 is required in cis for this regulation . (iii) Both the GCN4 gene and at least one copy of the repeated sequence are required for expression at the repressed level . (iv) The open reading frames in the 5' noncoding region are not required in either cis or trans for the regulation of HIS4.

Mol Cell Biol, 1984 Jul, 4(7), 1278 - 85
Structure of the SAD mutation and the location of control sites at silent mating type genes in Saccharomyces cerevisiae; Hicks J et al.; The SAD mutation, an extra mating type cassette, has been shown to arise from an unequal mitotic crossover between the MAT and HMR loci, resulting in the formation of a hybrid cassette and a duplication of the MAT-HMR interval . The SAD cassette contains the "a" information and left-hand flanking regions from the parental HMRa cassette and the right-hand flanking sequences of the parental MAT cassette . This arrangement of flanking sequences causes a leaky but reproducible mating phenotype correlated with a low-level expression of the cassette as measured by RNA blotting . This weak expression is attributed to the loss of one flanking control site normally present at the silent HM storage loci.

Mol Cell Biol, 1984 Jul, 4(7), 1246 - 51
Effects of Ty insertions on HIS4 transcription in Saccharomyces cerevisiae; Silverman SJ et al.; Insertion of two different Ty elements into the Saccharomyces cerevisiae HIS4 regulatory region eliminates transcription of HIS4 . Transcription can be restored by genetic rearrangements involving the Ty element inserted at HIS4 . Several deletions, an inversion, a translocation, and a gene conversion are capable of restoring HIS4 transcription . Some of the rearrangements result in new transcriptional initiation sites . One type of revertant of his4-912 results from recombination between the delta elements flanking the Ty element, leaving a solo delta in place of the complete Ty . Strains carrying a Ty912 delta at HIS4 are His- at 23 degrees C . Unlinked suppressors (SPT) lead to suppression of this His- phenotype and increase levels of the normal HIS4 transcript . These suppressor genes affect not only the amount of transcription from the normal HIS4 initiation site, but also that from new initiation sites within Ty sequences adjacent to HIS4.

J Biol Chem, 1984 Jun 25, 259(12), 7936 - 40
A DNA primase that copurifies with the major DNA polymerase from the yeast Saccharomyces cerevisiae; Singh H et al.; Biochemical fractionation of the yeast Saccharomyces cerevisiae has revealed a novel DNA primase activity that copurifies with the major DNA polymerase activity . In the presence of RNA precursors and single-stranded DNA (poly(dT), M13), the DNA primase synthesizes discrete length oligoribonucleotides (apparent length, 8-12 nucleotides) as well as longer RNA chains that appear to be multiples of a modal length of 11-12 nucleotides . When DNA precursors are also present, the oligoribonucleotides are utilized by the accompanying DNA polymerase as primers for DNA synthesis . Copurification of these two enzymatic activities suggests their association in a physical complex which may function in the synthesis of Okazaki fragments at chromosomal replication forks.

Biochim Biophys Acta, 1984 Jun 16, 782(2), 220 - 7
The effect of non-permissive temperature on met-tRNA synthetase in Saccharomyces cerevisiae temperature-sensitive mutant ts 7-45; Sadnik I et al.; The effects of incubation of yeast spheroplasts at elevated temperature (40 degrees C) on a number of activities involved in protein biosynthesis have been examined in preparations obtained from wild-type cells (wt A364A ) and a temperature-sensitive mutant (ts 7-45) derived from it . With wild-type cells, preincubation of spheroplasts at the elevated temperature had little or no effect on the following: the ribosomal subunit-polysome pattern; the translation of exogenous natural mRNA in postpolysomal extracts devoid of endogenous mRNA; the translation of poly(U) in postpolysomal extracts; the incorporation of methionine into 40 S preinitiation and 80 S initiation complexes; the synthesis of Met-tRNA in postribosomal (cytosol) extracts; and the formation of eIF-2 X GTP X Met-tRNAf ternary complex in the cytosol . With temperature-sensitive spheroplasts that had not been preincubated at the elevated temperature, the concentration of free, native 40 S subunits appeared to be lower and that of 60 S subunits higher than in wild-type cells; translation of exogenous natural mRNA in postpolysomal extracts was somewhat lower than in wild-type preparations, but all of the other reactions and components measured were comparable to those in wild-type preparations . Preincubation of temperature-sensitive spheroplasts at 40 degrees C resulted in: a further decrease in the level of 40 S subunits; disaggregation of polysomes; loss of ability to translate natural mRNA but not poly(U); decreased ability to form 40 S preinitiation intermediates; and production of an activity, found in the cytosol, that inhibited Met-tRNA synthetase reversibly . The inhibitor had the characteristics of a protein and did not appear to be a proteinase, nuclease, or nucleotidase.

Eur J Biochem, 1984 Jun 15, 141(3), 585 - 90
Methylated proteins and amino acids in the ribosomes of Saccharomyces cerevisiae; Lhoest J et al.; The occurrence of methylated proteins in the ribosomes of Saccharomyces cerevisiae was investigated by tracing the transfer of radioactive methyl groups from S-adenosyl methionine, taken up by growing cells, into the protein moiety of ribosomes . It was estimated that the large subunit contained about 10 protein-bound methyl groups distributed mainly among proteins YL23, YL32 and YL1 . The small subunit contained at most 2-4 methyl groups in proteins . Methyl groups could be transferred in vitro to proteins YL23 and YL32 in extracts from cultures of an S-adenosyl methionine auxotroph deprived of the methyl-group donor . In the most heavily methylated proteins the methylated amino acids formed in vitro were the same as those found in vivo (monomethyllysine and dimethyllysine in YL32; dimethyl and trimethyllsine in YL23) . It is concluded that the enzymatic reaction in vitro faithfully saturates with methyl groups the target amino acids which are normally fully methylated in vivo.

