S. cerevisiae

Carcinogenesis, 1986 Jul, 7(7), 1127 - 30
Specific inhibitors of the monooxygenase system of Saccharomyces cerevisiae modified the mutagenic effect of 4-nitroquinoline 1-oxide and the deethylation activity of the yeast; Del Carratore R et al.; A form of cytochrome P-450 is produced in Saccharomyces cerevisiae strain D7 during the logarithmic growth phase in 20% glucose liquid medium . This form was inhibited by metyrapone, tetrahydrofuran and by carbon monoxide, specific inhibitors of cytochrome P-450 in mammals . The inhibition was observed by means of the increase of the genetic activity of 4-nitroquinoline 1-oxide (4-NQO) on logarithmic growth phase cells of D7 strain, adding the inhibitors to the incubation mixture . 4-NQO is a strong direct mutagen on stationary phase cells that is detoxified by the monooxygenase system . The inhibition was measured by determining the decrease of the O-deethylation of 7-ethoxycoumarin in whole cells of yeast depending on different concentrations of inhibitors . A decrease of O-deethylation activity was found in the presence of tetrahydrofuran and metyrapone and this behaviour is typical of the cytochrome P-450 species induced by ethanol, as in mammals . Adding sodium phenobarbital to 0.5% glucose liquid medium, a form inhibited only by metyrapone was obtained . The presence of different inducible forms of cytochrome P-450 is evident.

Mol Cell Biol, 1986 Jul, 6(7), 2613 - 23
Saccharomyces cerevisiae PHO5 promoter region: location and function of the upstream activation site; Nakao J et al.; Saccharomyces cerevisiae repressible acid phosphatase (PHO5) is induced when inorganic phosphate in the culture medium is depleted . To study the mechanism of this regulation, we constructed various deletions in the 5'-flanking region of the PHO5 gene . Two elements were revealed by this analysis: an upstream activation site (UAS) and a downstream element, both playing parts in the expression of this gene . The UAS is located between -384 and -292 upstream of the initiation codon and activates expression of the gene when inorganic phosphate is depleted . It consists of two homologous regions (UAS I and UAS II) that contain CTGCACAAATG and an adenine-plus-thymine-rich sequence, either one of which suffices for the effect . The downstream element includes a putative TATA box at -100 from the ATG codon and is necessary for efficient transcription and expression of the normal-sized PHO5 transcript . The distance between the UAS and the downstream element can be altered without causing loss of expression efficiency, and the action of the UAS is not affected by its orientation . These results are consistent with a model wherein UAS acts as a site of activation for transcription by interaction with a protein factor(s) that becomes active when inorganic phosphate is depleted from the culture medium.

Mol Cell Biol, 1986 Jul, 6(7), 2490 - 9
PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors; Ammerer G et al.; The proteinase A structural gene of Saccharomyces cerevisiae was cloned by using an immunological screening procedure that allows detection of yeast cells which are aberrantly secreting vacuolar proteins (J . H . Rothman, C . P . Hunter, L . A . Valls, and T . H . Stevens, Proc . Natl . Acad . Sci . USA, 83:3248-3252, 1986) . A second cloned gene was obtained on a multicopy plasmid by complementation of a pep4-3 mutation . The nucleotide sequences of these two genes were determined independently and were found to be identical . The predicted amino acid sequence of the cloned gene suggests that proteinase A is synthesized as a 405-amino-acid precursor which is proteolytically converted to the 329-amino-acid mature enzyme . Proteinase A shows substantial homology to mammalian aspartyl proteases, such as pepsin, renin, and cathepsin D . The similarities may reflect not only analogous functions but also similar processing and intracellular targeting mechanisms for the two proteins . The cloned proteinase A structural gene, even when it is carried on a single-copy plasmid, complements the deficiency in several vacuolar hydrolase activities that is observed in a pep4 mutant . A strain carrying a deletion in the genomic copy of the gene fails to complement a pep4 mutant of the opposite mating type . Genetic linkage data demonstrate that integrated copies of the cloned proteinase A structural gene map to the PEP4 locus . Thus, the PEP4 gene encodes a vacuolar aspartyl protease, proteinase A, that is required for the in vivo processing of a number of vacuolar zymogens.

Mol Cell Biol, 1986 Jul, 6(7), 2443 - 51
Characterization and mutational analysis of a cluster of three genes expressed preferentially during sporulation of Saccharomyces cerevisiae; Percival-Smith A et al.; A differential hybridization screen of a genomic yeast DNA library previously identified 14 genes of Saccharomyces cerevisiae that are expressed preferentially during sporulation . Three of these sporulation-specific genes, SPS1, SPS2, and SPS3, have been shown to be closely linked . A mutational analysis has demonstrated that expression of the SPS1 gene, but not the SPS2 gene, is essential for the completion of sporulation . A diploid MATa/MAT alpha strain homozygous for a disruption of the SPS1 gene failed to form asci when subjected to sporulation conditions . The 3' end of the transcript encoded by the SPS1 gene was found to map only 185 base pairs from the 5' end of the SPS2 gene . The SPS1-SPS2 intergenic region was shown to contain all of the regulatory sequences necessary for the sporulation-specific activation of the SPS2 gene as assessed by expression of a translational SPS2-lacZ fusion gene present on a replicating, centromere-containing plasmid . The fusion gene was found to be expressed at the same time during sporulation as the chromosomal wild-type SPS2 gene.

Mol Cell Biol, 1986 Jul, 6(7), 2354 - 63
Fine-structure analysis of the DNA sequence requirements for autonomous replication of Saccharomyces cerevisiae plasmids; Bouton AH et al.; An autonomously replicating segment, ARS, is located 293 base pairs downstream from the histone H4 gene at the copy-I H3-H4 locus . The sequences needed for autonomous replication were defined by deletion analysis to include an ARS consensus sequence and an additional 3'-flanking region . External deletions into the 3'-flanking yeast sequences resulted in a loss of replication function . However, disruptions of the required 3'-flanking domain by either 10-base-pair linker-scanning substitutions or larger internal deletions did not impair autonomous replication . Thus, replication is dependent upon a flanking chromosome domain, but not an exact DNA sequence . The extent of the yeast sequences required in the 3'-flanking domain is variable depending on the nature of neighboring plasmid vector sequences . That is, there are certain vector sequences that prohibit replication when they are placed too close to the ARS consensus . These results suggest that the functional 3'-flanking domain of the H4 ARS is a specific DNA or chromatin structure or both.

Biochem Int, 1986 Jul, 13(1), 101 - 7
The nucleotide sequence of the mitochondrial DNA genome of an abundant petite mutant of Saccharomyces cerevisiae carrying the ori1 replication origin; Maxwell RJ et al.; We have determined the 903 bp nucleotide sequence of the mitochondrial DNA genome of a Saccharomyces cerevisiae petite mutant BB5 . This petite, containing the 265 nucleotide ori1 region, is representative of a class of petites arising at exceptionally high frequency within the population of spontaneous petites derived from a particular mit- strain Mb12 . The DNA sequences of both the ori1 region and the flanking intergenic regions have been compared to those of the corresponding regions of mtDNA in a previously reported petite strain, a1/1R/1 of Bernardi's laboratory, that has a similar (880 bp) repeat unit . The BB5 petite genome carries a canonical ori1 sequence that is identical in both petite mtDNAs, but the flanking intergenic sequences show significant differences between the two petite strains . The divergence is considered to arise from differences in the sequences flanking ori1 in the respective parent strains.

Genetics, 1986 Jul, 113(3), 531 - 50
Meiosis can induce recombination in rad52 mutants of Saccharomyces cerevisiae; Resnick MA et al.; The RAD52 and RAD50 genes have previously been shown to be required for normal meiotic recombination and for various types of recombination occurring in mitotic cells . Recent evidence suggests that rad52 mutants might be defective in an intermediate recombination step; we therefore examined recombination during meiosis in several rad52 mutants at several different loci and in genetic backgrounds that yield efficient sporulation and synchronous meiosis . Similar to previous reports, spores from rad52 diploids are inviable and meiotic recombination is greatly reduced by rad52 mutations . However, intragenic recombinants were detected when cells were plated on selective media during meiosis; rad52 mutants experience induction of recombination between homologues under these special conditions . The frequencies of recombination at four loci were considerably greater than the mitotic controls; however, they were still at least 20 times lower than corresponding Rad+ strains . The prototrophs induced by meiosis in rad52 mutants were not typical meiotic recombinants because incubation in nutrient-rich medium before plating to selective medium resulted in the complete loss of recombinants . We propose that previously observed single-strand breaks that accumulate in rad52 mutants may be associated with recombinational intermediates that are resolved when cells are returned to selective mitotic media and that the meiosis-induced recombination in rad52 cells does not involve double-strand breaks.

Arch Biochem Biophys, 1986 Jul, 248(1), 210 - 4
Role of exogenous hemin in the synthesis of hemoproteins and nonheme proteins during glucose repression in Saccharomyces cerevisiae; Gopalan G et al.; Exogenous addition of hemin to glucose-repressed cells of Saccharomyces cerevisiae stimulates the incorporation of amino acid into cytoplasmic proteins twofold . There was no significant change in the synthesis of total cytoplasmic RNA whereas a 40% increase in the synthesis of poly(A)-containing RNA was observed upon hemin treatment . Cell-free translation of cytoplasmic mRNAs and immunoprecipitation analysis of the translated products with antibodies against subunit V of cytochrome oxidase and the alpha and beta subunits of F1-ATPase reveals that there is an eightfold enrichment of the mRNA for subunit V of cytochrome oxidase upon hemin treatment . The effect of hemin on the alpha and beta subunits of F1-ATPase is only marginal, suggesting a differential role for heme in the synthesis of hemoproteins and nonheme proteins during glucose repression.

Nucleic Acids Res, 1986 Jun 25, 14(12), 4701 - 18
Cloning of the STA2 and SGA genes encoding glucoamylases in yeasts and regulation of their expression by the STA10 gene of Saccharomyces cerevisiae; Pardo JM et al.; The Saccharomyces STA2 and SGA genes, encoding the extracellular and intracellular sporulation-specific glucoamylase respectively, have been cloned and their transcription and regulation studied . The STA2 gene differs from the SGA gene in that it contains an extra piece of DNA, which encodes the domain for exportation of the extracellular glucoamylase . The STA2 gene produces a single 2.85 kb transcript . Transcription of the SGA gene is initiated from two different sites, yielding two transcripts of 1.95 and 2.40 kb . Transcription of both STA2 and SGA genes is repressed by the STA10 gene of Saccharomyces cerevisiae.

Experientia, 1986 Jun 15, 42(6), 607 - 8
Thiamine-binding activity of Saccharomyces cerevisiae plasma membrane; Nishimura H et al.; The specific binding activity to {14C}thiamine was found to be located in hte plasma membrane of Saccharomyces cerevisiae . The activity was inhibited by several thiamine analogs and it was hardly detectable in the plasma membrane from a thiamine transport mutant of Saccharomyces cerevisiae . Some properties of the thiamine-binding activity of yeast plasma membrane are discussed in connection with those of the thiamine transport system.

J Biol Chem, 1986 Jun 5, 261(16), 7558 - 65
Secretion of somatostatin by Saccharomyces cerevisiae . Correct processing of an alpha-factor-somatostatin hybrid; Green R et al.; Somatostatin is a 14-amino acid peptide hormone that is proteolytically processed from its precursor, prosomatostatin, by a paired-basic-specific protease localized in the Golgi apparatus and secretory vesicles . Yeast (Saccharomyces cerevisiae MAT alpha) synthesize an analogous peptide hormone precursor, pro-alpha-factor, that contains tandem repeats of alpha factor (13 amino acids) flanked by spacers that include paired basic residues . To investigate the role of these two pro regions in mediating intracellular transport and processing, cloned genes specific for preprosomatostatin and prepro-alpha-factor were used to generate recombinants encoding hybrids between the alpha-factor pro region (amino-terminal) and somatostatin (carboxyl-terminal) . These recombinants were inserted into yeast expression vectors under control of either the native alpha-factor promoter or the inducible yeast PHO5 (acid phosphatase) promoter . Yeast transformed with these plasmids expressed the hybrid messenger RNAs constitutively (alpha-factor promoter) or when induced in phosphate-deficient medium (PHO5 promoter) . Radioimmunoassay of culture media revealed the secretion of up to 200 ng of immunoreactive somatostatin/10(7) cells . Metabolic labeling with {35S}cysteine, followed by immunoprecipitation with anti-somatostatin antibodies revealed two forms of hybrid precursor intracellularly, one of Mr 25,000, containing core carbohydrates, and a second of Mr 11,000, which was unglycosylated . Translation of mRNA extracted from these transformants in the wheat germ cell-free system revealed that the Mr 11,000 form was the primary translation product, whereas the Mr 25,000 species could be generated in vitro by inclusion of mammalian rough microsomes . The secreted immunoreactive material was shown to be authentic somatostatin by high pressure liquid chromatography analysis and protein sequencing . These results demonstrate that the yeast processing enzymes recognize these chimeric precursors, resulting in the secretion of the mature peptide hormone.

Eur J Biochem, 1986 Jun 2, 157(2), 297 - 301
Metabolism of 2-oxoaldehydes in yeasts . Possible role of glycolytic bypath as a detoxification system in L-threonine catabolism by Saccharomyces cerevisiae; Murata K et al.; L-Threonine catabolism by Saccharomyces cerevisiae was studied to determine the role of glycolytic bypath as a detoxyfication system of 2-oxoaldehyde (methylglyoxal) formed from L-threonine catabolism . During the growth on L-threonine as a sole source of nitrogen, a large amount of aminoacetone was accumulated in the culture . The enzymatic analyses indicated that L-threonine was converted into either acetaldehyde and glycine by threonine aldolase or 2-aminoacetoacetate by NAD-dependent threonine dehydrogenase . Glycine formed was condensed with acetyl-CoA by aminoacetone synthase to form 2-aminoacetoacetate, a labile compound spontaneously decarboxylated into aminoacetone . The enzyme activities of the glycolytic bypath of the cells grown on L-threonine were considerably higher than those of the cells grown on ammonium sulfate as a nitrogen source . The result indicated the possible role of glycolytic bypath as a detoxification system of methylglyoxal formed from L-threonine catabolism.

Mol Cell Biol, 1986 Jun, 6(6), 2185 - 97
Isolation and functional analysis of sporulation-induced transcribed sequences from Saccharomyces cerevisiae; Gottlin-Ninfa E et al.; Strains of the yeast Saccharomyces cerevisiae that are heterozygous for the mating-type locus (MATa/MAT alpha) undergo meiosis and spore formation when they are starved for nitrogen and are provided with a nonfermentable carbon source such as potassium acetate . Haploids and diploids homozygous for the mating-type locus (MAT alpha/MAT alpha or MATa/MATa) are asporogenous and undergo neither meiosis nor spore formation when incubated under the same conditions . A small number of genes produce transcripts that appear to be induced specifically in sporulating cells . These transcripts either are not found or are present at much lower levels both in vegetatively growing cells and in cells from asporogenous strains that have been incubated in sporulation medium . Several genes complementary to these MATa/MAT alpha-dependent sporulation-induced transcripts were isolated from a gene-size insert yeast-lambda recombinant DNA library, by differential-plaque filter hybridization . An attempt was made to determine the function of three of these genes by mutating them in the yeast genome with in vitro mutagenesis and one-step gene replacement techniques . One gene was extensively disrupted by both a 0.3-kilobase deletion and the insertion of two large DNA sequences at different sites within the gene . Surprisingly, this compound mutation did not appear to affect meiosis or the production of viable ascospores, indicating that this gene was dispensable for differentiation . The other two genes were disrupted by simple insertion mutations at a site where it was possible that they might still possess some gene activity . These mutations also did not appear to affect sporulation . These results suggest that not all sporulation-induced genes are essential for meiosis and the production of viable ascospores under the conditions examined.

Mol Cell Biol, 1986 Jun, 6(6), 1855 - 65
Reciprocal regulation of the tandemly duplicated PHO5/PHO3 gene cluster within the acid phosphatase multigene family of Saccharomyces cerevisiae; Tait-Kamradt AG et al.; We characterized the organization and expression of PHO5 and PHO3, the tightly linked repressible and constitutive acid phosphatase genes of Saccharomyces cerevisiae . The "constitutive" gene, PHO3, is expressed only when PHO5 is not . Altering PHO5 expression, either through promoter deletions or through mutations in trans-acting regulatory genes, showed that PHO5 expression is sufficient to block transcription of PHO3 . An active genomic copy of PHO5 was able to block expression of PHO3 from a high-copy-number plasmid, showing that some trans-acting product of PHO5 is involved . This is probably a translation product, since the presence of a nontranslatable PHO5 RNA did not inhibit transcription of PHO3.

Arch Microbiol, 1986 Jun, 145(1), 27 - 31
Analysis of the energy metabolism after incubation of Saccharomyces cerevisiae with sulfite or nitrite; Hinze H et al.; After addition of 5 mM sulfite or nitrite to glucose-metabolizing cells of Saccharomyces cerevisiae a rapid decrease of the ATP content and an inversely proportional increase in the level of inorganic phosphate was observed . The concentration of ADP shows only small and transient changes . Cells of the yeast mutant pet 936, lacking mitochondrial F1 ATPase, after addition of 5 mM sulfite or nitrite exhibit changes in ATP, ADP and inorganic phosphate very similar to those observed in wild type cells . They key enzyme of glucose degradation, glyceraldehyde-3-phosphate dehydrogenase was previously shown to be the most sulfite- or nitrite-sensitive enzyme of the glycolytic pathway . This enzyme shows the same sensitivity to sulfite or nitrite in cells of the mutant pet 936 as in wild type cells . It is concluded that the effects of sulfite or nitrite on ATP, ADP and inorganic phosphate are the result of inhibition of glyceraldehyde-3-phosphate dehydrogenase and not of inhibition of phosphorylation processes in the mitochondria . Levels of GTP, UTP and CTP show parallel changes to ATP . This is explained by the presence of very active nucleoside monophosphate kinases which cause a rapid exchange between the nucleoside phosphates . The effects of the sudden inhibition of glucose degradation by sulfite or nitrite on levels of ATP, ADP and inorganic phosphate are discussed in terms of the theory of Lynen (1942) on compensating phosphorylation and dephosphorylation in steady state glucose metabolizing yeast.

Mol Gen Genet, 1986 Jun, 203(3), 538 - 43
Regulation of the RAD6 gene of Saccharomyces cerevisiae in the mitotic cell cycle and in meiosis; Kupiec M et al.; The regulation of the RAD6 gene at the mRNA level was investigated . The level of steady state RAD6 mRNA increases once every cell cycle, at late S/early G2 . This stage is the one at which rad6 mutants arrest, as do wild-type cells exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS), or cdc40 cells exposed to the restrictive temperature . This appears to be a repair-specific stage in the cell cycle . RAD6 mRNA levels increase when cells are treated with MMS, but this increase seems to be due to the arrest of the cells by MMS at the repair-specific stage; cells arrested at the same stage by HU or by the cdc40 lesion also show high levels of RAD6 mRNA . A much smaller increase in the level of RAD6 transcripts is seen following UV irradiation . During meiosis, RAD6 mRNA is more abundant before commitment to recombination . The differential increase of RAD6 mRNA during the S/G2 repair-specific stage of the cell cycle relates the RAD6 function to the normally occurring radioresistance found at this stage.

Proc Natl Acad Sci U S A, 1986 Jun, 83(12), 4433 - 7
In vitro L-A double-stranded RNA synthesis in virus-like particles from Saccharomyces cerevisiae; Fujimura T et al.; Most strains of Saccharomyces cerevisiae harbor L-A double-stranded RNA (dsRNA), 4.5 kilobases long, contained in virus-like particles (VLPs) . These L-A VLPs can be separated by CsCl density gradient centrifugation into a main peak of particles, containing full-length L-A dsRNA, which synthesizes only plus-strand single-stranded RNA (ssRNA), and a lighter fraction of VLPs, containing plus-strand ssRNA, which has L-A dsRNA-synthesizing activity . This dsRNA-synthesizing activity was present in particles from logarithmically growing cells but not from stationary-phase cells . The newly synthesized strand of dsRNA in the lightest particles was full-length minus strand . All or almost all of the new minus strand was synthesized in vitro, and the rate of chain elongation was approximately 100 nucleotides per minute . The lightest particles synthesized plus-strand ssRNA only after completion of dsRNA synthesis, indicating that the same particle contains dsRNA- and ssRNA-synthesizing enzyme(s) . We also observed dsRNA-synthesizing activity in L-BC dsRNA-containing particles similar to that in L-A VLPs.

Proc Natl Acad Sci U S A, 1986 Jun, 83(12), 4152 - 6
Carbon source regulation of RAS1 expression in Saccharomyces cerevisiae and the phenotypes of ras2- cells; Breviario D et al.; Transcriptional analysis of the yeast RAS genes in different culture conditions suggests that the inability of ras2 mutants to grow in nonfermentable carbon sources results from the regulation of RAS1 mRNA expression . The amount of RAS1 mRNA is significantly repressed in cultures grown on the nonfermentable carbon sources ethanol and acetate . As a result, low RAS function should be expressed under these conditions in a ras2 mutant . This can explain the inability of ras2- cells to grow on nonfermentable carbon sources . This interpretation is supported by the finding that an extragenic suppressor of ras2- (sra6-15), which restores growth on ethanol or acetate, also leads to an increase in the amount of RAS1 mRNA under these conditions . The sra6-15 mutation does not alter the level of RAS1 mRNA in cells grown on glucose . The pattern of transcriptional regulation described for the RAS1 gene is not shared by RAS2, indicating differential control of the functionally homologous yeast RAS genes at the level of gene expression.

J Bacteriol, 1986 Jun, 166(3), 905 - 13
Selection by genetic transformation of a Saccharomyces cerevisiae mutant defective for the nuclear uracil-DNA-glycosylase; Burgers PM et al.; A coliphage M13 chimer containing the Saccharomyces cerevisiae TRP1 gene and ARS1 replication origin (mPY2) was grown on an ung- dut- strain of Escherichia coli . The resulting single-stranded phage DNA had 13% of thymine residues substituted by uracil . This DNA failed to transform a delta trp1 yeast strain to prototrophy . However, when a mutagenized yeast stock was transformed with uracil-containing single-stranded mPY2 DNA, unstable transformants were obtained . After plasmid segregation, about half of these were retransformed at a high frequency by uracil-containing single-stranded mPY2 DNA . In vitro, these mutants were defective for uracil-DNA-glycosylase activity . They were designated ung1 . Strains containing the ung1 mutation have an increased sensitivity to sodium bisulfite and sodium nitrite but a wild-type sensitivity to methyl methanesulfonate, UV light, and drugs that cause depletion of the thymidylate pool . They have a moderate mutator phenotype for nuclear but not for mitochondrial genes . A low mitochondrial uracil-DNA-glycosylase activity was demonstrated in the mutant strains.

J Bacteriol, 1986 Jun, 166(3), 1123 - 7
Isolation of Saccharomyces cerevisiae mutants constitutive for invertase synthesis; Trumbly RJ; A new method for detecting invertase activity in Saccharomyces cerevisiae colonies was used to screen for mutants resistant to catabolite repression of invertase . Mutations causing the highest level of derepression were located in two previously identified genes, cyc8 and tup1 . Several of the cyc8 mutations, notably cyc8-10 and cyc8-11, were temperature dependent, repressed at 23 degrees C, and derepressed at 37 degrees C . The kinetics of derepression of invertase mRNA in cyc8-10 cells shifted from 23 to 37 degrees C was determined by Northern blots . Invertase mRNA was detectable at 5 min after the shift, with kinetics of accumulation very similar to that of wild-type cells shifted from high-glucose to low-glucose medium . Assays of representative enzymes showed that many but not all glucose-repressible enzymes are derepressed in both cyc8 and tup1 mutants . cyc8 and tup1 appear to be the major negative regulatory genes controlling catabolite repression in yeasts.

Yeast, 1986 Jun, 2(2), 129 - 39
Cloning and sequencing of the PHO80 gene and CEN15 of Saccharomyces cerevisiae; Toh-e A et al.; The PHO80 gene, which is one of the regulatory genes exerting negative control in the pho system of Saccharomyces cerevisiae, was cloned . The 1.8 kb DNA fragment carrying the PHO80 gene was sequenced and one open reading frame large enough to encode 293 amino acids was found in the sequence . Northern blot analysis of poly(A)+-RNA isolated from cells grown under repressed and derepressed conditions revealed that (i) the size of the PHO80 message was around 1.4 kb, (ii) the expression of the PHO80 gene was not affected by the presence or absence of inorganic phosphate in the medium, and (iii) the expression of the PHO80 gene was not affected by pho2, pho4, pho81, or by pho80 itself . A centromere sequence was found downstream of the PHO80 coding region.

Yeast, 1986 Jun, 2(2), 101 - 8
Mechanism of inactivation of UDP-glucose 4-epimerase from Saccharomyces cerevisiae by D-xylose and L-arabinose; Carmenes RS et al.; In a previous paper (Carmenes et al., 1984) we reported that UDP-glucose 4-epimerase from Saccharomyces was inactivated both in vivo and in vitro (crude extracts) by L-arabinose or D-xylose . In this paper, we report that pure epimerase requires the presence of UMP or UDP to be inactivated by sugars and that the inactivation is due to the reduction of the epimerase NAD+, which is essential for epimerase activity . The inactivation rate is directly proportional to epimerase and sugar concentrations and hyperbolically proportional to UMP concentration . In situ experiments made with permeabilized cells showed that epimerase is inactivated in the same way when it is inside the cell . In vivo studies showed that epimerase is inactivated to a smaller extent when 1% D-galactose is present in the culture medium than when 1% ethanol is the main carbon source.

J Gen Microbiol, 1986 Jun, 132 ( Pt 6), 1541 - 6
Some properties of a Saccharomyces cerevisiae mutant resistant to 2-amino-4-methyl-5-beta-hydroxyethylthiazole; Iwashima A et al.; A mutant of Saccharomyces cerevisiae highly resistant to 2-amino-4-methyl-5-beta-hydroxyethylthiazole (2-aminohydroxyethylthiazole), an antimetabolite of 4-methyl-5-beta-hydroxyethylthiazole (hydroxyethylthiazole), has been isolated . Its resistance to 2-aminohydroxyethylthiazole was about 10(4) times that of the sensitive parent strain . The amount of thiamin synthesized in the cells of the resistant strain grown in minimal medium was less than half of that of the sensitive strain . The ability to synthesize thiamin from 2-methyl-4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine) and hydroxyethylthiazole in the resistant strain was low compared with that of the sensitive strain . These results were found to be due to a deficiency of hydroxyethylthiazole kinase in the resistant strain: in sonic extracts of cells the enzyme activity was only 0.67% of that of the sensitive strain . Although the cells of the sensitive strain could accumulate exogenous hydroxyethylthiazole in the form of hydroxyethylthiazole monophosphate, no significant uptake of hydroxyethylthiazole by the cells of the resistant strain was observed . The possibilities that 2-aminohydroxyethylthiazole monophosphate may be the actual inhibitor of the growth of Saccharomyces cerevisiae, and that hydroxyethylthiazole may not be involved in the pathway of de novo synthesis of thiamin via hydroxyethylthiazole monophosphate, are discussed.

Mol Cell Biol, 1986 Jun, 6(6), 2106 - 14
Multiple regulation of STE2, a mating-type-specific gene of Saccharomyces cerevisiae; Hartig A et al.; The Saccharomyces cerevisiae STE2 gene, which is required for pheromone response and conjugation specifically in mating-type a cells, was cloned by complementation of the ste2 mutation . Transcription of STE2 is repressed by the MAT alpha 2 gene product, so that the 1.4-kilobase STE2 RNA is detected only in a or mat alpha 2 strains, not in alpha or a/alpha cells . However, STE2 RNA levels are also increased by the mating pheromone alpha-factor and decreased in strains bearing mutations in the nonspecific STE4 gene . Regulation of STE2 expression in a cells is therefore achieved by several mechanisms.

Genetics, 1986 Jun, 113(2), 247 - 64
Suppressors of the ras2 mutation of Saccharomyces cerevisiae; Cannon JF et al.; Saccharomyces cerevisiae contains two members of the ras gene family . Strains with disruptions of the RAS2 gene fail to grow efficiently on nonfermentable carbon sources . This growth defect can be suppressed by extragenic mutations called sra . We have isolated 79 independent suppressor mutations, 68 of which have been assigned to one of five loci . Eleven additional dominant mutations have not been assigned to a specific locus . Some sra1 and SRA4 and all SRA3 mutations were RAS independent, allowing growth of yeast cells that lack a functional RAS gene . Mutations in sra1, SRA3, SRA4 and sra6 are linked to his6, ino1, met3 and ade6, respectively . Some sra mutants have pleiotropic phenotypes that affect glycogen accumulation, sporulation, viability, respiratory capacity and suppression of two cell-division-cycle mutations, cdc25 and cdc35 . The proposed functions of many of the suppressor genes are consistent with the model in which RAS activates adenylate cyclase.

Genetics, 1986 Jun, 113(2), 229 - 46
Genetic and molecular analysis of the GAL3 gene in the expression of the galactose/melibiose regulon of Saccharomyces cerevisiae; Torchia TE et al.; During the galactose adaptation period of a Saccharomyces cerevisiae strain bearing a naturally occurring gal3 allele, we found a longer induction lag and slower rate of accumulation of GAL10 and MEL1 RNAs compared to wild-type strains . A strain of genotype gal3 gal1 gal7 is noninducible for MEL1 gene expression, but this expression block is bypassed by overexpression of the GAL4 gene or by deletion of the GAL80 gene, either of which causes a constitutive phenotype . An otherwise wild-type strain that bears a chromosomal gal3 gene disruption mutation does not produce wild-type GAL3 RNA and exhibits induction comparable to a strain bearing the naturally occurring gal3 . Based on this array of results, we conclude that the GAL3 gene product executes its function at a point before GAL4 mediated transcription of the GAL1-10-7 and MEL1 genes . Thus, the data are consistent with the previously advanced hypothesis that the GAL3 gene product functions to synthesize the inducer or coinducer molecule . In experiments in which the presence of either the plasmid-carried cloned GAL3 gene or the plasmid-carried cloned GAL1-10-7 genes allows MEL1 induction of a gal3 gal1 gal7 cell, we find that loss of the plasmid results in the shutoff of MEL1 expression even when galactose is continuously present . Either GAL3 function or GAL1-10-7 functions are therefore required for both the initiation and the maintenance of the induced state . Since the strains bearing either the naturally occurring gal3 allele or the gal3 disruption (null) allele do induce, the plasmid loss experiments indicate the existence of two completely independent induction initiation-maintenance pathways, one requiring GAL3 function, the other requiring GAL1-10-7 function . Finally, Northern blot analysis reveals two major GAL3 transcripts that differ in size by approximately 500 nucleotides.

Proc Natl Acad Sci U S A, 1986 Jun, 83(11), 3639 - 43
Regulated expression of Sindbis and vesicular stomatitis virus glycoproteins in Saccharomyces cerevisiae; Wen DZ et al.; cDNAs encoding either the structural proteins (capsid and glycoproteins E1 and E2) of Sindbis virus or the glycoprotein of vesicular stomatitis virus (VSV) were fused to the Saccharomyces cerevisiae galactokinase gene (GAL1) promoter and inserted into a yeast shuttle vector . After addition of galactose to yeast transformed with this vector, 2.5-3% of total yeast protein synthesis was detected as virus proteins by specific anti-virus protein antibodies . In cells containing the Sindbis virus structural genes, the virus capsid protein was effectively released from the nascent polypeptide and two endoglycosidase H-sensitive glycoproteins were produced . One of these was identical in its gel mobility to E1 and the other appeared to be p62, a precursor to E2 . A low level of E1 protein was detected on the cell's surface membranes . A single molecular weight species of glycosylated VSV glycoprotein was produced and half of the total protein could be detected at the surface membranes of yeast . Addition of long mannose chains and acylation of the virus proteins with fatty acids were not observed . Formation of virus proteins was also examined in yeast secretory mutants; one of these (sec53) failed to glycosylate the virus proteins.

J Bacteriol, 1986 Jun, 166(3), 914 - 23
Nucleotide sequence, transcript mapping, and regulation of the RAD2 gene of Saccharomyces cerevisiae; Madura K et al.; We determined the nucleotide sequence, mapped the 5' and 3' mRNA termini, and examined the regulation of the RAD2 gene of Saccharomyces cerevisiae . A long open reading frame within the RAD2 transcribed region encodes a protein of 1,031 amino acids with a calculated molecular weight of 117,847 . A disruption of the RAD2 gene that deletes the 78 carboxyl terminal codons results in loss of RAD2 function . The 5' ends of RAD2 mRNA show considerable heterogeneity, mapping 5 to 62 nucleotides upstream of the first ATG codon of the long RAD2 open reading frame . The longest RAD2 transcripts also contain a short open reading frame of 37 codons that precedes and overlaps the 5' end of the long RAD2 open reading frame . The RAD2 3' mRNA end maps 171 nucleotides downstream of the TAA termination codon and 20 nucleotides downstream from a 12-base-pair inverted repeat that might function in transcript termination . Northern blot analysis showed a ninefold increase in steady-state levels of RAD2 mRNA after treatment of yeast cells with UV light . The 5' flanking region of the RAD2 gene contains several direct and inverted repeats and a 44-nucleotide-long purine-rich tract . The sequence T G G A G G C A T T A A found at position -167 to -156 in the RAD2 gene is similar to a sequence present in the 5' flanking regions of the RAD7 and RAD10 genes.

