S. cerevisiae

Gene, 1990 May 31, 90(1), 79 - 85
Efficient expression of the Saccharomyces cerevisiae glycolytic gene ADH1 is dependent upon a cis-acting regulatory element (UASRPG) found initially in genes encoding ribosomal proteins; Tornow J et al.; The glycolytic form of alcohol dehydrogenase (ADHI) is encoded by the ADH1 gene of Saccharomyces cerevisiae . We found that efficient expression of the ADH1 gene requires a sequence between bp -635 and -615 with respect to the +1 mRNA start point; removal of this sequence reduced ADH1 mRNA levels 25-fold but did not affect carbon-source regulation . DNaseI footprinting analysis of the ADH1 promoter revealed the specific protection of a perfect match to UASRPG at -630 to -615 . UASRPG is thought to be responsible for activation of transcription, via binding of the translation upstream factor (TUF), of genes encoding components of the translational apparatus . In band retardation assays, the promoters for the elongation factor 1 alpha-encoding genes (TEF1 and TEF2) competed for binding of the protein to the copy of UASRPG in the ADH1 promoter . We conclude that TUF is probably involved in activation of the bulk of ADH1 transcription . Further, we propose that TUF has a role in the activation of many or most glycolytic genes . If so, it is essential for efficient expression of a wide variety of functionally disparate products that are required by yeast cells for rapid growth.

Eur J Biochem, 1990 May 31, 190(1), 39 - 44
GAP1, the general amino acid permease gene of Saccharomyces cerevisiae . Nucleotide sequence, protein similarity with the other bakers yeast amino acid permeases, and nitrogen catabolite repression; Jauniaux JC et al.; In Saccharomyces cerevisiae, mutations at the GAP1 locus selectively abolish the activity of the general amino acid transport system . This permease catalyses active transport of apparently all biological amino acids across the plasma membrane . We have determined the nucleotide sequence of the GAP1 gene . The sequence contains an open reading frame of 601 codons corresponding to a polypeptide of Mr 65578 . This polypeptide is strongly hydrophobic; it exhibits three potential glycosylation sites . Hydropathy analysis suggests 12 membrane-spanning regions . The N-terminal domain is charged, it does not resemble hydrophobic signal sequences found in secreted proteins . Hence the GAP1 gene encodes a protein with characteristics typical of integral membrane proteins translocating ligants across cellular membranes . The deduced amino acid sequence of GAP1 protein presents strong similarities to those of the yeast arginine, histidine and proline permeases, suggesting a common evolutionary origin for these amino acid permeases . Nitrogen-source regulation of the GAP1 permease is believed to occur at two distinct levels, i.e . permease synthesis and permease activity {Grenson (1983) Eur . J . Biochem . 133, 135-139} . Northern analysis of GAP1-specific transcripts in wild-type and in mutant strains is in agreement with these views and indicates that nitrogen catabolite repression of GAP1 synthesis occurs at the RNA level.

J Biol Chem, 1990 May 25, 265(15), 8377 - 81
Efficient expression and utilization of mutant 5 S rRNA in Saccharomyces cerevisiae; Van Ryk DI et al.; The expression of mutant 5 S rRNA genes in vivo is examined as a basis for further studies on the control, structure, and function of the ribosomal 5 S RNA . Specific single base substitutions (e.g . positions 98 or 99) or short insertions can result in substantial structural changes that can easily be detected by gel electrophoresis and permit the assay of mutant RNA synthesis and utilization . In addition, the use of high and low copy shuttle vectors as well as alternate growth conditions permits the adjustment of mutant RNA levels in vivo . Despite the high genomic copy number for the 5 S rRNA gene, under optimized conditions as much as 80% of the cellular 5 S RNA can be mutant, and RNA structure analyses indicate that some of these RNAs can readily be assembled into the ribosome structure resulting in an in vivo ribosome population which is also approximately 80% mutant . The results indicate that plasmid integrated 5 S rRNA genes are preferentially expressed and suggest that additional features of the chromosome structure regulate 5 S rRNA gene expression in vivo.

Biochemistry, 1990 May 15, 29(19), 4548 - 53
Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: physicochemical characteristics of the nucleotide binding site, as deduced from fluorescent spectroscopy measurements; Encinas MV et al.; Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase {ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49} is inactivated by the fluorescent sulfhydryl reagent N-(iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) . The inactivation reaction follows pseudo-first-order kinetics with respect to active enzyme to less than 10% remaining enzyme activity, with a second-order inactivation rate constant of 2.6 min-1 mM-1 at pH 7.5 and 30 degrees C . A stoichiometry of 1.05 mol of reagent incorporated per mole of enzyme subunit was found for the completely inactivated enzyme . Almost complete protection of the enzyme activity and of dansyl label incorporation are afforded by MnADP or MnATP, thus suggesting that 1,5-IAEDANS interacts with an enzyme sulfhydryl group at the nucleotide binding site . The fluorescence decay of the AEDANS attached to the protein shows a single-exponential behavior with a lifetime of 18 ns . A comparison of the fluorescence band position and the fluorescence decay with those of the adduct AEDANS-acetylcysteine indicates a reduced polarity for the microenvironment of the substrate binding site . The quenching of the AEDANS moiety in the protein can be described in terms of a collisional and a static component . The rate constant for the collisional component is much lower than that obtained for the adduct in a medium of reduced polarity . These last results indicate that the AEDANS moiety is considerably shielded from the solvent when it is covalently attached to PEPCK.

J Biol Chem, 1990 May 5, 265(13), 7596 - 603
Specific transcription of Saccharomyces cerevisiae 35 S rDNA by RNA polymerase I in vitro; Riggs DL et al.; Specific transcription of yeast 35 S rDNA by RNA polymerase I has been demonstrated using fractionated extracts prepared from whole cells of Saccharomyces cerevisiae . Determination of the 5'-nucleotides of the in vitro transcripts indicated that two apparent start sites, corresponding to the first (initiating) and fifth nucleotide of the in vivo transcript, were utilized . Production of the 35 S rDNA transcript in this system was not inhibited by alpha-amanitin . Specific transcription of both the 35 S and 5 S rDNA sequences contained on the same template occurred simultaneously in these extracts . Sequential template competition experiments demonstrated that 35 S and 5 S rDNA transcription required different transcription factors . Specific antisera raised against the largest subunit of RNA polymerase I significantly inhibited synthesis of the 35 S rDNA transcript, but had a negligible effect on 5 S rRNA synthesis by RNA polymerase III . Additionally, this 35 S rDNA transcriptional activity was present in extracts prepared from a strain deficient in the mitochondrial RNA polymerase . Experiments using truncated rDNA templates showed that in vitro no more than 206 base pairs of the sequence upstream of the initiation site are required for maximal activity in this system; the enhancer element did not stimulate 35 S rDNA transcription.

J Biol Chem, 1990 May 5, 265(13), 7464 - 71
Cloning and functional analysis of the arginyl-tRNA-protein transferase gene ATE1 of Saccharomyces cerevisiae; Balzi E et al.; Aminoacyl-tRNA-protein transferases (Arg-transferases) catalyze post-translational conjugation of specific amino acids to the amino termini of acceptor proteins . A function of these enzymes in eukaryotes has been shown to involve the conjugation of destabilizing amino acids to the amino termini of short-lived proteins, these reactions being a part of the N-end rule pathway of protein degradation (Gonda, D . K., Bachmair, A., Wunning, I., Tobias, J . W., Lane, W . S., and Varshavsky, A . (1989) J . Biol . Chem . 264, 16700-16712) . We have cloned the ATE1 gene of the yeast Saccharomyces cerevisiae which encodes arginyl-tRNA-protein transferase . ATE1 gives rise to a approximately 1.6-kilobase mRNA and codes for a 503-residue protein . Expression of the yeast ATE1 gene in Escherichia coli, which lacks Arg-transferases, was used to show that the ATE1 protein possesses the Arg-transferase activity . Null ate1 mutants are viable but lack the Arg-transferase activity and are unable to degrade those substrates of the N-end rule pathway that start with residues recognized by the Arg-transferase.

J Biol Chem, 1990 May 5, 265(13), 7278 - 83
The ferrochelatase from Saccharomyces cerevisiae . Sequence, disruption, and expression of its structural gene HEM15; Labbe-Bois R; The HEM15 gene in Saccharomyces cerevisiae encodes ferrochelatase (EC 4.99.1.1, protoheme ferrolyase), a mitochondrial inner membrane-bound enzyme which catalyzes the insertion of ferrous ion into protoporphyrin IX, the last step in protoheme biosynthesis . The gene was isolated by functional complementation of a hem15 mutant . Sequence analysis of a 2.9-kilobase genomic DNA fragment revealed an open reading frame of 1179 nucleotides, plus a gene coding for a tRNA(Val)(GUU) and delta elements downstream from the 3'-end of HEM15 . The open reading frame encodes a precursor form of the protein containing a 31-amino acid presequence . The mature enzyme contains 362 amino acid residues; its calculated molecular weight (40,900) and predicted amino-terminal sequence agree with those determined from the purified protein . It is relatively abundant in lysine (9%) and contains no apparent transmembrane segment . Disruption of the HEM15 gene led to non-viable cells in certain genetic background . Northern (RNA) analysis showed a slight (1.5-2-fold) repression of HEM15 expression by glucose.

J Biol Chem, 1990 May 5, 265(13), 7273 - 7
Mitochondrial import of cytochrome c oxidase subunit VIIa in Saccharomyces cerevisiae . Identification of sequences required for mitochondrial localization in vivo; Duhl DM et al.; Subunit VIIa of yeast cytochrome c oxidase is a small (59 amino acids) protein of the inner mitochondrial membrane that lacks a cleavable amino-terminal presequence . To identify regions within this polypeptide that are essential for its import, gene fusions were constructed using a leader peptide substitution vector (pLPS) developed in this laboratory (Glaser, S . M., Trueblood, C . E., Dircks, L . K., Poyton, R . O., and Cumsky, M . G . (1988) J . Cell . Biochem . 36, 275-287) . In this vector, oligonucleotide sequences encoding all or part of subunit VIIa were fused in-frame with the coding region of mature cytochrome c oxidase subunit Va . The plasmid pLPS is ideal for assaying protein sequences for their ability to direct mitochondrial import in vivo since subunit Va's leader peptide is essential for import and because subunit V is required for cytochrome c oxidase activity and respiration . Strains containing these fusions but lacking both subunit V genes (COX5a and COX5b) were analyzed to determine whether the chimeric protein is directed to mitochondria . Our findings indicate that the amino-terminal 17 amino acids of subunit VIIa are sufficient to localize subunit Va to the mitochondrion and that a 6-amino acid-long region within the amino terminus (Gly8-Arg13) is essential . In addition, some import (approximately 10% of wild type) is observed with the highly charged carboxyl terminus of subunit VIIa, suggesting that the subunit may contain redundancy in its import information.

J Biol Chem, 1990 May 5, 265(13), 7440 - 8
Biogenesis of vacuolar membrane glycoproteins of yeast Saccharomyces cerevisiae; Nishikawa S et al.; To investigate the biogenesis of the yeast vacuole, we have sought novel marker proteins localized to the vacuolar membrane . Glycoproteins were prepared from vacuolar membrane vesicles by concanavalin A-Sepharose column chromatography and used to raise monoclonal antibodies . The antibodies obtained recognize several vacuolar proteins that have N-linked oligosaccharide chains . A set of the antibodies reacts with a vacuolar glycoprotein with a major molecular species of 72 kDa (vgp72), which appears to associate peripherally with the vacuolar membrane . The biosynthesis of vgp72 has been examined in detail by pulse-chase experiments and by analyses using various secretory mutants (sec18, sec7, and sec1) and a vacuolar protease mutant (pep4) . vgp72 first appears in the endoplasmic reticulum as a 74-kDa species and is quickly modified in the Golgi apparatus to two distinct species: a 79-kDa form, and a heterogeneously glycosylated form (90-150 kDa) . Subsequently, both species are proteolytically processed in the vacuole giving rise to a 72-kDa species as well as heavily glycosylated form . Thus, the biogenesis of vgp72 utilizes the early part of the secretory pathway as is the case of vacuolar soluble enzymes . A unique feature is that two species that are different in the extent of glycosylation appear to follow the same destination to the vacuolar membrane.