Biochim Biophys Acta, 1984 Jun 15, 799(2), 181 - 6
Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae; Entian KD et al.; An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cells . (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased . This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis . (ii) In contrast, respiratory activity decreased after adding glucose . This decrease was clearly shown to be the result of repression of respiratory enzymes . A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ' Crabtree effect', was not observed in yeast . (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities . Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation . However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately . Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate dehydrogenase was subject to glucose inactivation.

Science, 1984 Jun 8, 224(4653), 1109 - 11
Saccharomyces cerevisiae produces a yeast substance that exhibits estrogenic activity in mammalian systems; Feldman D et al.; Partially purified lipid extracts of Saccharomyces cerevisiae contain a substance that displaces tritiated estradiol from rat uterine cytosol estrogen receptors . The yeast product induces estrogenic bioresponses in mammalian systems as measured by induction of progesterone receptors in cultured MCF-7 human breast cancer cells and by a uterotrophic response and progesterone receptor induction after administration to ovariectomized mice . The findings raise the possibility that bakers' yeast may be a source of environmental estrogens.

Nature, 1984 Jun 7-13, 309(5968), 523 - 7
Requirement of either of a pair of ras-related genes of Saccharomyces cerevisiae for spore viability; Tatchell K et al.; Cells of the yeast, Saccharomyces cerevisiae, containing disruptions of either of two genes that are members of the ras oncogene family are viable, but haploid yeast spores carrying disruptions of both genes fail to grow.

Biochem J, 1984 Jun 1, 220(2), 461 - 7
Defective processing of ribosomal precursor RNA in Saccharomyces cerevisiae; Mitlin JA et al.; Saccharomyces cerevisiae (strain A224A) has an abnormal distribution of cytoplasmic ribosomal subunits when grown at 36 degrees C, with sucrose-gradient analysis of extracts revealing an apparent excess of material sedimenting at 60 S . This abnormality is not observed at either 23 degrees C or 30 degrees C . At 36 degrees C the defect(s) is expressed as a slowed conversion of 20 S ribosomal precursor RNA to mature 18 S rRNA, although the corresponding maturation of 27 S ribosomal precursor RNA to mature 25 S rRNA is normal . Studies on this yeast strain and on mutants derived from it may help to elucidate the role(s) of individual ribosomal components in controlling ribosome biogenesis in eukaryotes.

Int J Radiat Biol Relat Stud Phys Chem Med, 1984 Jun, 45(6), 593 - 606
Common repair pathways acting upon U.V.- and X-ray induced damage in diploid cells of Saccharomyces cerevisiae; Nunes E et al.; Studies on X-ray sensitive mutants of Saccharomyces cerevisiae (Benathen 1973, Benathen and Beam 1977) show that the XS6, XS8 and XS9 genes are not only involved in the repair of X-ray-induced damage but also in the repair of U.V.-induced damage . Analysis of the U.V . sensitivity of multiple xs mutants indicates the participation of three repair pathways which differ from excision repair . Under conditions which can influence repair, such as plating of the U.V.-irradiated cells in the presence of caffeine, followed or not by hyperthermic incubation, the wild type strain shows a diphasic survival curve, consisting of an exponential component for low doses and a sigmoidal one for higher doses . Comparison with the survival curves obtained for the sensitive mutants suggests that the first component of the wild type survival curve corresponds to the inhibition of the XS6 and XS8 gene products while the appearance of a radio-resistant fraction in the population relies on the induction of another repair pathway . A sequential model of repair with two branching points is proposed to explain the results.

J Bacteriol, 1984 Jun, 158(3), 1152 - 6
Different structure-function relationships for alpha-factor-induced morphogenesis and agglutination in Saccharomyces cerevisiae; Baffi RA et al.; Eight synthetic analogs of the mating pheromone alpha-factor-induced morphogenesis and increased agglutinability in a cells . Most analogs induced increased agglutinability at lower concentrations than those at which they induced morphogenesis, but the ratio of the potencies for the two effects varied 140-fold among different analogs . Morphological response to pheromone required exposure for at least 90 min, but increased agglutinability followed exposures of 20 s . Two synthetic analogs induced neither response . In competition experiments, both of these analogs prevented induction of increased agglutinability and morphogenesis by active alpha factor . The inactive peptides blocked increased agglutinability at lower concentrations than those at which they blocked morphogenesis . alpha factors exhibited different structure-function relationships for morphogenesis as compared with agglutinability . Thus, response of Saccharomyces cerevisiae to alpha factor is complex and may be mediated by more than one receptor.

J Biochem (Tokyo), 1984 Jun, 95(6), 1677 - 90
Purification of a eukaryotic site-specific endonuclease, Endo.Sce I, from Saccharomyces cerevisiae and effectors on its specificity and activity; Watabe H et al.; A site-specific endonuclease (Endo.Sce I) which caused double-strand scission of DNA was highly purified from a eukaryote, Saccharomyces cerevisiae IAM4274 . The molecular weight of the active form of Endo.Sce I was estimated to be 120,000 and 110,000 by sedimentation analysis on a glycerol density gradient and gel filtration on Ultrogel AcA34, respectively . Analysis of the fractions from the last column chromatography by polyacrylamide gel-electrophoresis in the presence of sodium dodecyl sulfate and by an assay of the endonucleolytic activities suggested that Endo.Sce I consists of two non-identical subunits with molecular weights of 75,000 and 50,000 . Unlike restriction endonucleases, Endo.Sce I was active on chromosomal DNA of the cells which produced Endo.Sce I . Single-stranded DNA was not cleaved by Endo.Sce I, but inhibited the endonucleolytic activity of the enzyme on double-stranded DNA . The endonucleolytic activity of Endo.Sce I required the magnesium ions (Mg2+) as a sole cofactor; Mg2+ could not be replaced by Ca2+ or Zn2+ . When Mg2+ was replaced by manganese ions (Mn2+), extensively purified Endo.Sce I cleaved double-stranded DNA at many other sites in addition to the sites at which DNA was cleaved in the presence of Mg2+ . Experiments indicated that this is not the activation of contaminating endonuclease in the preparation of Endo.Sce I, but the result of relaxation in the site-specificity of cleavage.