J Biol Chem, 1986 May 5, 261(13), 5858 - 65
Protein secretion from Saccharomyces cerevisiae directed by the prepro-alpha-factor leader region; Zsebo KM et al.; The Saccharomyces cerevisiae secretory process was studied by evaluating secretion efficiency, processing efficiency, and the efficiency of protein folding for hybrid proteins containing the yeast prepro-alpha-factor leader region . Secretion of three proteins, beta-endorphin, calcitonin, and a consensus alpha-interferon (IFN-Con1), were compared in terms of secretion efficiency into the culture medium, beta-Endorphin and calcitonin, both small proteins, were found to be efficiently secreted from logarithmically grown cells . In contrast, the larger IFN-Con1 accumulated in the periplasmic space and cell wall . The glycosylated, unprocessed prepro-alpha-factor/IFN-Con1 fusion protein was also found to be secreted into the culture medium . The presence of (Glu-Ala) dipeptides in the alpha-factor spacer peptide increased the efficiency of cleavage at Lys-Arg in the prepro-alpha-factor/IFN-Con1 protein fusion . Purified secreted IFN-Con1 was structurally characterized to determine the effect of passage through the yeast secretory pathway on the fidelity and efficiency of protein folding . The disulfide structure of the secreted protein was found to be identical with that reported for the native human alpha-interferons.

Eur J Biochem, 1986 May 2, 156(3), 579 - 87
Purification and properties of coproporphyrinogen oxidase from the yeast Saccharomyces cerevisiae; Camadro JM et al.; Coproporphyrinogen oxidase has been located in the cytosol of yeast cells . The enzyme was purified to homogeneity from a heme mutant strain exhibiting a high specific activity (15-20 enzyme units/mg soluble protein compared to 1-2 enzyme units/mg soluble protein of by the wild-type strain) . The final preparation was homogeneous as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (Mr = 35,000) and isoelectrofocusing (pI = 6.2) . Gel filtration on AcA 44 gave a relative molecular mass of 70,000 . N-terminal amino-acid sequence analysis revealed a single polypeptide chain . Thus the enzyme appears to be a dimer with identical subunits . Two iron atoms/molecule of native protein were detected; they could not be removed by exhaustive dialysis or gel filtration on Sephadex G-25 . However the involvement of the iron atoms in the oxidative catalytic activity of the enzyme was not demonstrated . The Km value for coproporphyrinogen was 0.05 microM . The enzyme was active only when molecular oxygen was used as electron acceptor; no anaerobic activity could be detected . Thiol-directed reagents partially inhibited the enzyme, indicating that an SH group is required for activity . Yeast coproporphyrinogen oxidase was activated by phospholipids or neutral detergents as described for the bovine liver enzyme.

Eur J Biochem, 1986 May 2, 156(3), 511 - 9
The nucleotide sequence of the HEM1 gene and evidence for a precursor form of the mitochondrial 5-aminolevulinate synthase in Saccharomyces cerevisiae; Urban-Grimal D et al.; The biosynthesis of yeast 5-aminolevulinate (ALA) synthase, a mitochondrial protein encoded by the nuclear HEM1 gene, has been studied in vitro in a cell-free translation system and in vivo in whole cells . In vitro translation of mRNA hybrid-selected by the cloned HEM1 gene, or of total RNA followed by immunoprecipitation with anti-(ALA synthase) antibody yielded a single polypeptide of higher molecular mass than the purified ALA synthase . This larger form, also seen in pulse-labeled cells, can be post-translationally processed by isolated mitochondria . These results show that the cytoplasmically made ALA synthase is synthesized with a cleavable extension which was estimated to be about 3.5 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis . The complete nucleotide sequence of the HEM1 gene and its flanking regions was determined . The 5' ends of the HEM1 mRNAs map from -76 to -63 nucleotides upstream of the translation initiation codon . The open reading frame of 1644 base pairs encodes a protein of 548 amino acids with a calculated Mr of 59,275 . The predicted amino-terminal sequence of the protein is strongly basic (five basic and no acidic amino acids within the first 35 residues), rich in serine and threonine and must represent the transient presequence that targets this protein to the mitochondria . Comparison of deduced amino acid sequences indicates a clear homology between the mature yeast and chick embryo liver ALA synthases.

Mol Cell Biol, 1986 May, 6(5), 1820 - 9
New positive and negative regulators for general control of amino acid biosynthesis in Saccharomyces cerevisiae; Greenberg ML et al.; The biosynthesis of most amino acids in Saccharomyces cerevisiae is coregulated . Starvation for a single amino acid results in the derepression of amino acid biosynthetic enzymes in many unrelated pathways . This phenomenon, known as general control, is mediated by both positive (GCN) and negative (GCD) regulatory genes . In this paper we describe the identification and characterization of several new regulatory genes for this system, GCN6, GCN7, GCN8, GCN9, and GCD5 . A mutation in the negative regulator GCD5 was isolated on the basis of its suppression of a gcn2 mutation . The effect of gcd5 is a posttranscriptional increase in histidine biosynthetic enzyme activity . Suppressors of gcd5 which are deficient in derepression were in turn isolated . Eight such mutations, defining four new positive regulatory genes (GCN6 through GCN9), were obtained . These mutations are recessive, confer sensitivity to multiple amino acid analogs, and result in decreased mRNA levels for genes under general control . The GCN6 and GCN7 gene products were shown to be positive regulators for transcription of the GCN4 gene, the most direct-acting positive regulator thus far identified . The interaction of GCN6 and GCN7 with GCN4 is fundamentally different from that of previously isolated GCN genes . It should also be noted that these gcn selections gave a completely different nonoverlapping set of mutations from earlier selections which relied on analog sensitivity . Thus, we may have identified a new class of GCN genes which are functionally distinct from GCN1 through GCN5.

Mol Cell Biol, 1986 May, 6(5), 1633 - 9
mRNA transcription in nuclei isolated from Saccharomyces cerevisiae; Jerome JF et al.; We developed an improved method for the isolation of transcriptionally active nuclei from Saccharomyces cerevisiae, which allows analysis of specific transcripts . When incubated with alpha-32P-labeled ribonucleoside triphosphates in vitro, nuclei isolated from haploid or diploid cells transcribed rRNA, tRNA, and mRNAs in a strand-specific manner, as shown by slot blot hybridization of the in vitro synthesized RNA to cloned genes encoding 5.8S, 18S and 28S rRNAs, tRNATyr, and GAL7, URA3, TY1 and HIS3 mRNAs . A yeast strain containing a high-copy-number plasmid which overproduced GAL7 mRNA was initially used to facilitate detection of a discrete message . We optimized conditions for the transcription of genes expressed by each of the three yeast nuclear RNA polymerases . Under optimal conditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3) . We determined that the alpha-amanitin sensitivity of transcript synthesis in the isolated nuclei paralleled the sensitivity of the corresponding purified RNA polymerases; in particular, mRNA synthesis was 50% sensitive to 1 microgram of alpha-amanitin per ml, establishing transcription of mRNA by RNA polymerase II.

Mol Cell Biol, 1986 May, 6(5), 1590 - 8
Molecular characterization of cell cycle gene CDC7 from Saccharomyces cerevisiae; Patterson M et al.; The product of the CDC7 gene of Saccharomyces cerevisiae appears to have multiple roles in cellular physiology . It is required for the initiation of mitotic DNA synthesis . While it is not required for the initiation of meiotic DNA replication, it is necessary for genetic recombination during meiosis and for the formation of ascospores . It has also been implicated in an error-prone DNA repair pathway . Plasmids capable of complementing temperature-sensitive cdc7 mutations were isolated from libraries of yeast genomic DNA in the multicopy plasmid vectors YRp7 and YEp24 . The complementing activity was localized within a 3.0-kilobase genomic DNA fragment . Genetic studies that included integration of the genomic insert at or near the CDC7 locus and marker rescue of four cdc7 alleles proved that the cloned fragment contains the yeast chromosomal CDC7 gene . The RNA transcript of CDC7 is about 1,700 nucleotides . Analysis of the nucleotide sequence of a 2.1-kilobase region of the cloned fragment revealed the presence of an open reading frame of 1,521 nucleotides that is presumed to encode the CDC7 protein . Depending on which of two possible ATG codons initiates translation, the calculated size of the CDC7 protein is 58.2 or 56 kilodaltons . Comparison of the predicted amino acid sequence of the CDC7 gene product with other known protein sequences suggests that CDC7 encodes a protein kinase.

Mol Cell Biol, 1986 May, 6(5), 1552 - 61
Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis; Esteban R et al.; Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA) . The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin) . Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated . Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light) . M1-H and M1-L VLPs are present together in the same strains and in all those we tested . M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle . Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis . We show that M1-H VLPs are formed in vitro from the M1-L VLPs . We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand . We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).

Mol Gen Genet, 1986 May, 203(2), 316 - 9
Immunologically related proteins in cytoplasmic and mitochondrial ribosomes of yeast Saccharomyces cerevisiae; Sudarickov AB et al.; Ribosomal proteins from the cytoplasm and mitochondria of the yeast Saccharomyces cerevisiae were compared by immunoblotting techniques . Antibodies raised against cytoplasmic ribosomal proteins cross-react with five mitochondrial ribosomal proteins, four of which are located in the large and one in the small mitochondrial subunits . The possible existence of common ribosomal proteins for cytoplasmic and mitochondrial ribosomes is discussed.

Mutagenesis, 1986 May, 1(3), 207 - 9
DNA ethylations induced by ethylnitrosourea in the wild type, cdc4 and cdc7 strains of Saccharomyces cerevisiae; Fox JC et al.; The experiments reported here have investigated the induction of ethylations to DNA in yeast cells exposed to the chemical mutagen ethylnitrosourea . A similar level of alkylation was seen at the N7 and O6 of guanine and at the N3 of adenine in either log phase cells or in temperature-sensitive cdc4 and cdc7 cells growth arrested at their specific G1 positions . Hence the changes in chromosome structure associated with the above cdc phenotypes do not modify the amount of DNA damage induced by ethylnitrosourea.

Mol Cell Biol, 1986 May, 6(5), 1812 - 9
Saccharomyces cerevisiae secretes and correctly processes human interferon hybrid proteins containing yeast invertase signal peptides; Chang CN et al.; Synthetic oligonucleotides coding for the yeast invertase secretion signal peptide were fused to the gene for the mature form of human interferon (huIFN-alpha 2) . Two plasmids (E3 and F2) were constructed . E3 contained the invertase signal codons in a reading frame with the mature huIFN-alpha 2 gene . F2 had a deletion of the codon for alanine at amino acid residue-5 in the invertase signal and an addition of a methionine codon located between the coding sequences for the invertase signal and mature huIFN-alpha 2 . Both hybrid genes were located adjacent to the promoter from the 3-phosphoglycerate kinase gene on the multicopy yeast expression plasmid, YEp1PT . Yeast transformants containing these plasmids produced somewhat more IFN than did the same expression plasmid containing the IFN gene with its human secretion signal sequence . HuIFN-alpha 2, purified from the medium of yeast cells containing E3, was found to be processed at the correct site . The huIFN-alpha 2 made by plasmid F2 was found to be completely processed at the junction between the invertase signal (a variant) and the methionine of methionine-huIFN-alpha 2 . These results strongly suggested that the invertase signal (or its variant) attached to huIFN was efficiently recognized by the presumed signal recognition particle and was cleaved by the signal peptidase in the yeast cells . These results also suggested that amino acid changes on the right side of the cleavage site did not necessarily prevent cleavage or secretion.

Mol Cell Biol, 1986 May, 6(5), 1571 - 8
Activation of a cryptic TACTAAC box in the Saccharomyces cerevisiae actin intron; Cellini A et al.; We constructed a translational fusion between the Saccharomyces cerevisiae actin gene and the Escherichia coli beta-galactosidase structural gene such that expression of beta-galactosidase activity required accurate splicing of the actin intron . Using this chimeric gene, we generated a series of internal deletions which removed the TACTAAC box or, in addition, TACTAAC-like sequences within the intron . Analysis of the fusion transcripts produced in these deletions allowed us to conclude that the TACTAAC-like sequence TACTAAG can substitute, albeit inefficiently, for the authentic TACTAAC box in the splicing process . These results indicate that the yeast splicing machinery can utilize a cryptic TACTAAC box, but there are requirements for primary sequence and proper position.

Mol Cell Biol, 1986 May, 6(5), 1497 - 507
RAD7 gene of Saccharomyces cerevisiae: transcripts, nucleotide sequence analysis, and functional relationship between the RAD7 and RAD23 gene products; Perozzi G et al.; The RAD7 gene of Saccharomyces cerevisiae was cloned on a 4.0-kilobase (kb) DNA fragment and shown to provide full complementation of a rad7-delta mutant strain . The nucleotide sequence of a 2.2-kb DNA fragment which contains the complete RAD7 gene was determined . Transcription of the RAD7 gene initiates at multiple sites in a region spanning positions -61 to -8 of the DNA sequence . The 1.8-kb RAD7 mRNA encodes a protein of 565 amino acids with a predicted size of 63.7 kilodaltons . The hydropathy profile of the RAD7 protein indicates a highly hydrophilic amino terminus and a very hydrophobic region toward the carboxyl terminus . A RAD7 subclone deleted for the first 99 codons complements the rad7-delta mutation, but not the rad7-delta rad23-delta double mutation, indicating that the RAD23 protein can compensate for the function that is missing in the amino-terminally deleted RAD7 protein . The RAD7 and RAD23 genes in multicopy plasmids do not complement the rad23-delta and rad7-delta mutations, respectively . These observations could mean that although the two proteins might share a common functional domain, they must also perform distinct functions . Alternatively, an interaction between the RAD7 and RAD23 proteins could also account for these observations.

J Gen Microbiol, 1986 May, 132 ( Pt 5), 1143 - 51
Control of the cAMP pathway by the cell cycle start function, CDC25, in Saccharomyces cerevisiae; Tripp ML et al.; We investigated the relationship in Saccharomyces cerevisiae between the cell cycle start function, CDC25, and two mutants defining components of the cAMP pathway . The thermolabile adenylate cyclase mutant cyr1-2(ts) is phenotypically similar to the temperature-sensitive mutant cdc25(ts) in that both mutants, when shifted to the restrictive temperature, arrest in G1 of the cell cycle and permit the initiation of meiosis and sporulation . The mutant bcy1 {a lesion resulting in a low level of regulatory (R) subunit and a high level of active, catalytic (C) subunit of the cAMP-dependent protein kinase} suppresses the temperature-sensitive phenotype of cyr1-2(ts) and confers an asporogenous phenotype . We found that cdc25(ts) complemented cyr1-2(ts), and, unlike cyr1-2(ts), was not suppressible by bcy1, demonstrating that CYR1 and CDC25 must encode different functions . Also our results indicate that CDC25 does not encode the R subunit of the cAMP-dependent protein kinase . In addition, although the cdc25(ts)bcy1 double mutant was temperature sensitive like cdc25(ts), we found that the cdc25(ts)bcy1 homozygous diploid was asporogenous like bcy1/bcy1 . The inability of the cdc25(ts)bcy1 double mutant to sporulate demonstrated that CDC25 does not encode the C subunit of the cAMP kinase, and indicated that the CDC25 function modulates the cAMP pathway to control meiosis and sporulation . Further, the temperature-sensitive phenotype of the double mutant, and hence the inability of bcy1 to suppress cdc25(ts), suggested that a second CDC25 cell cycle function exists which is independent of the cAMP pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1986 May, 203(2), 300 - 4
Intron mutations that affect the splicing efficiency of the CYH2 gene of Saccharomyces cerevisiae; Swida U et al.; To define the extent of intervening sequences required for efficient splicing of the CYH2 gene in Saccharomyces cerevisiae, we have constructed a series of intron mutations . Artificial intron extensions of more than 300 bp of the natural intron lead to an inhibition of splicing whereas intron deletions lead to a drastic improvement of the splicing efficiency . It is shown that deletion of a 32 bp sequence element within the intron is responsible for this drastic improvement.

EMBO J, 1986 May, 5(5), 843 - 7
Heme regulates the expression in Saccharomyces cerevisiae of chimaeric genes containing 5'-flanking soybean leghemoglobin sequences; Jensen EO et al.; The TM1 yeast mutant was transformed with a 2 micron-derived plasmid (YEp24) which carries a chimaeric gene containing the Escherichia coli chloramphenicol acetyl transferase (CAT) gene fused to the 5'- and 3'-flanking regions of the soybean leghemoglobin (Lb) c3 gene . Expression of the chimaeric CAT gene is controlled specifically by heme at a post-transcriptional level, most likely by regulating the efficiencies of translation . Expression of another chimaeric gene consisting of the neomycin phosphotransferase (NPTII) gene fused to only the 5'-flanking region of the Lbc3 gene is regulated by heme in a similar way . Thus, in yeast, heme modulates the translation of the chimaeric mRNAs through interactions with the 5' Lbc3 non-coding region.

Mutat Res, 1986 May, 160(3), 207 - 14
The rad2 mutation affects the molecular nature of UV and acridine-mustard-induced mutations in the ADE2 gene of Saccharomyces cerevisiae; Ivanov EL et al.; We have studied the molecular nature of ade2 mutations induced by UV light and bifunctional acridine-mustard (BAM) in wild-type (RAD) and in excision-deficient (rad2) strains of the yeast, Saccharomyces cerevisiae . In the RAD strain, UV causes 45% GC----AT transitions among all mutations; in the rad2 strain this value is 77% . BAM was shown to be highly specific for frameshift mutagenesis: 60% frameshifts in the RAD strain, and as many as 84% frameshifts in the rad2 strain were induced . Therefore, the rad2 mutation affects the specificity of UV- and BAM-induced mutagenesis in yeast . Experimental data agree with the view that the majority of mutations in the RAD strain are induced by a prereplicative mechanism, whereas mutations in the RAD strain are induced by a prereplicative mechanism, whereas mutations in the rad2 strain are predominantly postreplicative events . Our results also suggest that: cytosine-containing photoproducts are the substances responsible for major premutational damage to cytosine-containing photoproducts are the substances responsible for major premutational damage to DNA; a fraction of the mutations may arise in the course of excision repair of UV photoproducts.

Biochem Biophys Res Commun, 1986 Apr 29, 136(2), 596 - 602
Release of a lectin from a fatty acid auxotroph of Saccharomyces cerevisiae grown in presence of oleic acid; Basu J et al.; The unsaturated fatty acid-requiring mutant KD 115 of Saccharomyces cerevisiae secretes a lectin when grown in presence of oleic acid . This lectin is homogeneous on PAGE at pH 8.3, has an approximate molecular weight of 320,000, pI of 4.2 and contains about 60% sugar . It agglutinates chicken and different mammalian erythrocytes, but lyses rabbit red cells only . It is D-galactose-specific . To our knowledge, this is the first report of a hemagglutinin from yeast.

Eur J Biochem, 1986 Apr 15, 156(2), 413 - 21
Expression of polyoma virus middle-T antigen in Saccharomyces cerevisiae; Belsham GJ et al.; The polyoma middle-T gene, lacking its intron, was inserted into a yeast expression plasmid containing the phosphoglycerate kinase promoter . Such plasmids transformed yeast at low frequency and these transformants expressed middle-T antigen at a level of approximately 0.1% cell protein . Furthermore, expression of this protein was frequently lost during growth in liquid culture and this loss of middle-T was accompanied by a twofold increase in the rate of growth . The spontaneous production of a truncated middle-T antigen, lacking the C terminus, was also observed; the expression of this protein did not inhibit the growth rate of the cells . Recovery and analysis of the expression plasmids encoding the truncated molecule showed that a single C X G base pair had been deleted from a run of nine consecutive C X G base pairs (Pyr nucleotide 1239--1247) within the middle-T coding region . This frame-shift mutation results in premature termination of the protein and loss of the strongly hydrophobic region of the molecule believed to be responsible for the membrane association of middle-T antigen.

J Biol Chem, 1986 Apr 15, 261(11), 4789 - 96
Expression and secretion of biologically active human atrial natriuretic peptide in Saccharomyces cerevisiae; Vlasuk GP et al.; A hybrid gene was constructed containing a fusion between the DNA sequences encoding the secretory precursor of the yeast mating pheromone alpha-factor and a synthetic sequence encoding a biologically active 24-amino acid carboxyl-terminal portion of the human atrial natriuretic peptide (hANP) precursor . Transformation of Saccharomyces cerevisiae with the hybrid gene resulted in the yeast cells secreting biologically active hANP into the extracellular medium . The secreted hANP was purified and found to be accurately processed at the junction in the chimeric alpha-factor/hANP protein, producing the desired mature hANP amino terminus . The secreted product was also folded correctly with respect to the single disulfide bond . However, the carboxyl terminus of the secreted hANP material was heterogeneous such that the major form lacked the last two amino acids of the peptide while the minor form was the full length material . The observed processing at the carboxyl terminus of the secreted hANP may reflect a normal processing event involved in alpha-factor peptide maturation.

Biochim Biophys Acta, 1986 Apr 14, 856(2), 189 - 92
Reversible loss of affinity induced by glucose in the maltose-H+ symport of Saccharomyces cerevisiae; Peinado JM et al.; Glucose represses and inactivates maltose transport in Saccharomyces cerevisiae . The inactivation has been described as an irreversible process involving proteolysis . We have studied the inactivation of the maltose-H+ symport in this yeast and have observed that the mechanism of inactivation depends on the physiological conditions . In resting cells there was a decrease in transport capacity . The rate of decrease was enhanced nonspecifically by the presence of a sugar, glucose being more effective than maltose . In growing cells, glucose induced a decrease in affinity of the H+-symport which could be recovered by starvation, even in the presence of cycloheximide; there was no loss in capacity or, if present, this loss could be explained fully by the dilution due to repression during growth on glucose . We submit that in growing cells inactivation consists in a reversible modification of the permease not involving proteolysis.

Nucleic Acids Res, 1986 Apr 11, 14(7), 3059 - 73
Isolation, physical characterization and expression analysis of the Saccharomyces cerevisiae positive regulatory gene PHO4; Legrain M et al.; The Saccharomyces cerevisiae PHO4 gene, which positively controls the expression of phosphatase genes, has been isolated by complementation of a pho4 mutation . The isolated DNA directed integration at the chromosomal PHO4 locus . The nucleotide sequence of PHO4 has a coding region of 930 nucleotides, flanked by sequences with typical transcription initiation and termination signals . The 5' region has characteristics of low-expression promoters and carries several uncommon elements, whose significance is not known . The predicted primary structure of the PHO4 protein, of 309 residues, does not show sequence elements typical of DNA-binding proteins . The transcription of PHO4 is independent of inorganic phosphate . Like other regulatory genes, PHO4 is transcribed at a very low level and the translation of its message uses preferentially several codons which are not employed for highly expressed genes.

Nucleic Acids Res, 1986 Apr 11, 14(7), 2939 - 49
Isolation and characterization of a Ty element inserted into the ribosomal DNA of the yeast Saccharomyces cerevisiae; Vincent A et al.; The yeast Saccharomyces cerevisiae has about 30 to 50 copies of a transposable element Ty . Most of these elements are located at the 5' ends of protein coding sequences and are flanked by a 5 bp duplication . We report below an insertion of a Ty element into one of the repeated ribosomal RNA (rRNA) genes of yeast . The element is located between the 3' ends of the divergentally transcribed 37S and 5S rRNA's and is not flanked by a 5 bp duplication . In addition, one end of the Ty insertion is contiguous with a 306 bp deletion of the sequences of the rRNA gene . We find that this insertion, unlike most Ty insertions, is mitotically unstable.

J Biol Chem, 1986 Apr 5, 261(10), 4629 - 37
Nucleotide sequence of the Saccharomyces cerevisiae ADE3 gene encoding C1-tetrahydrofolate synthase; Staben C et al.; The sequence of a cloned copy of the yeast ADE3 gene, which encodes the trifunctional enzyme C1-5,6,7,8-tetrahydrofolate (THF) synthase, was determined . Yeast cells transformed with a multicopy yeast plasmid containing this ADE3 gene overexpress C1-THF synthase 20-60-fold relative to wild-type yeast cells . C1-THF synthase from transformed cells is identical with that isolated from wild-type cells by all the criteria tested . The translated DNA sequence and amino-terminal protein sequences are identical and the amino acid composition predicted from the DNA sequence agrees closely with that determined by hydrolysis of C1-THF synthase protein . Correlation of the genetic map of the ADE3 region and of proteolysis experiments with the protein sequence suggests locations for two functional domains within yeast C1-THF synthase . The sequence of C1-THF synthase does not appear to be homologous to any other sequenced protein, including other proteins that use folate substrates . The 5' and 3' untranslated regions of the ADE3 gene suggest initiation and termination signals similar to transcription signals associated with other yeast genes . No special regulatory features have been associated with the ADE3 sequence . An unusual open reading frame that is encoded by a very unbiased set of codons follows the ADE3 gene.

Mol Cell Biol, 1986 Apr, 6(4), 1352 - 6
The Saccharomyces cerevisiae chromosome III left telomere has a type X, but not a type Y', ARS region; Button LL et al.; A yeast Saccharomyces cerevisiae telomeric region was isolated by chromosome walking from HML alpha, the most distal known gene on the chromosome III left (IIIL) end . The terminal heterodisperse 3.3-kilobase (kb) SalI fragment on chromosome IIIL, 8.6 kb distal to HML alpha, was cloned in a circular vector to generate a telomeric probe . Southern hybridization and DNA sequencing analyses indicated that 0.6 kb (+/- 200 base pairs) of 5'-C1-3A-3' simple tandem repeat sequence, adjacent to a 1.2-kb type X ARS region, constitutes the telomere on the chromosome IIIL end, and no type Y' ARS region homologies exist between HML alpha and the IIIL terminus.

Mol Cell Biol, 1986 Apr, 6(4), 1158 - 63
Tandemly duplicated upstream control sequences mediate copper-induced transcription of the Saccharomyces cerevisiae copper-metallothionein gene; Thiele DJ et al.; Transcription of the Saccharomyces cerevisiae copper-metallothionein gene, CUP1, inducible by copper . By analyzing deletion and fusion mutants in the CUP1 5'-flanking region, we identified two closely related, tandemly arranged copper regulatory elements . A synthetic version of one of these elements conferred efficient copper induction on a heterologous promoter when present in two tandem copies.

Mol Cell Biol, 1986 Apr, 6(4), 1148 - 57
Electron microscopic study of Saccharomyces cerevisiae rDNA chromatin replication; Saffer LD et al.; An electron microscopic study was made of the replication of rDNA chromatin of Saccharomyces cerevisiae . Two different methods were used to synchronize cells . cdc7-1 cells were raised to a restrictive temperature, whereas A364a cells were blocked with mating factor . Replication bubbles typically opened in the nontranscribed spacers of rDNA repeats in both cell types . The mean position of the center of these bubbles corresponds closely to a position where an autonomously replicating sequence previously has been mapped in an rDNA repeat . Clusters of replication bubbles containing up to four bubbles spaced one to three genes apart were seen opening in early S phase.

J Gen Microbiol, 1986 Apr, 132 ( Pt 4), 979 - 88
Isolation and characterization of Ca2+-sensitive mutants of Saccharomyces cerevisiae; Ohya Y et al.; Thirty Ca2+-sensitive (cls: calcium sensitive) mutants of Saccharomyces cerevisiae were isolated by replica-plating . These mutants, which each had a single recessive chromosomal mutation, were divided into 18 complementation groups . Some cls mutants showed a phenotype of specific sensitivity to Ca2+, while others showed phenotypes of sensitivities to several divalent cations . From measurements of the calcium contents and initial rates of Ca2+ uptake of the cls mutants, 16 of the 18 cls complementation groups were classified into four types: type I mutants (cls5, cls6, cls13, cls14, cls15, cls16, cls17, and cls18) had both elevated calcium contents and increased uptake activities . A type II mutant (cls4) had a normal calcium content and normal uptake activity; type III mutants (cls1, cls2 and cls3) had elevated calcium contents but normal initial rates of Ca2+ uptake; type IV mutants (cls8, cls9, cls10 and cls11) had normal calcium contents but increased initial rates of Ca2+ uptake . Two of the mutants (cls7 and cls12) had intermediate biochemical properties . The primary defects of these four types of cls mutants were considered in terms of the Ca2+ transport system(s) . Both type I and type III mutants, which had elevated calcium contents, simultaneously showed a trifluoperazine-sensitive phenotype, suggesting a close correlation of this phenotype with elevated calcium content . In addition, all type IV mutants were unable to utilize nonfermentable sugars . One CLS gene, CLS7, was located on the left arm of chromosome V.

DNA, 1986 Apr, 5(2), 129 - 36
Expression of cloned human haptoglobin and alpha 1-antitrypsin complementary DNAs in Saccharomyces cerevisiae; van der Straten A et al.; Nucleotide sequences coding either for human preprohaptoglobin or for prohaptoglobin have been placed under the control of yeast ARG3 expression signals . Recombinant plasmids pRIT12598 and pRIT12597 express the prepro- and the pro-form of alpha 2 beta haptoglobin respectively, but at very low levels . Comparison with the expression of human pre- and mature alpha 1-antitrypsin cDNAs, cloned in the same expression vector, reveals large differences in the levels of specific proteins produced in yeast, although specific mRNA levels are similar . It is shown that presence or absence of the signal sequence in the cDNA construction results in a 20- to 30-fold difference in the yields of heterologous products . However, since haptoglobin and alpha 1-antitrypsin behave differently, the difference in expression for prohaptoglobin compared with the expression of mature alpha 1-antitrypsin is about three orders of magnitude . In addition, we provide evidence that glycosylation of both proteins can occur in yeast only when the signal sequence is present in the DNA constructions.

Proc Natl Acad Sci U S A, 1986 Apr, 83(8), 2536 - 40
Sequences upstream of the STE6 gene required for its expression and regulation by the mating type locus in Saccharomyces cerevisiae; Wilson KL et al.; The STE6 gene of Saccharomyces cerevisiae is an a-specific gene; it is repressed in alpha cells by the alpha 2 product of the mating type locus . To study the role of sequences upstream of STE6 in its regulation and expression, we have determined the DNA sequence of the promoter region, identified the start sites for the STE6 transcript, and identified sequences governing its transcription . Deletions that remove DNA upstream of the STE6 gene were produced and assayed for effects on regulation and expression . The deletions defined two intervals upstream of the STE6 transcription initiation sites . One contains all or part of a negative element; the other contains all or part of a positive element . The negative element is required for repression of STE6 by alpha 2: deletions lacking this element express STE6 constitutively . Such deletions remove a 31-base-pair site, located 135 base pairs upstream of the first transcript start site, that is highly homologous to sites present in the upstream regions of four other genes repressed by alpha 2 . These sites are presumably responsible for repression of the a-specific genes by alpha 2 . The positive element (a putative upstream activation site) is required for expression of STE6 . The deletions define the left boundary of the proposed upstream activation site . Sequence homologies between STE6 and other a-specific genes are found in this region and may mediate activation of this set of genes.

Proc Natl Acad Sci U S A, 1986 Apr, 83(8), 2468 - 72
Components of microtubular structures in Saccharomyces cerevisiae; Pillus L et al.; Most studies of cytoskeletal organelles have concentrated on molecular analyses of abundant and biochemically accessible structures . In many of the classical cases, however, the nature of the system chosen has precluded a concurrent genetic analysis . The mitotic spindle of the yeast Saccharomyces cerevisiae is one example of an organelle that can be studied by both classical and molecular genetics . We show here that this microtubule structure also can be examined biochemically . The spindle can be isolated by selective extractions of yeast cells by using adaptations of methods successfully applied to animal cells . In this way, microtubule-associated proteins of the yeast spindle are identified.

Proc Natl Acad Sci U S A, 1986 Apr, 83(8), 2378 - 82
Sporulation of the yeast Saccharomyces cerevisiae is accompanied by synthesis of adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate; Jakubowski H; Two-dimensional TLC analysis of 32P-labeled nucleotides extracted from the yeast Saccharomyces cerevisiae reveals that two highly phosphorylated nucleotides are synthesized during sporulation . These nucleotides have been identified as adenosine 5'-tetraphosphate (ppppA) and adenosine 5'-pentaphosphate (pppppA) . The synthesis of ppppA and pppppA commences late in sporulation and follows formation of ascospores . The maximum concentration of ppppA and pppppA in sporulating yeast cultures was 2% and 1.5%, respectively, that of ATP . Adenosine 5'-tetraphosphate and 5'-pentaphosphate are unique to this stage of yeast development and are absent in vegetative yeast cells . Since these nucleotides are also absent in asporogenous a/a and alpha/alpha cells, it is reasonable to propose that they are signal nucleotides marking one of the stages of yeast development--i.e., ascospore formation.

Arch Biochem Biophys, 1986 Apr, 246(1), 306 - 20
Effect on gluconeogenesis of mutants blocking two mitochondrial transport systems in the yeast Saccharomyces cerevisiae; Wills C et al.; Two mutants of Saccharomyces cerevisiae, ccr1 and tpy1, have been found to interfere with the transport of small molecules across the inner mitochondrial membrane . Both also have the effect of interfering with the synthesis of a number of cytoplasmically located enzymes involved in gluconeogenesis, even when the cells are released from glucose repression . The ccr1 mutant, defective in the transport of dicarboxylic acids across the inner membrane, represses the synthesis of gluconeogenic enzymes almost totally, but synthesis can be induced on complete medium without a carbon source . This mutant has low levels of intracellular malate under all growth conditions tested . The tpy1 mutant, defective in the transport of pyruvate across the inner membrane, shows repression of gluconeogenesis enzymes under some growth conditions, particularly high levels of ethanol in the medium . These conditions also lead to low levels of malate in the cells . Intracellular levels of malate in these mutants, and in the wild type, are correlated with the levels of gluconeogenic enzymes present . The ability of isolated mutant mitochondria to phosphorylate ADP is shown to be consistent with the interpretation that they are defective in inner membrane transport, although as yet no evidence is available that these defects are the primary lesions in the two mutants . The data are consistent with two general models . In one, the exhaustion of an extramitochondrial corepressor or introduction of a coinducer by mitochondrial activity triggers the induction of gluconeogenic enzyme synthesis . In the second, the mitochondria themselves trigger this induction, but only when the tricarboxylic acid cycle is able to operate at a high level.

