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