EMBO J, 1984 Jun, 3(6), 1389 - 95
Expression of the clustered mitochondrial tRNA genes in Saccharomyces cerevisiae: transcription and processing of transcripts; Palleschi C et al.; The transcripts of a cluster of eight tRNA genes localized in the Cap-oxiI region of the mitochondrial genome of Saccharomyces cerevisiae were investigated by hybridization of gene-specific probes on Northern blots of mitochondrial RNA and by S1 mapping of the 5' termini of the transcripts . Two rho- mutants that lack mature tRNA species and accumulate precursors have been used to detect transcripts that are not detectable in wild-type (w.t.) mitochondria . The results have shown the existence of polygenic transcripts carrying at least 5-7 tRNA sequences, both in w.t . and in rho- strains . The existence of several alternative processing pathways, which involve cleavage at the 3' and 5' ends of the tRNA sequences and in the long intergenic regions (possibly at GC clusters), is suggested . Cleavage at the 5' ends of tRNA sequences is defective in the mutant strains . The transcripts of the genes for tRNAThrACN and tRNACys (the tRNA genes immediately downstream from the 21S rRNA gene) have been analyzed; the possibility that these species represent primary transcripts is considered, and potential sites for initiation of transcription of the clustered tRNA genes are discussed.

Nucleic Acids Res, 1984 May 25, 12(10), 4083 - 96
A minor class of 5S rRNA genes in Saccharomyces cerevisiae X2180-1B, one member of which lies adjacent to a Ty transposable element; Piper PW et al.; In Saccharomyces cerevisiae the majority of the genes for 5S rRNA lie within a 9kb rDNA sequence that is present as 100-200 tandemly-repeated copies on Chromosome XII . Following our observations that about 10% of yeast 5S rRNA exists as minor variant sequences, we screened a collection of yeast DNA fragments cloned in lambda gt for 5S rRNA genes whose flanking sequences differed from those adjacent to 5S rRNA genes of the rDNA repeat . Three variant 5S rRNA genes were isolated on the basis of such dissimilarity to rDNA repeat sequences . They display a remarkable conservation of their DNA in the vicinity of the 5S coding region, and are examples of a minor form of 5S rRNA coding sequence present in a small number of copies in the yeast genome . These variant sequences appear to be transcribed as efficiently as 5S rRNA genes of the rDNA repeat . In one of our isolates of the variant sequence a Ty transposable element is inserted 145bp upstream of the initiation point for 5S rRNA synthesis.

J Biol Chem, 1984 May 10, 259(9), 5745 - 51
Complete sequence of the heat shock-inducible HSP90 gene of Saccharomyces cerevisiae; Farrelly FW et al.; We have sequenced the yeast HSP90 gene, which encodes the (apparent) Mr = 90,000 heat shock-inducible protein of this organism . All sequences required for the heat shock-regulated expression of this gene reside on a segment of DNA containing no more than 273 nucleotides 5' to the transcription origin . Transcript mapping reveals that the mature hsp90 mRNA contains a 59-nucleotide 5' untranslated segment, a coding segment of 2130 nucleotides (sufficient to encode a protein of Mr = 81,419), and a 3' untranslated segment of 128 nucleotides . Although the sequences surrounding the coding region of the HSP90 gene are quite similar to those surrounding other sequenced yeast genes, we find as well a limited homology between sequences located 5' to this gene and the putative heat shock-regulatory sequences located 5' to the heat shock-inducible genes of Drosophila.

Ann Microbiol (Paris), 1984 May-Jun, 135A(3), 343 - 51
Molecular cloning of the tsm0185 gene responsible for adenylate cyclase activity in Saccharomyces cerevisiae; Masson P et al.; The gene of Saccharomyces cerevisiae responsible for adenylate cyclase activity was cloned by complementation of a thermosensitive tsm0185 mutation in yeast; it was also shown to complement the yeast cyr1 mutation . Preliminary results indicate the presence of a repeated sequence on the same genomic fragment.

Gene, 1984 May, 28(2), 229 - 35
Stability of recombinant plasmids containing the ars sequence of yeast extrachromosomal rDNA in several strains of Saccharomyces cerevisiae; Larionov V et al.; The mitotic stabilities of hybrid plasmid Rcp21/11, which contains the replicator of yeast rDNA, have been compared for four yeast host strains of different origins . In two related strains, Saccharomyces cerevisiae . A62-1G-P188 and 1A-P3812 from the Peterhof genetic stocks, the plasmid was much more stable than in strains DC5 and GRF18 from the USA stocks . The enhanced mitotic stability of Rcp21/11 in these two yeast strains is obviously attributable to a higher rate of integration of the plasmid into the chromosomal rDNA repeats of the hosts . The centromeric locus CEN3 was inserted into Rcp21/11 because it provides high mitotic and meiotic stability of plasmids with yeast replicators, due to an ordered distribution of plasmids throughout cell division . Using the new centromeric plasmid RcpCEN3, transformation of the four above-described yeast strains was carried out . It was found that, similarly to centromeric plasmids with other chromosomal replicators, RcpCEN3 remains in the cell as a single copy . In strains DC5, GRF18 and A62-1G-P188 the mitotic stability of RcpCEN3 was 20-50%, i.e., less than half that of plasmids containing locus CEN3 and other yeast replicators, ars1, ars2 and the 2mu DNA replicator . The mitotic stability of RcpCEN3 in strains 1A-P3812 (from the Peterhof genetic stocks) for individual clones reached 85%, i.e . close to that of the other plasmids . Genetic analysis showed that the capacity of strain 1A-P3812 to stably retain RcpCEN3 has a recessive polygenic character . We suggest that the observed differences in mitotic stability of centromeric plasmid RcpCEN3 between various yeast strains reflects the differences in activity of rDNA replicator in these strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1984 May, 28(2), 195 - 9
Plasmid-mediated complementation of a delta-aminolevulinic-acid-requiring Saccharomyces cerevisiae mutant; Bard M et al.; Recombinant plasmids able to complement the Saccharomyces cerevisiae ole3 mutation were isolated . The nucleotide sequence responsible for complementation was localized to a 3.5-kb region . The level of delta-aminolevulinate (ALV) synthase activity in wild-type cells was six-fold lower than in plasmid-transformed ole3 mutant cells . Certain clones secreted a compound that supported growth of a lawn of adjacent ole3 mutant cells.