J Bacteriol, 1986 Apr, 166(1), 328 - 30
Cloning of a gene encoding choline transport in Saccharomyces cerevisiae; Nikawa J et al.; By genetic complementation in a yeast choline transport mutant from a yeast gene library, we isolated plasmids encoding choline transport . The cloned plasmids contained a common 4.0-kilobase DNA fragment and also complemented an ethanolamine transport defect . The cloned sequence present in the yeast genome was possibly unique.

Eur J Biochem, 1986 Apr 1, 156(1), 15 - 22
Pyruvate carboxylase from Saccharomyces cerevisiae . Quaternary structure, effects of allosteric ligands and binding of avidin; Rohde M et al.; The quaternary structure of pyruvate carboxylase purified from Saccharomyces cerevisiae was investigated by electron microscopic examination of negatively stained samples . In the most frequently observed projection form four intensity maxima were arranged at the corners of a rhombus; a cleft along the longitudinal axis of individual protomers could often be discerned . The observation of occasional triangular and dual-intensity projections and the interconversion of all three projection forms in tilting studies indicates that this tetrameric enzyme has a structure very similar to the tetrahedron-like configuration previously proposed for pyruvate carboxylases from vertebrate sources {Mayer, F., Wallace, J . C . and Keech, D . B . (1980) Eur . J . Biochem . 112, 265-272} and Aspergillus nidulans {Osmani, S . A., Mayer, F., Marston, F . A . O., Selmes, I . P . and Scrutton, M . C . (1984) Eur . J . Biochem . 139, 509-518} . An improved structural preservation of the enzyme was observed in the presence of either of the activators acetyl-CoA (250 microM) and palmitoyl-CoA (1-5 microM) . At higher than 5 microM palmitoyl-CoA, although activity was further increased, dissociation of enzyme tetramers was evident, presumably because of the detergent effect of the long-chain acyl moiety . Two inhibitors of yeast pyruvate carboxylase, L-aspartate (10 mM) and 2-oxoglutarate (40 mM), added alone or together decreased significantly the proportion of intact tetramers even in the presence of acetyl-CoA or palmitoyl-CoA . When yeast pyruvate carboxylase was incubated with avidin, the formation of unbranched linear concatemers occurred at avidin:enzyme ratios between 2:1 and 1:2 . Avidin molecules were sometimes bound asymmetrically to the enzyme, appearing to complex only one biotin group on each side of the enzyme . This appeared to permit kinking and circularization of some concatemers.

Microbiol Sci, 1986 Apr, 3(4), 107 - 11, 114
Cellular control in the eukaryotic cell through action of proteinases: the yeast Saccharomyces cerevisiae as a model organism; Wolf DH; The involvement of proteinases in cellular control was neglected for many years since it was hard to imagine that such a vital class of macromolecules as the proteins, which are assembled at the expense of a lot of energy, could be broken down by the cell again . To many earlier researchers, proteinases seemed to be boring catalysts present in the cell only to annoy biochemists who wanted to purify proteins . In contrast, recent research has shown proteinases to be vital catalysts in the control of cellular events.

J Bacteriol, 1986 Apr, 166(1), 313 - 8
GAL2 codes for a membrane-bound subunit of the galactose permease in Saccharomyces cerevisiae; Tschopp JF et al.; The gene encoding the galactose permease of Saccharomyces cerevisiae (GAL2) was cloned . The clone restores galactose permease activity to gal2 yeasts and is regulated by galactose in a manner similar to other GAL gene products (GAL1, -7, and -10) . Experiments with temperature-conditional secretory mutants indicated that transport of the GAL2 gene product to the cell surface requires a functional secretory pathway . In addition, gene fusions were constructed between the GAL2 gene and the Escherichia coli lacZ gene . The GAL2-lacZ gene fusions code for galactose-regulated beta-galactosidase activity in yeasts . The beta-galactosidase activity was found to be membrane bound.

Mol Cell Biol, 1986 Apr, 6(4), 1218 - 27
Analysis of the essential and excision repair functions of the RAD3 gene of Saccharomyces cerevisiae by mutagenesis; Naumovski L et al.; The RAD3 gene of Saccharomyces cerevisiae, which is involved in excision repair of DNA and is essential for cell viability, was mutagenized by site-specific and random mutagenesis . Site-specific mutagenesis was targeted to two regions near the 5' and 3' ends of the coding region, selected on the basis of amino acid sequence homology with known nucleotide binding and with known specific DNA-binding proteins, respectively . Two mutations in the putative nucleotide-binding region and one in the putative DNA-binding region inactivate the excision repair function of the gene, but not the essential function . A gene encoding two tandem mutations in the putative DNA-binding region is defective in both excision repair and essential functions of RAD3 . Seven plasmids were isolated following random mutagenesis with hydroxylamine . Mutations in six of these plasmids were identified by gap repair of mutant plasmids from the chromosome of strains with previously mapped rad3 mutations, followed by DNA sequencing . Three of these contain missense mutations which inactivate only the excision repair function . The other three carry nonsense mutations which inactivate both the excision repair and essential functions . Collectively our results indicate that the RAD3 excision repair function is more sensitive to inactivation than is the essential function . Overexpression of wild-type Rad3 protein and a number of rad3 mutant proteins did not affect the UV resistance of wild-type yeast cells . However, overexpression of Rad3-2 protein rendered wild-type cells partially UV sensitive, indicating that excess Rad3-2 protein is dominant to the wild-type form . These and other results suggest that Rad3-2 protein retains its affinity for damaged DNA or other substrates, but is not catalytically active in excision repair.

Mol Cell Biol, 1986 Apr, 6(4), 1095 - 101
Transcription terminator-like element within a Saccharomyces cerevisiae promoter region; Yarger JG et al.; We analyzed a cloned fragment of the yeast URA3 promoter region that contains a sequence of DNA capable of functioning as a highly efficient transcription terminator . BAL 31 deletions have shown the signal for the transcription termination activity is less than or equal to 110 base pairs and resides between bases 45 and 155 upstream of the URA3 primary ATG codon at base 227 . In our in vivo assay system, the DNA fragment is able to terminate transcripts very efficiently in either orientation . The terminated transcripts bind to oligodeoxythymidylate cellulose columns and promote the synthesis of full-length cDNAs, suggesting that the transcripts are polyadenylated . The 110-base-pair region contains no sequence resembling terminator consensus sequences described by Zaret and Sherman (K.S . Zaret and F . Sherman, Cell, 28:563-573, 1982) or Henikoff and Cohen (S . Henikoff and E.H . Cohen, Mol . Cell . Biol., 4:1515-1520, 1984) . We discuss the possible physiological relevance of this sequence to bona fide termination of transcription and to URA3 regulation in Saccharomyces cerevisiae.

Bioorg Khim, 1986 Apr, 12(4), 555 - 8
{Nucleotide sequence of the ADE 1 gene of the yeast Saccharomyces cerevisiae}; Miasnikov AN et al.; The yeast ADE 1 gene has been cloned and sequenced . The primary structure deduced from the nucleotide sequence demonstrated that phosphoribosylaminoimidazole-succinocarboxamide synthetase is a protein with molecular weight of 34 500 D.

Biochemistry, 1986 Mar 25, 25(6), 1395 - 402
Coenzyme Q analogues reconstitute electron transport and proton ejection but not the antimycin-induced "red shift" in mitochondria from coenzyme Q deficient mutants of the yeast Saccharomyces cerevisiae; Beattie DS et al.; Mitochondria isolated from coenzyme Q deficient yeast cells had no detectable NADH:cytochrome c reductase or succinate:cytochrome c reductase activity but contained normal amounts of cytochromes b and c1 by spectral analysis . Addition of the exogenous coenzyme Q derivatives including Q2, Q6, and the decyl analogue (DB) restored the rate of antimycin- and myxothiazole-sensitive cytochrome c reductase with both substrates to that observed with reduced DBH2 . Similarly, addition of these coenzyme Q analogues increased 2-3-fold the rate of cytochrome c reduction in mitochondria from wild-type cells, suggesting that the pool of coenzyme Q in the membrane is limiting for electron transport in the respiratory chain . Preincubation of mitochondria from the Q-deficient yeast cells with DBH2 at 25 degrees C restored electrogenic proton ejection, resulting in a H+/2e- ratio of 3.35 as compared to a ratio of 3.22 observed in mitochondria from the wild-type cell . Addition of succinate and either coenzyme Q6 or DB to mitochondria from the Q-deficient yeast cells resulted in the initial reduction of cytochrome b followed by a slow reduction of cytochrome c1 with a reoxidation of cytochrome b . The subsequent addition of antimycin resulted in the oxidant-induced extrareduction of cytochrome b and concomitant oxidation of cytochrome c1 without the "red" shift observed in the wild-type mitochondria . Similarly, addition of antimycin to dithionite-reduced mitochondria from the mutant cells did not result in a red shift in the absorption maximum of cytochrome b as was observed in the wild-type mitochondria in the presence or absence of exogenous coenzyme Q analogues.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1986 Mar 25, 261(9), 3939 - 43
Amino acid sequence of the phosphorylation site of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase; Rittenhouse J et al.; Fructose-1,6-bisphosphatase from the yeast Saccharomyces cerevisiae has properties similar to other gluconeogenic fructose-1,6-bisphosphatases, but an unusual characteristic of the yeast enzyme is that it can be phosphorylated in vitro by cAMP-dependent protein kinase . Phosphorylation also occurs in vivo, presumably as part of a signalling mechanism for the enzyme's degradation . To probe the structural basis for the phosphorylation of yeast fructose-1,6-bisphosphatase, we have developed an improved procedure for the purification of the enzyme and then performed sequence studies with the in vitro-phosphorylated protein as well as with tryptic and chymotryptic peptides containing the phosphorylation site . As a result of these studies, we have determined that yeast fructose-1,6-bisphosphatase has the following 24-residue NH2-terminal amino acid sequence: Pro-Thr-Leu-Val-Asn-Gly-Pro-Arg-Arg-Asp-Ser-Thr-Glu-Gly- Phe-Asp-Thr-Asp-Ile-Ile-Thr-Leu-Pro-Arg . The site of phosphorylation is located at Ser-11 in the above sequence . The amino acid sequence around the site of phosphorylation contains the sequence - Arg-Arg-X-Ser- associated with many of the better substrates of cAMP-dependent protein kinase . The sequence of residues 15-24 above is highly homologous with the sequence of residues 6-15 of pig kidney fructose-1,6-bisphosphatase, showing 7 out of 10 residues in identical positions . The yeast enzyme, however, has a dissimilar NH2-terminal region which extends beyond the NH2 terminus of mammalian fructose-1,6-bisphosphatases and contains a unique phosphorylation site.

J Theor Biol, 1986 Mar 21, 119(2), 197 - 204
Copy number amplification of the 2 micron circle plasmid of Saccharomyces cerevisiae; Futcher AB; The 2 micron circle is a small double stranded DNA plasmid that occurs at about 60 copies per cell in the nuclei of virtually all strains of Saccharomyces cerevisiae . The plasmid has no apparent phenotypic effect on host cells, and is the basis of many useful vectors for the transformation of yeast . Under certain circumstances, the plasmid is apparently able to replicate more than once per cell cycle; this over-replication allows the maintenance of the plasmid at high copy number . The plasmid has two inverted repeat sequences, and encodes a product that catalyses intra-molecular recombination between these two repeats . Models are proposed whereby recombination leads to copy number amplification . In particular, it is proposed that intra-molecular recombination during replication flips the orientation of one replication fork with respect to the other, so that both forks travel in the same direction around a circular monomer template, generating a large multimer from a monomer and a single initiation of replication.

FEBS Lett, 1986 Mar 17, 198(1), 89 - 91
Null and electrophoretic mobility mutants in the structural gene for L-lactate dehydrogenase of Saccharomyces cerevisiae; Rush K et al.; A mutant lacking L-lactate dehydrogenase (EC 1.1.2.3) of Saccharomyces cerevisiae was isolated by its inability to grow on minimal medium with L-lactate as a carbon source . A simple activity gel assay for visualization of this enzyme and the two D-lactate dehydrogenases in this organism (EC 1.1.2.4 and 1.1.1.28) was developed . This enabled us to screen spontaneous and ethylmethanesulfonate-induced back mutants for electrophoretic mobility . Two mutants with a mobility faster than that of the wild type were isolated, and proved to be allelic to the L-lactate dehydrogenase negative mutant.

J Biol Chem, 1986 Mar 5, 261(7), 3178 - 83
Phosphatidylinositol synthase from Saccharomyces cerevisiae . Reconstitution, characterization, and regulation of activity; Fischl AS et al.; Purified membrane-associated phosphatidylinositol synthase (CDP diacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) from Saccharomyces cerevisiae was reconstituted into unilamellar phospholipid vesicles . Reconstitution of the enzyme was performed by removing detergent from an octylglucoside/phospholipid/Triton X-100/enzyme mixed micelle mixture by Sephadex G-50 superfine column chromatography . The average diameter of the vesicles was 40 nm and chymotrypsin treatment of intact vesicles indicated that over 90% of the reconstituted enzyme had its active site facing outward . The enzymological properties and reaction mechanism of reconstituted phosphatidylinositol synthase were determined in the absence of detergent . The reconstituted enzyme was used as a model system to study the regulation of activity . Phosphatidylinositol synthase was constitutive in wild type cells grown in the presence of water-soluble phospholipid precursors as determined by enzyme activity and immunoblotting . Reconstituted enzyme was not effected by water-soluble phospholipid precursors or nucleotides . Maximum activity was found when the enzyme was reconstituted into phosphatidylcholine: phosphatidylethanolamine: phosphatidylinositol: phosphatidylserine vesicles . Phosphatidylserine stimulated reconstituted activity, suggesting that the local phospholipid environment may regulate phosphatidylinositol synthase activity.

J Biol Chem, 1986 Mar 5, 261(7), 3054 - 9
Mapping and sequencing of the wild-type and mutant (G116-40) alleles of the tyrosyl-tRNA mitochondrial gene in Saccharomyces cerevisiae; Nobrega MP et al.; The Saccharomyces cerevisiae syn- mitochondrial mutant G116-40 isolated by Berlani et al . (Berlani, R . E., Pentella, C., Macino, G., and Tzagoloff, A . (1980) J . Bacteriol . 141, 1086-1097) is shown to have a mutation in the tyrosyl-tRNA gene by genetic data combined with restriction analysis and DNA sequencing of the appropriate rho- mitochondrial DNAs derived from wild-type and mutant strains . The new region sequenced spans 685 base pairs located between 9.5 and 10.4 map units, the gene being located at 10.0 units . The tRNA structure, as deduced from the DNA sequence, is in agreement with the data derived from sequencing the purified tyrosyl-tRNA reported by Sibler et al . (Sibler, A., Dirheimer, G., and Martin, R.P . (1983) FEBS Lett . 152, 153-156) . No in vitro tyrosyl-tRNA aminoacylation could be detected using mitochondrial RNA from the mutant . S1 nuclease mapping experiments showed that the mutant produces a transcript that is identical to the wild-type at its 5'-end . The same analysis carried out with the mitochondrial RNA from a rho- strain with the tyrosyl-tRNA region of mitochondrial DNA reveals a 5'-end shorter by about 3 nucleotides . The mutant gene has a single substitution (C----T) at the penultimate nucleotide near the 3'-end of the molecule creating an acceptor stem that lacks the two terminal Watson-Crick base pairs.

Genetika, 1986 Mar, 22(3), 390 - 8
{Genetic analysis of the mutant RD-50 in Saccharomyces cerevisiae . The role of nuclear and mitochondrial mutation}; Nevzgliadova OV; Inheritance of the mutant phenotype of respiratory deficience in RD-50 strain was studied . The deficience can be restored, giving respiratory sufficience, in crosses with rho0 testers . The "restorable" phenotype of the mutant, named RDc+, was shown to be determined by a nuclear pet-like mutation (pet50) . The restorable RDc+ phenotype is stabilized in the presence of the pet50 allele, but can remain as such in the presence of the wild-type allele PET50 . Restoration also takes place in cytoductants with the nucleus of kar partner . In order to explain the behaviour of the mitochondrial mutation mit50, we suppose it to be a microdeletion, capable of reversion, due to integration of a putative mt episome . Some features of the nuclear mutation pet50, particularly, its segregation in mitotic progeny of some revertants to respiratory competence point to its peculiarity . We suppose the mutation pet50 to be an insertion into the chromosomal PET50 gene . This insertion may be excised, remaining within the cell in the free state for some time, and then either eliminate or reintegrate into the chromosome.

Proc Natl Acad Sci U S A, 1986 Mar, 83(5), 1266 - 70
Processing and fatty acid acylation of RAS1 and RAS2 proteins in Saccharomyces cerevisiae; Fujiyama A et al.; We demonstrate the pathway for the biosynthesis of RAS1 and RAS2 gene products of Saccharomyces cerevisiae leading to their localization in membranes . The primary translation products of these genes are detected in a soluble fraction . Shortly after synthesis, these precursor molecules are converted to forms that migrate slightly faster than the precursor forms on a NaDodSO4/polyacrylamide gel . These processed proteins are further modified by fatty acid acylation, which is detected by {3H}palmitic acid labeling . The acylated derivatives are found exclusively in cell membranes, indicating the translocation of the RAS proteins from cytosol to membranes during maturation process . The attached fatty acids can be released by mild alkaline hydrolysis, suggesting that the linkage between the fatty acid and the protein is an ester bond . The site of the modification by fatty acid is presumably localized to the COOH-terminal portion of the RAS proteins . Fractionation of the membranes by sucrose gradient demonstrates that a majority of the fatty-acylated RAS proteins are localized in plasma membrane.

Mutat Res, 1986 Mar, 160(1), 19 - 26
Aneuploidy and other genetic effects induced by hydroxyurea in Saccharomyces cerevisiae; Mayer VW et al.; Hydroxyurea induces mitotic gene conversion, mitotic crossing-over, reverse mutation, respiration-deficient petite mutants and aneuploidy in growing cultures of Saccharomyces cerevisiae . Evidence is presented indicating that induction rather than selection is responsible for the increase in frequency of the genetic end points measured . Complications concerning the detection of aneuploidy in the presence of other genetic effects are described, and the need for following the complete protocol for confirmation of the aneuploids in any chemical screening program is emphasized.

Mutat Res, 1986 Mar, 160(1), 11 - 7
Effects of near-ultraviolet light on mutations, intragenic and intergenic recombinations in Saccharomyces cerevisiae; Machida I et al.; The effects of far (254 nm) and near (290-350 nm) ultraviolet (UV) light on mutations, intragenic and intergenic recombinations were compared in diploid strains of Saccharomyces cerevisiae . At equivalent survival levels there was not much difference in the induction of nonsense and missense mutations between far- and near-UV radiations . However, frameshift mutations were induced more frequently by near-UV than by far-UV radiation . Near-UV radiation induced intragenic recombination (gene conversion) as efficiently as far-UV radiation and the induced levels were similar in both radiations at equitoxic doses . A strikingly higher frequency was observed for the intergenic recombination induced by near-UV radiation than by far-UV radiation when compared at equivalent survival levels . Photoreactivation reduced the frequency only slightly in far-UV induced intergenic recombination and not at all in near-UV induction . These results indicate that near-UV damage involves strand breakage in addition to pyrimidine dimers and other lesions induced, whereas far-UV damage consists largely of photoreactivable lesions, pyrimidine dimers, and near-UV induced damage is more efficient for the induction of crossing-over.

Mutagenesis, 1986 Mar, 1(2), 151 - 5
Genetic effects of methylmethanesulphonate during meiosis in Saccharomyces cerevisiae; Kelly SL et al.; Enhanced mutagenic action after methylmethanesulphonate (MMS) treatment was found in pre-replicative meiotic yeast cells of the strain D7 . The level of gene conversion after MMS treatment rose above the spontaneous level during the period of commitment to meiotic recombination, but at later times into meiosis became indistinguishable from the full meiotic level . In contrast reciprocal recombination detected between ade 2 and the centromere of chromosome XV after MMS treatment remained above the spontaneous level up to commitment to meiotic cell division with relatively high levels through meiotic prophase I . These results are discussed in relation to MMS-induced damage and its repair, particularly double-strand break repair.

J Bacteriol, 1986 Mar, 165(3), 901 - 10
Subcellular and submitochondrial localization of phospholipid-synthesizing enzymes in Saccharomyces cerevisiae; Kuchler K et al.; Using highly enriched membrane preparations from lactate-grown Saccharomyces cerevisiae cells, the subcellular and submitochondrial location of eight enzymes involved in the biosynthesis of phospholipids was determined . Phosphatidylserine decarboxylase and phosphatidylglycerolphosphate synthase were localized exclusively in the inner mitochondrial membrane, while phosphatidylethanolamine methyltransferase activity was confined to microsomal fractions . The other five enzymes tested in this study were common both to the outer mitochondrial membrane and to microsomes . The transmembrane orientation of the mitochondrial enzymes was investigated by protease digestion of intact mitochondria and of outside-out sealed vesicles of the outer mitochondrial membrane . Glycerolphosphate acyltransferase, phosphatidylinositol synthase, and phosphatidylserine synthase were exposed at the cytosolic surface of the outer mitochondrial membrane . Cholinephosphotransferase was apparently located at the inner aspect or within the outer mitochondrial membrane . Phosphatidate cytidylyltransferase was localized in the endoplasmic reticulum, on the cytoplasmic side of the outer mitochondrial membrane, and in the inner mitochondrial membrane . Inner membrane activity of this enzyme constituted 80% of total mitochondrial activity; inactivation by trypsin digestion was observed only after preincubation of membranes with detergent (0.1% Triton X-100) . Total activity of those enzymes that are common to mitochondria and the endoplasmic reticulum was about equally distributed between the two organelles . Data concerning susceptibility to various inhibitors, heat sensitivity, and the pH optima indicate that there is a close similarity of the mitochondrial and microsomal enzymes that catalyze the same reaction.

Cytometry, 1986 Mar, 7(2), 132 - 41
Flow cytometry analysis of recombinant Saccharomyces cerevisiae populations; Srienc F et al.; A new fluorescent stain has been developed for detecting cloned beta-galactosidase activity in individual cells of Saccharomyces cerevisiae by flow cytometry . The staining reaction is based on enzymatic cleavage of alpha-naphthol-beta-D-galactopyranoside by intracellular beta-galactosidase and trapping of the liberated naphthol by hexazoniumpararosaniline yielding a fluorescent, insoluble end product . This stain, in connection with an appropriate host strain, has been applied for detecting plasmids encoding inducible beta-galactosidase in unstable recombinant cell populations carrying plasmids with different origins of replication . The method enables rapid determination of the fraction of plasmid-containing cells as well as quantitation of intracellular beta-galactosidase content by kinetic enzyme assay . Inducibility of the marker enzyme is important for maintaining correlation between enzyme and gene content.

J Biol Chem, 1986 Feb 25, 261(6), 2978 - 86
Saccharomyces cerevisiae tRNA ligase . Purification of the protein and isolation of the structural gene; Phizicky EM et al.; The tRNA ligase protein of Saccharomyces cerevisiae is one of the components required for splicing of yeast tRNA precursors in vitro . We have purified this protein to near homogeneity using an affinity elution chromatographic step . Purified tRNA ligase is a 90-kDa protein that, in addition to catalyzing the ligation of tRNA half-molecules in the coupled splicing reaction, will also ligate an artificial substrate . Using this artificial substrate, we provide evidence for the existence of a previously predicted activated intermediate in the ligation reaction . The amino acid sequence of the amino-terminal end of the protein was determined, and we have used this information to isolate the structural gene from a library of yeast DNA . We prove that this DNA encodes the tRNA ligase protein by DNA sequencing and by demonstrating overproduction of the protein.

J Biol Chem, 1986 Feb 25, 261(6), 2819 - 27
Partial purification and characterization of the Saccharomyces cerevisiae transcription factor TFIIIB; Klekamp MS et al.; Methods are described for the partial purification of the Saccharomyces cerevisiae class III gene transcription factor TFIIIB from yeast whole cell extracts . A major component (30% of the total protein) of our most highly purified TFIIIB preparation was a polypeptide with an apparent Mr = 60,000 when analyzed by denaturing polyacrylamide gel electrophoresis . This protein was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and polyclonal antibodies were raised against it . Using immunological methods it was shown that TFIIIB transcription factor activity was associated with this purified polypeptide . Furthermore, the polyclonal sera raised against the yeast TFIIIB polypeptide was also shown to specifically neutralize the activity of the human TFIIIB equivalent when it was added to a human KB cell S-100 in vitro transcription system.

J Mol Biol, 1986 Feb 5, 187(3), 363 - 78
Transcription initiation of the Saccharomyces cerevisiae iso-1-cytochrome c gene . Multiple, independent T-A-T-A sequences; McNeil JB et al.; The expression of the Saccharomyces cerevisiae CYC1 gene, which encodes iso-1-cytochrome c, produces a family of messenger RNAs whose 5' ends map in the region from position +7 to -93 relative to the first nucleotide at position +1 of the protein-coding DNA sequence . The mechanism of transcription initiation of the CYC1 gene has been examined by using linker-scanning deletions and gene fusions . The various CYC1 derivatives with mutations in the 5' non-coding region were constructed, reintroduced into yeast using a multicopy plasmid, and the mRNA starts mapped by primer extension . The results indicate that four, and possibly five T-A-T-A sequences are located within the 5' non-coding region of the CYC1 gene, and that each T-A-T-A is required for a specific subset of mRNA starts . This conclusion has been confirmed by oligonucleotide mutagenesis of a chromosomal CYC1 T-A-T-A sequence . A loose spatial relationship also exists between the T-A-T-A sequences and the mRNA start sites, and this distance relationship varies from 100 to 60 base-pairs (+/- 15 base-pairs).

Mol Cell Biol, 1986 Feb, 6(2), 723 - 9
A deletion that includes the segment coding for the signal peptidase cleavage site delays release of Saccharomyces cerevisiae acid phosphatase from the endoplasmic reticulum; Haguenauer-Tsapis R et al.; We studied ultrastructural localization of acid phosphatase in derepressed Saccharomyces cerevisiae cells transformed with a multicopy plasmid carrying either the wild-type PHO5 gene or a PHO5 gene deleted in the region overlapping the signal peptidase cleavage site . Wild-type enzyme was located in the cell wall, as was 50% of the modified protein, which carried high-mannose-sugar chains . The remaining 50% of the protein was active and core glycosylated, and it accumulated in the endoplasmic reticulum cisternae . The signal peptide remained uncleaved in both forms . Cells expressing the modified protein exhibited an exaggerated endoplasmic reticulum with dilated lumen.

Mol Cell Biol, 1986 Feb, 6(2), 530 - 8
Single base-pair mutations in centromere element III cause aberrant chromosome segregation in Saccharomyces cerevisiae; McGrew J et al.; In this paper we show that a 211-base pair segment of CEN3 DNA is sufficient to confer wild-type centromere function in the yeast Saccharomyces cerevisiae . We used site-directed mutagenesis of the 211-base pair fragment to examine the sequence-specific functional requirements of a conserved 11-base pair segment of centromere DNA, element III (5'-TGATTTATCCGAA-3') . Element III is the most highly conserved of the centromeric DNA sequences, differing by only a single adenine X thymine base pair among the four centromere DNAs sequenced thus far . All of the element III sequences contain specific cytosine X guanine base pairs, including a 5'-CCG-3' arrangement, which we targeted for single cytosine-to-thymine mutations by using sodium bisulfite . The effects of element III mutations on plasmid and chromosome segregation were determined by mitotic stability assays . Conversion of CCG to CTG completely abolished centromere function both in plasmids and in chromosome III, whereas conversion of CCG to TCG decreased plasmid and chromosome stability moderately . The other two guanine X cytosine base pairs in element III could be independently converted to adenine X thymine base pairs without affecting plasmid or chromosome stability . We concluded that while some specific nucleotides within the conserved element III sequence are essential for proper centromere function, other conserved nucleotides can be changed.

Mol Cell Biol, 1986 Feb, 6(2), 404 - 10
Thermolabile L-A virus-like particles from pet18 mutants of Saccharomyces cerevisiae; Fujimura T et al.; pet18 mutations in Saccharomyces cerevisiae confer on the cell the inability to maintain either L-A or M double-stranded RNAs (dsRNAs) at the nonpermissive temperature . In in vitro experiments, we examined the effects of pet18 mutations on the RNA-dependent RNA polymerase activity associated with virus-like particles (VLPs) . pet18 mutations caused thermolabile RNA polymerase activity of L-A VLPs, and this thermolability was found to be due to the instability of the L-A VLP structure . The pet18 mutations did not affect RNA polymerase activity of M VLPs . Furthermore, the temperature sensitivity of wild-type L-A RNA polymerase differed substantially from that of M RNA polymerase . From these results, and from other genetic and biochemical lines of evidence which suggest that replication of M dsRNA requires the presence of L-A dsRNA, we propose that the primary effect of the pet18 mutation is on the L-A VLP structure and that the inability of pet18 mutants to maintain M dsRNA comes from the loss of L-A dsRNA.

Mol Gen Genet, 1986 Feb, 202(2), 336 - 7
Effect of acrylonitrile on the transcription of specific genes in Saccharomyces cerevisiae; Thuroff E et al.; We have studied the effect of acrylonitrile on the transcription of specific genes of Saccharomyces cerevisiae . The results presented demonstrate that ACN disturbs the coordinated response of ribosomal protein genes and causes a dramatic induction of the LEU2 gene, which might be due to metabolites of ACN.

Arch Biochem Biophys, 1986 Feb 1, 244(2), 430 - 8
Deoxyribonucleotide biosynthesis in yeast: assay and properties of ribonucleotide reductase in permeabilized Saccharomyces cerevisiae cells; Lammers M et al.; Yeast cells permeabilized by freeze-thaw cycles in a sorbitol-containing medium provide an experimentally favorable system for the study of ribonucleotide reduction in a small number of cells or in mutant strains . Ribonucleotide reductase activities determined in such cells are about twice those found in cell extracts but properties of the enzyme, except pH optimum, are closely comparable in both assay procedures . In contrast with other organisms, the activities measured in permeabilized cells from both diploid or haploid strains exceed the demand for deoxyribonucleotide formation during replication of the yeast genome . The method has been applied to yeast cultures growing in the presence of the ribonucleotide reductase inhibitor hydroxyurea and a twofold increase of enzyme activity has been established in such cells . On the other hand, analysis of a series of hus mutants, selected for hydroxyurea sensitivity in the laboratory of Singer and Johnston did not reveal obvious alterations of the enzyme vs the parental strains, suggesting that the hus phenotype may be due to lesions other than in ribonucleotide reductase.

Mutat Res, 1986 Feb, 173(2), 117 - 20
Isolation of glutathione-deficient mutants of the yeast Saccharomyces cerevisiae; Kistler M et al.; Glutathione-deficient (gsh-) mutants of the yeast Saccharomyces cerevisiae were isolated after UV treatment using MNNG as selective agent . For genetic and biochemical characterization 5 mutant strains were chosen which exhibited considerably decreased residual GSH contents varying from 2 to 6% of the wild-type levels . All 5 isolates showed a 2:2 segregation of the gsh-:GSH+ phenotypes alluding to a monogenic recessive mutation . Complementation analysis indicates that all gsh- mutants belong to one complementation group.

Mol Cell Biol, 1986 Feb, 6(2), 688 - 702
Cloning and characterization of four SIR genes of Saccharomyces cerevisiae; Ivy JM et al.; Mating type in the yeast Saccharomyces cerevisiae is determined by the MAT (a or alpha) locus . HML and HMR, which usually contain copies of alpha and a mating type information, respectively, serve as donors in mating type interconversion and are under negative transcriptional control . Four trans-acting SIR (silent information regulator) loci are required for repression of transcription . A defect in any SIR gene results in expression of both HML and HMR . The four SIR genes were isolated from a genomic library by complementation of sir mutations in vivo . DNA blot analysis suggests that the four SIR genes share no sequence homology . RNA blots indicate that SIR2, SIR3, and SIR4 each encode one transcript and that SIR1 encodes two transcripts . Null mutations, made by replacement of the normal genomic allele with deletion-insertion mutations created in the cloned SIR genes, have a Sir- phenotype and are viable . Using the cloned genes, we showed that SIR3 at a high copy number is able to suppress mutations of SIR4 . RNA blot analysis suggests that this suppression is not due to transcriptional regulation of SIR3 by SIR4; nor does any SIR4 gene transcriptionally regulate another SIR gene . Interestingly, a truncated SIR4 gene disrupts regulation of the silent mating type loci . We propose that interaction of at least the SIR3 and SIR4 gene products is involved in regulation of the silent mating type genes.