J Biol Chem, 1990 May 5, 265(13), 7413 - 8
A glucose-repressible gene encodes acetyl-CoA hydrolase from Saccharomyces cerevisiae; Lee FJ et al.; Acetyl-CoA hydrolase, catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating the intracellular acetyl-CoA pool . Recently, a yeast acetyl-CoA hydrolase was purified to homogeneity from Saccharomyces cerevisiae and partially characterized (Lee, F.-J . S., Lin, L.-W., and Smith, J . A . (1989) Eur . J . Biochem . 184, 21-28) . In order to study the biological function and regulation of the acetyl-CoA hydrolase, we cloned and sequenced the full length cDNA encoding yeast acetyl-CoA hydrolase . RNA blot analysis indicates that acetyl-CoA hydrolase is encoded by a 2.5-kilobase mRNA . DNA blot analyses of genomic and chromosomal DNA reveal that the gene (so-called ACH1, acetyl-CoA hydrolase) is present as a single copy located on chromosome II . Acetyl-CoA hydrolase is established to be a mannose-containing glycoprotein, which binds concanavalin A . By measuring the levels of ACH1 mRNA and acetyl-CoA hydrolase activity in different growth phases and by examining the effects of various carbon sources, we have demonstrated that ACH1 expression is repressed by glucose.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1990 May, 23(2), 166 - 80
Isolation and characterization of temperature-sensitive mutants of Saccharomyces cerevisiae; Yang SS et al.; Temperature-sensitive (ts) mutants of Saccharomyces cerevisiae Y-196 were isolated by UV irradiation and/by EMS treatment . There were 163 ts mutants isolated from 66,957 colonies . These mutants were designated as AMY-1 to AMY-163 . These ts mutants grew normally at 30 degrees C, but did not grow at 38 degrees C . In an auxanographic study, we showed that AMY-46 was an absolute ts mutant, while AMY-10, AMY-15, AMY-16, AMY-49, AMY-66, AMY-88, AMY-114, AMY-126, AMY-127, AMY-129, AMY-136, AMY-150, AMY-157, AMY-158, AMY-159, AMY-161, and AMY-162 were auxotrophic ts mutants except AMY-16 had cell size 2-42% larger than that of the wild type . Cell aggregation phenomenon was found when the ts mutants were cultivated at 30 degrees C to stationary phase then cultured at 38 degrees C for 4 hr . The budding numbers at 30 degrees C were less than that at 38 degrees C . Mutant AMY-49 had the highest budding number . There was a positive correlation between the growth rate and the average budding number . However, there was a negative correlation between the growth rate at 30 degrees C and the length of long axis of cell or the size of cell.

Prikl Biokhim Mikrobiol, 1990 May-Jun, 26(3), 360 - 3
{Sterol level in Saccharomyces cerevisiae mutants with altered ergosterol biosynthesis}; Mikhailova NP et al.; The sterol content in Saccharomyces cerevisiae mutants defective in the synthesis of cyclic ergosterol precursors has been studied . It was found that strains with mutational blocks involving the stages of zymosterol side chain methylation at C24 and delta 8----delta 7 isomerization accumulated twice more sterols as compared to parent strains . Regulation of the ergosterol biosynthesis is discussed.

Mol Gen Mikrobiol Virusol, 1990 May, (5), 27 - 9
{Mutational analysis of the starch utilization system in the yeast Saccharomyces cerevisiae}; Kuchin SV et al.; Seven mutants of Saccharomyces cerevisiae deficient in production of extracellular glucoamylase have been analyzed . For each of the seven a monogenic pattern of inheriting the mutant phenotype has been observed . The mutations have been shown to map within five different genetic loci, three independent mutations affecting the STA2 locus and the other four residing in four formerly unidentified genes . As expected, the sta2 mutants recover the wild phenotype when transformed with a STA2-bearing multicopy plasmid . Such reversion has also been observed for the transformed stall mutant . Unlike the others, the sta16 mutant is unable to secrete heterologous alpha-amylase encoded by a plasmid-borne DNA fragment . All the mutants have a moderately reduced ability to secrete the invertase and acid phosphatase.

Mol Gen Mikrobiol Virusol, 1990 May, (5), 20 - 4
{Synthesis and secretion of recombinant protein--the product of v-sis oncogene in Saccharomyces cerevisiae cells}; Ratovitskii EA et al.; The recombinant plasmid DNA YEp secl-v-sis was constructed . This plasmid was able to code for the synthesis and secretion into the cultural medium of the protein-product of oncogene v-sis . Transcription, copy number and stability of the plasmid DNA were studied under the conditions of galactose induction . The v-sis protein was determined by gel electrophoresis and immunoblotting methods and assayed for cell-proliferation activity in the mammalian cell culture.

Mol Gen Genet, 1990 May, 221(3), 353 - 7
Repair of alkylation damage in Saccharomyces cerevisiae; Goth-Goldstein R et al.; Repair of methylated bases in Saccharomyces cerevisiae was measured by two methods: in vitro in cell extracts, and in vivo, by determining the loss of methylated bases from yeast DNA after treatment of stationary cultures with {3H}-N-methyl-N'-nitro-N-nitrosoguanidine . Whereas no repair activity could be detected by the in vitro method, the methylated bases were removed in vivo very efficiently . These contradictory results of in vitro and in vivo repair measurements suggest that either the repair enzymes of yeast are sufficiently different from those of bacteria and mammalian cells that they are not active in the in vitro assay, or that methylated bases are repaired in yeast by a different pathway.

FEMS Microbiol Lett, 1990 May, 57(1-2), 153 - 7
Biochemical and genetic analysis of an alpha-mannosidase mutant from Saccharomyces cerevisiae; Cueva R et al.; A yeast mutant lacking non-specific alpha-mannosidase activity was found as a background marker during our search for dap2 mutants (Suarez-Rendueles, P . and Wolf, D . H . (1987) J . Bacteriol . 169, 4041-4048) . The mutant (DPS-15) is characterized in detail . The mutation called amd1 segregates 2:2 in meiotic tetrads, indicating a single chromosomal gene mutation which is recessive . Diploids heterozygous for amd1 show gene dosage . Thus, it appears that AMD1 might be the structural gene for alpha-mannosidase . Results obtained with this mutant show that alpha-mannosidase is not a vital component of the vegetative cell cycle . The differentiation process of sporulation is not disturbed in homozygous mutant diploids . Mannose turnover does not seem to be altered in mutant cells.

Yeast, 1990 May-Jun, 6(3), 263 - 70
GABA transport in Saccharomyces cerevisiae; McKelvey J et al.; Gamma-aminobutyrate (GABA) accumulation in growing cultures of Saccharomyces cerevisiae was shown to occur by means of an active transport system that is inhibited by proton ionophores, azide, fluoride and arsenate ions . Transport occurred maximally at pH 5.0 and exhibited apparent Km values of 12 microM and 0.1 mM . Accumulated GABA did not efflux upon treatment with proton ionophores and exchanged with extracellular material only very slowly . However, release was complete upon treatment with nystatin . These observations raise the possibility that a major portion of intracellular GABA is sequestered in the vacuole . The response of GABA uptake to growth on various nitrogen sources suggested that uptake may be subject to several types of regulation.

Yeast, 1990 May-Jun, 6(3), 221 - 9
Bacterial plasmid pBR322 sequences serve as upstream activating sequences in Saccharomyces cerevisiae; Sidhu RS et al.; The expression of acid phosphatase (APase) from PHO5 and MF alpha-PHO5 hybrid genes is regulated by inorganic phosphate and mating type locus respectively, as well as the PHO4 and MAT alpha 1 gene products respectively . When PHO5 and MF alpha-PHO5 hybrid genes were cloned in the BamHI site of the pBR322 sequence of the yeast shuttle vectors (YRp7 or YEp9T), in one orientation they were regulated normally but in the other orientation their expression was not regulated but expressed constitutively . The pBR322 sequences present upstream of the inserted genes are responsible for the constitutive expression . By replacing the PHO5 upstream activating sequences (UAS) element with pBR322 fragments, we have identified three pBR322 sequences, from nucleotides 376 to 650, 2068 to 2116 and 2136 to 2247, which were able to promote expression of APase . A comparison of these three pBR322 fragments revealed 5' ATCGCGCGAG 3' and 5' CGGTGATGNCGG 3' to be the common sequences likely to act as UASs in Saccharomyces cerevisiae . By using synthetic oligonucleotides, it was found that both sequences are required for maximum expression of APase activity.

Yeast, 1990 May-Jun, 6(3), 205 - 12
Cell fusion of Saccharomyces cerevisiae fragile mutants; Philipova DH et al.; Fragile mutants of Saccharomyces cerevisiae are defective in the structure of the cell wall and plasma membrane . The mutant cells lyse in hypotonic solutions but grow exponentially when osmotic stabilizer is included in the medium . These mutants display a general increase in the permeability of the plasma membrane . We show here that fragile yeast cells of the same mating type can fuse without protoplast formation . The frequency of cell x cell fusion is lower than that observed for protoplast x protoplast fusion and can be significantly increased if the cells of one partner are converted to protoplasts . Microscopic observations and genetic analysis demonstrate that the hybrids obtained are fusion products . The fusion between fragile cells is explained in terms of the existence of local defects on their surface where the cell wall is thinner (or even missing), thus allowing a direct contact of cells by means of their plasma membranes.

Arch Biochem Biophys, 1990 May 1, 278(2), 467 - 74
Purification and characterization of human plasminogen activator inhibitor type I expressed in Saccharomyces cerevisiae; Gardell SJ et al.; The rapidly acting inhibitor of plasminogen activators, PAI-1, was produced intracellularly in Saccharomyces cerevisiae by using the ADH2 promoter to drive the expression of the human PAI-1 cDNA . Approximately 8 mg of human PAI-1 was produced per liter of confluent yeast culture . A purification scheme which resulted in 20% recovery of isolated PAI-1 from the broken yeast cell homogenate was devised . Yeast-derived human PAI-1 differs from endothelial-type PAI-1 isolated from HT1080 fibrosarcoma cells in that the recombinant inhibitor does not contain carbohydrate side chains . Nevertheless, the activity and other functional attributes of yeast-derived PAI-1 are similar to those exhibited by HT1080 fibrosarcoma cell-derived PAI-1 . Hence, this study demonstrates that expression of human PAI-1 in yeast is a viable strategy for the production of ample quantities of this key modulator of plasminogen activator-mediated proteolysis.

Mol Cell Biol, 1990 May, 10(5), 2437 - 41
The ABF1 factor is the transcriptional activator of the L2 ribosomal protein genes in Saccharomyces cerevisiae; Della Seta F et al.; The same factor, ABF1, binds to the promoters of the two gene copies (L2A and L2B) coding for the ribosomal protein L2 in Saccharomyces cerevisiae . In vitro binding experiments and in vivo functional analysis showed that the different affinities of the L2A and L2B promoters for the ABF1 factor are responsible for the differential transcriptional activities of the two gene copies . The presence of ABF1-binding sites in front of many housekeeping genes suggests a general role for ABF1 in the regulation of gene activity.

Mol Cell Biol, 1990 May, 10(5), 2308 - 14
Null alleles of SAC7 suppress temperature-sensitive actin mutations in Saccharomyces cerevisiae; Dunn TM et al.; Extragenic suppressors of a new temperature-sensitive mutation (act1-4) in the actin gene of Saccharomyces cerevisiae were isolated in an attempt to identify genes whose products interact directly with actin . One suppressor with a cold-sensitive growth phenotype defined the new gene, SAC7, which was mapped, cloned, sequenced, and disrupted . Genetic analysis of strains that are disrupted for SAC7 demonstrated that the protein is required for normal growth and actin assembly at low temperatures . Surprisingly, null mutations in SAC7 also suppressed the temperature-sensitive growth defect caused by the act1-1 and act1-4 mutations, whereas they were lethal in combination with the temperature-sensitive allele act1-2 . These results support the notion that the SAC7 gene product is involved in the normal assembly or function or both of actin.

Mol Cell Biol, 1990 May, 10(5), 2269 - 84
Identification and comparison of stable and unstable mRNAs in Saccharomyces cerevisiae; Herrick D et al.; We developed a procedure to measure mRNA decay rates in the yeast Saccharomyces cerevisiae and applied it to the determination of half-lives for 20 mRNAs encoded by well-characterized genes . The procedure utilizes Northern (RNA) or dot blotting to quantitate the levels of individual mRNAs after thermal inactivation of RNA polymerase II in an rpb1-1 temperature-sensitive mutant . We compared the results of this procedure with results obtained by two other procedures (approach to steady-state labeling and inhibition of transcription with Thiolutin) and also evaluated whether heat shock alter mRNA decay rates . We found that there are no significant differences in the mRNA decay rates measured in heat-shocked and non-heat-shocked cells and that, for most mRNAs, different procedures yield comparable relative decay rates . Of the 20 mRNAs studied, 11, including those encoded by HIS3, STE2, STE3, and MAT alpha 1, were unstable (t1/2 less than 7 min) and 4, including those encoded by ACT1 and PGK1, were stable (t1/2 greater than 25 min) . We have begun to assess the basis and significance of such differences in the decay rates of these two classes of mRNA . Our results indicate that (i) stable and unstable mRNAs do not differ significantly in their poly(A) metabolism; (ii) deadenylation does not destabilize stable mRNAs; (iii) there is no correlation between mRNA decay rate and mRNA size; (iv) the degradation of both stable and unstable mRNAs depends on concomitant translational elongation; and (v) the percentage of rare codons present in most unstable mRNAs is significantly higher than in stable mRNAs.