Mol Cell Biol, 1984 May, 4(5), 985 - 8
5-Methylcytosine is not detectable in Saccharomyces cerevisiae DNA; Proffitt JH et al.; We examined the DNA of Saccharomyces cerevisiae by both HpaII-MspI restriction enzyme digestion and high-performance liquid chromatography analysis for the possible presence of 5-methylcytosine . Both of these methods failed to detect cytosine methylation within this yeast DNA; i.e., there is less than 1 5-methylcytosine per 3,100 to 6,000 cytosine residues.

J Bacteriol, 1984 May, 158(2), 644 - 9
Isolation of a Saccharomyces cerevisiae mutant strain deficient in deoxycytidylate deaminase activity and partial characterization of the enzyme; McIntosh EM et al.; Deoxycytidylate deaminase activity in Saccharomyces cerevisiae has been partially characterized . The yeast enzyme was found to exhibit properties similar to those of dCMP deaminases isolated from higher eucaryotes . A mutant strain completely deficient in dCMP deaminase activity was isolated by selection for resistance to 5-fluoro-2'-deoxycytidylate followed by screening for cross sensitivity to 5-fluoro-2'-deoxyuridylate, a potent inhibitor of the yeast thymidylate synthetase . We have designated this new allele dcd1 . A strain exhibiting an auxotrophic requirement for dUMP was isolated after mutagenesis of a dcd1 tup7 haploid . Genetic analysis revealed that this auxotrophic phenotype resulted from a combination of the dcd1 allele and a second, unlinked, nuclear mutation that we designated dmp1 . This allele, which by itself conveys no readily discernible phenotype, presumably impairs efficient synthesis of dUMP from UDP . The auxotrophic requirement of dcd1 dmp1 tup7 strains also can be satisfied by exogenous dTMP but not deoxyuridine.

Mol Cell Biol, 1984 May, 4(5), 994 - 6
Toxicity of 2-deoxygalactose to Saccharomyces cerevisiae cells constitutively synthesizing galactose-metabolizing enzymes; Platt T; Analysis of 400 independent spontaneous mutations conferring 2-deoxygalactose resistance upon cells constitutive for the galactose pathway suggests that toxicity is due to 2-deoxygalactose-1-phosphate . Selection for and against growth on galactose in the same strain is now possible; application to systems with transcriptional or translational gene fusions to galactokinase are discussed.

Mol Cell Biol, 1984 May, 4(5), 939 - 46
Glucose represses transcription of Saccharomyces cerevisiae nuclear genes that encode mitochondrial components; Szekely E et al.; By Northern blot hybridization analysis, we demonstrated that the steady-state levels of mRNAs specifying the alpha subunit of ATPase, the beta subunit of ATPase, and the ATP/ADP translocator are all reduced in cells grown in glucose-rich medium . The extent to which glucose represses the levels of alpha, beta, and translocator mRNAs varies from strain to strain, from 2.5- to 7-fold . Furthermore, by hybridization experiments with an excess of DNA, we showed that glucose represses the rates of synthesis of these mRNAs . The kinetics of repression and depression of transcription were also studied . Finally, a mutant was characterized which appears to be defective in depression of transcription of the genes encoding the alpha and beta ATPase subunits as well as the ATP/ADP translocator.

Proc Natl Acad Sci U S A, 1984 May, 81(9), 2616 - 20
DNA topoisomerase II mutant of Saccharomyces cerevisiae: topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication; DiNardo S et al.; A temperature-sensitive DNA topoisomerase II mutant of the yeast Saccharomyces cerevisiae has been identified . Genetic analysis shows that a single recessive nuclear mutation is responsible for both temperature-sensitive growth and enzymatic activity . Thus, topoisomerase II is essential for viability and the mutation is most probably in the structural gene . Experiments with synchronized mutant cells show that at the nonpermissive temperature cells can undergo one, and only one, round of DNA replication . These cells are arrested at medial nuclear division . Analysis of 2-microns plasmid DNA from these cells shows it to be in the form of multiply intertwined catenated dimers . The results suggest that DNA topoisomerase II is necessary for the segregation of chromosomes at the termination of DNA replication.

J Bacteriol, 1984 May, 158(2), 530 - 4
Identification of a glutaminyl-tRNA synthetase mutation Saccharomyces cerevisiae; Mitchell AP et al.; Saccharomyces cerevisiae glutaminyl-tRNA synthetase mutants were isolated through systematic screening of tight Gln- derivatives of a leaky glutamine auxotroph . These mutations define a single nuclear gene, GLN4 . The gln4-1 mutation is specific for Gln-tRNA synthetase and shows a dosage effect in heterozygous diploids . The wild-type Gln-tRNA synthetase exhibits a Km for glutamine of 25 microM; the gln4-1 mutation increases this value 20-fold . These observations strongly suggest that GLN4 encodes the Gln-tRNA synthetase.