Mol Cell Biol, 1986 Feb, 6(2), 674 - 87
Tripartite upstream promoter element essential for expression of Saccharomyces cerevisiae ribosomal protein genes; Rotenberg MO et al.; To initiate a genetic analysis of yeast ribosomal protein gene promoters, we have constructed a gene fusion between the yeast ribosomal protein gene RP39A and the Escherichia coli lacZ gene . This gene fusion contains approximately 1,030 nucleotides of the 5' flanking region and the first 49 1/3 codons of RP39A fused in frame to a large 3' end fragment of lacZ . Whether it is introduced into yeast cells on a moderately high-copy-number plasmid, or integrated into the yeast genome at the RP39A locus, this RP39A-lacZ gene directs the synthesis of a hybrid transcript which encodes beta-galactosidase activity . Deletions in the 5' flanking region of RP39A-lacZ were constructed by linker insertion and BAL 31 mutagenesis . The expression of the mutant genes in yeast cells was assayed by measuring RP39A-lacZ mRNA and beta-galactosidase levels . By these means we have shown that the sequences between nucleotides -256 and -170 upstream of RP39A are essential for expression of this gene . Three sequence motifs, HOMOL1, RPG, and a T-rich region, which were found in that order 5'----3' upstream of most yeast ribosomal protein genes, were present within this interval . We found that substitution of the CYC1-lacZ upstream activation site with the fragment from nucleotides -298 to -172 upstream of RP39A, containing the HOMOL1-RPG-T-rich motif in that 5'----3' orientation, fully restored expression of the CYC1-lacZ gene . The essentially of HOMOL1, the RPG sequence, and the T-rich region for wild-type levels of expression of RP39A, the conserved location and order of these sequence motifs in yeast ribosomal protein genes, and the ability of a DNA fragment carrying these three sequence elements to substitute for the upstream activation site regions of CYC1 indicate that these three oligonucleotides may be essential to the transcription of yeast ribosomal protein genes.

Mol Cell Biol, 1986 Feb, 6(2), 494 - 501
A position effect on the expression of a tRNA gene mediated by the SIR genes in Saccharomyces cerevisiae; Schnell R et al.; The SIR genes of Saccharomyces cerevisiae are responsible for the position-dependent regulation of the a and alpha mating-type genes . Previous work by others has shown that the products of the SIR genes prevent the accumulation of stable transcripts of the a and alpha genes at HML and HMR . Results of this study establish that this regulation is a region-specific effect rather than a gene-specific effect since expression of a tRNA gene placed at HMR is repressed by the products of the SIR genes.

Antimicrob Agents Chemother, 1986 Feb, 29(2), 330 - 2
Polymyxin B nonapeptide inhibits mating in Saccharomyces cerevisiae; Boguslawski G; Polymyxin B nonapeptide enhanced susceptibility of yeast cells to various hydrophobic antibiotics and to mating pheromones . At much lower concentrations, the nonapeptide severely inhibited mating . The inhibition was caused by interference with sexual agglutination.

EMBO J, 1986 Feb, 5(2), 375 - 80
Characterization, cloning and sequence analysis of the CDC25 gene which controls the cyclic AMP level of Saccharomyces cerevisiae; Camonis JH et al.; The cell division cycle of the yeast Saccharomyces cerevisiae is triggered at the stage called 'START' . Many results strongly suggest that adenylate cyclase is an essential element of the control of START . We report here results arguing for a positive control of the cAMP level by the CDC25 gene, another gene of START . Firstly, cdc25 cells can be rescued by extracellular cAMP . Secondly, the cellular cAMP content drops when thermosensitive cdc25 mutant cells are shifted to restrictive temperature . We report the molecular cloning of the CDC25 gene by complementation of cdc25 mutant cells . The identity of the cloned gene was confirmed by site-specific gene re-integration experiments and segregation analysis: the isolated fragment is shown to integrate into the cdc25 gene locus . When transferred in cdc25 mutant cells this DNA prevents the drop of the cAMP level at restrictive temperature . This gene is transcribed in a 5200-nucleotides mRNA . We have determined the nucleotide sequence of a 5548-bp DNA fragment which shows an uninterrupted open reading frame (ORF) coding for a 1587-amino acid polypeptide chain . Only the C-terminal part of the ORF appears to be essential for the complementation of the cdc25-5 allele, suggesting a multidomain protein.

Mol Gen Genet, 1986 Feb, 202(2), 294 - 301
Primary structure of wild-type and mutant alleles of the PET494 gene of Saccharomyces cerevisiae; Costanzo MC et al.; The product of the yeast nuclear gene PET494 is required specifically for the translation of the mitochondrially encoded subunit III of cytochrome c oxidase . We have determined the DNA sequence of a 1.9 kb fragment carrying PET494 . The sequence contains a single long open reading frame of 489 codons . This open reading frame encodes the PET494 protein since the DNA sequence of the corresponding fragment derived from a strain with a known pet494 amber mutation contained an in frame UAG codon . The results of S1 nuclease protection experiments demonstrated that this region is transcribed and that the 5' ends of the major transcripts lie 30 to 40 base-pairs upstream of the first AUG codon in the PET494 reading frame . The predicted PET494 protein has a highly basic amino-terminal domain of 66 amino acids followed by a stretch of 32 uncharged residues, half of which are hydrophobic . The remainder of the protein is not unusual in amino acid composition or distribution except that the carboxyterminal region is notably basic . The phenotype of mutations generated in vitro around codon 119 by exonuclease digestion and linker insertion indicated that this region is dispensable for function . A mutation caused by deletion of 101 bp of coding sequence behaved like a simple frameshift when inserted into the chromosome: it was partially suppressed by the recessive non-group specific frameshift suppressor suf13 and reverted to Pet+ phenotype by mutations linked to PET494.

Proc Natl Acad Sci U S A, 1986 Feb, 83(3), 735 - 9
Shuttle mutagenesis: a method of transposon mutagenesis for Saccharomyces cerevisiae; Seifert HS et al.; We have extended the method of transposon mutagenesis to the eukaryote, Saccharomyces cerevisiae . A bacterial transposon containing a selectable yeast gene can be transposed into a cloned fragment of yeast DNA in Escherichia coli, and the transposon insertion can be returned to the yeast genome by homologous recombination . Initially, the cloned yeast DNA fragment to be mutagenized was transformed into an E . coli strain containing an F factor derivative carrying the transposable element . The culture was grown to allow transposition and cointegrate formation and, upon conjugation, recipients were selected that contained yeast sequences with transposon insertions . The yeast DNA was removed from the vector by restriction endonuclease digestion, and the transposon insertion was transformed into yeast . The procedure required a minimum number of manipulations, and each transconjugant colony contained an independent insertion . We describe 12 transposon Tn3 derivatives for this procedure as well as several cloning vectors to facilitate the method.

Science, 1986 Jan 24, 231(4736), 390 - 3
Protein-tyrosine kinase activity in Saccharomyces cerevisiae; Schieven G et al.; Saccharomyces cerevisiae was examined for tyrosine kinase activity in vitro because this organism offers molecular and genetic approaches for analyzing the role of tyrosine phosphorylation in cellular growth control that are unavailable in higher eukaryotes . Yeast extracts phosphorylated a random copolymer (glutamic acid:tyrosine, 80:20) at tyrosine in a reaction that was linear with respect to time and protein concentration . In the absence of added copolymer, phosphotyrosine was 0.1 percent of the total phosphoamino acids labeled with {gamma-32P}adenosine triphosphate in endogenous yeast proteins . However, specific activities of these reactions were low (approximately 1 percent of those in extracts of chick embryo fibroblasts) . Lack of significant incorporation of label from {alpha-32P}adenosine triphosphate into the copolymer or endogenous yeast proteins demonstrated that nucleotide interconversion, adenylylation, and subsequent hydrolysis could not account for the generation of phosphotyrosine observed.

FEBS Lett, 1986 Jan 20, 195(1-2), 159 - 63
Amino acid substitutions in mitochondrial ATPase subunit 9 of Saccharomyces cerevisiae leading to oligomycin or venturicidin resistance; Nagley P et al.; A series of isonuclear oligomycin-resistant mutants of Saccharomyces cerevisiae carrying mutations in the mitochondrial oli1 gene has been studied . DNA sequence analysis of this gene has been used to define the amino acid substitutions in subunit 9 of the mitochondrial ATPase complex . A domain of amino acids involved in oligomycin resistance can be recognized which encompasses residues in each of the two hydrophobic portions of the subunit 9 polypeptide that are thought to span the inner mitochondrial membrane . Certain amino acid substitutions also confer cross-resistance to venturicidin: these residues define an inner domain for venturicidin resistance . The expression of venturicidin resistance resulting from one particular substitution is modulated by nuclear genetic factors.

Eur J Biochem, 1986 Jan 15, 154(2), 307 - 11
Regulation and interconversion of the potassium transport systems of Saccharomyces cerevisiae as revealed by rubidium transport; Ramos J et al.; The kinetics of Rb+ transport in Saccharomyces cerevisiae depended on the K+ content of the cells and on K+ starvation, as follows . In cells with normal K+ (grown at millimolar K+), Rb+ transport was regulated by internal K+ . The loss of K+ first decreased the Km and later increased the Vmax of Rb+ transport . K+ starvation of normal-K+ cells for 4-5 h decreased the Km of Rb+ transport below the minimum observed after K+ loss . During this time Eadie-Hofstee plots of Rb+ transport suggest that the existing system was converted into a new one with a higher affinity . Growth at 10 microM K+ only required the system triggered by K+ loss, and the system expressed in K+-starved cells was not expressed under these conditions.

Biochemistry, 1986 Jan 14, 25(1), 212 - 9
31P and 13C NMR studies of intermediates of aerobic and anaerobic glycolysis in Saccharomyces cerevisiae; den Hollander JA et al.; The levels of intermediates of aerobic and anaerobic glycolysis were determined in perchloric acid extracts prepared from glycolyzing suspensions of Saccharomyces cerevisiae by 31P and 13C NMR spectroscopy . From 31P NMR measurements a small increase in the level of nucleoside triphosphates was found in derepressed cells upon oxygenation, while the ratio of nucleoside diphosphates to nucleoside triphosphates was a factor of 3 lower aerobically . Combined with the previous observation that the level of intracellular Pi is lower by a factor of 3 aerobically, this leads to the conclusion that the phosphate potential {NTP}/({NDP}{Pi}) is lower by an order of magnitude during anaerobic glycolysis than during aerobic glycolysis . There was no correlation between the level of glucose 6-phosphate and the rate of glucose utilization . We used 13C NMR to determine the scrambling of the 13C label from C1 to C6 in fructose 1,6-bisphosphate (Fru-P2) . There was more scrambling of the label during aerobic than during anaerobic glycolysis . Since the level of Fru-P2 did not change much upon oxygenation, this suggests that in aerobic glycolysis there is control of at least one enzyme in the lower part of the Embden-Meyerhof-Parnas pathway, below Fru-P2, which gives the 13C level more time to equilibrate between C1 and C6 of Fru-P2 . Previous 13C NMR measurements of glucose utilization rates had shown a 2-fold reduction upon oxygenation, reflecting control in the early stages of the pathway.

Gene, 1986, 50(1-3), 239 - 45
Expression of human salivary alpha-amylase gene in Saccharomyces cerevisiae and its secretion using the mammalian signal sequence; Nakamura Y et al.; A cDNA fragment coding for human salivary alpha-amylase precursor was joined to the promoter of the Saccharomyces cerevisiae PHO5 gene, and the recombinant gene was inserted into a vector plasmid capable of autonomous replication in yeast . Yeast cells transformed with this recombinant plasmid synthesized about 5 X 10(5) molecules of the enzyme per cell when synthesis was induced by deprivation of inorganic phosphate and released about half of the synthesized enzyme into the medium . The enzyme is stable, and exhibited the same specific activity as alpha-amylase in human saliva . The amylase-producing yeast grew on starch and produced alcohol.

Gene, 1986, 49(3), 283 - 93
The MET2 gene of Saccharomyces cerevisiae: molecular cloning and nucleotide sequence; Langin T et al.; A 5.1-kb DNA fragment from Saccharomyces cerevisiae, which complements a yeast met2 mutant strain, has been cloned . This fragment contains the wild-type MET2 gene which codes for the homoserine O-transacetylase, one of the methionine biosynthetic enzymes . The presence of the MET2 gene has been shown by integrative transformation experiments and genetic analyses of the resulting transformants . The complete nucleotide sequence of a 2826-bp DNA fragment carrying the MET2 gene has been determined . The sequence contains one major open reading frame of 438 codons, giving a calculated Mr of 48,370 for the encoded protein . We have identified the transcriptional product of the MET2 gene and estimated its size at 1650 nucleotides.

Basic Life Sci, 1986, 40, 499 - 510
Host factors in nuclear plasmid maintenance in Saccharomyces cerevisiae; Tye BK et al.; In yeast, the initiation of DNA replication on chromosomes is believed to occur at specific sequences known as autonomously replicating sequences (ARSs) . We previously isolated a number of mutants that are defective in the maintenance of minichromosomes . Analysis of these mutants suggests that although ARSs differ greatly from one another in their primary sequences, they appear to share a common enzyme complex for the initiation of DNA replication . However, this initiation enzyme complex probably binds with differential affinity to different ARSs . This idea is corroborated by our identification of an ARS-binding protein that binds to different ARSs with different efficiencies.

Basic Life Sci, 1986, 40, 165 - 71
Molecular characterization of chromosomal genes affecting double-stranded RNA replication in Saccharomyces cerevisiae; Icho T et al.; We cloned MAK11, MAK18, and MKT1 utilizing their genetic map positions . The MAK11 gene is close to CDC16 on chromosome XI . Both genes were cloned on a single 7-kb fragment, and both have now been sequenced . The MAK18 gene is located close to PET3 on chromosome VIII . A large plasmid carrying PET3 was obtained from R . Elder and R.E . Esposito and was found to also have the MAK18 gene . The MAK16 gene has been subcloned and sequenced starting with a clone provided by J . Crowley and D . Kaback . The MKT1 gene was mapped near the gene for topoisomerase II . The topoisomerase II clone was used as the starting point for chromosome-walking to isolate MKT1 . A deletion-insertion mutation (disruption) of MKT1 results in an inability to maintain M2, but does not affect M1 or L-A maintenance . Clones of SKI3 and SKI8 were selected using the cold sensitivity for cell growth of ski- M1 strains . The SKI8 gene was disrupted and found to be nonessential for cell growth in the absence of M double-stranded RNA (dsRNA) . The SKI3 and SKI8 genes were mapped using these clones . We have also obtained other clones suppressing the pathology caused by the high M titer in ski- strains . These clones are not the SKI genes themselves but somehow avoid the growth defect without repressing M copy number.

Basic Life Sci, 1986, 40, 149 - 63
Overview of double-stranded RNA replication in Saccharomyces cerevisiae; Wickner RB et al.; There are five families of double-stranded RNA (dsRNA) in strains of Saccharomyces cerevisiae, called L-A, L-BC, M, T, and W . Of these, L-A, L-BC, and M are found in intracellular virus-like particles (VLPs) . Their replication is controlled by over 40 chromosomal genes; some (called MAK genes) promote dsRNA replication or maintenance, others (called SKI genes) negatively control dsRNA replication . Extensive genetic interactions among the dsRNAs and the chromosomal genes are known . The VLPs containing dsRNA produce a message (+) strand RNA copy in vitro, while the VLPs containing a (+) strand synthesize a (-) strand copy to make dsRNA . The genes MAK10 and PET18 (= MAK31 + MAK32) are necessary for the structural stability of L-A dsRNA-containing particles, but not of those containing L-A (+) strand RNA . The M1 VLPs can have either one or two M1 dsRNA molecules per particle, a fact that we explain by a sort of "head-full" hypothesis . {D} (for disease) is a new cytoplasmic genetic element which, when introduced into a ski M1 strain, makes the strain unable to grow at 20 degrees C or at 37 degrees C . {D} is not located on L-A, L-BC, M, or W dsRNA . Element {D} is heat-curable, and chromosomal mutants unable to maintain {D} (mad-) have been isolated . They can maintain M1 and L-A . {B} is a cytoplasmic genetic element which suppresses the usual need of M1 for MAK11 and several other MAK genes . Element {B} is not located on L-A or M and is distinct from {D}.

Gene, 1986, 48(2-3), 267 - 75
Synthesis and processing of Semliki forest virus polyprotein in Saccharomyces cerevisiae: a yeast type glycosylation of E1 envelope protein; Keranen S; A cDNA coding for the structural proteins of Semliki Forest virus (SFV) was ligated between the ADC1 promoter and terminator in a yeast expression vector, pAAH5 . Synthesis of the SFV-specific proteins in Saccharomyces cerevisiae transformed with this vector was shown by immunoblotting and immunoprecipitation . Detection of the N-terminal and the C-terminal components of the viral polyprotein, capsid protein and E1 envelope protein, respectively, suggested that the entire polyprotein was translated in yeast . The capsid protein was effectively released from the polyprotein as a normal size polypeptide, but the following protein, p62 (E3, E2 precursor) was not detected, suggesting that it was rapidly degraded . Electrophoretic analyses indicated that the final protein, E1, entered the secretory pathway, the signal sequence was cleaved off and the protein became extensively and heterogeneously glycosylated . These data suggest that E1 was transported to the Golgi complex and that yeast-characteristic outer-chain glycans were added to the protein.

Gene, 1986, 48(1), 155 - 63
Expression of the hepatitis B virus large envelope protein in Saccharomyces cerevisiae; Dehoux P et al.; The yeast vector pPV2 has been constructed for inducible expression of non-fused proteins from the PHO5 promoter . Signals of the URA3 gene are used for transcription termination . The 226-amino-acid 'major' and the 389-amino-acid 'large' envelope protein of hepatitis B virus (HBV) have been produced in Saccharomyces cerevisiae following insertion of the S gene or of the entire pre-S region and the S gene, respectively, of HBV into pPV2 . Although normally only a minor constituent of the viral envelope, the 'large' protein forms particles with cellular lipids similar to those composed of the 'major' envelope protein . Such particles carry pre-S1, pre-S2, and S-encoded epitopes and, in addition, a receptor for polymerized human serum albumin.

Gene, 1986, 47(2-3), 155 - 77
The primary structure of the mitochondrial genome of Saccharomyces cerevisiae--a review; de Zamaroczy M et al.; We have collated and compiled all the available primary structure data on the mitochondrial genome of Saccharomyces cerevisiae . Data concern 78,500 bp, namely 92% of the 'long' genomes; they are derived from several laboratory strains . Interstrain differences belong to three classes: a small number of large deletions/additions, mainly concerning introns; a large number of small (10-150 bp) deletions/additions located in the intergenic sequences; 1-3 bp deletions/additions and point mutations; the interstrain sequence divergence due to the latter, is of the order of 2% for the strains compared; this low value is, however, an overestimate because of sequence mistakes.

Acta Biochim Pol, 1986, 33(3), 217 - 27
Intracellular localization of Aspergillus nidulans ornithine carbamoyltransferase in native host cells and in Saccharomyces cerevisiae cells harbouring its cloned structural gene; Maleszka R et al.; Differential centrifugation of the Aspergillus nidulans cell lysate shows that ornithine carbamoyltransferase (EC 2.1.3.3) appears mainly in the particulate (organellar) fraction . The enzyme was located to the mitochondria by co-sedimentation with cytochrome oxidase in isopycnic density gradient and by cytochemical-electron microscopic means . Arginase (EC 3.5.3.1) and ornithine delta-aminotransferase (E.C . 2.6.1.13) were found to reside in cytosol . The release of ornithine carbamoyltransferase from the organellar fraction by various agents indicates that the enzyme resides in the mitochondrial matrix . In Saccharomyces cerevisiae the plasmid pSAL43, carrying cloned Aspergillus nidulans ornithine carbamoyltransferase gene, directs the synthesis of the enzyme partially associated with yeast mitochondria even though the homologous yeast enzyme is exclusively cytosolic . The implications of these findings are discussed.

Gene, 1986, 46(2-3), 207 - 14
Molecular cloning and characterization of the MET6 gene of Saccharomyces cerevisiae; Csaikl U et al.; A yeast DNA fragment complementing the met6 mutation in yeast (Saccharomyces cerevisiae) was cloned in a shuttle vector, Yep13, by transforming a yeast host with plasmid DNA prepared from yeast gene bank CV13 of K . Nasmyth . A restriction map of a 6-kb Sau3A insert was constructed . A 2.6-kb fragment (Sau3A-BamHI) complementing the mutation was found by subcloning . Evidence that the DNA fragment contains the yeast MET6 gene was obtained by genomic integration . A 2.5-kb transcript is found both in wild-type (wt) and met6 yeast strains by Northern blotting experiments, indicating that the mutation acts at posttranscriptional level . The rate of transcription for the integrant lies between the values observed for the wt and mutant strains . The functional gene product seems to be involved in negative regulation of transcription of the MET6 gene.

Gene, 1986, 46(2-3), 181 - 6
Cloning of open reading frames and promoters from the Saccharomyces cerevisiae genome: construction of genomic libraries of random small fragments; Santangelo GM et al.; We have developed a novel efficient method, carrier-facilitated insertion, to insert small (150-600 bp) DNA fragments into plasmid vectors . This method employs a carrier segment of vector DNA to circumvent the difficulties in ligating two fragments together to generate a recombinant circle efficiently . We have used carrier-facilitated insertion to construct three genomic libraries of random (DNase I-generated) fragments from the Saccharomyces cerevisiae genome . One of these was an expression library, and the other two were promoter-cloning libraries . 87-90% of the Escherichia coli colonies in each library contained recombinant plasmids, and less than 3% of the recombinants contained more than one insert . Detection of open reading frames among the inserts in the expression library was accomplished by testing for beta-galactosidase activity . This methodology, unencumbered by the intrinsic disproportionality of cDNA libraries, can be used to identify and clone DNA that codes for a specific antigenic determinant . When used in combination with a method to detect and isolate random constitutive, repressible and inducible yeast promoters, these libraries should permit a comprehensive analysis of the yeast genome and its expression.

Microbios, 1986, 48(194), 7 - 16
Uptake of nystatin by protoplasts of sensitive and resistant cells of Saccharomyces cerevisiae; Beezer AE et al.; The uptake of nystatin by protoplasts derived from sensitive and resistant cells of Saccharomyces cerevisiae has been studied as a function of nystatin concentration, temperature and pH . The presence or absence of glucose in the uptake experiments was also studied . Activation energies (Ea) for nystatin uptake revealed profound differences between protoplasts derived from sensitive and resistant cells . Those for the latter closely resembled their whole cell counterparts . The values of Ea for the uptake of nystatin under all the conditions studied indicate the importance of the cell wall in the uptake process.

Microbios, 1986, 48(194), 17 - 26
Properties of glucose uptake in vegetative and sporulating cells of Saccharomyces cerevisiae; Ota A; The effect of digitonin, acetic acid, urea and ethanol treatment on the glucose uptake of vegetative cells and of sporulating cells (3 h after transfer to sporulation medium) was examined in Saccharomyces cerevisiae . Both glucose uptake activities decreased at a similar rate, and a slightly different rate, in treatment with various concentrations of digitonin and of acetic acid, respectively, at 25 degrees C for 10 min . The glucose uptake activity of the sporulating cells was much more stable to urea treatment than that of the vegetative cells; the activity decreased about 36% and 76% in the sporulating cells and the vegetative cells, respectively, under conditions of 2.5 M urea at 25 degrees C for 10 min . The glucose uptake activity of the vegetative cells was more stable to ethanol treatment than that of the sporulating cells; the activity decreased about 56% and 88% in the vegetative cells and the sporulating cells, respectively, in 25% ethanol at 25 degrees C for 10 min.

Mol Cell Biol, 1986 Jan, 6(1), 241 - 5
Structure and sequence of the centromeric DNA of chromosome 4 in Saccharomyces cerevisiae; Mann C et al.; The CEN4 sequences from chromosome 4 that impart mitotic stability to autonomously replicating (ARS) plasmids in yeast cells have been localized to a 1,755-base-pair (bp) fragment . This fragment could be cut in half to give two adjacent, nonoverlapping fragments, that each contained some mitotic stabilization sequences . One of the half-fragments worked as efficiently as the larger fragment from which it was derived, while the other half provided a much poorer degree of mitotic stabilization . Sequencing of 2,095 bp of DNA including this region revealed the presence of a centromere consensus sequence, elements I, II, and III (M . Fitzgerald-Hayes, L . Clarke, and J . Carbon, Cell 29:235-244, 1982), in the half-fragment providing high levels of mitotic stability . The poorly stabilizing half-fragment did not contain any obvious sequence homologies to other centromere sequences . Deletion analysis of the 1,755-bp fragment indicated that removal of the 14-bp element I plus 16 of the 82 bp of element II impaired mitotic stability . Removal of elements I and II eliminated the mitotic stability provided by the consensus sequence.

Mol Cell Biol, 1986 Jan, 6(1), 158 - 67
Tightly centromere-linked gene (SPO15) essential for meiosis in the yeast Saccharomyces cerevisiae; Yeh E et al.; We used DNA fragments from the centromere regions of yeast (Saccharomyces cerevisiae) chromosomes III and XI to examine the transcriptional activity within this chromosomal domain . DNA transcripts were found 200 to 300 base pairs from the 250-base-pair centromere core and lie within an ordered chromatin array . No transcripts were detected from the functional centromere region . We examined the cellular function of one of these tightly centromere-linked transcripts . (CEN11)L, by disrupting the coding sequences in vivo and analyzing the phenotype of the mutant yeast cell . Diploids heterozygous for the (CEN11)L disruption sporulated at wild-type levels, and the absence of the (CEN11)L gene product had no effect on the viability or mitotic growth of haploid cells . Diploids homozygous for the (CEN11)L disruption were unable to sporulate when induced by the appropriate nutritional cues . The mutant cells were competent for intragenic recombination and appeared to be blocked at the mononucleate stage . The temporal ordering of (CEN11)L function with respect to the sporulation mutant spo13 suggests that the (CEN11)L gene product may be required at both the first and second meiotic cell divisions . This new sporulation gene has been termed SPO15.

Microbios, 1986, 47(192-193), 165 - 76
Uptake of nystatin by sensitive and resistant cells of Saccharomyces cerevisiae; Beezer AE et al.; The uptake of nystatin by sensitive and by resistant cells of Saccharomyces cerevisiae was studied as a function of nystatin concentration, temperature and pH . The presence or absence of glucose in nystatin uptake experiments was also studied . The rate data, effects of glucose and the derived activation energies for nystatin uptake revealed significant differences in response between sensitive and resistant cells . The role of the cell wall in the uptake process is discussed.

Gene, 1986, 45(1), 67 - 75
Identification of a sequence containing the positive regulatory region of Saccharomyces cerevisiae gene ENO1; Uemura H et al.; A DNA fragment which contains the 5'-flanking region of the Saccharomyces cerevisiae enolase 1 gene (ENO1) and a portion of the coding sequence was cloned in a plasmid pMC1587 . This fragment was fused in frame to the lacZ gene of Escherichia coli . Many mutants which deleted a portion of the 5'-flanking region of ENO1 were isolated from this ENO1-lacZ fusion plasmid by in vitro recombination . Analysis of beta-galactosidase activity of these mutants indicated that the regulatory region responsible for an efficient expression of the ENO1-lacZ fused gene resides within an 86-bp sequence located at -487 to -402 upstream from the start codon of ENO1 . We found that the segment encompassing the 86-bp region worked equally well in an inverted orientation, but the tandem duplication of the sequence did not enhance the expression of the fused gene.

CRC Crit Rev Biochem, 1986, 21(3), 277 - 317
The general control of amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae; Hinnebusch AG; Enzymes in diverse amino acid biosynthetic pathways in Saccharomyces cerevisiae are subject to a general amino acid control in which starvation for any amino acid leads to increased levels of the mRNAs encoding these enzymes . The short nucleotide sequence TGACTC, found nontandemly repeated upstream from the coregulated structural genes, serves as a cis-acting site for positive regulation of transcription . Multiple trans-acting repressors and activators have been identified . Most of these factors act indirectly by regulating the level of an activator encoded by the GCN4 gene . This regulation occurs at the level of GCN4 translation and is mediated by sequences in the long 5' leader of GCN4 mRNA . The GCN4 protein is the most likely candidate for the transcriptional activator that interacts with the TGACTC sequences at the structural genes.

J Ultrastruct Mol Struct Res, 1986 Jan, 94(1), 37 - 51
Morphological observations on the formation and stability of the crystalline arrays in the plasma membrane of Saccharomyces cerevisiae; Sosinsky G et al.; Two-dimensional crystalline arrays of freeze-fracture particles are known to occur in abundant quantities in the plasma membrane of stationary state yeast cells . Although these crystalline arrays are seen only infrequently in cells during mid-exponential growth, we now observe that formation of crystalline arrays can be induced in such cells by a "metabolic starvation" protocol . Surprisingly, starvation-induced formation of crystalline patches can be prevented by inhibition of new protein synthesis during the starvation period . The size and quantity of crystalline arrays can be increased by removal of the cell wall prior to starvation . Induction of crystalline arrays in protoplasts has made it possible to investigate the surface morphology of the crystalline particles in isolated membranes as well as at the extracellular surface of intact protoplasts . The stability of isolated crystalline arrays to several detergents has been investigated and conditions have been found that result in improved morphological purity of the isolated crystalline patches.

Gene, 1986, 43(3), 273 - 9
Expression of synthetic human-lysozyme gene in Saccharomyces cerevisiae: use of a synthetic chicken-lysozyme signal sequence for secretion and processing; Jigami Y et al.; A multicopy plasmid was constructed to direct the synthesis and secretion of human lysozyme (HLY) in Saccharomyces cerevisiae . This plasmid contains a synthetic chicken-lysozyme signal sequence (SIG) and a synthetic HLY structural gene, both inserted between the yeast GAL10 promoter and 2 mu plasmid FLP (flip-flop recombination gene) terminator . The resulting plasmid directed the expression of the hybrid pre-lysozyme, with most of the HLY activity secreted into the culture medium and extracellular periplasmic space . The HLY activity in the culture medium increased with cell growth . The yeast accurately processed the hybrid precursor at the junction between the chicken SIG and the coding sequence downstream, yielding mature HLY . HLY purified from the culture medium was homogeneous and displayed specific activity identical to that of authentic HLY.

J Basic Microbiol, 1986, 26(2), 109 - 11
Oxidation of naphthalene by Saccharomyces cerevisiae and Candida utilis; Hofmann KH; The yeasts Saccharomyces cerevisiae 118 and Candida utilis 128 were examined for their ability to metabolize the polycyclic aromatic hydrocarbons naphthalene and anthracene . The two yeasts tested oxidized these aromatics . Metabolites were extracted and analyzed by thin layer chromatography and gas chromatography . The predominant oxidation product of naphthalene found in the culture medium was 1-naphthol.

J Cell Biochem, 1986, 30(4), 331 - 9
Properties of catalase activity in vegetative and sporulating cells of yeast Saccharomyces cerevisiae; Ota A; Properties of catalase activities have been examined in the intact cells of early stationary phase and cells 3 hr after transfer to sporulation medium in Saccharomyces cerevisiae . The catalase activities of the two cells had a broad optimal pH from 6 to 8 . Catalase activity in the intact cells increased throughout a 4-hr period of the observation following transfer to sporulation medium . Almost all the catalase activity in vegetative cells was lost by the treatment at 60 degrees C for 10 min . Catalase activities of both cells were inhibited by KCN, NaN3, o-phenanthroline, and PCMB . The catalase activity of the vegetative cells was slightly more inhibited and inactivated than that of the sporulating cells by the inhibitors and by the treatment with HCl or NaOH.

Folia Microbiol (Praha), 1986, 31(2), 129 - 37
Fed-batch cultivation of Saccharomyces cerevisiae with increased content of delta 5,7-sterols; Behalova B et al.; Cultivation of Saccharomyces cerevisiae with a linear nutrient feed was used to test the validity of a mathematical model describing changes in the physiological state of the culture . Markers of these changes were the concentrations of proteins and delta 5,7-sterols in yeast dry mass . The model was used to optimize the production of these sterols with regard to the magnitude and composition of nutrient feed.

Mol Gen Genet, 1986 Jan, 202(1), 68 - 74
Analysis of mutagenic DNA repair in a thermoconditional mutant of Saccharomyces cerevisiae . III . Dose-response pattern of mutation induction in UV-irradiated rev2ts cells; Siede W et al.; Recent studies regarding the influence of cycloheximide on the temperature-dependent increase in survival and mutation frequencies of a thermoconditional rev2 mutant lead to the suggestion that the REV2-coded mutagenic repair function is UV-inducible . In the present study we show that stationary-phase rev2ts cells are characterized by a biphasic linear-quadratic dose-dependence of mutation induction ("mutation kinetics") of ochre alleles at 23 degrees C (permissive temperature) but linear kinetics at the restrictive temperature of 36 degrees C . Mathematical analysis using a model based on Poisson statistics and a further mathematical procedure, the calculation of "apparent survival", support the assumption that the quadratic component of the reverse mutation kinetics investigated can be attributed to a UV-inducible component of mutagenic DNA repair controlled by the REV2 gene.

Mol Gen Genet, 1986 Jan, 202(1), 120 - 4
An altered ribosomal protein in an edeine-resistant mutant of Saccharomyces cerevisiae; Herrera F et al.; The r-proteins of an edeine-resistant mutant of Saccharomyces cerevisiae were compared to those of the wild-type strain by using two different two-dimensional electrophoretic techniques: (1) the Kaltschmidt-Wittmann method and, (2) the Kaltschmidt-Wittmann system, in the first dimension and the Na Dodecyl-SO4 system in the second . With the first technique, the results indicate that the patterns of basic ribosomal proteins are similar in the two strains . However, the pattern of acidic ribosomal proteins of the mutant revealed an additional protein band with respect to the normal one . Using the other technique, the patterns of basic and acidic ribosomal proteins of the mutant demonstrated a similarity to the corresponding pattern of the wild-type strain . The data disclose that an acidic ribosomal protein of the mutant may have two forms with different electrophoretic mobilities and similar molecular weights.