Mol Cell Biol, 1990 May, 10(5), 2224 - 36
Functional domains of a positive regulatory protein, PHO4, for transcriptional control of the phosphatase regulon in Saccharomyces cerevisiae; Ogawa N et al.; The PHO4 gene encodes a positive regulatory factor involved in regulating transcription of various genes in the phosphatase regulon of Saccharomyces cerevisiae . Besides its own coding region, the 1.8-kilobase PHO4 transcript contains a coding region for a mitochondrial protein which does not appear to be translated . Four functional domains were found in the PHO4 protein, which consists of 312 amino acid (aa) residues as deduced from the open reading frame of PHO4 . A gel retardation assay with beta-galactosidase::PHO4 fused protein revealed that the 85-aa C terminus is the domain responsible for binding to the promoter DNA of PHO5, a gene under the control of PHO4 . This region has similarities with the amphipathic helix-loop-helix motif of c-myc protein . Determination of the nucleotide sequences of four PHO4c mutant alleles and insertion and deletion analyses of PHO4 DNA indicated that a region from aa 163 to 202 is involved in interaction with a negative regulatory factor PHO80 . Complementation of a pho4 null allele with the modified PHO4 DNAs suggested that the N-terminal region (1 to 109 aa), which is rich in acidic aa, is the transcriptional activation domain . The deleterious effects of various PHO4 mutations on the constitutive transcription of PHO5 in PHO4c mutant cells suggested that the region from aa 203 to 227 is involved in oligomerization of the PHO4 protein.

Mol Cell Biol, 1990 May, 10(5), 2214 - 23
The SLP1 gene of Saccharomyces cerevisiae is essential for vacuolar morphogenesis and function; Wada Y et al.; The SLP1 gene, which is involved in the expression of vacuolar functions in the yeast Saccharomyces cerevisiae (K . Kitamoto, K . Yoshizawa, Y . Ohsumi, and Y . Anraku, J . Bacteriol . 170:2687-2691, 1988), has been cloned from a yeast genomic library by complementation of the slp1-1 mutation . The isolated plasmid has a 7.8-kilobase BamHI-BamHI fragment that is sufficient to complement several characteristic phenotypes of the slp1-1 mutation . The fragment was integrated at the chromosomal SLP1 locus, indicating that it contains an authentic SLP1 gene . By DNA sequencing of the SLP1 gene, an open reading frame of 2,073 base pairs coding for a polypeptide of 691 amino acid residues (Mr, 79,270) was found . Gene disruption of the chromosomal SLP1 did not cause a lethal event . Vacuolar proteins in the delta slp1 mutant are not processed to vacuolar forms but remain in Golgi-modified forms . Carboxypeptidase Y in the delta slp1 mutant is localized mainly to the outsides of the cells . delta slp1 mutant cells have no prominent vacuolar structures but contain numerous vesicles in the cytoplasm, as seen by electron microscopy . Genetic and molecular biological analyses revealed that SLP1 is identical to VPS33, which is required for vacuolar protein sorting as reported by Robinson et al . (J . S . Robinson, D . J . Klionsky, L . M . Banta, and S . D . Emr, Mol . Cell . Biol . 8:4936-4948, 1988) . These results indicate that the SLP1 (VPS33) gene is involved in the sorting of vacuolar proteins from the Golgi apparatus and their targeting to the vacuole and that it is required for the morphogenesis of vacuoles and subsequent expression of vacuolar functions.

Mol Cell Biol, 1990 May, 10(5), 2202 - 13
Courtship in Saccharomyces cerevisiae: an early cell-cell interaction during mating; Jackson CL et al.; During conjugation in Saccharomyces cerevisiae, two cells of opposite mating type (MATa and MAT alpha) fuse to form a diploid zygote . Conjugation requires that each cell locate an appropriate mating partner . To investigate how yeast cells select a mating partner, we developed a competition mating assay in which wild-type MAT alpha cells have a choice of two MATa cell mating partners . We first demonstrated that sterile MAT alpha 1 cells (expressing no a- or alpha-specific gene products) do not compete with fertile MATa cells in the assay; hence, wild-type MATa and MAT alpha cells can efficiently locate an appropriate mating partner . Second, we showed that a MATa strain need not be fertile to compete with a fertile MATa strain in the assay . This result defines an early step in conjugation, which we term courtship . We showed that the ability to agglutinate is not necessary in MATa cells for courtship but that production of a-pheromone and response to alpha-pheromone are necessary . Thus, MATa cells must not only transmit but must also receive and then respond to information for effective courtship; hence, there is a "conversation" between the courting cells . We showed that the only alpha-pheromone-induced response necessary in MATa cells for courtship is production of a-pheromone . In all cases tested, a strain producing a higher level of a-pheromone was more proficient in courtship than one producing a lower level . We propose that during courtship, a MAT alpha cell selects the adjacent MATa cell producing the highest level of a-pheromone.

Mol Cell Biol, 1990 May, 10(5), 2182 - 90
Disruption of single-copy genes encoding acidic ribosomal proteins in Saccharomyces cerevisiae; Remacha M et al.; Using the cloned genes coding for the ribosomal acidic proteins L44 and L45, constructions were made which deleted part of the coding sequence and inserted a DNA fragment at that site carrying either the URA3 or HIS3 gene . By gene disruption techniques with linearized DNA from these constructions, strains of Saccharomyces cerevisiae were obtained which lacked a functional gene for either protein L44 or protein L45 . The disrupted genes in the transformants were characterized by Southern blots . The absence of the proteins was verified by electrofocusing and immunological techniques, but a compensating increase of the other acidic ribosomal proteins was not detected . The mutant lacking L44 grew at a rate identical to the parental strain in complex as well as in minimal medium . The L45-disrupted strain also grew well in both media but at a slower rate than the parental culture . A diploid strain was obtained by crossing both transformants, and by tetrad analysis it was shown that the double transformant lacking both genes is not viable . These results indicated that proteins L44 and L45 are independently dispensable for cell growth and that the ribosome is functional in the absence of either of them.

Mol Cell Biol, 1990 May, 10(5), 2176 - 81
Isolation of mutant Saccharomyces cerevisiae strains that survive without sphingolipids; Dickson RC et al.; Sphingolipids comprise a large, widespread family of complex eucaryotic-membrane constituents of poorly defined function . The yeast Saccharomyces cerevisiae is particularly suited for studies of sphingolipid function because it contains a small number of sphingolipids and is amenable to molecular genetic analysis . Moreover, it is the only eucaryote in which mutants blocked in sphingolipid biosynthesis have been isolated . Beginning with a nonreverting sphingolipid-defective strain that requires the addition of the long-chain-base component of sphingolipids to the culture medium for growth, we isolated two strains carrying secondary, suppressor mutations that permit survival in the absence of exogenous long-chain base . Remarkably, the suppressor strains made little if any sphingolipid . A study of how the suppressor gene products compensate for the lack of sphingolipids may reveal the function(s) of these membrane lipids in yeast cells.

Mol Cell Biol, 1990 May, 10(5), 2104 - 10
Positive control of sporulation-specific genes by the IME1 and IME2 products in Saccharomyces cerevisiae; Mitchell AP et al.; In the yeast Saccharomyces cerevisiae, meiosis and spore formation require the induction of sporulation-specific genes . Two genes are thought to activate the sporulation program: IME1 and IME2 (inducer of meiosis) . Both genes are induced upon entry into meiosis, and IME1 is required for IME2 expression . We report here that IME1 is essential for expression of four sporulation-specific genes . In contrast, IME2 is not absolutely essential for expression of the sporulation-specific genes, but contributes to their rapid induction . Expression of IME2 from a heterologous promoter permits the expression of these sporulation-specific genes, meiotic recombination, and spore formation in the absence of IME1 . We propose that the IME1 and IME2 products can each activate sporulation-specific genes independently . In addition, the IME1 product stimulates sporulation-specific gene expression indirectly through activation of IME2 expression.

J Pharmacobiodyn, 1990 May, 13(5), 316 - 20
Singlet oxygen production and photobiological effects of pinacyanol chloride on yeast Saccharomyces cerevisiae; Iwamoto Y et al.; Photobiological activities of pinacyanol chloride (PC), which is known as a non-intercalating dye, were investigated . Irradiation of PC-sensitized yeast cells in the dark brought about marked decrease of survival and induction of "petites" which are respiration-deficient mutants caused by partial loss of mitochondrial deoxyribonucleic acid . Nuclear mutation represented by reversion from Trp- to Trp+ was also induced by photodynamic action of PC . This fact suggested photoactive dyes are not necessarily intercalated for inducing mitochondrial and nuclear mutation . Singlet oxygen production was determined in the photoirradiated PC solution by electron spin resonance spectrometry . Photobiological effects of PC might be brought about mainly by a type II photodynamic mechanism.

Mol Cell Biol, 1990 May, 10(5), 2367 - 78
SAS1 and SAS2, GTP-binding protein genes in Dictyostelium discoideum with sequence similarities to essential genes in Saccharomyces cerevisiae; Saxe SA et al.; We have identified two novel, very closely related genes, SAS1 and SAS2, from Dictyostelium discoideum . These encode small, approximately 20-kilodaton proteins with amino acid sequences thought to be involved in interaction with guanine nucleotides . The protein sizes, spacings of GTP-binding domains, and carboxyl-terminal sequences suggest their relationship to the ubiquitous ras-type proteins . Their sequences, however, are sufficiently different to indicate that they are not true ras proteins . More extensive sequence identity (approximately 55%) is shared with the YPT1 and SEC4 proteins from Saccharomyces cerevisiae . These yeast proteins are essential for growth and are believed to be involved in intracellular signaling associated with membrane function . SAS1 and SAS2 exhibit distinct patterns of genomic organization and developmentally regulated gene expression . SAS1 contains introns and is associated with a developmentally regulated repetitive element . SAS2 is colinear with its mRNA and does not appear to be closely linked with this repetitive element . Both genes are expressed during growth and throughout development . SAS1 is maximally expressed during cytodifferentiation, when two sizes of SAS1 mRNA are detectable . SAS2 mRNA levels are maximal during culmination . On the basis of the expression patterns of the SAS genes and their relationship to the YPT1 and SEC4 genes, we discuss possible functions of the SAS proteins.

Int J Radiat Biol, 1990 May, 57(5), 981 - 92
Identification of proteins whose synthesis in Saccharomyces cerevisiae is induced by DNA damage and heat shock; Gailit J; Protein synthesis in Saccharomyces cerevisiae after exposure to ultraviolet light (UV) was examined by two-dimensional gel electrophoresis of pulse-labelled proteins . The synthesis of 12 distinct proteins was induced by treatment with UV doses of 10-200 J/m2 . The induced proteins differed in the minimum dose necessary for induction, the maximum dose at which induction still occurred and the constitutive level present in unirradiated cells . A chemical mutagen, 4-nitroquinoline-1-oxide, induced synthesis of the same proteins . Induction after UV treatment was observed in seven different yeast strains, including three mutants deficient in DNA repair . Synthesis of five of the proteins was also induced by brief heat shock treatment . These five proteins may be members of a family of proteins whose synthesis is regulated by two different pathways responding to different types of stress.

J Biol Chem, 1990 Apr 25, 265(12), 6726 - 33
Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae; Hirata R et al.; Subunit a of the vacuolar membrane H(+)-translocating adenosine triphosphatase of the yeast Saccharomyces cerevisiae contains a catalytic site for ATP hydrolysis . N-terminal sequences of six tryptic peptides of the subunit were determined . Based on the peptide sequence information, a 39-base oligonucleotide probe was synthesized, and the gene encoding the subunit (VMA1) was isolated from a genomic DNA library by hybridization . The nucleotide sequence of the gene predicts a polypeptide of 1,071 amino acids with a calculated molecular mass of 118,635 daltons, which is much larger than the value 67 kDa estimated on sodium dodecyl sulfate-polyacrylamide gels . N- and C-terminal regions of the deduced sequence (residues 1-284 and 739-1,071) are very similar to those of the catalytic subunits of carrot (69 kDa) and Neurospora crassa (67 kDa) vacuolar membrane H(+)-ATPases (62 and 73% identity over 600 residues, respectively) . The homologous regions also show about 25% sequence identity over 400 residues with beta-subunits of F0F1-ATPases . In contrast, the internal region containing 454 amino acid residues (residues 285-738) shows no detectable sequence similarities to any known ATPase subunits and instead is similar to a yeast endonuclease encoded by the HO gene . None of the six tryptic peptides is located in this internal region . Northern blotting analysis detected a single mRNA of 3.5 kilobases, indicating that the gene has no introns . Although the reason for the discrepancy in molecular mass is unclear at present, these results suggest that a novel processing mechanism, which might involve a post-translational excision of the internal region followed by peptide ligation, operates on the yeast VMA1 product . The VMA1 gene has proven to be the same gene as the TFP1 gene (Shih, C.-K., Wagner, R., Feinstein, S., Kanik-Ennulat, C., and Neff, N . (1988) Mol . Cell . Biol . 8, 3094-3103) whose dominant mutant allele (TFP1-408) confers a dominant trifluoperazine resistance and Ca2(+)-sensitive growth . This and our findings suggest that the vacuolar membrane H(+)-ATPase participates in maintenance of cytoplasmic Ca2+ homeostasis.