J Biol Chem, 1984 Apr 25, 259(8), 5139 - 45
Purification and characterization of ornithine transcarbamoylase from Saccharomyces cerevisiae; Eisenstein E et al.; Ornithine transcarbamoylase (OTCase) has been purified in 100-mg quantities from a plasmid-containing, enzyme-overproducing strain of Saccharomyces cerevisiae . The specific activity of the homogeneous enzyme is 2.5-fold above that previously reported . The molecular weight and partial specific volume of OTCase were determined by sedimentation equilibrium in solutions containing H2O and D2O . Data from two rotor speeds were simultaneously fit using nonlinear least squares analysis with multiple independent variables giving a molecular weight of 110,000 +/- 2,200 and a partial specific volume of 0.732 +/- 0.006 ml g-1 . The ultraviolet absorption spectrum of OTCase gives a specific absorbance at 280 nm of 0.36 . This low value is consistent with a small number of aromatic residues . Amino acid analysis, fluorescence, and multicomponent analysis yield 1 tryptophan, 4 tyrosine, and 24 phenylalanine/polypeptide chain . From an analysis of the circular dichroic spectrum, it was determined that OTCase contained 22% alpha-helix, 43% beta-sheet, 8% beta-turn, and 27% random structure . The fluorescence of the single tryptophan/polypeptide chain has an emission maximum at 320 nm, indicating a hydrophobic environment.

Nucleic Acids Res, 1984 Apr 25, 12(8), 3611 - 8
Nucleotide sequence of two rasH related-genes isolated from the yeast Saccharomyces cerevisiae; Dhar R et al.; A complete nucleotide sequence of two ras-related yeast genes (c- rassc -1 and c- rassc -2) isolated from the yeast strain Saccharomyces cerevisiae is reported . They encode predicted polypeptides of 40,000 and 41,000 daltons, respectively . The N-terminal 170 amino acids from both genes show extensive amino acid homology to other ras genes from vertebrates, whereas their C-termini have diverged . These genes should be useful in the elucidation of a normal biological function of ras-related genes in a simple system like yeast.

Nucleic Acids Res, 1984 Apr 11, 12(7), 3143 - 54
Characteristic alteration in the nuclear DNA polymerase activity during the cell division cycle of Saccharomyces cerevisiae; Tsuchiya E et al.; Specific activity of the intranuclear DNA polymerase in cdc-mutant cells of Saccharomyces cerevisiae was found to be characteristically changed by arrest in their specific stage of cell division cycle without a notable alteration in the total cellular activity . The activities were low in the nuclei of cdc 25, cdc 28 and cdc 4, which were arrested in early to mid G1 phase by temperature shift-up, and in the nuclei of wild-type cells (A364A), which were arrested in early G1 phase by alpha-factor treatment, while high level of the activity was found in the nuclei of cdc 7 and cdc 8, which were arrested at late G1 and S phase, respectively . Activity-gel analysis of DNA polymerase in the nuclear extracts revealed the presence of two active peptides (120K and 72K), and the characteristic decrease in both active peptides was induced by arrest in early to mid G1 phase . Consequently, it is strongly suggested that intranuclear DNA polymerase activity alters in a dependent fashion on progression of cell division cycle . Subunit analysis indicated that the purified DNA polymerase I is constructed from two subunit peptides of 120K and 62K, and the large subunit possesses catalytic activity.

Eur J Biochem, 1984 Apr 2, 140(1), 183 - 9
Saccharomyces cerevisiae mating pheromones specifically inhibit the synthesis of proteins destined to be N-glycosylated; Orlean P et al.; alpha Factor specifically inhibits the synthesis of N-glycosylated proteins in Saccharomyces cerevisiae mating type a cells but not in alpha cells or in a/alpha diploids . a Factor has the same effect of alpha cells . The synthesis of O-glycosylated proteins is not inhibited . Although the mating pheromones act like a 'physiological tunicamycin', the mechanism of inhibition is different: not the glycosylation of proteins as such but rather the synthesis of those proteins destined to be N-glycosylated is inhibited . Thus none of a number of glycosylating enzymes tested in vitro is reduced in activity in alpha-factor-treated cells . The synthesis of the glycoprotein carboxypeptidase Y, on the other hand, is strongly inhibited by tunicamycin as well as by alpha factor; but only in the former case did carbohydrate-free protein accumulate in the cells . alpha Factor causes maximal inhibition of glycoprotein formation after as little as 30 min, long before all cells in the population are arrested in G1; moreover, release from this inhibition precedes the increase in budding index (resumption of cell division) . It is postulated, therefore, that N-glycosylated proteins are required for the G1/S-phase transition in the yeast cell cycle . This is supported by previous reports that first cycle arrest in G1 occurs when (a) tunicamycin is added to growing cultures, and (b) a temperature-sensitive N-glycosylation mutant is shifted to its restrictive temperature.

Eur J Biochem, 1984 Apr 2, 140(1), 163 - 71
Nuclear-magnetic-resonance studies on the conformations of tridecapeptide alpha-mating factor from yeast Saccharomyces cerevisiae and analog peptides in aqueous solution . Conformation-activity relationship; Higashijima T et al.; The conformation of tridecapeptide alpha-mating factor from yeast Saccharomyces cerevisiae in aqueous solution was analyzed, in comparison with those of active analog and inactive analog peptides . 270-MHz 1H-NMR spectra of these peptides were observed and the spectral patterns of main-chain N-H proton resonances were classified into three groups . alpha-mating factor and Trp1-bearing active peptides belong to the group A1, active des-Trp1-peptides belong to the group A2 while the peptides of group B are inactive . The main-chain N-H proton resonances of the groups A1 and A2 and side-chain N-H proton resonances were all assigned to individual residues . The 13C-NMR analysis of alpha-mating factor indicates that the Lys7-Pro8 and Gln10-Pro11 peptide bonds exclusively take the trans form . From the temperature and pH dependences of chemical shifts and Gd(III)-induced relaxation enhancements of amide proton resonances, alpha-mating factor is found to take partly a folded conformation in aqueous solution, with an alpha-helical form in the N-terminal domain and two beta-turn forms in the central and C-terminal domain . The pH dependence of fluorescence intensity indicates that, in this folded conformation, the C-terminal carboxylate group lies close to the N-terminal domain . The presence of the folded form in the N-terminal domain and the beta-turn form in the central domain correlates with the biological activity of alpha-mating factor and analog peptides . However, the folded conformation of alpha-mating factor is in equilibrium with predominantly unordered form, as found from the circular dichroism and NMR analyses . The N-H proton and C-alpha proton resonances of free alpha-mating factor as assigned in the present study allow the transferred nuclear Overhauser enhancement (NOE) analysis of the membrane-bound conformation that is more directly related with the activity.