Lipids, 1986 Jan, 21(1), 102 - 6
Evidence for facilitated transport in the absorption of sterols by Saccharomyces cerevisiae; Nes WR et al.; Saccharomyces cerevisiae is known to absorb sterols readily in the absence of air . As shown in this paper, yeast cells also will absorb sterols with and without various double bonds or an alkyl group at C-24 in the presence of air at a concentration (ca . 10% of the gas phase) which is growth-limiting due to limited sterol synthesis . However, if the growth conditions are changed to be fully aerobic, sterol is no longer absorbed to any significant extent even when the sterol in the medium (ergosterol) is the same as that present in the cells . This implies that sterol in the medium does not equilibrate passively with sterol in the plasma membrane and that some sort of facilitated transport, which can be turned on and off, is responsible for the entry of sterol when it occurs as a response to an inadequate endogenous supply of sterol . In agreement with facilitated transport mediated by protein binding, yeast cells in an auxotrophic state for sterol exhibit a high degree of stereoselectivity with respect to the orientation of the side chain around the C-17(20)-bond . For instance, E-17(20)- but not Z-17(20)-dehydrocholesterol is absorbed by cells undergoing limited growth with 10% air.

Arch Microbiol, 1986 Jan, 143(4), 387 - 95
Assembly of microfibrils in vivo and in vitro from (1----3)-beta-D-glucan synthesized by protoplasts of Saccharomyces cerevisiae; Kopecka M et al.; Polymer chains of (1----3)-beta-D-glucan were dissolved with 1 M NaOH at 4 degrees C from native microfibrillar protoplast nets . The chains associated into microfibrils during NaOH neutralization or dialysis . In contrast to the native microfibrils which are of uniform width individually (10 to 20 nm) and arranged in flat bundles, the microfibrils formed in vitro showed no band formation and consisted of fibrous spindle-shaped subunits of variable width or loose elementary fibrils about 1.7 nm wide . X-ray diagrams of native nets indicated a fairly high crystallinity and were different for wet and dry specimens . They corresponded to those of paramylon . Precipitated glucans produced diagrams different from the former and revealing a lower crystallinity especially with the dry samples . The X-ray pattern, combined with other data, allowed the precipitated microfibrils to be identified as aggregates of molecular strands composed each of three intertwined helical glucan chains . Since these triple helical chains are about 1.7 nm wide the elementary fibrils of this width can represent only single triple-helical strands . These helices have 7 glucose residues per turn and therefore a low symmetry which explains the poor crystallizing properties . The 7 membered helix represents a basic difference with the well crystallized native glucan which is built of highly symmetrical triple helices with 6 glucose residues per turn . Since 6(1) helical conformation is not formed in vitro at normal temperatures its generation in vivo must be due to the action of synthesizing enzymes at the protoplast membrane . The intertwining of these helices and crystallization of the strands are determined by their symmetry and physical properties of the chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Microbiol, 1986 Jan, 143(4), 337 - 44
Ethanol-sensitive mutants of Saccharomyces cerevisiae; Aguilera A et al.; Saccharomyces cerevisiae mutants unable to grow at ethanol concentrations at which the wild type strain S288C does grow, have been isolated . Some of them show additional phenotypic alterations in colony size, temperature sensitivity and viability in ethanol, which cosegregate with the growth sensitivity in ethanol . 21 selected monogenic ethanol-sensitive mutants define 20 complementation groups, denominated ETA1 to ETA20, which indicates that there is a high number of genes involved in the ethanol tolerance/sensitivity mechanism . Out of 21 selected monogenic mutants, 20 are not altered in the glycolytic pathway since, when maintained in glucose-supplemented medium, they can produce as much ethanol as the wild type and at about the same velocity . Nor do any of the mutants seem to be altered in the lipid biosynthetic pathway since, whether grown in the absence or in the presence of ethanol, their concentration of fatty acids and ergosterol is similar to that of the wild type under the same conditions . Therefore growth sensitivity to ethanol does not seem necessarily to be related to carbohydrate or lipid metabolism.

Mutat Res, 1986 Jan-Mar, 167(1-2), 47 - 60
The detection of chemically induced aneuploidy in Saccharomyces cerevisiae: an assessment of mitotic and meiotic systems; Resnick MA et al.; Several systems have been evaluated for their ability to detect aneuploidy . Chromosome gain can be detected in mitotic haploid cells as well as meiotically derived haploid spores . Both chromosome gain and loss are detectable in mitotic diploid cells . Several chemicals have been identified that clearly induce aneuploidy in at least one or more of the systems.

J Bacteriol, 1986 Jan, 165(1), 293 - 6
Asparaginase II of Saccharomyces cerevisiae: selection of four mutations that cause derepressed enzyme synthesis; Kamerud JQ et al.; A positive selection method was used to isolate four Saccharomyces cerevisiae mutations that cause derepressed synthesis of asparaginase II . The four mutations (and1, and2, and3, and4) were neither closely linked to each other nor linked to previously characterized mutations (asp3, asp6) which cause the complete loss of asparaginase II activity . One of the new mutations (and4) was shown to be allelic to gdh-CR, a pleiotropic mutation which causes derepressed synthesis of a number of enzymes of nitrogen catabolism.

J Bacteriol, 1986 Jan, 165(1), 28 - 33
Calcium-sensitive cls4 mutant of Saccharomyces cerevisiae with a defect in bud formation; Ohya Y et al.; A calcium-sensitive cls4 mutant of Saccharomyces cerevisiae ceased dividing in the presence of 100 mM CaCl2, producing large, round, unbudded cells . Since its DNA replication and nuclear division still continued after interruption of normal budding, the cls4 mutant had a defect in bud formation in Ca2+-rich medium . Its calcium content and calcium uptake activity were the same as those of the wild-type strain, suggesting that the primary defect of the mutation was not in a Ca2+ transport system . Genetic analysis showed that the cls4 mutation did not complement the cdc24-1 mutation, which is known to be a temperature-sensitive mutation affecting bud formation and localized cell surface growth at a restrictive temperature . Moreover, cls4 was tightly linked to cdc24, and a yeast 3.4-kilobase-pair DNA fragment carrying both the CLS4 and CDC24 genes was obtained . These results suggest that the cls4 mutation is allelic to the cdc24 mutation . Thus, Ca2+ ion seems to control bud formation and bud-localized cell surface growth.

Princess Takamatsu Symp, 1986, 17, 253 - 60
Exploring the function of RAS oncogenes by studying the yeast Saccharomyces cerevisiae; Toda T et al.; The RAS oncogenes comprise a family of genes found to be activated in perhaps 10-20% of human cancers and which have been highly conserved in evolution . Homologs of the mammalian RAS exist in the yeast Saccharomyces cerevisiae (RAS1 and RAS2) . We have shown that human ras proteins can complement the loss of RAS1 and RAS2 proteins in yeast, and hence are functionally homologous . Both human and yeast RAS proteins can stimulate the magnesium and guanine nucleotide-dependent adenylate cyclase activity present in yeast membranes . However, RAS proteins do not appear to stimulate adenylate cyclase in vertebrate cells . Our studies indicate that although RAS proteins are essential controlling elements of adenylate cyclase in yeast, they have other essential functions in that organisms . RAS proteins are themselves probably controlled by growth regulatory proteins.

Curr Genet, 1986, 11(3), 205 - 10
DNA repair genes of Saccharomyces cerevisiae: complementing rad4 and rev2 mutations by plasmids which cannot be propagated in Escherichia coli; Siede W et al.; The RAD4 gene of yeast required for the incision step of DNA excision repair and the REV2 (= RAD5) gene involved in mutagenic DNA repair could not be isolated from genomic libraries propagated in E . coli regardless of copy number of the shuttle vector in yeast . Transformants with plasmids conferring UV resistance to a rad4-4 or a rev2-1 mutant were only recovered if yeast was transformed directly without previous amplification of the gene bank in E . coli . DNA preparations from these yeast clones yielded no transformants in E . coli but retransformation of yeast was possible . This lead to the isolation of a defective derivative of the rad4 complementing plasmid . The modified plasmid was now capable of transforming E . coli but still interfered significantly with its growth.

Curr Genet, 1986, 11(1), 79 - 82
Transformation of psi- Saccharomyces cerevisiae to psi+ with DNA co-purified with 3 micron circles; Dai H et al.; DNA enriched for supercoiled plasmids prepared from the 3 micron plasmid-enriched, {psi+}, {2 micron degrees} strain 6-1G-P188 and from the {2 micron+} {psi+} strain LL20 can be used to transform a psi- recipient strain to psi+ . Fractionation of the former preparation by electrophoresis showed that the 3 micron plasmid band contained the transforming activity.

Curr Genet, 1986, 10(9), 665 - 70
Genetic mapping of nuclear mucidin resistance mutations in Saccharomyces cerevisiae . A new pdr locus on chromosome II; Subik J et al.; In the yeast Saccharomyces cerevisiae, two nuclear pleiotropic drug resistance mutations pdr3-1 (former designation mucPR) and pdr3-2 (former designation DRI9/T7) have been selected as resistant to mucidin and as resistant to chloramphenicol plus cycloheximide, respectively . The pdr3 mutations were found not to affect the plasma membrane ATPase activity measured in a crude membrane fraction . Meiotic mapping using strains with standard genetic markers revealed that mutation pdr3-1 is centromere linked on the left arm of chromosome II at a distance of 5.9 +/- 3.3 cM from its centromere and 11.6 +/- 3.1 cM from the marker pet9 . The centromere linked pdr3-2 mutation exhibited also genetic linkage to pet9 with a map distance of 9.8 +/- 3.2 cM . These results indicate that pdr3-1 and pdr3-2 are alleles of the same pleiotropic drug resistance locus PDR3 which is involved in the control of the plasma membrane permeability in yeast.

Curr Genet, 1986, 10(9), 657 - 64
Identification and characterization of four new GCD genes in Saccharomyces cerevisiae; Niederberger P et al.; Mutant strains, resistant against the amino acid analogues 5-methyltryptophan, 5-fluorotryptophan and canavanine were isolated, starting with a trp2 leaky auxotrophic strain . Of 10 such strains, only four turned out to be of the "general control derepressed" (gcd) mutant type . Three other isolates were shown to be defective in the general amino acid permease system, while the remaining three strains displayed low spore viability and were not further investigated . Complementation tests amongst the four new gcd-mutant strains, including strain RH558 gcd2-1 isolated earlier, yielded five complementation groups: GCD2, GCD3, GCD4, GCD5, and GCD6 . All mutant strains showed a dual phenotype, which was not separable by wild type backcrosses: "constitutive derepression" and "slow growth" . Epistasis of all gcd mutations over gcn1-1, gcn2-1 and gcn3-1 was found with respect to both phenotypes, except for gcd5-1, which was lethal in these combinations . On the other hand gcn4-101 was found to be epistatic over all gcd mutations, but only with respect to the "constitutive derepression" phenotype, and not to "slow growth"; again the combination with gcd5-1 was lethal . Mutation gcd2-1 was mapped on chromosome VII, 50 cM from leu1 and 22 cM from ade6 . A new model is discussed, in which GCD-genes are involved in the amino acid uptake into the vacuoles.

Curr Genet, 1986, 10(9), 643 - 6
Clones from two different genomic regions complement the cdc25 start mutation of Saccharomyces cerevisiae; Daniel J et al.; We have cloned the CDC25 gene of Saccharomyces cerevisiae whose product is required for traversing the Go phase of the cell cycle . A preliminary physical characterization of the CDC25 gene region is presented . In addition, we show that another gene, when cloned in a high-copy number plasmid, is able to partially suppress growth thermosensitivity of a strain carrying the cdc25 mutation . We briefly discuss the possible interaction of these gene products with adenylate cyclase encoded by the CDC35 gene.

Curr Genet, 1986, 10(12), 943 - 5
Alpha-factor enhancement of hybrid formation by protoplast fusion in Saccharomyces cerevisiae II; Curran BP et al.; When Mat a cells are treated with alpha-factor prior to being protoplasted and fused, the frequency of karyogamy is higher than in unarrested controls.

Curr Genet, 1986, 10(12), 879 - 85
The CDC25 "Start" gene of Saccharomyces cerevisiae: sequencing of the active C-terminal fragment and regional homologies with rhodopsin and cytochrome P450; Daniel JH; The CDC25 Start gene whose product appears to be required for traversing the Go phase of the cell cycle in Saccharomyces cerevisiae, has been previously cloned (J . Daniel and G . Simchen (1986), Curr Genet 10:643-646) . By nucleotide sequencing of an active subclone, we found that only a region of the gene that codes for the C-terminal portion of the CDC25 protein was required for full suppression of the cdc25 mutation . The codon usage in this region indicates a poor translation of the transcript compared to genes encoding abundant proteins . The derived CDC25 protein fragment contains two regions of homology, one with the rhodopsin family, the other with the cytochrome P450 family . Strikingly, these two regions of homology are adjacent on the CDC25 protein . In view of the likely involvement of the CDC25 protein in the regulation of adenylate cyclase activity, a working hypothesis is proposed that accounts for the observed homologies.

Curr Genet, 1986, 10(12), 871 - 8
Analysis of mutagenic DNA repair in a thermoconditional mutant of Saccharomyces cerevisiae . IV . Influence of DNA replication and excision repair on REV2 dependent UV-mutagenesis and repair; Siede W et al.; A double mutant being thermoconditionally defective in mutation induction as well as in repair of pre-lethal UV-induced DNA damage (rev2ts) and deficient in excision repair (rad3-2) was studied in temperature-shift experiments . The influence of inhibitors of DNA replication (hydroxyurea, aphidicolin) was determined . Additionally, an analysis of the dose-response pattern of mutation induction ("mutation kinetics") at several ochre alleles was carried out . It was concluded that the UV-inducible REV2 dependent mutagenic repair process is not induced in excision-deficient cells . In excision-deficient cells, REV2 dependent mutation fixation is slow and mostly post-replicative though not dependent on DNA replication . The REV2 mediated mutagenic process could be separated from the repair function.

Curr Genet, 1986, 10(10), 713 - 23
The oli1 gene and flanking sequences in mitochondrial DNA of Saccharomyces cerevisiae: the complete nucleotide sequence of a 1.35 kilobase petite mitochondrial DNA genome covering the oli1 gene; Ooi BG et al.; As part of our genetic and molecular analysis of mutants of Saccharomyces cerevisiae affected in the oli1 gene (coding for mitochondrial ATPase subunit 9) we have determined the complete nucleotide sequence of the mtDNA genome of a petite (23-3) carrying this gene . Petite 23-3 (1,355 base pairs) retains a continuous segment of the relevant wild-type (J69-1B) mtDNA genome extending 983 nucleotides upstream, and 126 nucleotides downstream, of the 231 nucleotide oli1 coding region . There is a 15-nucleotide excision sequence in petite 23-3 mtDNA which occurs as a direct repeat in the wild-type mtDNA sequence flanking the unique petite mtDNA segment (interestingly, this excision sequence in petite 23-3 carries a single base substitution relative to the parental wild-type sequence) . The putative replication origin of petite 23-3 is considered to be in its single G,C rich cluster, which differs in just one nucleotide from the standard oriS sequence . The DNA sequences in the intergenic regions flanking the oli1 gene of strain J69-1B (and its derivatives) have been systematically compared to those of the corresponding regions of mtDNA in strains derived from the D273-10B parent (sequences from the laboratory of A . Tzagoloff) . The nature and distribution of the sequence divergences (base substitutions, base deletions or insertions, and more extensive rearrangements) are considered in the context of functions associated with mitochondrial gene expression which are ascribed to specialized sequences in the intergenic regions of the yeast mitochondrial genome.

Curr Genet, 1986, 10(8), 565 - 71
A direct study of the relative synthesis of petite and grande mitochondrial DNA in zygotes from crosses involving suppressive petite mutants of Saccharomyces cerevisiae; Chambers P et al.; Work in recent years has produced indirect evidence to support the view that the phenomenon of suppressiveness in yeast is the result of the ability of the petite mtDNA to out-replicate the wild-type genome . We have developed a method, based on fluorography of gels containing restriction fragments of radioactively labelled zygotic mtDNA, by which it has been possible to follow directly the incorporation of label into the two mtDNA species and hence their relative synthesis . Four petite isolates of 70%, 43%, 23% and 12% suppressiveness were tested by this method in crosses with a grande strain . Only the mtDNA from the 70% suppressive petite showed a replicative advantage over the grande mtDNA . The mtDNA from the 43% and 23% suppressive actually appeared to undergo, if anything, less replication in the zygote than the grande mtDNA . It is concluded that while some petites may exhibit suppressiveness as a result of enhanced replicative efficiency of their mtDNA, this cannot be the explanation for all suppressive petite strains.

Curr Genet, 1986, 10(7), 495 - 501
A new negative control gene for amino acid biosynthesis in Saccharomyces cerevisiae; Skvirsky RC et al.; Enzyme levels in multiple amino acid biosynthetic pathways in yeast are coregulated . This control is effected largely at the transcriptional level by a number of regulatory genes . We report the isolation and characterization of a new negative regulatory gene, GCD4, for this general control system . GCD4 mutations are recessive and define a single Medelian gene on chromosome III . A gcd4 mutation results in resistance to different amino acid analogs and elevated, but fully inducible, mRNA levels of genes under general control . Epistasis analysis indicates that GCD4 acts more directly than the positive regulators GCN1, GCN2, GCN3 and GCN5, but less directly than GCN4, on the transcription of the amino acid biosynthetic genes . These data imply that GCD4 is a negative regulator of the positive effector, GCN4 . Although GCD4 occupies the same position relative to the GCN genes as other GCD genes, it produces a unique phenotype . These results illustrate the diversity of function of negative regulators in general control.

Curr Genet, 1986, 10(6), 487 - 9
The RAD50 gene of Saccharomyces cerevisiae is not essential for vegetative growth; Kupiec M; The gene RAD50 was located by the ability of subclones to restore the Rad+ phenotype following transformation of a rad50-1 mutant . Disruption of the gene was achieved by directed integration of a plasmid carrying a fragment internal to RAD50 . Haploids with the disrupted gene are viable and do not differ in growth rate or plating efficiency from isogenic rad50-1 or Rad+ strains.

Curr Genet, 1986, 10(6), 435 - 41
Cloned mitochondrial DNA from the zygomycete Absidia glauca promotes autonomous replication in Saccharomyces cerevisiae; Burmester A et al.; We have cloned fragments from mitochondrial and chromosomal DNA of the zygomycete Absidia glauca in Saccharomyces cerevisiae using the ARS selection vector YIp5 . Though it has not been possible to select ARS elements from chromosomal DNA, we succeeded in isolating two clones of mitochondrial origin that support autonomous replication in bakers' yeast . DNA from these plasmids has been shown to hybridize with mitochondrial DNA from both mating types . Generation times of the transformed yeast strain in selective medium are around 20 h . In liquid minimal medium only 6% of the cells contain the plasmid; in complete medium a mitotic stability of 50% has been determined.

Curr Genet, 1986, 10(6), 425 - 33
The REC46 gene of Saccharomyces cerevisiae controls mitotic chromosomal stability, recombination and sporulation: cell-type and life cycle stage-specific expression of the rec46-1 mutation; Esposito MS et al.; The recessive hyperrecombination mutation rec46-1, isolated by ultraviolet light mutagenesis of the MAT alpha n+1 chromosome VII disomic strain LBL1 (Esposito et al . 1982), enhances the mitotic rates of spontaneous gene conversion, intergenic recombination and restitution of haploidy (due to chromosomal loss or mitotic nondisjunction) in MAT alpha n+1 chromosome VII disomic strains . The rec46-1 mutation does not prevent HO directed homothallic interconversion of mating types . MATa/MaT alpha rec46-1/rec46-1 diploids exhibit the same degree of hyperrecombinational activity as MAT alpha rec46-1 n+1 chromosome VII disomics with respect to gene conversion and intragenic recombination resulting in prototrophy . When compared to MAT alpha rec46-1 n+1 disomics however, MATa/MAT alpha rec46-1/rec46-1 diploids exhibit a ten fold reduced level of hyperrecombinational activity with respect to intergenic recombination and present no evidence of chromosomal loss or nondisjunction resulting in 2n-1 monosomic segregants . MATa/MAT alpha rec46-1/rec46-1 diploids are sporulation-deficient . The results obtained demonstrate that the REC46 gene product modulates mitotic chromosomal stability and recombination and is essential for sporulation (meiosis and ascospore formation).

Curr Genet, 1986, 10(5), 353 - 7
Genetic characterization of an alpha-specific gene responsible for sexual agglutinability in Saccharomyces cerevisiae: mapping and gene dose effect; Suzuki K et al.; A recessive ag alpha 1 mutation leads to specific defect in sexual agglutinability specifically in alpha cells of the yeast Saccharomyces cerevisiae . The cryptopleurine resistance gene cryR 1, closely linked to the mating type locus, was used to select alpha/alpha strains which emerged from alpha/alpha strains by mitotic nonreciprocal recombination, to genetically analyse ag alpha 1, since ag alpha 1 is expressed only in alpha mating type . The ag alpha 1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv 3 at 12 cM on chromosome X . Sexual agglutinability of alpha cells was shown to be dependent on the dose of the AG alpha 1 gene, using alpha/alpha isogenic strains carrying AG alpha 1/AG alpha 1, AG alpha 1/ag alpha 1 or ag alpha 1/ag alpha 1 . The sst2-1 mutation did not suppress the ag alpha 1 mutation . Based on these results, function of the AG alpha 1 gene is discussed.

Mutagenesis, 1986 Jan, 1(1), 21 - 8
Disomic and diploid meiotic products induced in Saccharomyces cerevisiae by the salts of 27 elements; Sora S et al.; The effects of salts of 27 elements on recombination and on the production of disomic and/or diploid spores during meiosis of Saccharomyces cerevisiae has been investigated . Be(NO3)2, MgSO4, FeSO4, CuSO4, AgNO3, Na2HAsO4 were inactive on the events studied during meiotic cell division . AuCl4, CdCl2, C4H6O4Pb, SnCl2, K2Cr2O7, RbCl induced both disomic and diploid spores . LiCl acted similarly and also affected recombination . Activity in the induction of disomic spores was shown by MnSO4, HgCl2 and SrCl2 . CsCl, CaCl2, Na2MoO4, NiCl2, K2PtCl4 increased the frequency of diploid spores, while NaWO4, VOSO4, KCl, BaCl2 already increased recombination frequency . NaBiO3 showed an effect on meiotic recombination only . A decrease in the occurrence of both diploid and disomic spores was suggested by the data obtained with CoCl2.

Gene, 1986, 45(2), 149 - 58
An efficient chloramphenicol-resistance marker for Saccharomyces cerevisiae and Escherichia coli; Hadfield C et al.; Chloramphenicol (Cm) was demonstrated to be a suitable selective agent for the plasmid-mediated transformation of haploid and polyploid strains of Saccharomyces cerevisiae . A yeast/Escherichia coli shuttle Cm-resistance (CmR) marker was constructed by inserting the CAT coding sequence from Tn9, and its associated bacterial ribosome-binding site, between a modified yeast ADC1 promoter and CYC1 terminator . When present on a 2 microns-based replicating plasmid, this marker transformed yeast as efficiently as the auxotrophic markers TRP1 and LEU2 . When included in an integrating vector, single-copy transformants were formed as efficiently as with LEU2 and HIS3 . Industrial yeast strains were transformed with both the replicating and integrating plasmids . The CmR marker could also efficiently transform E . coli . This versatile and efficient performance is currently unique for a yeast dominant marker.

Mol Cell Biol, 1986 Jan, 6(1), 246 - 56
Duplicate upstream activating sequences in the promoter region of the Saccharomyces cerevisiae GAL7 gene; Tajima M et al.; We constructed a series of deletions in the 5' noncoding region of the Saccharomyces cerevisiae GAL7 gene, fused them to the Escherichia coli gene lacZ, and introduced them into yeasts by using a multicopy vector . We then studied the effect of the deletions on beta-galactosidase synthesis directed by the gene fusions in media with various carbon sources . This analysis identified a TATA box and two upstream activating sequences as necessary elements for galactose-controlled GAL7 transcription . Two upstream activating sequences exhibiting 71% homology with each other were located 255 and 168 base pairs, respectively, upstream of the GAL7 transcription start point . Each sequence consists of 21 base pairs, displaying an approximate rotational symmetry with a core consensus sequence of GAA--AGCTGCTTC--CGCG . At least one of the two sequences is required for galactose induction and also for glucose repression of the GAL7'-lac'Z gene . Analysis with host regulatory mutants delta gal14 and delta gal180 suggests that these sequences are the site at which the GAL4 product exerts its action to activate the GAL7 gene . We also observed that a deletion lacking both upstream activation sequences allowed the gene fusion to be expressed in the absence of galactose at about 10% of the fully induced level of the intact fusion . This constitutive expression depended on the presence of the TATA box of GAL7 in cis but not on a functional GAL4 gene . The level of the uncontrolled expression was decreased by increasing the distance between the TATA box and the pBR322 sequence in the vector plasmid.

Gene, 1986, 44(2-3), 337 - 40
Expression of human T-cell leukemia virus type I envelope protein in Saccharomyces cerevisiae; Kuga T et al.; The entire envelope gene of human T-cell leukemia virus type I (HTLV-I) was inserted into an expression vector and expressed under the control of the repressible acid phosphatase promoter in yeast (Saccharomyces cerevisiae) . The product in yeast cells was glycosylated into heterodisperse proteins.

Gene, 1986, 42(2), 193 - 9
Molecular cloning and genetic mapping of the DNA topoisomerase II gene of Saccharomyces cerevisiae; Voelkel-Meiman K et al.; The structural gene for DNA topoisomerase II from the yeast Saccharomyces cerevisiae has been cloned . The clones were selected from a YEp13 plasmid bank of yeast DNA by complementing a temperature-sensitive mutation (top2-1) in the topoisomerase II gene, TOP2 . Chromosomal integrants of the clone were derived by homologous recombination in strains lacking the 2 mu circle plasmid . Genetic analysis of these integrants indicates that we have cloned the TOP2 gene and not an extragenic suppressor . A YEp13-TOP2 hybrid plasmid integrant was used to localize the TOP2 gene to the left arm of chromosome XIV by the 2 mu circle-directed marker loss method . Results from standard meiotic mapping experiments indicate that TOP2 is about 16 centi-Morgans to the centromere proximal side of MET4 . Northern blot analysis of TOP2 RNA isolated from a wild-type strain and from an rna2 mutant shows the RNA to be 4.5 kb long in both cases, thus indicating that the TOP2 gene has no large introns.

Mol Gen Genet, 1986 Jan, 202(1), 83 - 9
Isolation and molecular analysis of the phosphoglucose isomerase structural gene of Saccharomyces cerevisiae; Aguilera A et al.; The PGI1 gene of Saccharomyces cerevisiae coding for the glycolytic enzyme phosphoglucose isomerase has been cloned by complementation of a mutant strain (pgi1) with a strongly reduced phosphoglucose isomerase activity . A genomic library constructed in the yeast multicopy vector YEp13 (Nasmyth and Tatchell 1980) was used . Four plasmids containing an overlapping region of 4.1 kb were isolated and characterized by restriction endonuclease mapping . Southern analysis of genomic digests prepared with different restriction enzymes confirmed the same pattern for the chromosomal sequences . Transformants with the isolated plasmids had a phosphoglucose isomerase activity increased by a factor of 7 . The cloned sequence hybridized to a constitutively synthesized 2.2 kb RNA in Northern analysis . The coding region includes a 2.05 kb EcoRI fragment common to all four inserts . A fragment including part of the PGI1 region was subcloned into vector YRp7 and used to induce integration at the PGI1 locus . Genetical and Southern analysis of stable transformants showed that single as well as tandem integration took place at this locus . This showed that the PGI1 gene had been isolated . Finally, and in contrast to the results of Kempe et al . (1974a, b) who reported three isoenzymes in yeasts, only one copy of the PGI1 gene per genome was found in several laboratory strains tested by Southern analysis.

Curr Genet, 1986, 11(2), 93 - 6
Chromosomal mapping of the uracil permease gene of Saccharomyces cerevisiae; Weber E et al.; The gene FUR4, coding for the uracil permease in Saccharomyces cerevisiae, was mapped on chromosome II, at a distance of 7.8 cM from the centromere on the right arm of the chromosome . In a first step, we used the chromosome loss mapping method developed by Falco and Botstein (1983) to determine on which chromosome the gene mapped . After the observation that FUR4 was closely linked to GAL10, one of the three genes forming the gal cluster (Bassel and Mortimer 1971), we could determine precisely the position of the gene on chromosome II.

Histochemistry, 1986, 84(1), 87 - 96
Ultracytochemical localization of X-prolyl-dipeptidyl (amino)peptidase in microglobules and endoplasmic membranes accumulated in pep4-3 mutant of Saccharomyces cerevisiae; Vorisek J; The ultracytochemical localization of X-prolyl-dipeptidyl (amino)peptidase (DPP) activity was studied in a late exponential culture of a haploid (alpha) wild-type strain of Saccharomyces cerevisiae and its pep4-3 mutant . Yeast cells were fixed for 20 min in cold 1% glutaraldehyde buffered with 50 mM TES buffer to pH 7.0 and then incubated for 80 min with 1.2 mM L-alanyl-L-proline-4-methoxy-2-naphthylamide (Ala-Pro-MNA) or Lys-Pro-MNA as cytochemical substrates plus 0.06% hexazonium p-rosaniline (HPR) buffered with 160 mM cacodylate to pH 7.0 . The osmiophilic azoindoxyl complex was formed by coupling HPR with MNA liberated by DPP activity and was then osmicated during an overnight post-fixation of cells in cold 1% OsO4 . In the wild-type strain, conspicuous deposits of DPP reaction product were observed in vacuolar membranes . When compared with the parent strain, the pep4-3 mutant cells were enriched in endoplasmic reticulum (ER), cytoplasmic lipoprotein, and microcompartments: membranous vesicles and microglobules . In the mutant, DPP reaction product was found in about 50% of non-vacuolated cells at the following sites: the nuclear envelope, polar layers of ER sheets and of membranous vesicles (diameter, 40-90 nm), the surface or the lumen of these vesicles, the cytoplasmic membrane (under some bud scars) and the periplasmic space . The largest amount of reaction product was found in microglobules (diameter, 20-50 nm) that were mainly observed in the cytoplasmic matrix but were also present in nuclei (nucleoli) and mitochondria . These microglobules had a single-line boundary and appeared to be composed of lipoprotein . The surface ultrastructure of sectioned microglobules in the cytoplasmic matrix was similar to that of the coated vesicles found in mammalian cells . Only sparse amounts of DPP reaction product were seen in budding yeast . In all pep4-3 cells with electron-lucent vacuoles, the reaction product was confined to the vacuolar membranes (i.e . homologous to the ER), microglobules and the periplasmic space . Polysaccharides with free vic-groups were shown by the cytochemical reaction to be present on the surface of ER membranes, in microglobules, in the periplasmic space and in the cell wall . Our cytochemical results indicate that microglobules participate in the exocytosis of both DPP and glycoproteins, and reveal new features of vacuolar morphogenesis in yeast.

FEBS Lett, 1986 Jan 1, 194(1), 131 - 8
Codon reading patterns in Saccharomyces cerevisiae mitochondria based on sequences of mitochondrial tRNAs; Sibler AP et al.; The sequences of Saccharomyces cerevisiae mitochondrial tRNA Arg1, tRNA Arg2, tRNA Gly, tRNA Lys2, tRNA Leu amd tRNA Pro are reported . Special structural features were found in tRNA Pro, which has A8, C21, A48 instead of the constant residues U8, A21 and pyrimidine 48, and in tRNA Lys2, which has a U excluded from base-paring and bulging out from the TpsiC stem . The tRNA Arg1, tRBA Lys2 and tRNA Leu, which belong to two-codon families ending in a purine, have a modified uridine in the wobble position, which prevents misreading of C and U . It is likely to be 5-carboxymethylaminomethyluridine . tRNA Gly and tRNA Pro have an unmodified uridine in the wobble position allowing the reading of all four codons of a four-codon family . However, tRNA Arg2, which is a minor species and belongs to the CGN four-codon family, has an unmodified A in the wobble position . This unusual feature raises the problem of the mechanism by which the codons CGA, CGG and CGC are recognized.

Biochem Biophys Res Commun, 1985 Dec 31, 133(3), 844 - 50
Stimulation of cell proliferation and polyphosphoinositide metabolism in Saccharomyces cerevisiae GL7 by ergosterol; Dahl JS et al.; The effect of ergosterol on cell division and phospholipid metabolism was investigated in Saccharomyces cerevisiae strain GL7, a sterol and unsaturated fatty acid auxotroph . Cells growing poorly on cholesterol were stimulated to grow more rapidly by supplementing the medium with 100 ng of ergosterol per ml . Within 10 min after ergosterol addition to cells prelabeled with 32Pi or {3H}inositol the isotope content of the polyphosphoinositides increases markedly followed by an equally striking and rapid decrease . Subsequently upon continuous labeling, 32P incorporation into phosphatidylinositol and, to a lesser degree, other phospholipids increased . Finally 3h after ergosterol addition the growth rate increased . Only stimulation of the first process, i.e . polyphosphoinositide metabolism, upon ergosterol addition is resistant to inhibition by cycloheximide.

Biochem Biophys Res Commun, 1985 Dec 31, 133(3), 917 - 22
Decrease of the plasma membrane H+-ATPase activity during late exponential growth of Saccharomyces cerevisiae; Tuduri P et al.; During the last cell division of exponential growth, the H+-ATPase activity from the yeast plasma membrane decreases by a factor of two to three . This "arrest growth control" of ATPase activity is not accompanied by modification of the sensitivity to vanadate.