Biochim Biophys Acta, 1990 Apr 19, 1038(2), 253 - 9
Iron-reductases in the yeast Saccharomyces cerevisiae; Lesuisse E et al.; Several NAD(P)H-dependent ferri-reductase activities were detected in sub-cellular extracts of the yeast Saccharomyces cerevisiae . Some were induced in cells grown under iron-deficient conditions . At least two cytosolic iron-reducing enzymes having different substrate specificities could contribute to iron assimilation in vivo . One enzyme was purified to homogeneity: it is a flavoprotein (FAD) of 40 kDa that uses NADPH as electron donor and Fe(III)-EDTA as artificial electron acceptor . Isolated mitochondria reduced a variety of ferric chelates, probably via an 'external' NADH dehydrogenase, but not the siderophore ferrioxamine B . A plasma membrane-bound ferri-reductase system functioning with NADPH as electron donor and FMN as prosthetic group was purified 100-fold from isolated plasma membranes . This system may be involved in the reductive uptake of iron in vivo.

Gene, 1990 Apr 16, 88(2), 297 - 301
Secretion of N-glycosylated human recombinant interleukin-1 alpha in Saccharomyces cerevisiae; Livi GP et al.; We have expressed fragments of the cDNA coding for mature human interleukin-1 alpha (hIL-1 alpha) in Saccharomyces cerevisiae . Mature hIL-1 alpha contains one potential N-linked glycosylation site that is not recognized in mammalian cells . Translational fusions to either one of three yeast signal sequences resulted in secretion of bioactive, N-glycosylated hIL-1 alpha . The extent of glycosylation was significantly reduced using the alpha-factor signal sequence, which itself contains three N-linked glycosylation sites known to be core glycosylated . N-glycosylation has no effect on biological specific activity.

Gene, 1990 Apr 16, 88(2), 149 - 57
The FUR1 gene of Saccharomyces cerevisiae: cloning, structure and expression of wild-type and mutant alleles; Kern L et al.; The FUR1 gene of Saccharomyces cerevisiae encodes uracil phosphoribosyltransferase (UPRTase) which catalyses the conversion of uracil into uridine 5'-monophosphate (UMP) in the pyrimidine salvage pathway . The FUR1 gene is included in a 2.1 kb genomic segment of DNA and is transcribed into a 1 kb poly(A)+mRNA . Sequencing has determined a 753 bp open reading frame capable of encoding a protein of 251 amino acids . The FUR1 genes for three recessive fur1 alleles, having different sensibilities to 5-fluorouridine (5-FUR) but identical levels of resistance to 5-fluorouracil (5-FU), were cloned and sequenced . Single bp changes located in different regions of the gene were found in each mutant . Two in vitro-constructed deletions of the FUR1 gene have been integrated at the chromosomal locus, giving strains with 5-FURR and 5-FURR mutant phenotype . Assays of UPRTase, uridine kinase, uridine ribohydrolase and uridine 5'-monophosphate nucleotidase enzymatic activities, in extracts of strains where the FUR1 gene is overexpressed or deleted, indicate that the FUR1 encoded protein possesses only UPRTase activity.

Gene, 1990 Apr 16, 88(2), 253 - 7
Isolation of the ERG12 gene of Saccharomyces cerevisiae encoding mevalonate kinase; Oulmouden A et al.; The ERG12 gene of Saccharomyces cerevisiae has been cloned by complementation of an erg12-1 mutation affecting mevalonate kinase . From the 2.8-kb insert isolated, the functional gene has been localized on a DNA fragment of 2.1 kb . The mRNA is 1.45 kb long . Gene disruption shows that the ERG12 gene is essential in yeast, both for spore germination and vegetative growth.

Nucleic Acids Res, 1990 Apr 11, 18(7), 1783 - 7
Properties of the transcriptional enhancer in Saccharomyces cerevisiae telomeres; Runge KW et al.; Saccharomyces cerevisiae chromosomes end with the sequence C2-3A(CA)1-4, commonly abbreviated as C1-3A . These sequences can function as upstream activators of transcription (UAS's) when placed in front of a CYC1-lacZ fusion gene . When C1-3A sequences are placed between the GAL1,10 UAS and the CYC1-lacZ fusion, the C1-3A UAS still functions and the amount of beta-galactosidase produced in cells grown on glucose is as much or more than that for cells grown on either glycerol medium, or cells grown on glucose medium containing a plasmid with just the C1-3A UAS . These data indicate that the UAS is immune from glucose repression from the upstream GAL1,10 UAS . Because C1-3A sequences are bound in vitro by the transcription factor RAP1, the UAS activity of yeast telomere sequences was compared with that of a similar UAS from the tightly regulated ribosomal protein gene RP39A, which also contains a RAP1 binding site . While transcription from the ribosomal protein gene UAS was responsive to cell density, the amount of transcription from the C1-3A UAS was nearly the same at all cell densities tested . These data show that the transcriptional activation by C1-3A sequences is not regulated by cell density.

FEBS Lett, 1990 Apr 9, 263(1), 93 - 8
Isolation and RNA sequence analysis of cytochrome b mutants resistant to funiculosin, a center i inhibitor of the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae; di Rago JP et al.; Funiculosin is a well-known inhibitor of the mitochondrial respiratory chain, probably acting at the ubiquinone reducing site or center i of QH2-cytochrome c reductase . We report here the isolation, mapping and RNA sequence analysis of yeast apo-cytochrome b mutants resistant to this inhibitor . Funiculosin-resistance was found to be conferred, in 4 independent isolates, upon replacement of a leucine residue by phenylalanine in position 198 of the cytochrome b polypeptide chain.

J Gen Microbiol, 1990 Apr, 136 ( Pt 4), 733 - 7
The relationship of growth rate and catabolite repression with WHI2 expression and cell size in Saccharomyces cerevisiae; Mountain HA et al.; Mixtures of D-glucosamine and glucose were used to slow the growth of wild-type and whi2 mutant strains of Saccharomyces cerevisiae without affecting the level of catabolite repression . The following observations were made . Firstly, mutant cells were found to be partially resistant to the inhibitory effect of glucosamine . Secondly, slow growth induced by glucosamine resulted in cells becoming larger, in direct contrast to the effect of slowing growth by glucose limitation in a chemostat or by carbon source substitution . It is concluded that the level of repression/derepression, rather than absolute growth rate, is responsible for controlling cell size . Thirdly, when WHI2 transcript levels were measured it was found that expression was correlated with growth rate rather than the level of repression . These results are interpreted in terms of a model which envisages that the WHI2 product acts as a negative regulator of catabolite repression . A test of this model is reported: it is shown that mutant cells respired more actively in the presence of glucose and grew more rapidly on glycerol, whereas overexpression of WHI2 from multicopy plasmids prevented growth on glycerol and depressed respiration.

J Gen Microbiol, 1990 Apr, 136 ( Pt 4), 727 - 32
Regulation of the Saccharomyces cerevisiae WHI2 gene; Mountain HA et al.; WHI2 mRNA levels were followed through the growth cycle in WHI2 mutant and wild-type cells of Saccharomyces cerevisiae . Levels were high during the first (glucose) phase of growth, and were reduced sharply during the second (ethanol) phase of growth . Transcript levels of the glycolytic genes PDC1 and PYK1 were also measured; they each showed a pattern similar to that of WHI2, whereas transcript levels of the CDC7 gene remained constant throughout the cycle, showing that a decrease in transcription is not a general feature of genes . These results make it unlikely that the WHI2 product acts as an inhibitor of cell proliferation which is activated upon carbon starvation . No difference was observed between the pattern of expression of mutant and wild-type strains, showing that the mutant phenotype was not the result of a change in regulation at the transcriptional level.

Mol Gen Genet, 1990 Apr, 221(2), 176 - 86
Initiation of meiosis and sporulation in Saccharomyces cerevisiae requires a novel protein kinase homologue; Yoshida M et al.; SME1 was cloned due to its high copy number effect: it enabled MATa/MAT alpha diploid cells to undergo meiosis and sporulation in a vegetative medium . Disruption of SME1 resulted in a recessive Spo- phenotype . These results suggest that SME1 is a positive regulator for meiosis . DNA sequencing analysis revealed an open reading frame of 645 amino acids . An amino terminal peptide of ca 400 amino acids in the deduced protein was similar to known protein kinases . Transcription of SME1 was regulated negatively by nitrogen and glucose and positively by MATa/MAT alpha and IME1, another positive regulator gene of meiosis . By complementation analysis, SME1 was found to be identical to IME2, which had been shown to be important in meiosis . These results suggest that IME1 product stimulates meiosis by activating transcription of SME1 (IME2) and that protein phosphorylation is required for initiation of meiosis.

Mol Microbiol, 1990 Apr, 4(4), 585 - 96
The purine-cytosine permease gene of Saccharomyces cerevisiae: primary structure and deduced protein sequence of the FCY2 gene product; Weber E et al.; A 2.1 kb DNA segment carrying the purine-cytosine permease gene (FCY2) of Saccharomyces cerevisiae was sequenced, the primary structure of the protein (533 amino acids) deduced and a folding pattern in the membrane is proposed for the permease protein . Expression of the FCY2 gene product requires a functional secretory pathway and is reduced in mnn9, a mutant strain deficient in outer chain glycosylation . The FCY2 gene was mapped on the right arm of chromosome V close to the HIS1 gene.

Curr Genet, 1990 Apr, 17(4), 275 - 80
The effect of DNA replication on mutation of the Saccharomyces cerevisiae CDC8 gene; Zaborowska D et al.; Incubation in YPD medium under permissive conditions when DNA replication is going on, strongly stimulates the induction of cdc+ colonies of UV-irradiated cells of yeast strains HB23 (cdc8-1/cdc8-3), HB26 (cdc8-3/cdc8-3) and HB7 (cdc8-1/cdc8-1) . Inhibition of DNA replication by hydroxyurea, araCMP, cycloheximide or caffeine or else by incubation in phosphate buffer pH 7.0, abolishes this stimulation . Thus the replication of DNA is strongly correlated with the high induction of cdc+ colonies by UV irradiation . It is postulated that these UV-induced cdc+ colonies arise as the result infidelity in DNA replication.

Genetics, 1990 Apr, 124(4), 817 - 31
The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway; Schiestl RH et al.; rad6 mutants of Saccharomyces cerevisiae are defective in the repair of damaged DNA, DNA damage induced mutagenesis, and sporulation . In order to identify genes that can substitute for RAD6 function, we have isolated genomic suppressors of the UV sensitivity of rad6 deletion (rad6 delta) mutations and show that they also suppress the gamma-ray sensitivity but not the UV mutagenesis or sporulation defects of rad6 . The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of gamma-ray sensitivity . The six suppressor mutations we isolated are all alleles of the same locus and are also allelic to a previously described suppressor of the rad6-1 nonsense mutation, SRS2 . We show that suppression of rad6 delta is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6 delta SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes . Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into the RAD52 DNA repair pathway are discussed.

EMBO J, 1990 Apr, 9(4), 1253 - 8
Temperature sensitive synthesis of transfer RNAs in vivo in Saccharomyces cerevisiae; Marschalek R et al.; Dictyostelium discoideum tRNA genes can be expressed efficiently in vivo in yeast, and transcription products are processed to mature tRNAs . However, primary transcripts of a variant tRNA(Val)(UAC) gene are processing deficient under standard growth conditions (30 degrees C), due to a slightly altered 5' flanking region . A stable extended amino acid acceptor stem, which seems to be required to compensate a G5-G68 mismatch, cannot form . This mismatch destabilizes secondary and probably tertiary structures to such an extent that recognition of processing enzyme(s) under normal conditions (30 degrees C) is impaired . Growing yeast cells at reduced temperature (22 degrees C) can phenotypically complement the processing defect . This observation provides a new concept for the temperature dependent expression of protein coding genes which carry a nonsense codon . Translation of corresponding messages can be controlled by products of a temperature sensitive su-tRNA gene . We successfully tested this concept with two amber suppressors derived from a tRNA(Glu)(UUC) gene from D . discoideum . One of the variant tRNA genes codes for a product with a destabilized amino acid acceptor stem . Primary transcripts of this particular su-tRNA(Glu)(CUA) gene are processed only at reduced growth temperatures and consequently function as temperature sensitive suppressors only under these conditions.

EMBO J, 1990 Apr, 9(4), 1245 - 52
Accumulation of pre-tRNA splicing '2/3' intermediates in a Saccharomyces cerevisiae mutant; Ho CK et al.; In an effort to identify genes involved in the excision of tRNA introns in Saccharomyces cerevisiae, temperature-sensitive mutants were screened for intracellular accumulation of intron-containing tRNA precursors by RNA hybridization analysis . In one mutant, tRNA splicing intermediates consisting of the 5' exon covalently joined to the intron ('2/3' pre-tRNA molecules) were detected in addition to unspliced precursors . The mutant cleaves pre-tRNA(Phe) in vitro at the 3' exon/intron splice site, generating the 3' half molecule and 2/3 intermediate . The 5' half molecule and intron are not produced, indicating that cleavage at the 5' splice site is suppressed . This partial splicing activity co-purifies with tRNA endonuclease throughout several chromatographic steps . Surprisingly, the splicing defect does not appreciably affect cell growth at normal or elevated temperatures, but does confer a pseudo cold-sensitive phenotype of retarded growth at 15 degrees C . The mutant falls into the complementation group SEN2 previously defined by the isolation of mutants defective for tRNA splicing in vitro {Winey, M . and Culbertson, M.R . (1988) Genetics, 118, 609-617}, although its phenotypes are distinct from those of the previous sen2 isolates . The distinguishing genetic and biochemical properties of this new allele, designated sen2-3, suggests the direct participation of the SEN2 gene product in tRNA endonuclease function.