Mutat Res, 1984 Apr, 126(2), 145 - 51
Petite and sectored induction in Saccharomyces cerevisiae by propidium iodide: synergistic effect of sodium dodecyl sulfate; Iwamoto Y et al.; Sodium dodecyl sulfate (SDS) was examined for its effect on petite and sectored colony induction in Saccharomyces cerevisiae by propidium iodide (PI) and ethidium bromide (EB) . 4-h cultivation with 100 microM PI and 100 micrograms/ml SDS resulted in virtually all plated cells growing as sectored colonies with no decrease in viability . Sectored colonies are mixed colonies comprised of respiratory deficient and competent cells believed to be derived from an unstable respiratory deficient cell . Further cultivation with PI and SDS prior to plating led to induction of complete petite colonies with a rapid decrease in viable cells . PI alone at this concentration exhibited weak induction of sectored colonies (maximum 12.3% at 8 h) and petite colonies (maximum 10.8% at 12 h), but SDS alone caused induction of neither . 50 microM PI had almost the same activity as 100 microM except for a delay in the induction of sectored colonies in the initial stage, and a decreased rate of petite colony induction . The effects of 20 microM PI and SDS were much lower than that by 50 microM and no inhibition of growth was observed . 10 microM PI was quite inactive even in the presence of SDS . Under resting conditions, 10 approximately 100 microM PI and 100 micrograms/ml SDS induced about 60% sectored colonies at 12 h incubation and more than 60% petite colonies at 24 h . After 6 h incubation, decrease in survival was also observed.

Mol Cell Biol, 1984 Apr, 4(4), 761 - 70
Superkiller mutations in Saccharomyces cerevisiae suppress exclusion of M2 double-stranded RNA by L-A-HN and confer cold sensitivity in the presence of M and L-A-HN; Ridley SP et al.; In an mktl host, L-A-HN double-stranded RNA excludes M2 double-stranded RNA at 30 degrees C but not at 20 degrees C . Recessive mutations suppressing the exclusion of M2 by L-A-HN in an mktl host include six ski (superkiller) genes, three of which (ski6, ski7 and ski8) are new genes . The dominant mutations in one gene (MKS50) and recessive mutations in at least two genes (mks1 and mks2) suppress M2 exclusion by L-A-HN but do not show other characteristics of ski mutations and thus define a new class of killer-related chromosomal genes . Mutations in ski2, ski3, ski4, ski6, ski7, and ski8 result in increased M copy number at 30 degrees C and prevent the cells from growing at 8 degrees C . Elimination of M double-stranded RNA from a cold-sensitive ski- strain results in the loss of cold sensitivity . ski- {KIL-sd1} strains lack L-A-HN, carry L-A-E, and have a lower M1 copy number than do ski- {KIL-k1} strains and are only slightly cold sensitive . The LTS5 (=MAK6) product is required both for low temperature growth and for M1 maintenance or replication . We propose that the elevated levels of M in ski- strains divert the host LTS5 product away from the host and to the M replication process . We also suggest that the essential role of L-A in M replication is protection of M double-stranded RNA from the negative influence of SKI+ products.

Mol Cell Biol, 1984 Apr, 4(4), 695 - 702
Developmental changes in translatable RNA species associated with meiosis and spore formation in Saccharomyces cerevisiae; Weir-Thompson EM et al.; During Saccharomyces cerevisiae sporulation distinct changes in translatable mRNA species have been detected by two-dimensional gel electrophoresis of the polypeptides produced in a messenger-dependent, cell-free rabbit reticulocyte lysate primed with RNA prepared from a/alpha, a/a, and alpha/alpha isogenic diploids at different stages of sporulation . The availability of functional mRNA increased by about 25% during the first 4 h after transfer of either sporulating (a/alpha), or nonsporulating (a/a and alpha/alpha) diploids to sporulation medium . Thereafter functional mRNA decreased such that in the a/alpha strain after 24 h there was only about 50% of the amount in vegetative cells; a less marked decrease was observed in the a/a and alpha/alpha strains . Of 750 mRNA species detected, 43 underwent alterations only during sporulation in the a/alpha strain, whereas 36 changes were common to all three strains and one mRNA specific to alpha/alpha vegetative cells was detected . Only four of the sporulation-specific changes were due to the de novo appearance of translatable species, and two of these became predominant species of the total population . The majority of the specific changes were due to either permanent or transient increases in the concentration of individual mRNA species; 11 decreases were found . Changes were found at most stages of sporulation, although many occurred in either of two stages, one early (before 2 h) and the other later (between 6 and 8 h) when commitment to meiotic segregation was beginning . The results provide evidence for both quantitative and, to a lesser extent, qualitative transcriptional control of gene expression during sporulation.

Mol Cell Biol, 1984 Apr, 4(4), 591 - 8
Repetitive Dictyostelium heat-shock promotor functions in Saccharomyces cerevisiae; Cappello J et al.; The Dictyostelium genome contains 40 copies of a 4.7-kilobase repetitive and apparently transposable DNA sequence (DIRS-1) and about 250 smaller elements that appear to be deletions or rearrangements of DIRS-1 . Transcripts of these sequences are induced during differentiation and also by heat shock treatment of growing cells . We showed that one such cloned element, pB41.6 (2.5 kilobases) contains a nucleotide sequence identical to the Drosophila consensus heat shock promotor . To test whether this sequence might indeed control the expression of DIRS-1-related RNAs, we have cloned this genomic segment into yeast cells . In yeast cells, 41.6 directs synthesis of a 1.7-kilobase RNA that is induced at least 10-fold by heat shock . Transcription initiates at about 124 bases 3' of the putative promotor sequence and terminates within the 41.6 insert . A 381-base-pair subclone that contains the putative promotor sequence is sufficient to induce the heat shock response of 41.6 in yeast cells.