J Biol Chem, 1985 Dec 25, 260(30), 16367 - 74
Multiple species of single-stranded nucleic acid-binding proteins in Saccharomyces cerevisiae; Jong AY et al.; DNA affinity chromatography has been used to identify the major single-stranded nucleic acid binding proteins (SSBs) of Saccharomyces cerevisiae . There are five abundant species having molecular masses of 50, 45, 31, 23, and 20 kDa . Four of these proteins are cytoplasmic and one is mitochondrial . To date, three of the proteins have been purified to homogeneity . The purified proteins are designated SSB-m, SSB-1, and SSB-2, with molecular masses of 20, 45, and 50 kDa, respectively . SSB-m is found only in mitochondrial subcellular fractions . SSB-1 stimulates purified yeast DNA polymerase I, while SSB-2 inhibits DNA polymerase I . An antibody against SSB-1 has been prepared in rabbits and purified by SSB-1-Sepharose affinity chromatography . The purified antibody specifically inhibits DNA synthesis in an in vitro replication system, suggesting that SSB-1 may be involved in DNA replication in vivo . SSB-2 has the highest affinity for single-stranded DNA of all three proteins . It may represent a new class of eukaryotic SSB, on the basis of molecular weight, inhibition of DNA polymerase and antigenicity . Antibodies have also been prepared against SSB-2 . The immunological reagents have been used to show that SSB-1, SSB-2, and SSB-m are antigenically distinct, as well as to study the relationship of these three SSBs to other proteins in yeast.

EMBO J, 1985 Dec 16, 4(13A), 3549 - 52
Nucleotide sequence of the RAD10 gene of Saccharomyces cerevisiae; Reynolds P et al.; The RAD10 gene is one of several genes in Saccharomyces cerevisiae required for incision of u.v.-irradiated or cross-linked DNA . We have determined the nucleotide sequence of the RAD10 gene and its flanking regions . The RAD10 nucleotide sequence presented here differs significantly from that recently reported . The RAD10 protein predicted from the nucleotide sequence contains 210 amino acids with a calculated mol . wt . of 24 310 . The middle portion of the RAD10 protein, which is highly basic and also contains eight of the total of 10 tyrosine residues present in the protein, may be involved in DNA binding by ionic interactions and tyrosine intercalation between the bases of DNA . A genomic deletion of the entire RAD10 gene does not affect viability; however, the rad10 deletion mutant is highly u.v . sensitive.

Biochim Biophys Acta, 1985 Dec 4, 837(3), 336 - 43
Multiple functions for sterols in Saccharomyces cerevisiae; Rodriguez RJ et al.; Analyses with a yeast sterol auxotroph indicated that there are at least four different levels of function for sterol which have been designated sparking, critical domain, domain and bulk . Growth of yeast sterol auxotrophs on cholestanol is precluded unless minute amounts of ergosterol are available . We have designated this phenomenon the sparking of growth, in which cholestanol satisfies an overall membrane sterol requirement and ergosterol fulfills a high specificity sparking function . The critical domain role for sterol is observed under conditions of lanosterol supplementation where low levels of ergosterol (10-times those necessary for sparking on cholestanol) are required for growth . The sterol functions designated domain and bulk are illustrated by assessing cellular free sterol levels and plasma membrane properties of a sterol auxotroph after growth on different concentrations of exogenously supplied sterol . Plasma membranes isolated from auxotrophs grown on domain or bulk levels of sterol underwent no lipid thermotropic transitions, while plasma membranes from cells grown on critical domain levels of sterol underwent a lipid thermotropic transition, when analyzed by steady-state fluorescence anisotropy.

Eur J Biochem, 1985 Dec 2, 153(2), 243 - 7
Metabolism of 2-oxoaldehydes in yeasts . Purification and characterization of lactaldehyde dehydrogenase from Saccharomyces cerevisiae; Inoue Y et al.; NAD-dependent lactaldehyde dehydrogenase, catalyzing an oxidation of lactaldehyde to lactate, was purified approximately 70-fold from cell extracts of Saccharomyces cerevisiae with a 28% yield of activity . The enzyme was homogeneous on polyacrylamide gel electrophoresis . The relative molecular mass of the enzyme was estimated to be 40 000 on Sephadex G-150 column chromatography and on sodium dodecyl sulfate/polyacrylamide gel electrophoresis . The enzyme was most active at pH 6.5, 60 degrees C and specifically oxidized L-lactaldehyde to L-lactate in the presence of NAD . The Km values for L-lactaldehyde and NAD were 10 mM and 2.9 mM, respectively . The purest enzyme was extremely unstable and almost completely inactivated during storage at -20 degrees C, pH 7.5 . For the reactivation of the enzyme, halide ions such as Cl-, I- and Br- were required.

FEBS Lett, 1985 Dec 2, 193(2), 189 - 93
In vivo 31P nuclear magnetic resonance saturation transfer measurements of phosphate exchange reactions in the yeast Saccharomyces cerevisiae; Campbell SL et al.; 31P saturation transfer techniques have been used to measure phosphate kinetics in the yeast Saccharomyces cerevisiae . The phosphate consumption rate observed in acetate grown mid-log cells was combined with measurements of O2 consumption to yield P/O ratios of 2.2 and 2.9, for cells respiring on glucose and ethanol, respectively . However, no phosphate consumption activity was observed in saturation transfer experiments on anaerobic glucose fed cells . The phosphate consumption rates measured by saturation transfer in cells respiring on glucose and ethanol was attributed to the unidirectional rates of mitochondrial ATP synthesis.

Mol Cell Biol, 1985 Dec, 5(12), 3436 - 42
Posttranscriptional regulation and assembly into ribosomes of a Saccharomyces cerevisiae ribosomal protein-beta-galactosidase fusion; Gritz L et al.; To study the regulation of ribosomal protein genes, we constructed a 'lacZ fusion of the Saccharomyces cerevisiae RP51A gene, containing the first 64 codons of RP51A . In a strain lacking an intact RP51A gene (cells are viable due to the presence of an active RP51B gene), beta-galactosidase activity is 10-fold greater than in a strain containing RP51A . RP51A-lacZ mRNA levels are equal in the two strains, indicating that regulation is posttranscriptional . In the absence of the RP51A gene, the fusion protein is predominantly cytoplasmic and associated with polysomes, whereas in the presence of RP51A, the fusion protein is predominantly nuclear, and none is associated with polysomes . Deletions were made in the RP51A-coding portion of the fusion gene . The most extensively deleted gene, containing only the first seven RP51A codons fused to lacZ, produced a high level of beta-galactosidase activity in both the presence and the absence of the RP51A gene . In both cases, little or none of this shorter fusion protein was found associated with polysomes . Thus, a regulatory site (or sites) lies in the protein-coding region of RP51A . We suggest that posttranscriptional regulation of the rp51 fusion protein is related to assembly of the protein into ribosomes.

Yeast, 1985 Dec, 1(2), 159 - 71
The PET18 locus of Saccharomyces cerevisiae: a complex locus containing multiple genes; Toh-e A et al.; The basis of pleiotropy shown by the pet18 mutants of Saccharomyces cerevisiae (rho-0,KIL-0 and temperature sensitive growth) was examined by cloning the fragment which complements the defect in growth at 37 degrees C of the pet18 mutants . The cloned DNA could complement the defect in the maintenance of the killer plasmid but did not give the cell the ability to maintain mitochondrial DNA . Sequence analysis of the cloned DNA revealed the presence of four open reading frames, at least two of which are necessary for the complementation activity . By using the cloned DNA as a probe, we found that two independent pet18 mutants have a deletion covering the entire sequence contained in the probe . From these results we predict that the traits of the pet18 mutants that concern temperature sensitivity and killer of the pet18 mutants are controlled by a separate gene(s) from that which participates in the maintenance of mitochondrial DNA.

Mol Cell Biol, 1985 Dec, 5(12), 3545 - 51
Saccharomyces cerevisiae CYC1 mRNA 5'-end positioning: analysis by in vitro mutagenesis, using synthetic duplexes with random mismatch base pairs; McNeil JB et al.; Expression of the Saccharomyces cerevisiae CYC1 gene produces mRNA with more than 20 different 5' ends . A derivative of the CYC1 gene (CYC1-157) was constructed with a deletion of a portion of the CYC1 5'-noncoding region, which includes the sites at which many of the CYC1 mRNAs 5' ends map . A 54-mer double-stranded oligonucleotide homologous with the deleted sequence of CYC1-157 and which included a low level of random base pair mismatches (an average of two mismatches per duplex) was used to construct mutants of the CYC1 gene and examine the role of the DNA sequence at and immediately adjacent to the mRNA 5' ends in specifying their locations . The effect of these mutations on the site selection of mRNA 5' ends was examined by primer extension . Results indicate that there is a strong preference for 5' ends which align with an A residue (T in the template DNA strand) preceded by a short tract of pyrimidine residues.

Mol Cell Biol, 1985 Dec, 5(12), 3532 - 44
Meiotic exchange within and between chromosomes requires a common Rec function in Saccharomyces cerevisiae; Wagstaff JE et al.; We used haploid yeast cells that express both the MATa and MAT alpha mating-type alleles and contain the spo13-1 mutation to characterize meiotic recombination within single, unpaired chromosomes in Rec+ and Rec- Saccharomyces cerevisiae . In Rec+ haploids, as in diploids, intrachromosomal recombination in the ribosomal DNA was detected in 2 to 6% of meiotic divisions, and most events were unequal reciprocal sister chromatid exchange (SCE) . By contrast, intrachromosomal recombination between duplicated copies of the his4 locus occurred in approximately 30% of haploid meiotic divisions, a frequency much higher than that reported in diploids; only about one-half of the events were unequal reciprocal SCE . The spo11-1 mutation, which virtually eliminates meiotic exchange between homologs in diploid meiosis, reduced the frequency of intrachromosomal recombination in both the ribosomal DNA and the his4 duplication during meiosis by 10- to greater than 50-fold . This Rec- mutation affected all forms of recombination within chromosomes: unequal reciprocal SCE, reciprocal intrachromatid exchange, and gene conversion . Intrachromosomal recombination in spo11-1 haploids was restored by transformation with a plasmid containing the wild-type SPO11 gene . Mitotic intrachromosomal recombination frequencies were unaffected by spo11-1 . This is the first demonstration of a gene product required for recombination between homologs as well as recombination within chromosomes during meiosis.

Mol Cell Biol, 1985 Dec, 5(12), 3517 - 24
Mutations in cognate genes of Saccharomyces cerevisiae hsp70 result in reduced growth rates at low temperatures; Craig EA et al.; Expression of two Saccharomyces cerevisiae genes (YG101 and YG103) that are related to the gene encoding inducible 70K protein (hsp70) is repressed upon heat shock . Mutations of the two genes were constructed in vitro and substituted into the yeast genome in place of the wild-type alleles . No phenotypic effect of single mutations of either gene was detected . However, cells containing both YG101 and YG103 mutations showed altered growth properties; double-mutation cells possess an optimal growth temperature of 37 degrees C rather than 30 degrees C and grow increasingly poorly as the temperature is lowered . Mutations of two other members of this hsp70-related multigene family, YG100 and YG102, have been analyzed (E . A . Craig and K . Jacobsen, Cell 38:841-849, 1984) . Cells containing both YG100 and YG102 mutations cannot form colonies at 37 degrees C . Fusions between the YG101 and YG102 promoter regions and the YG100 and YG101 structural genes, respectively, were constructed . The YG101 promoter-YG100 structural gene fusion was not able to restore normal growth properties to the yg101- yg103- mutant . Also, yg100- yg102- cells containing the YG102 promoter-YG101 structural gene fusion were unable to grow at 37 degrees C . Failure of the protein products of related genes to rescue the relative cold sensitivity of growth suggests that members of the hsp70 multigene family are functionally distinct.

Mol Cell Biol, 1985 Dec, 5(12), 3429 - 35
Effect of RP51 gene dosage alterations on ribosome synthesis in Saccharomyces cerevisiae; Abovich N et al.; The Saccharomyces cerevisiae ribosomal protein rp51 is encoded by two interchangeable genes, RP51A and RP51B . We altered the RP51 gene dose by creating deletions of the RP51A or RP51B genes or both . Deletions of both genes led to spore inviability, indicating that rp51 is an essential ribosomal protein . From single deletion studies in haploid cells, we concluded that there was no intergenic dosage compensation at the level of mRNA abundance or mRNA utilization (translational efficiency), although phenotypic analysis had previously indicated a small compensation effect on growth rate . Similarly, deletions in diploid strains indicated that no strong mechanisms exist for intragenic dosage compensation; in all cases, a decreased dose of RP51 genes was characterized by a slow growth phenotype . A decreased dose of RP51 genes also led to insufficient amounts of 40S ribosomal subunits, as evidenced by a dramatic accumulation of excess 60S ribosomal subunits . We conclude that inhibition of 40S synthesis had little or no effect on the synthesis of the 60S subunit components . Addition of extra copies of rp51 genes led to extra rp51 protein synthesis . The additional rp51 protein was rapidly degraded . We propose that rp51 and perhaps many ribosomal proteins are normally oversynthesized, but the unassembled excess is degraded, and that the apparent compensation seen in haploids, i.e., the fact that the growth rate of mutant strains is less depressed than the actual reduction in mRNA, is a consequence of this excess which is spared from proteolysis under this circumstance.

Mol Cell Biol, 1985 Dec, 5(12), 3410 - 6
Cloning and molecular analysis of the HAP2 locus: a global regulator of respiratory genes in Saccharomyces cerevisiae; Pinkham JL et al.; We report here the cloning of the HAP2 gene, a locus required for the expression of many cytochromes and respiratory functions in Saccharomyces cerevisiae . The cloned sequences were found to direct integration of a marked vector to the chromosomal HAP2 locus, and derivatives of these sequences were shown to yield chromosomal disruptions with a Hap2- phenotype . The gene maps 18 centimorgans centromere proximal to ade5 on the left arm of chromosome VII, distinguishing it from any other previously characterized nuclear petite locus . The HAP2 locus encodes a 1.3-kilobase transcript which is present at extremely low levels and which is derepressed in cells grown in media containing nonfermentable carbon sources . Levels of HAP2 mRNA are not reduced in strains bearing a mutation at the HAP3 locus, which is also required for expression of respiratory functions . Models outlining possible interactions of the products of the HAP2 and HAP3 genes are presented.

J Gen Microbiol, 1985 Dec, 131 ( Pt 12), 3219 - 27
Discrimination by heat and proteinase treatments between flocculent phenotypes conferred on Saccharomyces cerevisiae by the genes FLO1 and FLO5; Hodgson JA et al.; The effects of elevated temperature and of digestion with a variety of proteinases on the flocforming ability of flocculent strains of Saccharomyces cerevisiae, both genetically defined (FLO1 and FLO5) laboratory and genetically undefined brewing strains, have been determined . This has permitted classification of the flocculent phenotypes of these strains according to criteria other than quantitative grading of flocculence . The flocculent phenotypes conferred by both the FLO1 and the FLO5 gene were irreversibly lost upon treatment with pronase, proteinase K, trypsin or 2-mercaptoethanol treatments . However, the floc-forming ability of cells of the FLO1 strain ABXL-1D was destroyed by chymotrypsin digestion and was stable to incubation at 70 degrees C, whereas the floc-forming ability of cells of the FLO5 strain ABXR-11A was resistant to the action of chymotrypsin and was heat labile . Tetrad analysis of a cross of these FLO1 and FLO5 strains indicated that the chymotrypsin and heat sensitivity phenotypes were FLO-gene determined . It appears that expression of the FLO1 and FLO5 genes leads to the production of different and characteristic cell-wall proteins underlying their respective flocculent phenotypes.

J Gen Microbiol, 1985 Dec, 131 ( Pt 12), 3185 - 91
Further studies on the subcellular distribution of Cd2+ in Cd-sensitive and Cd-resistant strains of Saccharomyces cerevisiae; Joho M et al.; When a Cd-resistant strain (301 N) and a Cd-sensitive strain (101 N) of Saccharomyces cerevisiae were incubated in medium containing Cd2+, a large proportion of the cellular Cd2+ was found in the cytosol of strain 301 N, but not in that of strain 101 N . Approximately 65% of the cellular Cd2+ was released from strain 301 N after treatment with chitosan, which affects cell membrane permeability . About 80% of the cellular Cd2+ released from strain 301 N by chitosan treatment was detected in a 30 000-10 000 molecular weight fraction prepared by ultrafiltration . The distribution of Cd2+ into the cytosol in strain 301 N was inhibited in the presence of cycloheximide . The proportion of cellular Cu2+ or Zn2+ present in the cytosol after incubation with these ions was similar for the two strains (about 40%).

Proc Natl Acad Sci U S A, 1985 Dec, 82(24), 8557 - 61
The relationship between the "TATA" sequence and transcription initiation sites at the HIS4 gene of Saccharomyces cerevisiae; Nagawa F et al.; Transcription of the HIS4 gene begins at a single site (I) at position -60 from the ATG that begins translation . We have made linker insertions/deletions in the 5' noncoding region to identify the elements required for the specificity of transcription initiation . Although there are four sequences that begin TATA and are near the start of transcription (-170, -132, -123, and -102) only the sequence at -123 (TATA-123) is required for transcription initiation . By inserting synthetic oligonucleotides into a mutant from which TATA-123 had been deleted, we found that just TATA or TATAA does not work but that TATAAA functions almost as well as the wild-type sequence . This hexamer does not work in the opposite orientation (TTTATA) . When a synthetic TATA sequence is placed upstream from the normal site, the site of initiation also moves upstream in a roughly cometric way even when TATA-123 is present . Analysis of transcripts in strains where the distance between the TATA sequence and the wild-type site of transcription initiation (I site) has been altered shows that in yeast, unlike higher cells, transcription does not initiate at a strictly defined distance from the TATA sequence . Constructions that alter the distance between the TATA and the I site or remove the I site change the pattern of transcription initiation without affecting the level of HIS4 expression . Deletions that eliminate the I site produce heterogeneous transcripts and deletions that substantially shorten the distance between TATA-123 and the I site initiate at multiple sites downstream from the I site . Thus, both the TATA and the sequences downstream from it determine the pattern of transcription initiation.

Mutat Res, 1985 Dec, 158(3), 169 - 75
Photodynamic mutagenic action of acridine compounds on yeast Saccharomyces cerevisiae; Iwamoto Y et al.; The photodynamically produced mutagenicity and toxicity of 8 acridine compounds were compared in Saccharomyces cerevisiae under resting and growing conditions . Without irradiation none of the acridines induced respiratory-deficient ('petite') colonies, indicative of mitochondrial DNA damage, in resting cells; and only acriflavine and proflavine induced 'petites' in growing cells . Also, without irradiation none of the acridines were significantly toxic or mutagenic for nuclear DNA under resting or growing conditions . However, with irradiation, acriflavine, proflavine, acridine yellow and rivanol became effective 'petite'-inducing mutagens and highly toxic for resting cells, while acriflavine, proflavine, and acridine orange became effective nuclear mutagens for resting cells . Acridine, quinacrine and 9-aminoacridine were not at all biologically effective with irradiation for resting cells . The results presented here indicate that singlet oxygen is generated by a photodynamic mechanism when acriflavine is irradiated, and further, that acridine, quinacrine and 9-aminoacridine are ineffective photosensitizers, because they are incapable of generating singlet oxygen with irradiation.

Int J Radiat Biol Relat Stud Phys Chem Med, 1985 Dec, 48(6), 987 - 96
Thermal aspects of biological effects of microwaves in Saccharomyces cerevisiae; Dardalhon M et al.; The formation of zygotes between two haploid strains of yeast (Saccharomyces cerevisiae) was determined under treatment with microwaves of 9.4 and 17 GHz at power levels up to 50 and 60 mW/cm2 and a specific absorption rate below 24 mW/g, or with conventional heating . Microwave treatments at 9.4 GHz or 17 GHz at a power density of 10 mW/cm2 produced an increase in zygote formation equivalent to that produced by conventional heating in an incubator, i.e . equivalent to a rise in temperature of 0.5 or 1 degrees C . At higher power densities zygote formation was slightly increased by microwaves at 17 GHz as compared to microwaves at 9.4 GHz probably due to the higher absorption of microwaves at 17 GHz by intracellular water molecules . Under these conditions, microwaves had no effect on cell survival or the induction of cytoplasmic 'petite' mutations.

Genetics, 1985 Dec, 111(4), 745 - 58
SUM1, an apparent positive regulator of the cryptic mating-type loci in Saccharomyces cerevisiae; Klar AJ et al.; The mating-type information residing at the HML and HMR loci in Saccharomyces cerevisiae is kept unexpressed by the action of at least four MAR (or SIR) loci . To determine possible interactions between the MAR/SIR gene products and to find new regulatory loci, we sought extragenic suppressors of the mar1-1 mutation . A strain with the genotype HMLa MAT alpha HMRa mar1-1 is unable to mate because of the simultaneous expression of a and alpha information . A mutant of this strain was isolated that exhibits an alpha phenotype and, therefore, presumably fails to express the HML and HMR loci . We designate the new locus SUM1 (suppressor of mar) . The mutation is recessive, centromere unlinked and does not correspond to the MAT, HML, HMR, SIR1, MAR1, MAR2 (SIR3) or SIR4 loci . The sum1 mutation affects expression of both a and alpha information at the HM loci . Suppression by sum1-1 is neither allele specific nor locus specific as it suppresses a deletion mutation of the MAR1 locus and mutations in SIR3 and SIR4 . The sum1-1 mutation has no discernible phenotype in a Mar+ strain . We propose that the MAR/SIR gene products negatively regulate the SUM1 locus, the gene product of which is necessary for expression of the HM loci.

Mol Cell Biol, 1985 Dec, 5(12), 3451 - 7
Deletions extending from a single Ty1 element in Saccharomyces cerevisiae; Downs KM et al.; Chromosomal rearrangements associated with one Ty1 element in the iso-1-cytochrome c (CYC1) region of Saccharomyces cerevisiae yeast cells were examined . Most of the rearrangements were deletions of the three linked genes, CYC1, OSM1, and RAD7, and resulted from recombination involving the single Ty1 element and a solo delta in the same orientation . These deletions differed by the number of Ty1 elements (zero, one, or two) remaining after deletion and by restriction site heterogeneities associated with these elements . A single Ty1 element remained at the deletion junction point much more frequently than no Ty1 . Apparently the Ty1-associated delta element nearer to the solo delta was involved more often in recombination than the more distal Ty1-associated delta element . The restriction site data implicate gene conversion and suggest that site-specific recombination within the deltas, if occurring, is not the only mechanism of delta-delta recombination . Three other rearrangements bore deletions which began at the end of the Ty1 element and extended into regions not bearing Ty1 or delta sequences . Two of these deletions eliminated 7 kilobases of DNA, although they differed by an associated reciprocal translocation . The third involved a deletion of 14.7 kilobases of DNA associated with an overlapping inversion.

Can J Microbiol, 1985 Dec, 31(12), 1095 - 102
Activity changes of three nucleolytic enzymes during the life cycle of Saccharomyces cerevisiae; Van Ryk DI et al.; Conditions were established for the assay of three nucleolytic enzymes: a Mg2+-independent endoribonuclease, a Mg2+-dependent endonuclease, and a Mg2+-dependent 5'-exonuclease in Saccharomyces cerevisiae cell extracts . The changes in the activities of these enzymes were determined throughout the life cycle of the organism . As the cells progressed from the exponential to the stationary growth phase, the specific activities of the Mg2+-independent endoribonuclease and of the Mg2+-dependent 5'-exonuclease increased, whereas the Mg2+-dependent endonuclease decreased . During sporulation the Mg2+-independent endoribonuclease and the Mg2+-dependent 5'-exonuclease increased several-fold over the first 10 h, but, since a similar increase was seen in nonsporulating control cells, the increases did not appear to be related to sporulation . However, the specific activity of the Mg2+-dependent endonuclease showed a sporulation-related increase during the first 3 h of sporulation, with a subsequent decline to very low levels . The specific activity of this enzyme increased again during germination to the levels seen in exponential phase cells . The Mg2+-independent endoribonuclease and the Mg2+-dependent 5'-exonuclease showed little change during germination of the ascospores . The high specific activity of the Mg2+-independent endoribonuclease during periods of nutrient deprivation is in agreement with the proposed role for this enzyme in the degradation of rRNA under these conditions.

Arch Microbiol, 1985 Dec, 143(3), 216 - 9
Differential sensitivities to glucose and galactose repression of gluconeogenic and respiratory enzymes from Saccharomyces cerevisiae; Herrero P et al.; The synthesis of isocitrate lyase was induced by the presence of ethanol in the chemostat reaching a specific activity of 200 mU X mg-1 at this induced state . In glucose-limited, derepressed cells, 20 mU X mg-1 were detected and under repressed conditions isocitrate lyase activity was not detected . The sensitivity of gluconeogenic enzymes: cytoplasmic malate dehydrogenase; fructose 1,6-bisphosphatase and isocitrate lyase as well as the mitochondrial enzymes NADH dehydrogenase and succinate cytochrome c oxidase to glucose and galactose repression were studied in chemostat cultures . Our results show that galactose was less effective as a repressor than glucose . Malate dehydrogenase was completely inactivated by glucose, whereas galactose only produced a 78% decrease of specific activity . Fructose 1,6-bisphosphatase and isocitrate lyase were completely inactivated by both sugars but at different rate . Glucose produced an 85% decrease of specific activity of the mitochondrial enzymes whereas galactose only decrease an 67%.

Proc Natl Acad Sci U S A, 1985 Dec, 82(24), 8562 - 6
Each of three "TATA elements" specifies a subset of the transcription initiation sites at the CYC-1 promoter of Saccharomyces cerevisiae; Hahn S et al.; Transcription initiation of the yeast iso-1-cytochrome c gene (CYC-1) occurs in six major clusters at positions +1, +10, +16, +25, +34, and +43 . Potential "TATA elements" lie upstream at positions -154, -106, -52, and -22 . Analysis of the TATA region suggests that three of these TATA sequences are functional and contribute to initiation at CYC-1, with the -106 TATA promoting initiation at +1, +10, and +16; the -52 TATA, at +16, +25, +34, and +43; and the -22 TATA, at +34 and +43 . Deletions changing the spacing between the TATA sequences and the region of transcription initiation do not change the location of the CYC-1 transcription start points . This finding suggests that at least part of the information determining mRNA initiation sites is encoded within the DNA sequence at the site of transcription initiation . Analysis of 18 yeast RNA polymerase II promoters suggests that two classes of DNA sequences serve as preferred sites for transcription initiation . To test this possibility, we replaced some of the normal CYC-1 start sites with one of these sequences, TCGA, and found that transcription initiates at this newly introduced sequence . These results are in contrast to those from higher eukaryotes, where RNA polymerase II typically initiates transcription a fixed distance downstream from the TATA element . The presence of multiple, functional TATA sequences at CYC-1 is inconsistent with the idea that RNA polymerase or another transcription factor attaches to the template at an upstream activation site and scans for the nearest TATA element.

Nucleic Acids Res, 1985 Nov 25, 13(22), 8231 - 46
Sequence of the Saccharomyces cerevisiae PHR1 gene and homology of the PHR1 photolyase to E . coli photolyase; Sancar GB; The nucleotide sequence of a 2301 base pair region of Saccharomyces cerevisiae DNA containing the PHR1 gene is reported . Within this region a single open reading frame of 1695 base pairs was found; using the insertional inactivation technique it was shown that part or all of this open reading frame specifies the PHR1-encoded photolyase . The amino acid sequence of the 565 amino acid long polypeptide predicted from the PHR1 nucleotide sequence was compared to the amino acid sequence of E . coli photolyase . Overall the sequence homology was 36.5%; however, two short regions near the amino terminus as well as the carboxy-terminal 150 amino acids display significantly greater sequence homology . The presence of these strongly conserved regions suggests that the yeast and E . coli photolyase possess common structural and functional domains involved in substrate and/or chromophore binding.

J Mol Biol, 1985 Nov 20, 186(2), 307 - 19
Fructose bisphosphatase of Saccharomyces cerevisiae . Cloning, disruption and regulation of the FBP1 structural gene; Sedivy JM et al.; Fructose bisphosphatase catalyzes a key reaction of gluconeogenesis . We have cloned the fructose bisphosphatase (FBP1) structural gene from Saccharomyces cerevisiae by screening a genomic library for complementation of an Escherichia coli fbp deletion mutation . The cloned DNA expresses in E . coli a fructose bisphosphatase activity which is precipitable with antibodies specific for the yeast enzyme and is sensitive to inhibition by fructose 2,6-bisphosphate . Evidence is presented demonstrating that the entire gene, including all cis-acting regulatory sequences, has been cloned . A substitution mutation that disrupts FBP1 was incorporated into the yeast genome by transplacement to construct a fructose bisphosphatase null mutation . The fbp1 mutant strain is a hexose auxotroph, otherwise growing normally . Southern blot hybridization analysis confirmed the structure of the transplacement and demonstrated that FBP1 is present in single copy in the haploid genome . Northern blot hybridization analysis revealed an mRNA of about 1350 nucleotides, whose presence was repressible by glucose in the medium . Fructose bisphosphatase activity was not greatly overproduced when the FBP1 gene was present on a multicopy vector in yeast.

Biochem Biophys Res Commun, 1985 Nov 15, 132(3), 1116 - 23
The role of adenine nucleotide translocation in the energization of the inner membrane of mitochondria isolated from rho + and rho degree strains of Saccharomyces cerevisiae; Dupont CH et al.; The energization of rho + and rho degrees isolated mitochondria was measured using a tetraphenylphosphonium selective electrode . In both strains translocase mediated ATP/ADP exchange energization was observed . This energization was more sensitive to uncoupler than that induced by respiration in rho + mitochondria . This observation is in accordance with the hypersensitivity of rho - cell growth to uncoupler.

J Biol Chem, 1985 Nov 15, 260(26), 14214 - 23
A multicomponent mitochondrial RNA polymerase from Saccharomyces cerevisiae; Winkley CS et al.; Using a whole cell extract from Saccharomyces cerevisiae (bakers' yeast) we have been able to detect a selective RNA polymerase activity originally identified in purified yeast mitochondria (Levens, D., Morimoto, R., and Rabinowitz, M . (1981) J . Biol . Chem . 256, 1466-1473) . We have shown that in in vitro transcription reactions this activity recognizes a consensus mitochondrial promoter sequence ATA-TAAGTA (Osinga, K . A., DeHaan, M., Christianson, T., and Tabak, H . F . (1982) Nucleic Acids Res . 10, 7993-8006) in the upstream region of the nuclear GAL10 gene as well as promoters from yeast mitochondrial DNA . Using these promoter-containing templates for in vitro assays, we have chromatographically separated the mitochondrial specific RNA polymerase activity from the three nuclear RNA polymerases (I, II, and III) . Further characterization has revealed that this preparation has distinctive properties on two different types of DNA templates, poly{d(AT)} and cloned DNA containing mitochondrial promoters . Salt and divalent cation optima and substrate saturation kinetics are different for the two types of templates . Using promoter-containing DNA as an assay template, we have chromatographically dissociated the RNA polymerase activity into two nonfunctional components . Selective transcription of the GAL10 template is restored when the two components are recombined . It is possible that the RNA polymerase active on poly{d(AT)} is a nonspecific component of the selective transcription apparatus or that two distinct RNA polymerases are present in the preparation.

Biochem J, 1985 Nov 15, 232(1), 205 - 9
Upstream activation of ribosomal RNA biosynthesis in Saccharomyces cerevisiae; Quincey RV et al.; Yeast was transformed with eight recombinants that contained an rRNA minigene and upstream elements of rDNA in different orientations in the multi-copy yeast-Escherichia coli shuttle vector, pJDB207 . The effect of these elements of upstream rDNA on the initiation of transcription of the minigene at the site for rRNA biosynthesis was determined by using an S1 nuclease mapping procedure to measure the abundance of the minigene transcript in RNA from the yeast transformants . Transcription of the minigene was enhanced 3-fold by DNA within a 2.2 kb element more than 1.5 kb upstream from the initiation site . Inversion of the 2.2 kb element decreased expression of the minigene by 40% . This 2.2 kb element contained approx . 500 bp from the 25S rRNA coding region at the 3' end of the preceding rRNA gene and 1 kb of adjacent nontranscribed spacer rDNA . The enhancing activity was independent of interference from readthrough that might have contributed to the 7-fold decrease in minigene expression caused by removing all rDNA upstream from -209 bp.

Eur J Biochem, 1985 Nov 4, 152(3), 709 - 14
DNA sequence analysis of the oli1 gene reveals amino acid changes in mitochondrial ATPase subunit 9 from oligomycin-resistant mutants of Saccharomyces cerevisiae; Ooi BG et al.; The nucleotide sequence of the oli1 gene encoding mitochondrial ATPase subunit 9 (76 amino acids) has been determined for five oligomycin-resistant mutants of Saccharomyces cerevisiae . Three of the mutations affect amino acids in the vicinity of the glutamic acid residue 59 at which dicylohexyl carbodiimide binds . Two other mutations lead to substitution of amino acid 23, which would lie very close to residue 59 in the folded hairpin conformation that this protein is thought to adopt in the inner mitochondrial membrane . The apposition of residues 23 and those adjacent to residue 59, lying respectively in the two hydrophobic membrane-spanning arms of subunit 9, is considered to constitute an oligomycin-binding domain . By consideration of the amino acid substitutions in those mutants cross-resistant to venturicidin, a domain of resistance for venturicidin is defined to lie within the oligomycin-binding domain, also centered on residues 23 and 59 . These data also clarify the genetic recombination behaviour of alleles previously defined to form part of the oli3 locus (mutants characterized by resistance to both oligomycin and venturicidin) together with alleles defined to form part of the oli1 locus (mutants not cross-resistant to venturicidin) . The oli1 and oli3 loci can now be seen to form two overlapping extended groups within the oli1 gene, with sequenced oli3 mutations being as far apart as 125 nucleotides within the subunit 9 coding region of 231 nucleotides.