Carcinogenesis, 1990 Apr, 11(4), 633 - 8
Analysis of mutations induced in the SUP4-o gene of Saccharomyces cerevisiae by cis-diammine dichloroplatinum(II); Mis JR et al.; A collection of 196 mutations induced in the SUP4-o gene of yeast by treatment with cis-diammine dichloroplatinum(II) (cis-DDP) was characterized by DNA sequencing . All possible types of base pair substitution were identified as well as deletions, insertions and double mutations . Base pair changes at G.C sites predominated and were distributed throughout the gene . The majority of substitutions occurred at 5'-GG-3' and 5'-GA-3' sequences, potential sites of cis-DDP adducts . However, mutations were also detected at a number of other DNA sequences where cis-DDP has been found to bind in vitro or form adducts in vivo including 5'-AA-3', 5'-AG-3', 5'-GNG-3' and 5'-AAA-3' . The site specificity of cis-DDP mutagenesis argues that some of these sequences are significant targets for the induction of mutation in vivo despite the fact that they were considered to be weak binding sites for cis-DDP in vitro . In addition, the distribution of the substitutions within the SUP4-o gene indicates that DNA sequence context influences cis-DDP mutagenesis in vivo . Finally, our results suggest that intrastrand cross-links formed by cis-DDP might facilitate the gain or loss of single base pairs by stabilizing strand misalignments that template these events.

Antonie Van Leeuwenhoek, 1990 Apr, 57(3), 159 - 64
Determination of the role of polyphosphate in transport-coupled phosphorylation in the yeast Saccharomyces cerevisiae; Schuddemat J et al.; The role of polyphosphate in 2-deoxy-D-glucose transport was studied in yeast cells, pulse-labeled with {32P}orthophosphate, by comparing the concentrations and specific activities of polyphosphate, orthophosphate and 2-dGlc-phosphate . When 2-dGlc transport was measured under aerobic conditions, it appeared that polyphosphate replenished the orthophosphate pool, indicating that polyphosphate has, at least mainly, an indirect role in sugar phosphorylation . Also in cells with a reduced respiratory capacity, due to a treatment with antimycin A, no direct role for polyphosphate in 2-dGlc transport could be detected . Under these conditions, only a very limited breakdown of polyphosphate occurred, probably because of the small decrease in the orthophosphate concentration.

Mol Cell Biol, 1990 Apr, 10(4), 1743 - 53
Identification of a Saccharomyces cerevisiae DNA-binding protein involved in transcriptional regulation; Wang H et al.; A DNA-binding protein has been identified from extracts of the budding yeast Saccharomyces cerevisiae which binds to sites present in the promoter regions of a number of yeast genes transcribed by RNA polymerase II, including SIN3 (also known as SDI1), SWI5, CDC9, and TOP1 . This protein also binds to a site present in the enhancer for the 35S rRNA gene, which is transcribed by RNA polymerase I, and appears to be identical to the previously described REB1 protein (B . E . Morrow, S . P . Johnson, and J . R . Warner, J . Biol . Chem . 264:9061-9068, 1989) . When oligonucleotides containing a REB1-binding site are placed between the CYC1 upstream activating sequence and TATA box, transcription by RNA polymerase II in vivo is substantially reduced, suggesting that REB1 acts as a repressor of RNA polymerase II transcription . The in vitro levels of the REB1 DNA-binding activity are reduced in extracts prepared from strains bearing a mutation in the SIN3 gene . A greater reduction in REB1 activity is observed if the sin3 mutant strain is grown in media containing galactose as a carbon source.

Mol Cell Biol, 1990 Apr, 10(4), 1622 - 32
Self-regulation of 70-kilodalton heat shock proteins in Saccharomyces cerevisiae; Stone DE et al.; To determine whether the 70-kilodalton heat shock proteins of Saccharomyces cerevisiae play a role in regulating their own synthesis, we studied the effect of overexpressing the SSA1 protein on the activity of the SSA1 5'-regulatory region . The constitutive level of Ssa1p was increased by fusing the SSA1 structural gene to the GAL1 promoter . A reporter vector consisting of an SSA1-lacZ translational fusion was used to assess SSA1 promoter activity . In a strain producing approximately 10-fold the normal heat shock level of Ssa1p, induction of beta-galactosidase activity by heat shock was almost entirely blocked . Expression of a transcriptional fusion vector in which the CYC1 upstream activating sequence of a CYC1-lacZ chimera was replaced by a sequence containing a heat shock upstream activating sequence (heat shock element 2) from the 5'-regulatory region of SSA1 was inhibited by excess Ssa1p . The repression of an SSA1 upstream activating sequence by the SSA1 protein indicates that SSA1 self-regulation is at least partially mediated at the transcriptional level . The expression of another transcriptional fusion vector, containing heat shock element 2 and a lesser amount of flanking sequence, is not inhibited when Ssa1p is overexpressed . This suggests the existence of an element, proximal to or overlapping heat shock element 2, that confers sensitivity to the SSA1 protein.

Mol Cell Biol, 1990 Apr, 10(4), 1399 - 405
Citrate synthase encoded by the CIT2 gene of Saccharomyces cerevisiae is peroxisomal; Lewin AS et al.; The product of the CIT2 gene has the tripeptide SKL at its carboxyl terminus . This amino acid sequence has been shown to act as a peroxisomal targeting signal in mammalian cells . We examined the subcellular site of this extramitochondrial citrate synthase . Cells of Saccharomyces cerevisiae were grown on oleate medium to induce peroxisome proliferation . A fraction containing membrane-enclosed vesicles and organelles was analyzed by sedimentation on density gradients . In wild-type cells, the major peak of citrate synthase activity was recovered in the mitochondrial fraction, but a second peak of activity cosedimented with peroxisomes . The peroxisomal activity, but not the mitochondrial activity, was inhibited by incubation at pH 8.1, a characteristic of the extramitochondrial citrate synthase encoded by the CIT2 gene . In a strain in which the CIT1 gene encoding mitochondrial citrate synthase had been disrupted, the major peak of citrate synthase activity was peroxisomal, and all of the activity was sensitive to incubation at pH 8.1 . Yeast cells bearing a cit2 disruption were unable to mobilize stored lipids and did not form stable peroxisomes in oleate . We conclude that citrate synthase encoded by CIT2 is peroxisomal and participates in the glyoxylate cycle.

Mol Cell Biol, 1990 Apr, 10(4), 1358 - 66
The product of the Saccharomyces cerevisiae cell cycle gene DBF2 has homology with protein kinases and is periodically expressed in the cell cycle; Johnston LH et al.; Several Saccharomyces cerevisiae dbf mutants defective in DNA synthesis have been described previously . In this paper, one of them, dbf2, is characterized in detail . The DBF2 gene has been cloned and mapped, and its nucleotide sequence has been determined . This process has identified an open reading frame capable of encoding a protein of molecular weight 64,883 (561 amino acids) . The deduced amino acid sequence contains all 11 conserved domains found in various protein kinases . DBF2 was periodically expressed in the cell cycle at a time that clearly differed from the time of expression of either the histone H2A or DNA polymerase I gene . Its first function was completed very near to initiation of DNA synthesis . However, DNA synthesis in the mutant was only delayed at 37 degrees C, and the cells blocked in nuclear division . Consistent with this finding, the execution point occurred about 1 h after DNA synthesis, and the nuclear morphology of the mutant at the restrictive temperature was that of cells blocked in late nuclear division . DBF2 is therefore likely to encode a protein kinase that may function in initiation of DNA synthesis and also in late nuclear division.

J Bacteriol, 1990 Apr, 172(4), 1840 - 5
O2-dependent methionine auxotrophy in Cu,Zn superoxide dismutase-deficient mutants of Saccharomyces cerevisiae; Chang EC et al.; Mutant strains of the yeast Saccharomyces cerevisiae which lack functional Cu,Zn superoxide dismutase (SOD-1) do not grow aerobically unless supplemented with methionine . The molecular basis of this O2-dependent auxotrophy in one of the mutants, Dscd1-1C, has been investigated . Sulfate supported anaerobic but not aerobic mutant growth . On the other hand, cysteine and homocysteine supported aerobic growth while serine, O-acetylserine, and homoserine did not, indicating that the interconversion of cysteine and methionine (and homocysteine) was not impaired . Thiosulfate (S2O3(2-} and sulfide (S2-) also supported aerobic growth; the activities of thiosulfate reductase and sulfhydrylase in the aerobic mutant strain were at wild-type levels . Although the levels of SO4(2-) and adenosine-5'-sulfate (the first intermediate in the SO4(2-) assimilation pathway) were elevated in the aerobically incubated mutant strain, this condition could be attributed to a decrease in protein synthesis caused by the de facto sulfur starvation and not to a block in the pathway . Therefore, the activation of SO4(2-) (to form 3'-phosphoadenosine-5'-phosphosulfate) appeared to be O2 tolerant . Sulfite reductase activity and substrate concentrations {( NADPH} and {SO3(2-)}) were not significantly different in aerobically grown mutant cultures and anaerobic cultures, indicating that SOD-1- mutant strains could reductively assimilate sulfur oxides . However, the mutant strain exhibited an O2-dependent sensitivity to SO3(2-) concentrations of less than 50 microM not exhibited by any SOD-1+ strain or by SOD-1- strains supplemented with a cytosolic O2(-)-scavenging activity . This result suggests that the aerobic reductive assimilation of SO4(2-) at the level of SO3(2-) may generate a cytotoxic compound(s) which persists in SOD-(1-) yeast strains.

Exp Cell Res, 1990 Apr, 187(2), 315 - 9
Changes in the protein synthesis pattern during a nutritional shift-down transition in Saccharomyces cerevisiae; Rodriguez F et al.; In Saccharomyces cerevisiae cells (strain A364A) during a shift-down from glucose to raffinose, a rapid reduction in the rate of RNA accumulation was observed whereas the rate of protein accumulation was unaffected for at least 2 h . Following the transition the percentage of unbudded cells slightly increased and the cell volume distribution showed a newly formed subpopulation of smaller cells . To study the effects of the shift-down on the protein synthesis pattern, total {35S}-methionine pulse-labeled extracts were fractionated by high-resolution two-dimensional gel electrophoresis . The synthesis of two classes of proteins (I and II) was modulated during the transitory state of growth: one positively, the other negatively . Two polypeptides of 57 kDa showed the most dramatic increase in synthesis during the shift-down . Also a heat-shock protein (HSP 256) appeared to be positively correlated to the shift-down transition.

Genetics, 1990 Apr, 124(4), 797 - 806
Comparison of thermosensitive alleles of the CDC25 gene involved in the cAMP metabolism of Saccharomyces cerevisiae; Petitjean A et al.; The CDC25 gene from Saccharomyces cerevisiae is an essential component of the RAS-adenylate cyclase pathway . Genetic and biochemical evidence has led to the proposal that the gene product may act upstream of RAS, possibly as a guanine nucleotide exchange factor . We report here the cloning, sequencing and characterization of four mutations in the CDC25 gene . All four are missense mutations which reside within the carboxy-terminal quarter of the single open reading frame found within the gene . Three of the four are missense mutations in the same amino acid codon . A search of protein data bases reveals that the carboxy terminus of the putative CDC25 gene product is similar to that of LTE1, a gene required for growth at low temperature and SCD25, a suppressor of cdc25 . Taken together these data indicate that the carboxy terminus of CDC25 plays a critical role in the function of the CDC25 gene product and that other proteins, such as LTE1 or SCD25, may have related activities.

Proc Natl Acad Sci U S A, 1990 Apr, 87(7), 2695 - 9
Mutation of cysteine-88 in the Saccharomyces cerevisiae RAD6 protein abolishes its ubiquitin-conjugating activity and its various biological functions; Sung P et al.; The RAD6 gene of Saccharomyces cerevisiae is required for DNA repair, DNA damage-induced mutagenesis, and sporulation . RAD6 protein is a ubiquitin-conjugating enzyme (E2) that has been shown to attach multiple molecules of ubiquitin to histones H2A and H2B . We have now examined whether the E2 activity of RAD6 is involved in its various biological functions . Since the formation of a thioester adduct between E2 and ubiquitin is necessary for E2 activity, the single cysteine residue (Cys-88) present in RAD6 was changed to alanine or valine . The mutant proteins were overproduced in yeast cells and purified to near homogeneity . We show that the rad6 Ala-88 and rad6 Val-88 mutant proteins lack the capacity for thioester formation with ubiquitin and, as a consequence, are totally devoid of any E2 activity . The rad6 Ala-88 and rad6 Val-88 mutations confer a defect in DNA repair, mutagenesis, and sporulation equivalent to that in the rad6 null allele . We suggest that the biological functions of RAD6 require its E2 activity.