J Cell Biol, 1984 Apr, 98(4), 1185 - 93
Specific early-G1 blocks accompanied with stringent response in Saccharomyces cerevisiae lead to growth arrest in resting state similar to the G0 of higher eucaryotes; Iida H et al.; Growth arrests of Saccharomyces cerevisiae cells in early G1 phase brought by various means were classified into two types according to the mode of growth recovery after release of the restraints against growth . The first type, including arrests caused by cdc25, cdc33, cdc35, and ils1 mutations at the nonpermissive temperature and also by sulfur starvation, showed a subsequent delay in the onset of budding when shifted back to permissive conditions . The length of the delay was positively correlated with the time that cells had been arrested . The second type, including those caused by cdc28 and cdc24 mutations and by alpha factor, did not affect the mode of growth recovery after the shift to permissive conditions irrespective of the time that cell proliferation had been restricted . Growth arrests of the first type seem to allow yeast cells to enter a resting state equivalent to the G0 state of higher eucaryotes because features of the G0 shown with lymphocytes and other cultured cells including unusually long delay before the growth recovery (L.H . Augenlicht and R . Baserga, 1974, Exp . Cell Res., 89:255-262; and Kumagai, J., H . Akiyama, S . Iwashita, H . lida, and I . Yahara, 1981, J . Immunol., 126:1249-1254) appeared to be associated with this type . We have noted that arrests of the first type were always accompanied with a stringent response of macromolecular synthesis and its partial release by cycloheximide . Mapping of arrest points along the path of the cell cycle by the reciprocal shift experiment suggested that arrest points in G1 that led to the G0-like arrest precede or are near the step sensitive to alpha-factor.

J Bacteriol, 1984 Apr, 158(1), 29 - 35
Saccharomyces cerevisiae mutants provide evidence of hexokinase PII as a bifunctional enzyme with catalytic and regulatory domains for triggering carbon catabolite repression; Entian KD et al.; A selection system has been devised for isolating hexokinase PII structural gene mutants that cause defects in carbon catabolite repression, but retain normal catalytic activity . We used diploid parental strains with homozygotic defects in the hexokinase PI structural gene and with only one functional hexokinase PII allele . Of 3,000 colonies tested, 35 mutants (hex1r) did not repress the synthesis of invertase, maltase, malate dehydrogenase, and respiratory enzymes . These mutants had additional hexokinase PII activity . In contrast to hex1 mutants (Entian et al., Mol . Gen . Genet . 156:99-105, 1977; F.K . Zimmermann and I . Scheel, Mol . Gen . Genet . 154:75-82, 1977), which were allelic to structural gene mutants of hexokinase PII and had no catalytic activity (K.-D . Entian, Mol . Gen . Gent . 178:633-637, 1980), the hex1r mutants sporulated hardly at all or formed aberrant cells . Those ascospores obtained were mostly inviable . As the few viable hex1r segregants were sterile, triploid cells were constructed to demonstrate allelism between hex1r mutants and hexokinase PII structural gene mutants . Metabolite concentrations, growth rate, and ethanol production were the same in hex1r mutants and their corresponding wild-type strains . Recombination of hexokinase and glucokinase alleles gave strains with different specific activities . The defect in carbon catabolite repression was strongly associated with the defect in hexokinase PII and was independent of the glucose phosphorylating capacity . Hence, a secondary effect caused by reduced hexose phosphorylation was not responsible for the repression defect in hex1 mutants . These results, and those with the hex1r mutants isolated, strongly supported our earlier hypothesis that hexokinase PII is a bifunctional enzyme with (i) catalytic activity and (ii) a regulatory component triggering carbon catabolite repression (Entian, Mol . Gen . Genet . 178:633-637, 1980; K.-D . Entian and D . Mecke, J . Biol . Chem . 257:870-874, 1982).

Genetics, 1984 Apr, 106(4), 577 - 89
Semidominance of rad18-2 for several phenotypic characters in Saccharomyces cerevisiae; Mayer VW et al.; Inbred diploid yeast strains heterozygous or homozygous for the rad18-2 allele and carrying markers for detection of mitotic recombination were constructed . The homozygous rad18-2/rad 18-2 strain was highly sensitive to killing by UV light, showed greatly elevated frequencies of spontaneous and induced mitotic recombination and was more sensitive to trimethoprim than the wild-type diploid . The heterozygous strain RAD18/rad 18-2 was intermediate in its response for these same phenotypic characters . These findings are discussed in the light of other studies in which incomplete dominance of genes involved in some aspect of DNA repair has been reported.

Dev Biol, 1984 Apr, 102(2), 438 - 51
Initiation of meiosis and sporulation of Saccharomyces cerevisiae by sulfur or guanine deprivation; Freese EB et al.; Homothallic Saccharomyces cerevisiae, growing exponentially in a synthetic acetate medium, could be initiated to undergo meiosis and subsequent sporulation by removal of sulfur from the medium or by partial purine deprivation of purine auxotrophs or, most efficiently, by guanine deprivation of a guanine auxotroph . In contrast, partial uracil deprivation of uracil auxotrophs did not cause sporulation . Under any of the above and other sporulation conditions, the intracellular concentrations of GTP and, usually at some time later, S-adenosylmethionine (SAM) decreased; the concentrations of the other nucleoside triphosphates decreased under some but increased under other sporulation conditions . The addition of 1 mM methionine or, more effectively, of SAM or the combination of adenine plus methionine greatly increased the intracellular concentration of SAM and reduced or prevented sporulation, even when GTP decreased . However, differentiation can be inhibited by an excess of many metabolites which do not specifically control the initiation process; in particular, SAM is known to inhibit yeast metabolism (e.g., transamination) . Therefore, we cannot yet decide whether the deficiency of GTP or SAM (or related compounds) serves as a signal for the initiation of meiosis/sporulation.