Mol Cell Biol, 1985 Nov, 5(11), 3069 - 73
Transcriptional control of the sporulation-specific glucoamylase gene in the yeast Saccharomyces cerevisiae; Yamashita I et al.; In the yeast Saccharomyces cerevisiae, glucoamylase activity appears specifically in sporulating cells heterozygous for the mating-type locus (MAT) . We identified a sporulation-specific glucoamylase gene (SGA) and show that expression of SGA is positively regulated by the mating-type genes, both MATa1 and MAT alpha 2 . Northern blot analysis revealed that control of SGA is exerted at the level of RNA production . Expression of SGA or the consequent degradation of glycogen to glucose in cells is not required for meiosis or sporulation, since MATa/MAT alpha diploid cells homozygous for an insertion mutation at SGA still formed four viable ascospores.

Arch Microbiol, 1985 Nov, 143(2), 143 - 6
Regulation of beta-exoglucanase activity production by Saccharomyces cerevisiae in batch and continuous culture; Olivero I et al.; The rate of synthesis and secretion of exo-1-3-beta-glucanase activity closely paralleled the specific rate of growth in exponentially growing Saccharomyces cerevisiae cells in batch culture . When the stationary phase was reached both synthesis and secretion stopped . No activity was synthesized when the cells were maintained in carbon sources that did not allow them to grow . Studies in continuous culture indicate a strong relationship between the synthesis of exoglucanase activity and the specific growth rate . These results are taken as evidence of an essential role of this activity during the yeast budding cycle.

Mol Cell Biol, 1985 Nov, 5(11), 3139 - 48
General amino acid control and specific arginine repression in Saccharomyces cerevisiae: physical study of the bifunctional regulatory region of the ARG3 gene; Crabeel M et al.; To characterize further the regulatory mechanism modulating the expression of the Saccharomyces cerevisiae ARG3 gene, i.e., the specific repression by arginine and the general amino acid control, we analyzed by deletion the region upstream of that gene, determined the nucleotide sequence of operator-constitutive-like mutations affecting the specific regulation, and examined the behavior of an ARG3-galK fusion engineered at the initiating codon of ARG3 . Similarly to what was observed in previous studies on the HIS3 and HIS4 genes, our data show that the general regulation acts as a positive control and that a sequence containing the nucleotide TGACTC, between positions -364 and -282 upstream of the transcription start, functions as a regulatory target site . This sequence contains the most proximal of the two TGACTC boxes identified in front of ARG3 . While the general control appears to modulate transcription efficiency, the specific repression by arginine displays a posttranscriptional component (F . Messenguy and E . Dubois, Mol . Gen . Genet . 189:148-156, 1983) . Our deletion and gene fusion analyses confirm that the specific and general controls operate independently of each other and assign the site responsible for arginine-specific repression to between positions -170 and +22 . In keeping with this assignment, the two operator-constitutive-like mutations were localized at positions -80 and -46, respectively, and thus in a region which is not transcribed . We discuss a hypothesis accounting for the involvement of untranscribed DNA in a posttranscriptional control.

Mol Cell Biol, 1985 Nov, 5(11), 2887 - 93
Identification and characterization of the centromere from chromosome XIV in Saccharomyces cerevisiae; Neitz M et al.; A functional centromere located on a small DNA restriction fragment from Saccharomyces cerevisiae was identified as CEN14 by integrating centromere-adjacent DNA plus the URA3 gene by homologous recombination into the yeast genome and then by localizing the URA3 gene to chromosome XIV by standard tetrad analysis . DNA sequence analysis revealed that CEN14 possesses sequences (elements I, II, and III) that are characteristic of other yeast centromeres . Mitotic and meiotic analyses indicated that the CEN14 function resides on a 259-base-pair (bp) RsaI-EcoRV restriction fragment, containing sequences that extend only 27 bp to the right of the element I to III region . In conjunction with previous findings on CEN3 and CEN11, these results indicate that the specific DNA sequences required in cis for yeast centromere function are contained within a region about 150 bp in length.

J Gen Microbiol, 1985 Nov, 131 ( Pt 11), 2909 - 18
Mutants of Saccharomyces cerevisiae partially defective in the last steps of the haem biosynthetic pathway: isolation and genetical characterization; Kurlandzka A et al.; A novel method for the isolation of Saccharomyces cerevisiae mutants partially defective in haem synthesis is described . Mutant clones were identified by their fluorescence under UV light due to the accumulation of porphyrins in cells, and by their ability to grow on nonfermentable carbon sources due to their preserved haemoprotein synthesis . Thirteen such mutants were obtained by this procedure . The defects in haem synthesis and accumulation of porphyrins in all the mutants were confirmed by spectrophotometric analysis . Complementation tests with biochemically defined, haem-less strains showed that in seven mutants uroporphyrinogen decarboxylase was affected and that in three mutants the defect concerned ferrochelatase . The defects in the remaining three mutants were not defined.

J Biochem (Tokyo), 1985 Nov, 98(5), 1301 - 7
Comparison of beta-glucan structures in a cell wall mutant of Saccharomyces cerevisiae and the wild type; Shiota M et al.; A mutant of Saccharomyces cerevisiae defective in the cell wall beta-glucan structure was obtained . The mutant cells are extremely sensitive to (beta 1-3)-glucanase digestion and mild alkali treatment . Structural analysis revealed that the alkali-insoluble, skeletal glucan from wild type cells contains two components, a (beta 1-3) linked glucan with a laminated structure, and a highly branched glucan containing predominantly (beta 1-6) linkages . The mutant cells lack the latter component.

Mol Biol (Mosk), 1985 Nov-Dec, 19(6), 1579 - 84
{Structural organization of double-stranded RNA from killer yeasts Saccharomyces cerevisiae by the thermal denaturation method}; Duzhak AB et al.; The thermal denaturation method for studying the structural organization of double-stranded RNA (dsRNA) from virus-like particles of killer yeasts Saccharomyces cerevisiae was used . High resolution derivative denaturation profiles of total dsRNA and its L- and M-types were obtained . Comparative analysis of these data with those on phage DNA denaturation demonstrated that the processes of denaturation of dsRNA and phage DNA were identical in quality . Increase of thermostability, interval of thermal denaturation and width of local helix-to-coil transitions in dsRNA as compared with phage DNA are caused by the differences of corresponding thermodynamic parameters . Derivative denaturation profiles of L- and M-types of yeasts dsRNA were shown to have certain identical local transitions . Low melting transition, consisting of three local thermalites, is due to the denaturation of AU-rich region (about 200 n.b.p.) in M-dsRNA.

J Bacteriol, 1985 Nov, 164(2), 964 - 8
Regulation of inorganic phosphate transport systems in Saccharomyces cerevisiae; Tamai Y et al.; A kinetic study of Pi transport with 32Pi revealed that Saccharomyces cerevisiae has two systems of Pi transport, one with a low Km value (8.2 microM) for external Pi and the other with a high Km value (770 microM) . The low-Km system was derepressed by Pi starvation, and the activity was expressed under the control of a genetic system which regulates the repressible acid and alkaline phosphatases . The function of the PHO2 gene, which is essential for the derepression of repressible acid phosphatase but not for the derepression of repressible alkaline phosphatase, was also indispensable for the derepression of the low-Km system.

J Bacteriol, 1985 Nov, 164(2), 611 - 7
Isolation and characterization of vanadate-resistant mutants of Saccharomyces cerevisiae; Willsky GR et al.; Cellular vanadium metabolism was studied in Saccharomyces cerevisiae by isolating and characterizing vanadate {VO4(3-), V(V)}-resistant mutants . Vanadate growth inhibition was reversed by the removal of the vanadate from the medium, and vanadate resistance was found to be a recessive trait . Vanadate-resistant mutants isolated from glucose-grown cells were divided into five complementation classes containing more than one mutant . Among the vanadate-resistant mutants isolated in maltose medium, the majority of mutants were found in only two complementation groups . Three of the classes of vanadate-resistant mutants were resistant to 2.5 mM vanadate but sensitive to 5.0 mM vanadate in liquid media . Two classes of vanadate-resistant mutants were resistant to growth in media containing up to 5.0 mM vanadate . Electron spin resonance studies showed that representative strains of the vanadate-resistant complementation classes contained more cell-associated vanadyl {VO2+, V(IV)} than the parental strains . 51 Vanadium nuclear magnetic resonance studies showed that one of the vanadate resonances previously associated with cell toxicity (G . R . Willsky, D . A . White, and B . C . McCabe, J . Biol . Chem . 259:13273-132812, 1984) did not accumulate in the resistant strains compared with the sensitive strain . The amount of vanadate remaining in the media after growth was larger for the sensitive strain than for the vanadate-resistant strains . All of the strains were able to accumulate phosphate, vanadate, and vanadyl.

Genetics, 1985 Nov, 111(3), 389 - 402
Elevated levels of petite formation in strains of Saccharomyces cerevisiae restored to respiratory competence . I . Association of both high and moderate frequencies of petite mutant formation with the presence of aberrant mitochondrial DNA; Evans RJ et al.; When recently arisen spontaneous petite mutants of Saccharomyces cerevisiae are crossed, respiratory competent diploids can be recovered . Such restored strains can be divided into two groups having sectored or unsectored colony morphology, the former being due to an elevated level of spontaneous petite mutation . On the basis of petite frequency, the sectored strains can be subdivided into those with a moderate frequency (5-16%) and those with a high frequency (greater than 60%) of petite formation . Each of the three categories of restored strains can be found on crossing two petites, suggesting either that the parental mutants contain a heterogeneous population of deleted mtDNAs at the time of mating or that different interactions can occur between the defective molecules . Restriction endonuclease analysis of mtDNA from restored strains that have a wild-type petite frequency showed that they had recovered a wild-type mtDNA fragmentation pattern . Conversely, all examined cultures from both categories of sectored strains contained aberrant mitochondrial genomes that were perpetuated without change over at least 200 generations . In addition, sectored colony siblings can have different aberrant mtDNAs . The finding that two sectored, restored strains from different crosses have identical but aberrant mtDNAs provides evidence for preferred deletion sites from the mitochondrial genome . Although it appears that mtDNAs from sectored strains invariably contain duplications, there is no apparent correlation between the size of the duplication and spontaneous petite frequency.

Mol Cell Biol, 1985 Nov, 5(11), 3325 - 9
Behavior of a Drosophila melanogaster transposable element in Saccharomyces cerevisiae; Hoshizaki DK et al.; The Drosophila melanogaster transposable element 412 is transiently unstable in Saccharomyces cerevisiae when present on a freely replicating plasmid . The 412 element undergoes recombination to form two circular molecules, a 412 deletion plasmid and, presumably, a 412 circle . The 412 deletion plasmid contains a single long terminal repeat which most likely is the result of homologous recombination within the long terminal repeats . This recombination occurs at or shortly after transformation and is independent of both the RAD52 gene product and the Flp gene of 2 micron DNA.

Mol Cell Biol, 1985 Nov, 5(11), 3035 - 40
Cloning of hexokinase structural genes from Saccharomyces cerevisiae mutants with regulatory mutations responsible for glucose repression; Entian KD et al.; The regulatory hexokinase PII mutants isolated previously (K.-D . Entian and K.-U . Frohlich, J . Bacteriol . 158:29-35, 1984) were characterized further . These mutants were defective in glucose repression . The mutation was thought to be in the hexokinase PII structural gene, but it did not affect the catalytic activity of the enzyme . Hence, a regulatory domain for glucose repression was postulated . For further understanding of this regulatory system, the mutationally altered hexokinase PII proteins were isolated from five mutants obtained independently and characterized by their catalytic constants and bisubstrate kinetics . None of these characteristics differed from those of the wild type, so the catalytic center of the mutant enzymes remained unchanged . The only noticeable difference observed was that the in vivo modified form of hexokinase PII, PIIM, which has been described recently (K.-D . Entian and E . Kopetzki, Eur . J . Biochem . 146:657-662, 1985), was absent from one of these mutants . It is possible that the PIIM modification is directly connected with the triggering of glucose repression . To establish with certainty that the mutation is located in the hexokinase PII structural gene, the genes of these mutants were isolated after transforming a hexokinaseless mutant strain and selecting for concomitant complementation of the nuclear function . Unlike hexokinase PII wild-type transformants, glucose repression was not restored in the hexokinase PII mutant transformants . In addition mating experiments with these transformants followed by tetrad analysis of sporulated diploids gave clear evidence of allelism to the hexokinase PII structural gene.

Mol Cell Biol, 1985 Nov, 5(11), 2951 - 8
Point mutations implicate repeated sequences as essential elements of the CYC7 negative upstream site in Saccharomyces cerevisiae; Wright CF et al.; The transcription of the CYC7 gene of Saccharomyces cerevisiae, encoding the iso-2-cytochrome c protein, is controlled by two upstream regulatory elements, a positive element and a negative element . The nature of the DNA sequences in the negative element were investigated in a two-part approach . The first involved the construction of a CYC7-galK fusion gene which placed the coding sequence of the Escherichia coli galactokinase gene under the regulation of the CYC7 upstream sequences . This fusion allowed the quantitation by galactokinase enzyme assays of the effects on gene expression of a variety of previously isolated deletion mutations within the negative site . The results suggested that the negative site contained three related sequences . This hypothesis was tested in the second part of these studies, the selection of point mutations within the region of the negative site which led to increased CYC7 expression . Point mutations were introduced by a technique which induced mutations within a localized region at high efficiency . All but one of the mutations involved more than a single base-pair change . The mutations followed the pattern that multiple base-pair changes occurred in one repeat or single base-pair changes occurred in two repeats, with the exception of one mutant, which had a single base-pair change in one repeat . This pattern of mutations and the base pairs that were altered strongly supported the hypothesis that the repeats are integral elements of the negative site.

Genetics, 1985 Nov, 111(3), 403 - 32
Elevated levels of petite formation in strains of Saccharomyces cerevisiae restored to respiratory competence . II . Organization of mitochondrial genomes in strains having high and moderate frequencies of petite mutant formation; Evans RJ et al.; Restriction enzyme analysis of aberrant mtDNA molecules in restored strains of Saccharomyces cerevisiae that displays an elevated level of petite formation has shown the occurrence of novel junction fragments and nonstoichiometric amounts for some unaltered bands . Five aberrant mitochondrial genomes from high-frequency petite-forming (hfp) strains (greater than 60% petites per generation) contain like-oriented duplications and single copy regions . High-frequency petite formation is postulated to arise from increased intramolecular recombination between duplicated segments . Mitochondrial DNA structures in two other hfp strains cannot be easily interpreted and might arise from intramolecular recombination . Mitochondria DNA from moderate-frequency petite-forming (mfp) strains (5-16% petites per generation) contains inverted duplications in two cases . The elevated petite formation is postulated to arise from homologous recombination between directly repeated sequences . In mtDNA from one mfp strain, deletion end-points have been shown to overlap . Such deletion endpoint overlap is postulated to be required for the maintenance of the tandem duplication in hfp strains . Two regions of the wild-type mtDNA (between cyb and oli2 and between SrRNA and oxi2) appear to be dispensable for mitochondrial function.

Mol Cell Biol, 1985 Nov, 5(11), 3024 - 34
Isolation and DNA sequence of ADH3, a nuclear gene encoding the mitochondrial isozyme of alcohol dehydrogenase in Saccharomyces cerevisiae; Young ET et al.; The Saccharomyces cerevisiae nuclear gene, ADH3, that encodes the mitochondrial alcohol dehydrogenase isozyme ADH III was cloned by virtue of its nucleotide homology to ADH1 and ADH2 . Both chromosomal and plasmid-encoded ADH III isozymes were repressed by glucose and migrated heterogeneously on nondenaturing gels . Nucleotide sequence analysis indicated 73 and 74% identity for ADH3 with ADH1 and ADH2, respectively . The amino acid identity between the predicted ADH III polypeptide and ADH I and ADH II was 79 and 80%, respectively . The open reading frame encoding ADH III has a highly basic 27-amino-acid amino-terminal extension relative to ADH I and ADH II . The nucleotide sequence of the presumed leader peptide has a high degree of identity with the untranslated leader regions of ADH1 and ADH2 mRNAs . A strain containing a null allele of ADH3 did not have a detectably altered phenotype . The cloned gene integrated at the ADH3 locus, indicating that this is the structural gene for ADH III.

Mol Cell Biol, 1985 Nov, 5(11), 3274 - 9
The FLP recombinase of the Saccharomyces cerevisiae 2 microns plasmid attaches covalently to DNA via a phosphotyrosyl linkage; Gronostajski RM et al.; The FLP recombinase, encoded by the 2 micron plasmid of Saccharomyces cerevisiae, promotes efficient recombination in vivo and in vitro between its specific target sites (FLP sites) . It was previously determined that FLP interacts with DNA sequences within its target site (B . J . Andrews, G . A . Proteau, L . G . Beatty, and P . D . Sadowski . Cell 40:795-803, 1985), generates a single-stranded break on both DNA strands within the FLP site, and remains covalently attached to the 3' end of each break . We now show that the FLP protein is bound to the 3' side of each break by an O-phosphotyrosyl residue and that it appears that the same tyrosyl residue(s) is used to attach to either DNA strand within the FLP site.

Biochemistry, 1985 Oct 22, 24(22), 6085 - 9
Purification and characterization of protein synthesis initiation factor eIF-4E from the yeast Saccharomyces cerevisiae; Altmann M et al.; A 24 000-dalton protein {yeast eukaryotic initiation factor 4E (eIF-4E)} was purified from yeast Saccharomyces cerevisiae postribosomal supernatant by m7GDP-agarose affinity chromatography . The protein behaves very similarly to mammalian protein synthesis initiation factor eIF-4E with respect to binding to and elution from m7GDP-agarose columns and cross-linking to oxidized reovirus mRNA cap structures . Yeast eIF-4E is required for translation as shown by the strong and specific inhibition of cell-free translation in a yeast extract by a monoclonal antibody directed against yeast eIF-4E.

Mol Cell Biol, 1985 Oct, 5(10), 2521 - 6
Upstream region of the SUC2 gene confers regulated expression to a heterologous gene in Saccharomyces cerevisiae; Sarokin L et al.; The SUC2 gene produces two differently regulated mRNAs that encode two forms of invertase . The 1.9-kilobase mRNA encoding secreted invertase is regulated by glucose (carbon catabolite) repression, and the 1.8-kilobase mRNA encoding intracellular invertase is synthesized constitutively . Previous work has shown that the 5' noncoding region between -650 and -418 is required for derepression of secreted invertase in response to glucose deprivation . We show here that this upstream region can confer glucose-repressible expression to a heterologous gene, a LEU2-lacZ gene fusion, that is not normally regulated by glucose repression . This expression was found to respond appropriately to mutations in trans-acting genes that affect regulation of SUC2 expression . Mutations in the SNF1 through SNF6 loci reduced derepression of beta-galactosidase, and a mutation at the SSN6 locus caused constitutive expression . These findings indicate that the SUC2 upstream region mediates the regulatory effects of these genes and suggest that regulation occurs at the level of transcription . In addition, the upstream region was partially active in the inverted orientation.

Biochem J, 1985 Oct 1, 231(1), 157 - 63
Characteristics of alanine: glyoxylate aminotransferase from Saccharomyces cerevisiae, a regulatory enzyme in the glyoxylate pathway of glycine and serine biosynthesis from tricarboxylic acid-cycle intermediates; Takada Y et al.; Alanine: glyoxylate aminotransferase (EC 2.6.1.44), which is involved in the glyoxylate pathway of glycine and serine biosynthesis from tricarboxylic acid-cycle intermediates in Saccharomyces cerevisiae, was highly purified and characterized . The enzyme had Mr about 80 000, with two identical subunits . It was highly specific for L-alanine and glyoxylate and contained pyridoxal 5'-phosphate as cofactor . The apparent Km values were 2.1 mM and 0.7 mM for L-alanine and glyoxylate respectively . The activity was low (10 nmol/min per mg of protein) with glucose as sole carbon source, but was remarkably high with ethanol or acetate as carbon source (930 and 430 nmol/min per mg respectively) . The transamination of glyoxylate is mainly catalysed by this enzyme in ethanol-grown cells . When glucose-grown cells were incubated in medium containing ethanol as sole carbon source, the activity markedly increased, and the increase was completely blocked by cycloheximide, suggesting that the enzyme is synthesized de novo during the incubation period . Similarity in the amino acid composition was observed, but immunological cross-reactivity was not observed among alanine: glyoxylate aminotransferases from yeast and vertebrate liver.

Mol Cell Biol, 1985 Oct, 5(10), 2770 - 80
Signals for transcription initiation and termination in the Saccharomyces cerevisiae plasmid 2 micron circle; Sutton A et al.; By S1 nuclease protection experiments and primer extension analysis, we determined precisely the cap and polyadenylation sites of transcripts from the four genes of the yeast 2 micron circle plasmid, as well as those of other plasmid transcripts of unknown function . In addition, we used deletion analysis to identify sequences necessary for polyadenylation in plasmid transcripts . Our results indicate that plasmid genes constitute independent transcription units and that plasmid mRNAs are not derived by extensive processing of precursor transcripts . In addition, we found that the D coding region of 2 micron circle is precisely encompassed by a polyadenylated transcript, suggesting that this coding region constitutes a functional plasmid gene . Our identification of the position of plasmid polyadenylation sites and of sequences necessary for polyadenylation provides support for a tripartite signal for polyadenylation as proposed by Zaret and Sherman (K.S . Zaret and F . Sherman, Cell 28:563-573, 1982) . Finally, these data highlight salient features of the transcriptional regulatory circuitry that underlies the control of plasmid maintenance in the cell.

J Gen Microbiol, 1985 Oct, 131 ( Pt 10), 2705 - 9
Inhibition and inactivation of glucose-phosphorylating enzymes from Saccharomyces cerevisiae by D-xylose; Fernandez R et al.; Three glucose-phosphorylating enzymes were separated from cell-free extracts of Saccharomyces cerevisiae by hydroxylapatite chromatography . Variations in the amounts of these enzymes in cells growing on glucose and on ethanol showed that hexokinase PI was a constitutive enzyme, whereas synthesis of hexokinase PII and glucokinase were regulated by the carbon source used . Glucokinase proved to be a glucomannokinase with Km values of 0.04 mM for both glucose and mannose . D-Xylose produced an irreversible inactivation of the three glucose-phosphorylating enzymes depending on the presence or absence of ATP . Hexokinase PI inactivation required ATP, while hexokinase PII was inactivated by D-xylose without ATP in the reaction mixture . Glucokinase was protected by ATP from this inactivation . D-Xylose acted as a competitive inhibitor of hexokinase PI and glucokinase and as a non-competitive inhibitor of hexokinase PII.

J Gen Microbiol, 1985 Oct, 131 ( Pt 10), 2555 - 64
Active extrusion of potassium in the yeast Saccharomyces cerevisiae induced by low concentrations of trifluoperazine; Eilam Y et al.; Trifluoperazine (TFP), the antipsychotic drug, induces substantial K+ efflux, membrane hyperpolarization and inhibition of H+-ATPase in the yeast Saccharomyces cerevisiae . Investigations on the mechanism of these effects revealed two different processes observed at different incubation conditions . At an acidic pH of 4.5 and an alkaline pH of 7.5, K+ efflux was accompanied by substantial proton influx which led to intracellular acidification and dissipation of delta psi formed by cation efflux . The results indicated nonspecific changes in membrane permeability . Similar results were also observed when cells were incubated at pH 5.5-6.0 with higher concentrations of TFP (above 75 microM) . On the other hand, low concentrations of TFP (30-50 microM) at pH 5.5-6.0 caused marked membrane hyperpolarization and K+ efflux unaccompanied by the efflux of other cations and by H+ influx . Our experiments indicate that under these conditions K+ efflux was an active process . (1) K+ efflux proceeded only in the presence of a metabolic substrate and was inhibited by metabolic inhibitors . (2) When 0.3-0.9 mM-KCl was present in the medium at pH 6.0, the concentration of K+ within the cells (measured at the end of the incubation with TFP) was much lower than the theoretical concentration of Kin+ if the distribution of K+ between medium and cell water was at equilibrium (at zero electrochemical gradient) . (3) Valinomycin decreased the net K+ efflux and decreased the membrane hyperpolarization induced by TFP, probably by increasing the flux of K+ into the cells along its electrochemical gradient . (4) Conditions which led to active K+ efflux also led to a marked decrease in cellular ATP level . The results indicate that under a specific set of conditions TFP induces translocation of K+ against its electrochemical gradient.

Proc Natl Acad Sci U S A, 1985 Oct, 82(19), 6445 - 9
Cleavage of cruciform DNA structures by an activity from Saccharomyces cerevisiae; West SC et al.; Protein extracts from Saccharomyces cerevisiae have been fractionated to reveal a nuclease activity that cleaves cruciform structures in DNA . Negatively supercoiled plasmids that contain inverted repeats that are extruded into cruciform structures have been used as DNA substrates . The sites of cleavage of pColIR215 DNA are located within the extruded cruciform stems and are symmetrically opposed to each other across the cruciform junction . Neither relaxed duplex DNA nor single-stranded DNA serve as substrates . The native molecular weight of the activity was estimated to be approximately equal to 200,000 by gel filtration.

Mutat Res, 1985 Oct, 144(2), 67 - 71
Phenobarbital induces aneuploidy in Saccharomyces cerevisiae and stimulates the assembly of porcine brain tubulin; Albertini S et al.; Phenobarbital (PB) specifically induces mitotic chromosomal malsegregation in the diploid Saccharomyces cerevisiae strain D61.M but no other genetic events such as mitotic recombination or point mutations . In accordance with the hypothesis that PB exerts its genotoxic activity by an interaction with tubulin, it stimulates the GTP-promoted assembly of porcine brain tubulin in vitro . This process is reversible thus excluding an unspecific denaturation of the tubulin protein by PB.

J Bacteriol, 1985 Oct, 164(1), 57 - 62
Pyrimidine-specific cleavage by an endoribonuclease of Saccharomyces cerevisiae; Stevens A; An endoribonuclease with pyrimidine cleavage site specificity was isolated from Saccharomyces cerevisiae . The enzyme had a pH optimum of 6 to 7 and did not require a divalent cation . It was inhibited by 5 X 10(-5) M ethidium bromide, although it appeared to be single strand specific . The enzyme gave a limited cleavage of yeast mRNA and rRNA, yielding products that were terminated with pyrimidine nucleoside 2',3'-cyclic phosphate . The bonds between pyrimidine and A residues constituted more than 90% of the scission sites when the average product size was 50 nucleotides . Homopolyribonucleotides were cleaved poorly . Poly(A,U) was cleaved rapidly, and analysis of the products of poly(A,U) hydrolysis showed a very stringent cleavage of U-A bonds.

J Gen Microbiol, 1985 Oct, 131 ( Pt 10), 2565 - 71
NADP+-dependent glutamate dehydrogenase activity is impaired in mutants of Saccharomyces cerevisiae that lack aconitase; Gonzalez A et al.; A mutant of Saccharomyces cerevisiae lacking aconitase did not grow on minimal medium (MM) and had five- to tenfold less NADP+-dependent glutamate dehydrogenase (GDH) activity than the wild-type, although its glutamine synthetase (GS) activity was still inducible . When this mutant was incubated with glutamate as the sole nitrogen source, the 2-oxoglutarate content rose, and the NADP+-dependent GDH activity increased . Furthermore, carbon-limited cultures showed a direct relation between NADP+-dependent GDH activity and the intracellular 2-oxoglutarate content . We propose that the low NADP+-dependent GDH activity found in the mutant was due to the lack of 2-oxoglutarate or some other intermediate of the tricarboxylic acid cycle.

Genetics, 1985 Oct, 111(2), 243 - 58
The GLN1 locus of Saccharomyces cerevisiae encodes glutamine synthetase; Mitchell AP; Among 41 yeast glutamine auxotrophs, complementation analysis defined a single gene, GLN1, on chromosome 16 between MAK3 and MAK6 . Half of the alleles fell into two intragenic complementation classes . No clustering of complementing alleles was found in a fine structure map . Altered glutamine synthetase subunits, including nonsense fragments and charge variants, were identified in several of the mutants, indicating that GLN1 is the structural gene for this enzyme . Negative complementation was observed for almost every allele associated with a protein product and all gln1/+ heterozygotes displayed reduced susceptibility to ammonia repression of the remaining glutamine synthetase activity . This latter observation is explained by the hypothesis that ammonia represses the enzyme only through its metabolism to glutamine . A basis for the two gln1 complementation classes is proposed.

J Biol Chem, 1985 Sep 25, 260(21), 11831 - 7
Molecular characterization of the CAN1 locus in Saccharomyces cerevisiae . A transmembrane protein without N-terminal hydrophobic signal sequence; Hoffmann W; The complete DNA sequence of the CAN1 locus of the yeast Saccharomyces cerevisiae is presented . The predicted primary translation product consists of 590 amino acids . From the hydropathic profile of the amino acid sequence (as calculated by the algorithm of Kyte and Doolittle (Kyte, J., and Doolittle, R . F . (1982) J . Mol . Biol . 157, 105-132)), one can divide the protein into two distinct regions . The 93-amino acid long N-terminal domain is extremely hydrophilic and does not exhibit any cleavable signal sequence . The rest of the protein (from amino acids 94 to 590) shows features typical for an integral membrane protein . The proposal for the N terminus of the primary translation product is based on results obtained by S1 mapping, insertion mutagenesis, and gene fusion experiments.

Eur J Biochem, 1985 Sep 16, 151(3), 631 - 6
Metabolism of 2-oxoaldehyde in yeasts . Purification and characterization of NADPH-dependent methylglyoxal-reducing enzyme from Saccharomyces cerevisiae; Murata K et al.; An enzyme catalyzing the reduction of methylglyoxal was isolated from Saccharomyces cerevisiae and its enzymatic properties were analyzed . The enzyme, specifically eluted from a blue-dextran--Sepharose CL-6B column by the substrate, methylglyoxal, was homogeneous on polyacrylamide gel electrophoresis . The enzyme consisted of single polypeptide chain with a relative molecular mass of 43 000 . The enzyme was glycoprotein and contained 6.6% carbohydrate . NADPH was specifically required for activity and the Km for NADPH was 2.0 X 10(-7) M . The enzyme was active on various glyoxals such as glyoxal, methylglyoxal (Km = 5.88 mM) and phenylglyoxal (Km = 1.54 mM) . The reaction catalyzed by the enzyme was virtually irreversible . The activity was inhibited by sulfhydryl agents and activated by reducing agents such as glutathione . Intermediates in glycolysis, nucleosides, nucleotides, polyamines and various metal ions showed little inhibitory or activating effects on enzyme activity . Tricarboxylic acids showed a slight inhibitory effect . The activity of the enzyme was strongly inhibited by anionic detergents . The enzyme was rapidly inactivated by incubating with the substrates probably because of the non-enzymatic interaction between glyoxals and NH2 groups in arginine residues in the enzyme . NADP, one of the reaction products, also inhibited the enzyme activity and the Ki for NADP was about 0.07 mM . We tentatively designated the enzyme methylglyoxal reductase.

Nucleic Acids Res, 1985 Sep 11, 13(17), 6249 - 63
The Ty transposon of Saccharomyces cerevisiae determines the synthesis of at least three proteins; Mellor J et al.; Two new Ty determined proteins have been identified by placing a Ty transcriptional unit under the control of a high efficiency yeast expression vector . One of these proteins is the product of a post-translational processing event and it binds nucleic acids . A previously identified protein, pl (Tyl-15), has also been shown to bind nucleic acids and to be modified by phosphorylation.

J Biol Chem, 1985 Sep 5, 260(19), 10482 - 6
Specific induction of Ca2+ transport activity in MATa cells of Saccharomyces cerevisiae by a mating pheromone, alpha factor; Ohsumi Y et al.; Incubation with a high concentration of a mating pheromone, alpha factor, of Saccharomyces cerevisiae induced the accumulation of Ca2+ ion in MATa cells, but not in MAT alpha or MATa/alpha cells, after a lag of 30-40 min . The alpha factor did not cause a nonspecific lesion of the membrane barrier, but induced Ca2+ transport activity specifically . This induction of Ca2+ transport activity correlated with formation of a projection on the cells, or with localized cell elongation, but not with G1 arrest or agglutinin induction . The increased Ca2+ transport activity was maintained only in the continuous presence of a high concentration of alpha factor and de novo protein synthesis . Kinetic studies of induction of Ca2+ transport by alpha factor and effects of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and Ca2+ ionophore, A23187, on mating suggested an essential role of this physiological reaction in the initiation of sexual conjugation.

Eur J Biochem, 1985 Sep 2, 151(2), 275 - 81
The redox interconversion mechanism of Saccharomyces cerevisiae glutathione reductase; Pinto MC et al.; The changes undergone by pure yeast glutathione reductase during redox interconversion have been studied . Both the active and inactive forms of the enzyme had similar molecular masses, suggesting that the inactivation is probably due to intramolecular modification(s) . The glutathione reductase and transhydrogenase activities were similarly inactivated by NADPH and reactivated by GSH, while the diaphorase activity remained unaltered during redox interconversion of glutathione reductase . These results suggest that the inactivation site could be located far from the NADPH-binding site, although interfering with transhydrogenase activity, perhaps by conformational changes . The inactivation of glutathione reductase by 0.2 mM NADPH at pH 8 was paralleled by a gradual decrease in the absorbance at 530 nm and a simultaneous increase in the absorbance at 445 nm, while the reactivation promoted by GSH was initially associated with reversal of these spectral changes . The inactive enzyme spectrum retained some absorbance between 500 nm and 700 nm, showing a shoulder at 580-600 nm . Upon treatment of the enzyme with NADPH at pH 6.5 the spectrum remained unchanged, while no redox inactivation was observed under these conditions . It is suggested that the redox inactivation could be associated with the disappearance of the charge-transfer complex between the proximal thiolate and oxidized FAD in the two-electron-reduced enzyme . The inactive enzyme was reactivated by low GSSG concentrations, moderate dithiol concentrations, and high monothiol concentrations . These results and the spectral changes described above support the hypothesis attributing the redox interconversion to formation/disappearance of an erroneous disulfide between one of the half-cystines located at the GSSG-binding site and another cysteine nearby.