J Bacteriol, 1990 Apr, 172(4), 2005 - 12
Repression of choline kinase by inositol and choline in Saccharomyces cerevisiae; Hosaka K et al.; The regulation of choline kinase (EC 2.7.1.32), the initial enzyme in the CDP-choline pathway, was examined in Saccharomyces cerevisiae . The addition of myo-inositol to a culture of wild-type cells resulted in a significant decrease in choline kinase activity . Additional supplementation of choline caused a further reduction in the activity . The coding frame of the choline kinase gene, CK1, was joined to the carboxyl terminus of lacZ and expressed in Escherichia coli as a fusion protein, which was then used to prepare an anti-choline kinase antibody . Upon Western (immuno-) and Northern (RNA) blot analyses using the antibody and a CK1 probe, respectively, the decrease in the enzyme activity was found to be correlated with decreases in the enzyme amount and mRNA abundance . The molecular mass of the enzyme was estimated to be 66 kilodaltons, in agreement with the value predicted previously from the nucleotide sequence of the gene . The coding region of CK1 was replaced with that of lacZ, and CK1 expression was measured by assaying beta-galactosidase . The expression of beta-galactosidase from this fusion was repressed by myo-inositol and choline and derepressed in a time-dependent manner upon their removal . The present findings indicate that yeast choline kinase is regulated by myo-inositol and choline at the level of mRNA abundance.

Antimicrob Agents Chemother, 1990 Apr, 34(4), 660 - 2
Use of Saccharomyces cerevisiae expressing beta-galactosidase to screen for antimycotic agents directed against yeast cell wall biosynthesis and possible application to pathogenic fungi; Zaworski PG et al.; Saccharomyces cerevisiae expressing beta-galactosidase was used to develop a screen for compounds active against formation of the yeast cell wall . The screen detailed here is based on the release of beta-galactosidase from cells which had received an osmotic shock.

Agric Biol Chem, 1990 Apr, 54(4), 979 - 84
Cloning and nucleotide sequence of the KHR killer gene of Saccharomyces cerevisiae; Goto K et al.; The KHR gene cloned from a genomic library was on 4.7-kbp DNA fragment and was inserted into YCpG11 vector (KHR-YCp) and YEp vector (KHP-YEp) . Transformants with KHR-YEp could secrete 3-4 times as much killer toxin into the media as the donor strain . The complete nucleotide sequence of the KHR gene was analyzed . It was found that the KHR gene consisted of 888 bp . It was suggested that this protein was processed before being secreted into the media, because its molecular mass presumed from the nucleotide sequence was larger than that of the mature killer toxin.

Biochemistry, 1990 Mar 27, 29(12), 3119 - 26
Characterization of the RAD10 gene of Saccharomyces cerevisiae and purification of Rad10 protein; Bardwell L et al.; The RAD10 gene of Saccharomyces cerevisiae is one of at least five genes required for damage-specific incision of DNA during nucleotide excision repair . This gene was previously cloned and sequenced {Weiss, W . A., & Friedberg, E . C . (1985) EMBO J . 4, 1575-1582; Reynolds et al . (1985) EMBO J . 4, 3549-3552} . In the present studies, we have mapped one major and three minor transcriptional start sites in the RAD10 gene . The locations of these sites relative to the translational start codon are remarkably similar to those previously identified in the yeast RAD2 gene {Nicolet et al . (1985) Gene 36, 225-234} . The two genes also share common sequences in these regions . However, in contrast to RAD2 {Robinson et al . (1986) Proc . Natl . Acad . Sci . U.S.A . 83, 1842-1846}, RAD10 is not induced following exposure of cells to the DNA-damaging agent 4-nitroquinoline 1-oxide . Native RAD10 protein and also two different Rad10 fusion proteins are rapidly degraded in most Escherichia coli strains . However, following overexpression of the cloned RAD10 gene in yeast, native Rad10 protein was purified to greater than 90% homogeneity . A catalytic function has not been identified for the purified protein . RAD10 cells (untransformed with the cloned gene) contain fewer than 500 molecules per cell . This is similar to the levels of the UvrA, UvrB, and UvrC nucleotide excision repair proteins in E . coli.

Nucleic Acids Res, 1990 Mar 25, 18(6), 1521 - 9
Cloning and characterization of nuclear genes for two mitochondrial ribosomal proteins in Saccharomyces cerevisiae; Kitakawa M et al.; The genes for two large subunit proteins, YmL8 and YmL20, of the mitochondrial ribosome of Saccharomyces cerevisiae were cloned by hybridization with synthetic oligonucleotide mixtures corresponding to their N-terminal amino acid sequences . They were termed MRP-L8 and MRP-L20, respectively, and their nucleotide sequences were determined using a DNA sequencer . The MRP-L8 gene was found to encode a 26.8-kDa protein whose deduced amino acid sequence has a high degree of similarity to ribosomal protein L17 of Escherichia coli . The gene MRP-L20 was found to encode a 22.3-kDa protein with a presequence consisting of 18 amino acid residues . By Southern blot hybridization to the yeast chromosomes separated by field-inversion gel electrophoresis, the MRP-L8 and MRP-L20 genes were located on chromosomes X and XI, respectively . Gene disruption experiments indicate that their products, YmL8 and YmL20 proteins, are essential for the mitochondrial function and the absence of these proteins causes instability of the mitochondrial DNA.

J Biol Chem, 1990 Mar 25, 265(9), 5095 - 103
Purification and characterization of Saccharomyces cerevisiae transcription factor TFIIIC . Polypeptide composition defined with polyclonal antibodies; Parsons MC et al.; The class III gene transcription factor termed TFIIIC has been extensively purified from Saccharomyces cerevisiae . Three polypeptides of 138, 131, and 95 kDa consistently copurified with TFIIIC transcription factor activity . These polypeptides were present in approximately equimolar quantities in all TFIIIC preparations . To determine which, if any, of these polypeptides were involved in TFIIIC activity, rabbit polyclonal antibodies were generated against each of these three polypeptides purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Immunoblot analyses showed that each of the three antiserum preparations reacted uniquely with the respective polypeptide to which they had been elicited . This lack of cross-reactivity by any of the antiserum preparations suggested that these three polypeptides represented distinct unrelated gene products . Each of the three specific antiserum preparations decreased the mobility of TFIIIC-tDNA complexes in a DNA mobility shift assay . More importantly, all three antiserum preparations directly inhibited the transcription factor activity of TFIIIC . In addition, all three antiserum preparations depleted a solution of TFIIIC transcription factor activity . These results indicated that each of these three polypeptides of Mr = 138,000, 131,000, and 95,000 was a distinct and necessary component of yeast TFIIIC . Immunoblot analyses of immunoaffinity-purified TFIIIC fractions indicated that each of the three antiserum preparations alone could deplete the solution of all three polypeptides . These results suggested that these three polypeptides were tightly associated with one another in solution.

Biochem Biophys Res Commun, 1990 Mar 16, 167(2), 628 - 36
DNA binding properties of Saccharomyces cerevisiae chromatin associated ras-related protein Yp20; Valansi C et al.; Yp20 is an abundant 20 kDa chromatin associated protein which has been shown to be related antigenically to genuine Hras products . Using Southwestern blots we have demonstrated that Yp20 is a DNA binding protein . It is also shown that protein Yp20 like protein HM (an abundant thermostable 20 kDa DNA binding protein isolated from mitochondria) and like the 21 kDa autonomously replicating sequence binding factor II (ABFII) is able to introduce superhelical turns into circular relaxed DNA in the presence of DNA topoisomerase I activity . We suggest that this protein may be important for chromatin structure and function.

Gene, 1990 Mar 15, 87(2), 157 - 66
Nucleotide sequence of the COR region: a cluster of six genes in the yeast Saccharomyces cerevisiae; Melnick L et al.; We have determined the nucleotide (nt) sequence of the 7.5-kb COR segment that encompasses a cluster of six genes (CYC1, UTR1, UTR3, OSM1, tRNA(Gly) and RAD7) located on chromosome X of the yeast Saccharomyces cerevisiae . This sequence revealed five open reading frames and a tRNA gene which correspond in position, size and orientation to the transcripts previously identified by Barry et al . {Mol . Cell . Biol . 7 (1987) 632-638} . The extensively studied CYC1 gene encodes iso-1-cytochrome c; the UTR1 and UTR3 genes encode dispensible proteins whose functions are unknown; the OSM1 gene encodes a protein required for growth on hypertonic media; the tRNA(Gly) gene encodes a glycine tRNA; and the RAD7 gene encodes a protein required for repair of UV-induced damage . The OSM1 protein contains a signal sequence for secretion and a region similar to GTP-binding domains . The RAD7 protein displays 5'-untranslated elements similar to those of the stress-inducible gene UB14 . The nt sequence upstream from the tRNA(Gly) gene contains a diverged copy of the sigma repeated element . This cluster of COR genes appears to have an ancestral relationship with the cluster of ARC genes on chromosome V.

J Biol Chem, 1990 Mar 15, 265(8), 4736 - 45
The Saccharomyces cerevisiae INO4 gene encodes a small, highly basic protein required for derepression of phospholipid biosynthetic enzymes; Hoshizaki DK et al.; The INO4 gene product is believed to be a positive regulatory factor in a complex cascade of positive and negative factors that coordinates the synthesis of phospholipids in the yeast Saccharomyces cerevisiae . Mutations at the INO4 locus result in a decrease in phosphatidylcholine synthesis and an inability to derepress the structural genes for inositol-1-phosphate synthase and phosphatidylserine synthase . In the present study, the transcript encoding the INO4 gene product has been identified and a transcription map of the INO4 region has been constructed . An ino4 deletion mutant was constructed by in vitro gene disruption and the deletion mutant was shown to be viable but auxotrophic for inositol . The deletion mutant expressed repressed levels of inositol-1-phosphate synthase (INO1) mRNA and exhibited reduced phosphatidylcholine biosynthesis, a phenotype similar to previously characterized ino4 mutants . The INO4 gene has been mapped to chromosome 15 and is tightly linked to the SUF1 tRNA gene . Translation of the DNA sequence of the INO4 gene results in a very basic protein of molecular weight 17,378 . Computer analysis of the INO4 protein sequence identified several potential phosphorylation sites as well as several regions that contained significant similarities with the lupus LA antigen and with the helix-loop-helix region of the Myc family of proteins.

Eur J Biochem, 1990 Mar 10, 188(2), 393 - 403
Purification and properties of uroporphyrinogen decarboxylase from Saccharomyces cerevisiae . Yeast uroporphyrinogen decarboxylase; Felix F et al.; Uroporphyrinogen decarboxylase (EC 4.1.1.37) was purified about 14000-fold to homogeneity from the yeast Saccharomyces cerevisiae with a 70% overall yield . The purification included affinity chromatography on uroporphyrin-I-Affi-Gel 102 . The specific activity of the final preparation was 1750 nmol coproporphyrinogen formed.h-1.(mg protein)-1 at pH 7.5 and 37 degrees C using 4 microM uroporphyrinogen I as substrate . The purified enzyme has a minimum molecular mass of 38 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and 46 kDa by gel filtration, suggesting that yeast uroporphyrinogen decarboxylase is a monomer . Chromatofocusing gave a pI of 6.0 . Enzyme activity was inhibited by metals, such as Cu2+, Zn2+, Fe2+, Fe3+ and by sulfhydryl-specific reagents, but no cofactor requirement could be demonstrated . The optimum pH was pH 5.7 for uroporphyrinogens I and III and heptacarboxylate porphyrinogen I as estimated by coproporphyrinogen formation . The optimum pH for substrate decarboxylation was pH 5.7 for uroporphyrinogen I, but pH 6.8 for the two other substrates . The Km values at pH 5.7 were 10 nM for uroporphyrinogen I, 6 nM for uroporphyrinogen III and 7 nM for heptacarboxylate porphyrinogen I as measured by coproporphyrinogen formation . The pattern of accumulation of intermediate and final decarboxylation products and the rates of the successive decarboxylations were determined for the three substrates at different concentrations at pH 5.7 and pH 6.8 . The rate-limiting step at 4 microM substrate concentration was the elimination of the second carboxyl group of uroporphyrinogen III and the fourth carboxyl of uroporphyrinogen I . An antiserum to purified yeast uroporphyrinogen decarboxylase was used to characterize the protein in several mutants.

Science, 1990 Mar 9, 247(4947), 1213 - 6
An intron in the genes for U3 small nucleolar RNAs of the yeast Saccharomyces cerevisiae; Myslinski E et al.; The origin of the intervening sequences (introns), which are removed during RNA maturation, is currently unknown . They are found in most genes encoding messenger RNAs, but are lacking in almost all small nuclear (sn)RNAs . One exceptional snRNA (U6) is part of the spliceosomal machinery that is involved in messenger RNA maturation . It has been suggested that its intron arose as a result of incorrect splicing of a messenger RNA precursor . This study revealed the presence of an intron, with the characteristic features of nuclear introns from precursors to messenger RNA, in the two genes coding for Saccharomyces cerevisiae U3 snRNA . The branch point was GACTAAC instead of the TACTAAC sequence found in all yeast introns examined so far . As U3 is a nucleolar snRNA required for maturation of ribosomal RNA, its intron could not have been acquired from aberrant messenger RNA processing in a spliceosome.