Exp Cell Res, 1984 Apr, 151(2), 542 - 56
Synchronous cell growth occurs upon synchronizing the two regulatory steps of the Saccharomyces cerevisiae cell cycle; Moore SA; There are two known asynchronous steps in the budding yeast Saccharomyces cerevisiae cell cycle, where an asynchronous step is one which is completed in different lengths of time by different cells in an isogenic population . It is shown here that elimination of the asynchrony due to cell size by preincubation of cells with the mating pheromone alpha-factor, and decreasing the asynchrony in the cdc28 'start' step by lowering the pH, yields highly synchronous cell growth measured as the time period between the emergence of buds . In one experiment, cell budding for 92% of cells occurred within a 12-min period for at least two generations . Under identical conditions, cell number increase is not as synchronous as bud emergence indicating that there is a third asynchronous step, which is concluded to be at cell separation . These results are consistent with there being two--and only two--asynchronous steps in the cell cycle, measured from bud emergence to bud emergence . Surprisingly, these two steps are also the two major regulatory steps of the cell cycle . It is concluded that asynchrony may be a general feature of cell cycle regulatory steps . The asynchrony in the completion of the cdc28 'start' step which occurs in the first cell cycle after alpha-factor washout is shown here to be almost or entirely eliminated for the second passage through this step after alpha-factor washout . The 'true' time between the onset of budding and the point where 50% of cells have budded (called t50BE) is 17 and less than or equal to 2 min for the first and second budding, respectively, after alpha-factor washout . The cell cycle models requiring a transition probability, or asynchrony, at 'start' for every cell cycle are therefore incorrect.

Proc Natl Acad Sci U S A, 1984 Apr, 81(8), 2431 - 4
Ty-mediated gene expression of the LYS2 and HIS4 genes of Saccharomyces cerevisiae is controlled by the same SPT genes; Simchen G et al.; Five Ty insertion mutations were isolated at the LYS2 locus of Saccharomyces cerevisiae . Genetic and physical analyses show that four Ty insertions are in the 5' noncoding region of LYS2 and one is within the structural gene . Three of these Ty elements have been cloned and characterized . The Ty mutations differ from each other in restriction pattern, phenotypic effects on LYS2, reversion frequency, and the nature of reversion events . Spt2 and spt3 mutations, known to suppress Ty insertions and their solo delta derivatives at HIS4, can also suppress at least one of the Ty insertions (Ty61) at LYS2 and can also suppress the Lys- phenotype of a solo delta derivative of another Ty insertion (Ty128) at LYS2 . These results demonstrate that spt mutations can suppress Ty and delta mutations at both HIS4 and LYS2, suggesting that they are general for their effects on Ty and delta elements.

J Bacteriol, 1984 Apr, 158(1), 94 - 101
Physiology of a temperature-sensitive mutant of Saccharomyces cerevisiae defective in phosphofructokinase activity; Banerjee S et al.; In this paper, we describe a temperature-sensitive mutant of the yeast Saccharomyces cerevisiae (P5-9) which at a restrictive temperature (36 degrees C) shows a pleiotropic defect for transport of many different metabolites . The temperature sensitivity of the mutant is closely related to a reduction in phosphofructokinase activity . This conclusion is based on the following criteria . (i) Both the primary isolate, designated P5-9 (ts {rho-} Ino-), which is an inositol auxotroph and respiration deficient, and a purified derivative, SB4 (ts {rho+} Ino+ ), which is respiration competent and capable of growing in the absence of inositol, are temperature sensitive for growth and ethanol production in media containing glucose or fructose as the sole carbon source . (ii) The respiration-competent derivative SB4 is not temperature sensitive in media containing glycerol or glycerol-pyruvate; glucose inhibits its growth at 36 degrees C in these media . (iii) Assays of glycolytic enzymes in P5-9 and SB4 extracts, prepared from cells incubated for 1 to 2 h at 36 degrees C before harvesting, show selective reduction in phosphofructokinase activity . Analysis of tetrads derived from the cross of mutant and nonmutant haploids indicates that temperature sensitivity for growth is due to a single gene or to two closely linked genes . The biochemical analysis of spores from seven such tetrads revealed a uniform cosegregation of temperature sensitivity for growth and phosphofructokinase activity . Transport and ATP levels were drastically reduced in SB4 cells incubated at 36 degrees C for 1 to 2 h with glucose as the carbon source, but not when glycerol-pyruvate or lactate was the energy source . Therefore, depletion of energy as a result of phosphofructokinase inactivation appears to be the cause of the pleiotropic transport defect observed in the mutant.

FEBS Lett, 1984 Mar 26, 168(2), 245 - 8
Quantitative characterization of pyrimidine dimer excision from UV-irradiated DNA (excision capacity) by cell-free extracts of the yeast Saccharomyces cerevisiae; Bekker ML et al.; Cell-free extracts from wild-type yeast (RAD+) and from rad mutants belonging to the RAD3 epistatic group (rad1-1, rad2-1, rad3-1, rad4 -1) contain activities catalyzing the excision of pyrimidine dimers (PD) from purified ultraviolet-irradiated DNA which was not pre-treated with exogenous UV-endonuclease . The level of these activities in cell-free extracts from rad mutants did not differ from that in wild-type extract and was close to the in vivo excision capacity of the latter calculated from the LD37 (about 10(4) PD per haploid genome).

What Is Pcr?, What Is MIC?, What Is Staphylococcus Aureus?, What Is Anthrax?, What Is Genome?, a, Microorganisms, a, Microbes, c, Microbe, n, Bacteriology, n, Bacterium, s, Streptococcal, s, Bacillus, i, Lactobacillus, i, Antibiotic prophylaxis, o, Meningococcus, s, Staphylococcus, c, Antibiotics, i, Bacteriological, s, Sepsis, c, Campylobacter, c, Acinetobacter, n, Pathogenic bacterium, e, Yeasts, n, Antibiotics, e, Enterococci, a, Escherichia coli, c, S. cerevisiae, a, Escherichia coli, c, Escherichia coli, a, Escherichia coli, n, Yeasts

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005