Mol Cell Biol, 1985 Sep, 5(9), 2190 - 6
Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products; Veit BE et al.; We have used gene disruptions and nuclease probes to assess the roles of yeast 2 micron plasmid genes in plasmid chromatin organization . The chromatin structure at the replication origin is not dependent on any of the four major open reading frames, A, B, C, or D . While stable plasmid maintenance is known to depend on a cis-acting locus STB and genes B and C, we find that only gene B influences STB chromatin . Other interactions between plasmid gene products and sequences may reflect gene regulation: the chromatin organization at the 5' end of gene A, which codes for a site-specific recombinase, depends on both gene B and gene C . Since disruption of gene C results in an increase in plasmid copy number that is dependent on gene A, we propose that gene C (and probably gene B) control copy number by regulating the level of the gene A recombinase.

Appl Environ Microbiol, 1985 Sep, 50(3), 713 - 6
Survival of Saccharomyces cerevisiae Y5 during starvation in the presence of osmotic supports; Peled ON; The viability of starved or dying Saccharomyces cerevisiae Y5 cells was extended for up to 1,500 h by adding osmotic supports (0.6 M sorbitol or mannitol) to the starvation media . Replacement of these polyols with 0.3 M KCl, 0.5 M MgSO4, or 0.2 M MgCl2 postponed the death of cells incubated in N-free medium, but accelerated it in distilled water.

Yeast, 1985 Sep, 1(1), 57 - 65
On the mechanism of exclusion of M2 double-stranded RNA by L-A-E double-stranded RNA in Saccharomyces cerevisiae; Hannig EM et al.; L-A-E double-stranded RNA (dsRNA), when introduced into cells carrying L-A-H and M2 dsRNAs, does not eliminate the L-A-H dsRNA, but (i) L-A-E does lower the copy number of L-A-H dramatically and (ii) L-A-E eliminates M2 dsRNA from the cell . That these two effects of L-A-E are related is shown by the fact that mutants of a strain carrying L-A-H and M2 selected for their resistance to exclusion of M2 by L-A-E {effect (ii)} have an altered L-A-H whose copy number is not lowered by L-A-E {effect (i)} . Although the L-A in K1 strains (L-A-HN in all cases examined) differs significantly both genetically and physically from the L-A in the K2 strain studied (L-A-H), the L-A-HN from the K1 strains can maintain M2 dsRNA, and the L-A-H from the K2 strains can maintain M1 dsRNA.

Yeast, 1985 Sep, 1(1), 39 - 47
Partial restoration of meiosis in an apomictic strain of Saccharomyces cerevisiae: a model system for investigation of nucleomitochondrial interactions during sporulation; Marmiroli N et al.; In an apomictic strain of Saccharomyces cerevisiae (ATCC 4117-H2) which undergoes a single nuclear division during sporulation and consequently forms asci containing two uninucleate diploid spores, a study was undertaken to investigate the effects of cultivation in three presporulation media (YPA; YNB; SMM) on nuclear division and ascosporogenesis in sporulation medium . Comparison of effects of presporulation culture in these media on the number of spores formed per ascus showed that a marked induction (30 +/- 4.3 per cent) of three- and four-spored asci could occur in sporulation medium following cultivation in a defined YNB medium supplemented with a 1 per cent solution of vitamins and containing decreased ammonium sulphate and increased glucose levels . Experiments in which the concentrations of glucose and of ammonium sulphate were varied simultaneously indicated that the initial presporulation carbon to nitrogen source ratio is an important factor in determining tetrad formation in sporulation medium . Nuclear staining demonstrated two classes of asci: binucleate (one- and two-spored) and tetranucleate (three- and four-spored) . Genetic evidence and data concerning effects of inclusion in sporulation medium of a meiotic inhibitor (glucose) indicated spores in tetrads were haploid rather than diploid . This ability to condition a significant number of cells for meiotic rather than apomictic differentiation made possible investigation of effects of mitochondrial inhibitors on both developmental processes simultaneously . It was found possible to selectively inhibit meiotic development by inclusion in sporulation medium of appropriate concentrations of specific inhibitors . Moreover, the data suggest meiotic sporulation is more strictly dependent than apomictic sporulation on mitochondrial function.

Mol Cell Biol, 1985 Sep, 5(9), 2466 - 75
Properties of REP3: a cis-acting locus required for stable propagation of the Saccharomyces cerevisiae plasmid 2 microns circle; Jayaram M et al.; Stable propagation of the yeast plasmid 2 microns requires an origin of replication, a cis-active locus designated REP3, and two plasmid-encoded proteins which are the products of the REP1 and REP2 genes . The three REP loci appear to constitute a partitioning system, ensuring equal distribution of plasmid molecules to mother and daughter cells after mitosis . We have localized the REP3 site completely within a segment of five-and-one-half direct tandem repeats of a 62-base-pair unit, bordered by HpaI and AvaI restriction sites within the large unique region of the 2 microns genome . In addition, we find that the repeated elements are functionally distinct . Only a subset of the repeats is necessary to promote full partitioning activity . The other repeats appear to promote plasmid transcription . These results are discussed in the context of a model of plasmid copy control involving titration of a plasmid-specific protein by the repeated elements within REP3.

Mol Cell Biol, 1985 Sep, 5(9), 2361 - 8
Plasmid recombination intermediates generated in a Saccharomyces cerevisiae cell-free recombination system; Symington LS et al.; We have developed an assay utilizing Saccharomyces cerevisiae cell extracts to catalyze recombination in vitro between homologous plasmids containing different mutant alleles of the tet gene . Electrophoretic analysis of product DNA indicated that a number of novel DNA species were formed during the reaction . These species migrated through agarose gels as distinct bands with decreased electrophoretic mobility compared with the substrate DNA . The DNA from each individual band was purified and shown to be enriched 5- to 100-fold for tetracycline-resistant recombinants by using a transformation assay . The structure of the DNA molecules present in these bands was determined by electron microscopy . Recombination between circular substrates appeared to involve the formation and processing of figure-eight molecules, while recombination between circular and linear substrates involved the formation of molecules in which a circular monomer had a monomer-length linear tail attached at a region of homology.

Mol Cell Biol, 1985 Sep, 5(9), 2279 - 88
Identification of the ureidoglycolate hydrolase gene in the DAL gene cluster of Saccharomyces cerevisiae; Yoo HS et al.; This report describes the isolation of the genes encoding allantoicase (DAL2) and ureidoglycolate hydrolase (DAL3), which are components of the large DAL gene cluster on the right arm of chromosome IX of Saccharomyces cerevisiae . During this work a new gene (DAL7) was identified and found to be regulated in the manner expected for an allantoin pathway gene . Its expression was (i) induced by allophanate, (ii) sensitive to nitrogen catabolite repression, and (iii) responsive to mutation of the DAL80 and DAL81 loci, which have previously been shown to regulate the allantoin degradation system . Hybridization probes generated from these cloned genes were used to analyze expression of the allantoin pathway genes in wild-type and mutant cells grown under a variety of physiological conditions . When comparison was possible, the patterns of mRNA and enzyme levels observed in various strains and physiological conditions were very similar, suggesting that the system is predominantly regulated at the level of gene expression . Although all of the genes seem to be controlled by a common mechanism, their detailed patterns of expression were, at the same time, highly individual and diverse.

Biochimie, 1985 Sep, 67(9), 999 - 1006
{Characterization of gamma-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae}; Penninckx MJ et al.; In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase . The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell . Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100 . The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration . As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven . Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected . The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione . Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme . The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase . Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme . The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.

Radiobiologiia, 1985 Sep-Oct, 25(5), 612 - 6
{Effect of rad 54 mutation on the ability of Saccharomyces cerevisiae cells to recover from radiation and thermal injury}; Glazunov AV et al.; The diploid cells of Saccharomyces cerevisiae carrying rad 54 homozygous mutation do not exhibit an ability for any considerable "rapid" postirradiation and post-hyperthermic recovery . A pretreatment with high temperature (50 degrees C) increases the radiation response of mutant cells . Survival of cells overheated before gamma-irradiation is increased by keeping them in water for 2-6 h at 28 degrees C, while the corresponding value of survival for cells treated by each of the factors delivered separately remains constant in these conditions.

J Gen Microbiol, 1985 Sep, 131 ( Pt 9), 2153 - 64
The metabolism of 5'-methylthioadenosine and 5-methylthioribose 1-phosphate in Saccharomyces cerevisiae; Marchitto KS et al.; Cordycepin sensitive mutants of Saccharomyces cerevisiae, which are permeable to 5'-deoxy-5'-methylthioadenosine (MTA), were used to study the fate of the methylthioribose carbons of this purine nucleoside . Evidence is presented for the recycling of the methylthio group and part of the ribose portion of MTA in a biosynthetic pathway which leads to the synthesis of methionine . The main pathway involves the phosphorylytic cleavage of MTA by MTA phosphorylase yielding 5-methylthioribose 1-phosphate and adenine as products . Loss of the phosphate group of 5-methylthioribose 1-phosphate, concurrent with the rearrangement of the ribose carbons, leads to the synthesis of 2-keto-4-methylthiobutyric acid . In the final step of the sequence, 2-keto-4-methylthiobutyric acid is converted to methionine via transamination . Several compounds not directly associated with the biosynthesis of methionine were also isolated . These compounds, which may arise through the degradation of intermediates in the pathway, were: 5'-methylthioinosine, a deaminated catabolite of MTA; 5-methylthioribose, a result of the phosphorylysis of 5-methylthioribose 1-phosphate, and 3-methylthiopropionaldehyde, 3-methylthiopropionic acid and 2-hydroxy-4-methylthiobutyric acid, all arising from the catabolism of 2-keto-4-methylthiobutyric acid.

Genetics, 1985 Sep, 111(1), 7 - 22
Rad52-independent mitotic gene conversion in Saccharomyces cerevisiae frequently results in chromosomal loss; Haber JE et al.; We have examined spontaneous, interchromosomal mitotic recombination events between his4 alleles in both Rad+ and rad52 strains of Saccharomyces cerevisiae . In Rad+ strains, 74% of the His+ prototrophs resulted from gene conversion events without exchange of flanking markers . In diploids homozygous for the rad52-1 mutation, the frequency of His+ prototroph formation was less than 5% of the wild-type value, and more than 80% of the gene conversion events were accompanied by an exchange of flanking markers . Most of the rad52 intragenic recombination events arose by gene conversion accompanied by an exchange of flanking markers and not by a simple reciprocal exchange between the his4A and his4C alleles . There were also profound effects on the kinds of recombinant products that were recovered . The most striking effect was that RAD52-independent mitotic recombination frequently results in the loss of one of the two chromosomes participating in the gene conversion event.

Genetics, 1985 Sep, 111(1), 1 - 6
Two recessive suppressors of Saccharomyces cerevisiae cho1 that are unlinked but fall in the same complementation group; Atkinson KD; Phenotypic reversion of ethanolamine-requiring Saccharomyces cerevisiae cho1 mutants is predominantly due to recessive mutations at genes unlinked to the chromosome V cho1 locus . The recessive suppressors do not correct the primary cho1 defect in phosphatidylserine synthesis but circumvent it with a novel endogenous supply of ethanolamine . One suppressor (eam1) was previously mapped to chromosome X, and 135 suppressor isolates were identified as eam1 alleles by complementation analysis . Additional meiotic recombination studies have identified a second genetic locus, eam2, that falls in the eam1 complementation group but maps close to the centromere of chromosome IV . Although the normal EAM1 and EAM2 alleles are fully dominant over recessive mutant alleles, their dominance fails in diploids heterozygous for defects in both genes simultaneously . The unusual complementation pattern could be explained by interaction of the gene products in formation of the same enzyme.

Yeast, 1985 Sep, 1(1), 67 - 77
Primary structure of the Saccharomyces cerevisiae GAL7 gene; Tajima M et al.; We present the nucleotide sequence of a 1599-base pair (bp) DNA fragment containing the entire GAL7 gene that encodes galactose-1-phosphate uridyltransferase of Saccharomyces cerevisiae . The deduced peptide was composed of 364 amino acid residues . The expected molecular weight was 42,005 daltons, which agreed with the observed value for the purified enzyme . The 3'-end of the GAL7 transcript mapped at a position 82 bp downstream from the UAA termination codon by the S1 nuclease protection experiment . We constructed a GAL7'-lac'Z fusion on various types of yeast plasmid vectors . The fused gene on any type of vector was induced by galactose and repressed by glucose as for the GAL7 gene on the chromosome . The response of GAL7'-lac'Z fusion to gal4 delta and gal80 delta regulatory mutations was also similar to the response of the chromosomal GAL7 gene . By using various deletions in the 5'-flanking region of the gene fusion, we delimited the sequence essential for galactose controlled expression with a 180 bp-fragment of DNA lying 92 bp upstream of the transcription initiation site.

Mol Cell Biol, 1985 Sep, 5(9), 2247 - 56
Suppressible and nonsuppressible +1 G-C base pair insertions induced by ICR-170 at the his4 locus in Saccharomyces cerevisiae; Mathison L et al.; Fifteen independent ICR-170-induced his4 mutations in Saccharomyces cerevisiae were examined by DNA sequence analysis . All of the mutations contained a +1 G-C base pair addition in the HIS4 coding region . Eleven different sites of insertion were identified . Combined with previous DNA sequence data, 21 ICR-170-induced his4 mutations distributed at 16 different sites were analyzed . The insertions were always located in a consecutive run of two or more G-C base pairs, with all base pairs in each run having identical orientation . Long consecutive G-C runs were preferred target sites over short runs . Although some consecutive G-C runs appeared to be preferred target sites over others of identical length, such preference was not due to any particular type of nucleotide pair immediately adjacent to a given target site . In addition, DNA sequence analyses of the his4 mutations provided a basis for examining the mechanism of mRNA sequence recognition by extragenic suppressors of ICR-170-induced mutations . The implications of these results for mechanisms of frameshift suppression are discussed.

Biochem Biophys Res Commun, 1985 Aug 30, 131(1), 190 - 8
Characterization of methylglyoxal synthase in Saccharomyces cerevisiae; Murata K et al.; Methylglyoxal synthase in Saccharomyces cerevisiae was purified approximately 300 folds from cell extracts with 20% of activity yield . During purification procedures, polymorphic behaviours of the enzyme were observed . The purified enzyme was homogeneous on polyacrylamide gel electrophoresis and consisted of a single polypeptide chain of Mr = 26,000 . The enzyme was most active at pH 9.5-10.5 and strictly specific to dihydroxyacetone phosphate with Km = 3 mM . Phosphoenolpyruvate, glyceraldehyde-3-phosphate, orthophosphate and thiol compounds were potent inhibitors of the enzyme.

Biochim Biophys Acta, 1985 Aug 22, 836(1), 89 - 95
Inhibition of sterol biosynthesis by ergosterol and cholesterol in Saccharomyces cerevisiae; Pinto WJ et al.; When accumulation of squalene was used as a measure of the flow of carbon into the sterol pathway in whole cells of semi-anaerobic Saccharomyces cerevisiae, both ergosterol and cholesterol were found to be inhibitory . However, at equivalent concentrations in the medium ergosterol was substantially the more potent inhibitor . Marked differences found in the absorption and esterification of the two sterols failed to account for the observed difference in their capacities to act as feedback agents . Cholesterol was much more effectively absorbed as well as esterified, but, when the abilities of the two sterols to lower the squalene level were calculated on the basis of free sterol in the cells, ergosterol remained more effective by a factor of four.

Mol Cell Biol, 1985 Aug, 5(8), 2154 - 8
Homothallic switching of Saccharomyces cerevisiae mating type genes by using a donor containing a large internal deletion; Weiffenbach B et al.; Homothallic switching of the mating type genes of Saccharomyces cerevisiae occurs by a gene conversion event, replacing sequences at the expressed MAT locus with a DNA segment copied from one of two unexpressed loci, HML or HMR . The transposed Ya or Y alpha sequences are flanked by homologous regions that are believed to be essential for switching . We examined the transposition of a mating type gene (hmr alpha 1-delta 6) which contains a 150-base-pair deletion spanning the site where the HO endonuclease generates a double-stranded break in MAT that initiates the gene conversion event . Despite the fact that the ends of the cut MAT region no longer share homology with the donor hmr alpha 1-delta 6, switching of MATa or MAT alpha to mat alpha 1-delta 6 was efficient . However, there was a marked increase in the number of aberrant events, especially the formation of haploid-inviable fusions between MAT and the hmr alpha 1-delta 6 donor locus.

Mol Cell Biol, 1985 Aug, 5(8), 2131 - 41
Regulation of repressible acid phosphatase gene transcription in Saccharomyces cerevisiae; Lemire JM et al.; We examined the genetic system responsible for transcriptional regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase {acid optimum, EC 3.1.3.2}) in Saccharomyces cerevisiae at the molecular level by analysis of previously isolated and genetically well-defined regulatory gene mutants known to affect APase expression . These mutants identify numerous positive- (PHO4, PHO2, PHO81) and negative-acting (PHO80, PHO85) regulatory loci dispersed throughout the yeast genome . We showed that the interplay of these positive and negative regulatory genes occurs before or during APase gene transcription and that their functions are all indispensible for normal regulation of mRNA synthesis . Biochemical evidence suggests that the regulatory gene products they encode are expressed constitutively . More detailed investigation of APase synthesis is a conditional PHO80(Ts) mutant indicated that neither PHO4 nor any other protein factor necessary for APase mRNA synthesis is transcriptionally regulated by PHO80 . Moreover, in the absence of PHO80, the corepressor, presumed to be a metabolite of Pi, did not inhibit their function in the transcriptional activation of APase.

Mol Cell Biol, 1985 Aug, 5(8), 1878 - 86
Molecular cloning and characterization of the STE7 and STE11 genes of Saccharomyces cerevisiae; Chaleff DT et al.; In the yeast Saccharomyces cerevisiae, haploid cells occur in one of the two cell types, a or alpha . The allele present at the mating type (MAT) locus plays a prominent role in the control of cell type expression . An important consequence of the elaboration of cell type is the ability of cells of one mating type to conjugate with cells of the opposite mating type, resulting in yet a third cell type, an a/alpha diploid . Numerous genes that are involved in the expression of cell type and the conjugation process have been identified by standard genetic techniques . Molecular analysis has shown that expression of several of these genes is subject to control on the transcriptional level by the MAT locus . Two genes, STE7 and STE11, are required for mating in both haploid cell types; ste7 and ste11 mutants are sterile . We report here the molecular cloning of STE7 and STE11 genes and show that expression of these genes is not regulated transcriptionally by the MAT locus . We also have genetically mapped the STE11 gene to chromosome XII, 40 centimorgans from ura4.

EMBO J, 1985 Aug, 4(8), 2087 - 92
Mitochondrial protein synthesis is required for maintenance of intact mitochondrial genomes in Saccharomyces cerevisiae; Myers AM et al.; The genes of Saccharomyces cerevisiae coding for the mitochondrial threonine and tryptophan tRNA synthetases and for a putative mitochondrial ribosomal protein have been cloned . These, and the previously cloned gene for a mitochondrial elongation factor, were used to disrupt or partially delete the wild-type chromosomal copies of the genes in the respiratory-competent strain W303 . In each case, inactivation of a gene whose product is required for mitochondrial protein synthesis causes an instability in mitochondrial DNA . Although intact mitochondrial genomes are rapidly and quantitatively eliminated in the protein synthesis defective strains, specific rho- genomes can be maintained stably over many generations . These results indicate that mitochondrial protein synthesis is required for the propagation of wild-type mitochondrial DNA in yeast.

Genetics, 1985 Aug, 110(4), 609 - 46
Meiotic gene conversion mutants in Saccharomyces cerevisiae . I . Isolation and characterization of pms1-1 and pms1-2; Williamson MS et al.; The pms1 mutants, isolated on the basis of sharply elevated meiotic prototroph frequencies for two closely linked his4 alleles, display pleiotropic phenotypes in meiotic and mitotic cells . Two isolates carrying recessive mutations in PMS1 were characterized . They identify a function required to maintain low postmeiotic segregation (PMS) frequencies at many heterozygous sites . In addition, they are mitotic mutators . In mutant diploids, spore viability is reduced, and among survivors, gene conversion and postmeiotic segregation frequencies are increased, but reciprocal exchange frequencies are not affected . The conversion event pattern is also dramatically changed in multiply marked regions in pms1 homozygotes . The PMS1 locus maps near MET4 on chromosome XIV . The PMS1 gene may identify an excision-resynthesis long patch mismatch correction function or a function that facilitates correction tract elongation . The PMS1 gene product may also play an important role in spontaneous mitotic mutation avoidance and correction of mismatches in heteroduplex DNA formed during spontaneous and UV-induced mitotic recombination . Based on meiotic recombination models emphasizing mismatch correction in heteroduplex DNA intermediates, this interpretation is favored, but alternative interpretations involving longer recombination intermediates in the mutants are also considered.

Genetics, 1985 Aug, 110(4), 569 - 89
A mapping method for Saccharomyces cerevisiae using rad52-induced chromosome loss; Schild D et al.; Saccharomyces cerevisiae diploids homozygous for the rad52-1 mutation have previously been shown to lose chromosomes mitotically . Spontaneous events and events following low levels of X-ray or methyl methanesulfonate treatment result in monosomic diploids, whereas higher levels of treatment result in near haploidization . This rad52-1-dependent chromosome loss has been used to develop a new mapping method which can be used to assign a previously unmapped gene to a chromosome . Chromosome loss mapping can be done in either of two ways: if a diploid, homozygous for rad52-1 but heterozygous for a variety of other recessive markers, is constructed with an unmapped recessive mutation in coupling with known chromosomal markers, chromosome loss will result in the coordinate expression of the mutation and other recessive markers on the same chromosome; if, however, the diploid is constructed with the unmapped mutation in repulsion to chromosomal markers, then even haploidization will never result in the coordinate expression of the unmapped mutation and other markers on the same homologous chromosome pair--This mapping method and subsequent tetrad analyses have been used to locate hom6 on chromosome X, ade4 on chromosome XIII and cdc31 on chromosome XV and to demonstrate that met5, previously assigned to chromosome V, actually maps to chromosome X; the met- marker on chromosome V has been shown to be met6 . GAL80 and SUP5, previously assigned to an unmapped fragment, have now been mapped to the right arm of chromosome XIII.

Arch Biochem Biophys, 1985 Aug 1, 240(2), 613 - 20
Effects of sinefungin on rRNA production and methylation in the yeast Saccharomyces cerevisiae; Li AW et al.; The antifungal agent, Sinefungin (SF), has been shown to be an inhibitor of transmethylation reactions . We report here the effects of SF on the production and methylation of rRNA in the yeast, Saccharomyces cerevisiae . Under conditions of SF treatment which have been shown to affect the regulation of cell proliferation in this yeast, pulse-chase labeling experiments using {methyl-3H}methionine and {3H}uracil indicated that methyl incorporation into rRNA during a short labeling period was inhibited, and stable 18 S rRNA production was differentially decreased . Other experiments quantitating modified nucleotides in newly produced rRNA showed that stable molecules were methylated . Taken together, these results suggest that SF slows methylation of rRNA, and is associated with differential loss of undermethylated 18 S rRNA species.

Mol Cell Biol, 1985 Aug, 5(8), 2029 - 38
Recombination hot spot in the human beta-globin gene cluster: meiotic recombination of human DNA fragments in Saccharomyces cerevisiae; Treco D et al.; We describe a novel system for the analysis of sequence-specific meiotic recombination in Saccharomyces cerevisiae . A comparison of three adjacent restriction fragments from the human beta-globin locus revealed that one of them, previously hypothesized to contain a relative hot spot for genetic recombination, engages in reciprocal exchange during yeast meiosis significantly more frequently than either of the other two fragments . Removal of the longest of four potential Z-DNA-forming regions from this fragment does not affect the high frequency of genetic recombination.

Mol Cell Biol, 1985 Aug, 5(8), 1839 - 46
A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome c; Baim SB et al.; The CYC1-239-O mutation in the yeast Saccharomyces cerevisiae produces a -His-Leu- replacement of the normal -Ala-Gly- sequence at amino acid positions 5 and 6, which lie within a dispensable region of iso-1-cytochrome c; this mutation can accommodate the formation of a hairpin structure at the corresponding site in the mRNA . The amount of the altered protein was diminished to 20% of the wild-type level, whereas the amount of the mRNA remained normal . However, in contrast to the normal CYC1+ mRNA that is associated mainly with four to seven ribosomes, the bulk of the CYC1-239-O mRNA is associated with one to four ribosomes . These results suggest that the stable secondary structure within the translated region of the CYC1 mRNA diminishes translation by inhibiting elongation.

J Bacteriol, 1985 Aug, 163(2), 763 - 8
Cloning of GLN4: an essential gene that encodes glutaminyl-tRNA synthetase in Saccharomyces cerevisiae; Ludmerer SW et al.; The structural gene for glutaminyl-tRNA synthetase has been isolated from a gene bank of Saccharomyces cerevisiae chromosomal DNA . Cloning was achieved by complementation of a recently described yeast strain that is auxotrophic for glutamine . A multicopy recombinant plasmid with a 5-kilobase-pair genomic insert conferred sixfold elevation in glutaminyl-tRNA synthetase activity and restored a Gln+ phenotype to strains that were Gln- by virtue of a mutant gln4 allele . Subfragments of the 5-kilobase insert directed integration of URA3 to GLN4 . Further experiments established that GLN4 is an essential gene that is located on chromosome XV . RNA blots with a GLN4-specific probe detected a single transcript of approximately 2,900 nucleotides.

J Biol Chem, 1985 Jul 5, 260(13), 8173 - 81
Purification of a DNA primase activity from the yeast Saccharomyces cerevisiae . Primase can be separated from DNA polymerase I; Wilson FE et al.; A primase activity which permits DNA synthesis by yeast DNA polymerase I on a single-stranded circular phi X174 or M13 DNA or on poly(dT)n has been extensively purified by fractionation of a yeast enzyme extract which supports in vitro replication of the yeast 2-microns plasmid DNA (Kojo, H., Greenberg, B . D., and Sugino, A . (1981) Proc . Natl . Acad . Sci . U.S.A . 78, 7261-7265) . Most of this DNA primase activity was separated from DNA polymerase activity, although a small amount remained associated with DNA polymerase I . The primase, active as a monomer, has a molecular weight of about 60,000 . The primase synthesizes oligoribonucleotides of discrete size, mainly eight or nine nucleotides, in the presence of single-stranded template DNA and ribonucleoside 5'-triphosphates; it utilizes deoxyribonucleoside 5'-triphosphates as substrate with 10-fold lower efficiency . Product size, chromatographic properties, alpha-amanitin resistance, and molecular weight of the primase activity distinguish it from RNA polymerases I, II, and III . The DNA products synthesized by both primase and DNA polymerase I on a single-stranded DNA template were 200-500 nucleotides long and covalently linked to oligoribonucleotides at their 5'-ends . Addition of yeast single-stranded DNA-binding protein (Arendes, J., Kim, K . C., and Sugino, A . (1983) Proc . Natl . Acad . Sci . U.S . A . 80, 673-677) stimulated the DNA synthesis 2-3-fold.

Nature, 1985 Jul 4-10, 316(6023), 83 - 5
Identification of myosin heavy chain in Saccharomyces cerevisiae; Watts FZ et al.; Motility in biological systems is expressed in a variety of ways, such as cytoplasmic streaming, cell shaping, nuclear migration and muscle contraction . These functions are thought to be mediated by structural proteins, for example, myosin, actin and tubulin . The involvement of myosin in muscle contraction is well documented and this protein is implicated in generating the cleavage forces during cytokinesis in some non-muscle cells . Here, we report the isolation of a protein similar to myosin as judged by its biochemical and immunological properties, from the yeast Saccharomyces cerevisiae . Parts of the protein have been conserved through evolution at the protein and DNA sequence levels . The presence of this protein in the region bordering mother cell and bud, as revealed by immunofluorescence, suggests that it is involved in cell division.

Biochemistry, 1985 Jul 2, 24(14), 3540 - 7
Evidence for overlapping active sites in a multifunctional enzyme: immunochemical and chemical modification studies on C1-tetrahydrofolate synthase from Saccharomyces cerevisiae; Appling DR et al.; The relationship of the active sites which catalyze the three reactions in the trifunctional enzyme C1-tetrahydrofolate synthase (C1-THF synthase) from Saccharomyces cerevisiae has been examined with immunochemical and chemical modification techniques . Immunotitration of the enzyme with a polyclonal antiserum resulted in identical inhibition curves for the dehydrogenase and cyclohydrolase activities which were distinctly different from the inhibition curve for the synthetase activity . During chemical modification with diethyl pyrocarbonate (DEPC), the three activities were inactivated at significantly different rates, indicating that at least three distinct essential residues are involved in the reaction with DEPC . The pH dependence of the reaction with DEPC was consistent with the modification of histidyl residues . Treatment of C1-THF synthase with N-ethylmaleimide (NEM) resulted in significant inactivation of only the dehydrogenase and cyclohydrolase activities, with the cyclohydrolase at least an order of magnitude more sensitive than the dehydrogenase . Inactivation of cyclohydrolase was biphasic at NEM concentrations above 0.1 mM, suggesting two essential cysteinyl residues were being modified . NADP+, a dehydrogenase substrate, protected both dehydrogenase and cyclohydrolase activities, but not synthetase activity, against inactivation by either reagent . Synthetase substrates had no protective ability . Pteroylpolyglutamates and p-aminobenzoic acid polyglutamates exhibited some protection of all three activities . The p-aminobenzoic acid polyglutamate series showed progressive protection with increasing chain length . These results are consistent with an overlapping site for the dehydrogenase and cyclohydrolase reactions, independent from the synthetase active site . Possible active-site configurations and the role of the polyglutamate tail in substrate binding are discussed.

J Gen Microbiol, 1985 Jul, 131 ( Pt 7), 1797 - 806
Molecular cloning of WHI2, a gene involved in the regulation of cell proliferation in Saccharomyces cerevisiae; Saul DJ et al.; The WHI2 gene of Saccharomyces cerevisiae plays a key role in coordinating cell proliferation and nutrient availability . A 2.6 kb yeast DNA sequence has been cloned which fully suppresses the whi2 mutation . Integration of this sequence to demonstrate that the structural gene itself had been cloned proved difficult . Integration occurred only rarely and only at the LEU2 locus which was also present on the integrating plasmid . To circumvent these difficulties an adjacent sequence, present on the original isolate from the gene library, was subcloned onto the integrating vector YIp5, after which directed integration proved straightforward . The integrated sequence was closely linked to WHI2, confirming that the structural gene had been cloned . A chromosomal restriction map of the WHI2 region is presented; no gross changes were observed in the region as cells entered stationary phase.

J Biochem (Tokyo), 1985 Jul, 98(1), 167 - 75
Characterization of rat cytochrome P-450MC synthesized in Saccharomyces cerevisiae; Sakaki T et al.; Rat cytochrome P-450MC cDNA was expressed in Saccharomyces cerevisiae AH22, SHY3 and NA87-11A cells under the control of the yeast ADH1 promoter and terminator . Although the three yeast strains transformed with the constructed expression plasmid, pAMC1, contained approximately three copies of the plasmid, the levels of both P-450MC mRNA and the corresponding protein in the AH22 cells carrying plasmid pAMC1 were 1.4- to 1.7-fold and 2-fold higher than in the other two strains, respectively . The P-450MC protein was purified from the microsomal fraction of AH22 cells carrying pAMC1 by a rapid purification method . The apparent molecular weight, chromatographic behavior, spectral properties, substrate specificity and immunochemical properties of the purified P-450MC protein were indistinguishable from those of rat liver P-450MC-I and P-450MC-II (Sasaki, T., et al . (1984) J . Biochem . 96, 117-126) . The NH2-terminal amino acid sequence of the purified protein up to 10 residues was the same as those of P-450MC-I and P-450MC-II . In addition, HPLC analysis of the microsomal fraction of AH22 cells containing pAMC1 indicated that the synthesized P-450MC protein corresponds to P-450MC-II, but not P-450MC-I . With another purification method, we obtained the cleaved P-450MC protein which lacked the NH2-terminal 30 amino acids of intact P-450MC . The spectral properties and monooxygenase activities towards benzo(a)pyrene and 7-ethoxycoumarin of the cleaved P-450MC were nearly the same as those of intact P-450MC.

Mol Cell Biol, 1985 Jul, 5(7), 1676 - 84
Effect of ARS1 mutations on chromosome stability in Saccharomyces cerevisiae; Srienc F et al.; We have used a set of deletion mutations in the ARS1 element of Saccharomyces cerevisiae to measure their effect on chromosome stability . This work establishes the previously proposed existence of three domains in ARS1 . Domain C, which we have previously inferred, but not proved, to be a part of ARS1, is now established . In addition, we show that increasingly large deletions of the domain have increasingly large effects, which was not realized before . Furthermore, we have provided the first positive evidence for the central importance of a 14-base-pair core sequence containing the ARS consensus element by showing that it has the ability to act as a replicator on a plasmid containing no other ARS1 flanking sequence . The method of analyzing plasmid stability used in our study employs a novel and sensitive flow cytometry assay for beta-galactosidase . We discuss ways in which flow cytometry, based on this assay, could be generalized beyond its particular application in this work to studying other aspects of the cell biology of yeast and higher cells . The actual flow cytometry method will be described in detail elsewhere.

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