J Biol Chem, 1990 Mar 5, 265(7), 4072 - 83
Purification and characterization of DNA polymerase II from the yeast Saccharomyces cerevisiae . Identification of the catalytic core and a possible holoenzyme form of the enzyme; Hamatake RK et al.; We have purified yeast DNA polymerase II to near homogeneity as a 145-kDa polypeptide . During the course of this purification we have detected and purified a novel form of DNA polymerase II that we designate as DNA polymerase II . The most highly purified preparations of DNA polymerase II are composed of polypeptides with molecular masses of 200, 80, 34, 30, and 29 kDa . Immunological analysis and peptide mapping of DNA polymerase II and the 200-kDa subunit of DNA polymerase II indicate that the 145-kDa DNA polymerase II polypeptide is derived from the 200-kDa polypeptide of DNA polymerase II . Activity gel analysis shows that the 145- and the 200-kDa polypeptides have catalytic function . The polypeptides present in the DNA polymerase II preparation copurify with the polymerase activity with a constant relative stoichiometry during chromatography over five columns and co-sediment with the activity during glycerol gradient centrifugation, suggesting that this complex may be a holoenzyme form of DNA polymerase II . Both forms of DNA polymerase II possess a 3'-5' exonuclease activity that remains tightly associated with the polymerase activity during purification . DNA polymerase II is similar to the proliferating cell nuclear antigen (PCNA)-independent form of mammalian DNA polymerase delta in its resistance to butylpheny-dGTP, template specificity, stimulation of polymerase and exonuclease activity by KCl, and high processivity . Although calf thymus PCNA does not stimulate the activity of DNA polymerase II on poly(dA):oligo(dT), possibly due to the limited length of the template, the high processivity of yeast DNA polymerase II on this template can be further increased by the addition of PCNA, suggesting that conditions may exist for interactions between PCNA and yeast DNA polymerase II.

J Biol Chem, 1990 Mar 5, 265(7), 3793 - 802
Glucose oxidase from Aspergillus niger . Cloning, gene sequence, secretion from Saccharomyces cerevisiae and kinetic analysis of a yeast-derived enzyme; Frederick KR et al.; The gene for Aspergillus niger glucose oxidase (EC 1.1.3.4) has been cloned from both cDNA and genomic libraries using oligonucleotide probes derived from the amino acid sequences of peptide fragments of the enzyme . The mature enzyme consists of 583 amino acids and is preceded by a 22-amino acid presequence . No intervening sequences are found within the coding region . The enzyme contains 3 cysteine residues and 8 potential sites for N-linked glycosylation . The protein shows 26% identity with alcohol oxidase of Hansenuela polymorpha, and the N terminus has a sequence homologous with the AMP-binding region of other flavoenzymes such as p-hydroxybenzoate hydroxylase and glutathione reductase . Recombinant yeast expression plasmids have been constructed containing a hybrid yeast alcohol dehydrogenase II-glyceraldehyde-3-phosphate dehydrogenase promoter, either the yeast alpha-factor pheromone leader or the glucose oxidase presequence, and the mature glucose oxidase coding sequence . When transformed into yeast, these plasmids direct the synthesis and secretion of between 75 and 400 micrograms/ml of active glucose oxidase . Analysis of the yeast-derived enzymes shows that they are of comparable specific activity and have more extensive N-linked glycosylation than the A . niger protein.

J Biol Chem, 1990 Mar 5, 265(7), 3603 - 6
Identification of methionine Nalpha-acetyltransferase from Saccharomyces cerevisiae; Lee FJ et al.; N alpha-Acetylation is the most frequently occurring chemical modification of the alpha-NH2 group of eukaryotic proteins and was believed until now to be catalyzed by a single N alpha-acetyltransferase . The transfer of an acetyl group from acetyl coenzyme A to the alpha-amino group of five NH2-terminal residues (serine, alanine, methionine, glycine, and threonine) in proteins accounts for approximately 95% of acetylated residues . We have found that a crude lysate from Saccharomyces cerevisiae mutant (aaa1) deficient in N alpha-acetyltransferase activity can effectively transfer an acetyl group to peptides containing NH2-terminal methionine but not to serine or alanine . This methionine N alpha-acetyltransferase has been extensively purified, and this purified enzyme can selectively transfer an acetyl group to various model peptides containing an NH2-terminal methionine residue and a penultimate aspartyl, asparaginyl, or glutamyl residue . Such specificity of N alpha-acetylation of methionine has been previously observed based on the analysis of eukaryotic protein sequences (Persson, B., Flinta, C., Heijne, G., and Jornvall, H . (1985) Eur . J . Biochem . 152, 523-527; Arfin, S.M., and Bradshaw, R . A . (1988) Biochemistry 27, 7979-7984) . The indentification of this methionine N alpha-acetyltransferase provides an explanation as to why two distinct classes of N alpha-acetylated proteins exist in nature: (i) those whose initiator methionine is acetylated and (ii) those whose penultimate residue is acetylated after cleavage of the initiator methionine.

Mol Cell Biol, 1990 Mar, 10(3), 1226 - 33
Arginine-specific repression in Saccharomyces cerevisiae: kinetic data on ARG1 and ARG3 mRNA transcription and stability support a transcriptional control mechanism; Crabeel M et al.; A specific repression mechanism regulates arginine biosynthesis in Saccharomyces cerevisiae . The involvement of regulatory proteins displaying DNA-binding features and the location of an operator region between the TATA box and the transcription start of the structural gene ARG3 suggest that this mechanism operates at the level of transcription . A posttranscriptional mechanism has, however, been proposed to account for the conspicuous lack of proportionality between ARG3 mRNA steady-state levels (as determined by Northern {RNA} assays; F . Messenguy and E . Dubois, Mol . Gen . Genet . 189:148-156, 1983) and the cognate enzyme activities . In this work, we have analyzed the time course of the incorporation of radioactive precursors into ARG1 and ARG3 mRNAs and the kinetics of their decay under different regulatory statuses . The results (expressed in terms of relative mRNA levels, relative transcription rates, and mRNA half-lives) give the picture expected from a purely transcriptional control . A similar analysis of expression of the gene CPA1, for which a translational regulation by arginine has been clearly demonstrated (M . Werner, A . Feller, F . Messenguy, and A . Pierard, Cell 49:805-813, 1987), indicates that this gene is also partly regulated at the transcriptional level by the ARGR repressor system . Moreover, the half-life of CPA1 mRNA is reduced twofold in the presence of excess arginine; we suggest that this could be inherent in the mechanism of translational regulation of CPA1.

Mol Cell Biol, 1990 Mar, 10(3), 1145 - 52
Depletion of U14 small nuclear RNA (snR128) disrupts production of 18S rRNA in Saccharomyces cerevisiae; Li HD et al.; Repression of an essential nucleolar small nuclear RNA (snRNA) gene of Saccharomyces cerevisiae was shown to result in impaired production of 18S rRNA . The effect, observed for an snRNA species of 128 nucleotides (snR128), was evident within one generation after the onset of SNR128 gene repression and correlated well with depletion of the snRNA . The steady-state mass ratio of 18S RNA to 25S RNA decreased eightfold over the course of the analysis . Results from pulse-chase assays revealed the basis of the imbalance to be underaccumulation of 18S RNA and its 20S precursor . This effect appears to result from impairment of processing of the 35S rRNA transcript at sites that define the 20S species coupled with rapid turnover of unstable intermediates . Possible bases for the effects observed are discussed . A common U14 designation is proposed for the structurally related yeast snRNA and 4.5S hybRNAs from amphibians and mammals.

Mol Cell Biol, 1990 Mar, 10(3), 1105 - 15
Mutational analysis of the SNF3 glucose transporter of Saccharomyces cerevisiae; Marshall-Carlson L et al.; The SNF3 gene of Saccharomyces cerevisiae encodes a high-affinity glucose transporter that is homologous to mammalian glucose transporters . Point mutations affecting the function of the transporter were recovered from the genomes of four snf3 mutants and characterized . Two of the mutations introduced a charged amino acid into the first and second predicted membrane-spanning regions, respectively . The analogs of a bifunctional SNF3-lacZ fusion containing these two mutations were constructed, and the mutant fusion proteins were not localized to the plasma membrane, as judged by immunofluorescence microscopy . The third mutation produced a valine-to-isoleucine substitution in hydrophobic region 8, and the corresponding mutant fusion protein was correctly localized . The finding that this conservative change causes a transport defect is consistent with the possibility that this transmembrane region, which could exist as an amphipathic alpha-helix, forms part of the glucose channel through the membrane . The fourth snf3 allele harbored an ochre mutation midway through the coding sequence . We have also constructed mutations in the cloned SNF3 gene . A major difference between the yeast SNF3 protein and mammalian glucose transporters is the presence in the SNF3 protein of an additional 303 amino acids at the C terminus . Analysis of a series of C-terminal deletions and fusions to lacZ showed that this C-terminal region is important, but not essential, for transport function . We also report the genetic mapping of the SNF3 locus on the left arm of chromosome IV.

J Gen Microbiol, 1990 Mar, 136 ( Pt 3), 419 - 23
Localization of dipeptidyl aminopeptidase yscIV in the plasma membrane of Saccharomyces cerevisiae; Bordallo C et al.; The subcellular distribution of dipeptidyl aminopeptidase activity was studied in protoplast lysates of Saccharomyces cerevisiae that were virtually free from vacuolar contamination . Dipeptidyl aminopeptidase yscIV, the STE13 gene product, was found to be associated with plasma membrane vesicles after sucrose gradient isopycnic centrifugation . Another dipeptidyl aminopeptidase activity, not yet fully characterized, was localized in a microvesicular population co-sedimenting with chitosomes.

J Gen Microbiol, 1990 Mar, 136 ( Pt 3), 413 - 8
Changes in the incorporation of carbon derived from glucose into cellular pools during the cell cycle of Saccharomyces cerevisiae; Oehlen LJ et al.; The rate of incorporation of 14C derived from {U-14C}glucose into cells of Saccharomyces cerevisiae X2180(1B) was investigated as a function of the cell cycle . After pulse-labelling of exponentially growing populations, centrifugal elutriation was used to isolate various cell fractions of increasing cell size, representing successive stages of the cell cycle . The total amount of 14C incorporated per cell was found to increase continuously during the cell cycle along with cellular protein content and Coulter counter cell volume . This pattern supports the model of exponential cell growth . In order to evaluate changes in intracellular carbon flow during the cell cycle, chemical extraction procedures were used to obtain four cellular fractions enriched in either low-molecular-mass components, lipid material, polysaccharides or proteins . The distribution of 14C among these cellular fractions varied during successive stages of the cell cycle, indicating cell-cycle-dependent fluctuations in intracellular carbon flow . During the G1 phase the flow of 14C into the low-molecular-mass pool increased markedly; concurrently, the rate of incorporation into the polysaccharide-enriched pool decreased.

J Gen Microbiol, 1990 Mar, 136 ( Pt 3), 405 - 12
Energetics of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures; Verduyn C et al.; The energetics of Saccharomyces cerevisiae were studied in anaerobic glucose-limited chemostat cultures via an analysis of biomass and metabolite production . The observed YATP was dependent on the composition of the biomass, the production of acetate, the extracellular pH, and the provision of an adequate amount of fatty acid in the medium . Under optimal growth conditions, the YATP was approximately 16 g biomass (mol ATP formed)-1 . This is much higher than previously reported for batch cultures . Addition of acetic acid or propionic acid lowered the YATP . A linear correlation was found between the energy required to compensate for import of protons and the amount of acid added . This energy requirement may be regarded as a maintenance energy, since it was independent of the dilution rate at a given acid concentration.

Photochem Photobiol, 1990 Mar, 51(3), 331 - 42
Repair of UV-damaged incoming plasmid DNA in Saccharomyces cerevisiae; Keszenman-Pereyra D; A whole-cell transformation assay was used for the repair of UV-damaged plasmid DNA in highly transformable haploid strains of Saccharomyces cerevisiae having different repair capabilities . Six rad alleles were selected from the three epistasis groups: rad 1-1 and rad2-1 from the RAD3 group, rad6-1 and rad18-2 from the RAD6 group, and rad52-1 and rad54-1 from the RAD52 group . Cells carrying single, double and triple rad alleles were transformed to uracil prototrophy by centromeric plasmid DNA (YCp19) modified in vitro with UV (254 nm) . Surviving fractions were calculated as the number of transformants at each fluence relative to the number of transformants with unirradiated plasmid DNA . The sensitivity of incoming DNA in single rad mutants shows that most repair is carried out by excision repair and a RAD18-dependent process . In the rad52-1 host, the sensitivity of incoming DNA was intermediate between those found in RAD+ and rad2-1 hosts, suggesting the involvement of a recombinational repair process . Non-epistatic interactions were observed between rad alleles belonging to different epistasis groups . This provides validation for the classification of the three epistasis groups concerning the repair of chromosomal DNA for UV-incoming DNA . In both rad1-1 rad6-1 and rad1-1 rad18-2 rad54-1 hosts, the mean fluence for one lethal event corresponds approximately to one pyrimidine dimer per plasmid molecule, indicating that they are absolute repairless hosts for incoming DNA . A comparison between cell and plasmid survival reveals that there are differences in the repairability of both chromosomal and incoming DNA . The large effect of rad6-1 mutation on cell survival and the small effect on incoming DNA suggest that, in the RAD+ strain, the RAD6 product may be essential for the repair processes which act on chromosomal DNA, but not for those which act on incoming DNA . It is proposed that in yeasts postreplication repair of incoming DNA is limited to supercoiled molecules with 1-2 pyrimidine dimers that can initiate replication.

Mol Microbiol, 1990 Mar, 4(3), 337 - 43
Aging and senescence of the budding yeast Saccharomyces cerevisiae; Jazwinski SM; The budding yeast Saccharomyces cerevisiae has a limited life span, defined by the number of times an individual cell divides . Longevity in this organism involves a genetic component . Several morphological and physiological changes are associated with yeast aging and senescence . One of these, an increase in generation time with age, provides a 'biomarker' for the aging process . This increase in generation time has revealed the operation of a 'senescence factor(s)', which is likely to be a product of age-specific gene expression . The Cell Spiral Model indicates coordination of successive cell cycles to be inherent in the determination of life span . It is proposed that life expectancy depends on the function of a stochastic trigger during aging that sets in motion a programme leading to cell senescence and death.

DNA Cell Biol, 1990 Mar, 9(2), 111 - 8
Expression of alcohol-inducible rabbit liver cytochrome P-450 3a (P-450IIE1) in Saccharomyces cerevisiae with the copper-inducible CUP1 promoter; Fujita VS et al.; The expression of the cDNA for alcohol-inducible rabbit liver microsomal cytochrome P-450 form 3a (P450IIE1) in Saccharomyces cerevisiae, with the use of the copper-inducible yeast metallothionein (CUP1) promoter and the ADH1 promoter, is described . Strains 50.L4 and PP1002 were compared for optimal levels of expressed protein . Immunoblot analysis showed that a much higher level of expression of cytochrome P-450 3a is obtained with strain 50.L4, and that the uninduced levels of expressed protein are similar with the two promoters . With the CUP1 promoter, transcription of the cDNA is strongly induced in the presence of cupric ions, and the amount of immunoreactive protein expressed in increased 20-fold in strain 50.L4, such that it constitutes 0.8% of the total cellular protein . The cytochrome P-450 holoenzyme content of these cells, calculated from the reduced CO difference spectrum, is about 0.02 nmole/mg of protein, or 0.1% of the total cellular protein . The holoenzyme content of microsomes prepared from these cells is up to 0.06 nmole/mg of protein, or 0.4% of the microsomal protein . Microsomal assays for ethylene formation from N-nitrosodiethylamine and for aniline p-hydroxylation, two reactions typical of purified rabbit cytochrome P-450 form 3a, showed that the cytochrome synthesized in yeast catalyzes both reactions . Furthermore, polyclonal anti-3a IgG completely inhibits the reactions with both substrates in yeast microsomes . A comparison of the product ratios from these substrates showed that the cytochrome P-450 3a expressed in yeast has catalytic activities similar to those of the authentic rabbit protein.

Curr Genet, 1990 Mar, 17(3), 261 - 4
Application of the beta-glucuronidase gene fusion system to Saccharomyces cerevisiae; Schmitz UK et al.; Bacterial beta-glucuronidase (GUS) has been described as a useful reporter enzyme for gene fusion studies in bacteria and plants . Here we report the expression of GUS in yeast to illustrate further applications of this enzyme as a quantitative tool for measuring gene activity, as a colour selection marker and as a versatile system for protein targeting studies . There is no intrinsic GUS activity in any yeast strain tested . GUS was expressed in transgenic yeast on a multiple-copy vector under the control of the alcohol dehydrogenase 1 (ADH1) promoter . The enzyme is stable in yeast and its activity may be monitored by very sensitive colorimetric or fluorometric methods in extracts, or by the histochemical reagent 5-bromo-4-chloro-3-indolylglucuronide (X-Gluc) on plates . To test the efficacy of GUS as a reporter for targeting proteins into different subcellular compartments in vivo, we fused the presequence of the mitochondrial tryptophanyl-tRNA-synthetase gene (MSW) to the amino terminus of GUS . The activity of the fusion protein is not substantially impaired and it is imported efficiently into yeast mitochondria.

Yeast, 1990 Mar-Apr, 6(2), 159 - 69
A K2 neutral Saccharomyces cerevisiae strain contains a variant K2 M genome; Wingfield BD et al.; K2 neutral strain Saccharomyces cerevisiae USM12 was identified and characterized . This strain carried an M double-stranded RNA (dsRNA) genome encoding for resistance to K2 toxin . The M dsRNA was larger than the K2 killer yeast M dsRNA and homoduplex analysis of denatured and reannealed K2 neurtal M dsRNA revealed an inverted duplication . Heteroduplex analysis showed that two thirds of the K2 M genome had homology with the K2 neutral M genome . Hybridization showed that the USM12 M dsRNA had significant homology with the K2 M dsRNA . Protein profiles of extracellular proteins from USM12 and a cured strain indicated that USM12 did not secrete any toxin . This is the first time that a K2 neutral yeast strain has been characterized.

Genetics, 1990 Mar, 124(3), 547 - 59
Mitotic recombination among subtelomeric Y' repeats in Saccharomyces cerevisiae; Louis EJ et al.; Y's are a dispersed family of repeats that vary in copy number, location and restriction fragment lengths between strains but exhibit within-strain homogeneity . We have studied mitotic recombination between members of the subtelomeric Y' repeated sequence family of Saccharomyces cerevisiae . Individual copies of Y's were marked with SUP11 and URA3 which allowed for the selection of duplications and losses of the marked Y's . Duplications occurred by ectopic recombinational interactions between Y's at different chromosome ends as well as by unequal sister chromatid exchange . Several of the ectopic duplications resulted in an originally Y'-less chromosome end acquiring a marked Y' . Among losses, most resulted from ectopic exchange or conversion in which only the marker sequence was lost . In some losses, the chromosome end became Y'-less . Although the two subsets of Y's, Y'-longs (6.7 kb) and Y'-shorts (5.2 kb), share extensive sequence homology, a marked Y' recombines highly preferentially within its own subset . These mitotic interactions can in part explain the maintenance of Y's and their subsets, the homogeneity among Y's within a strain, as well as diversity between strains.

Genetics, 1990 Mar, 124(3), 533 - 45
The subtelomeric Y' repeat family in Saccharomyces cerevisiae: an experimental system for repeated sequence evolution; Louis EJ et al.; The subtelomeric Y' repeated sequence families in two divergent strains of the yeast Saccharomyces cerevisiae have been characterized in terms of copy number, location and restriction site differences . The strain YP1 has 26 to 30 Y's that fall into two previously described, long (6.7 kb) and short (5.2 kb), size classes . These Y's reside at 19 of the 32 chromosome ends and are concentrated in the higher molecular weight chromosomes . Five ends contain tandem arrays, each of which has only one size class of Y's . There is restriction site homogeneity among the Y's of YP1 even between size classes . The Y's of strain Y55 contrast sharply with the Y's of YP1 in terms of copy number, location and sequence differences . There are 14 to 16 Y's, both long and short, most of which are found at different chromosome ends than those of YP1 . None of these are tandemly arrayed . Four to six of the Y's appear degenerate in that they have homology with a telomere distal end Y' probe but no homology with sequences at the telomere proximal end . The majority of the Y55 Y's have the same restriction sites as in YP1 . Despite the conservation of restriction sites among Y's, a great deal of restriction fragment length heterogeneity between the strains is observed . The characterized Y' repeated sequence families provide an experimental system in which repeated sequence interactions and subsequent evolution can be studied.

Genetics, 1990 Mar, 124(3), 515 - 22
Isolation and characterization of omnipotent suppressors in the yeast Saccharomyces cerevisiae; Wakem LP et al.; Approximately 290 omnipotent suppressors, which enhance translational misreading, were isolated in strains of the yeast Saccharomyces cerevisiae containing the psi+ extrachromosomal determinant . The suppressors could be assigned to 8 classes by their pattern of suppression of five nutritional markers . The suppressors were further distinguished by differences in growth on paromomycin medium, hypertonic medium, low temperatures (10 degrees), nonfermentable carbon sources, alpha-aminoadipic acid medium, and by their dominance and recessiveness . Genetic analysis of 12 representative suppressors resulted in the assignment of these suppressors to 6 different loci, including the three previously described loci SUP35 (chromosome IV), SUP45 (chromosome II) and SUP46 (chromosome II), as well as three new loci SUP42 (chromosome IV), SUP43 (chromosome XV) and SUP44 (chromosome VII) . Suppressors belonging to the same locus had a wide range of different phenotypes . Differences between alleles of the same locus and similarities between alleles of different loci suggest that the omnipotent suppressors encode proteins that effect different functions and that altered forms of each of the proteins can effect the same function.

Genetics, 1990 Mar, 124(3), 497 - 504
srd1, a Saccharomyces cerevisiae suppressor of the temperature-sensitive pre-rRNA processing defect of rrp1-1; Fabian GR et al.; We define a new gene, SRD1, involved in the processing of pre-rRNA to mature rRNA . The SRD1 gene was identified by selecting for second-site suppressors of the previously described rrp1-1 mutation . The rrp1-1 mutation causes temperature-sensitive growth, a conditional defect in processing of 27S pre-rRNA to mature 25S rRNA, and a nonconditional increase in sensitivity to several aminoglycoside antibiotics . All srd1 alleles identified are recessive and apparently specific to the rrp1-1 mutation . Although a mutation of SRD1 suppresses the pre-rRNA processing defect, drug sensitivity and thermolethality of a point mutation of RRP1, it is unable to suppress a rrp1-disruption allele . We suggest that the SRD1 gene product either interacts with or regulates the RRP1 product.

Genetics, 1990 Mar, 124(3), 483 - 95
Genetic characterization of the Saccharomyces cerevisiae translational initiation suppressors sui1, sui2 and SUI3 and their effects on HIS4 expression; Castilho-Valavicius B et al.; Saccharomyces cerevisiae strains containing mutations of the HIS4 translation initiation AUG codon were studied by reversion analysis in an attempt to identify components of the translation initiation complex that might participate in initiation site selection during the scanning process . The genetic characterization of these revertants identified three unlinked suppressor loci: SUI1, SUI2 and sui3, which when mutated restored the expression of the HIS4 allele despite the absence of the AUG initiator codon . Both sui1 and sui2 are recessive and cause temperature-sensitive growth on enriched medium . The temperature-sensitive phenotype and the ability to restore HIS4 expression associated with either sui1 or sui2 mutations cosegregate in crosses . SUI3 mutations are dominant and do not alter the thermal profile for growth . None of the mutations at the three loci suppresses known frameshift, missense or nonsense mutations . Each is capable of suppressing the nine different point mutations of the initiator codon at HIS4 or HIS4-lacZ as well as a two base change (ACC) and a three base deletion of the AUG codon, suggesting that the site of suppression resides outside the normal initiator region . sui1 and sui2 suppressor mutations were mapped to chromosomes XIV and X, respectively . Suppression by sui1, sui2 and SUI3 mutations results in 14-, 11- and 47-fold increases, respectively, relative to isogenic parent strains, in the expression of a HIS4 allele lacking the initiator AUG codon . Part of this increase in the HIS4 expression by sui2 and SUI3 can be attributed to increases of HIS4 mRNA levels, presumably mediated by perturbation of the general amino acid control system of yeast.

EMBO J, 1990 Mar, 9(3), 663 - 73
Intermediates of recombination during mating type switching in Saccharomyces cerevisiae; White CI et al.; We have identified two novel intermediates of homothallic switching of the yeast mating type gene, from MATa to MAT alpha . Following HO endonuclease cleavage, 5' to 3' exonucleolytic digestion is observed distal to the HO cut, creating a 3'-ended single-stranded tail . This recision is more extensive in a rad52 strain unable to switch . Surprisingly, the proximal side of the HO cut is protected from degradation; this stabilization depends on the presence of the silent copy donor sequences . A second intermediate was identified by a quantitative application of the polymerase chain reaction (PCR) . The Y alpha-MAT distal covalent fragment of the switched product appears 30 min prior to the appearance of the MAT proximal Y alpha junction . No covalent joining of MAT distal to HML distal sequences is detected . We suggested that the MAT DNA distal to the HO cut invades the intact donor and is extended by DNA synthesis . This step is prevented in a rad52 strain . These intermediates are consistent with a model for MAT switching in which only the distal side of the HO cut is initially active in strand invasion and transfer of information from the donor.

Int J Pept Protein Res, 1990 Mar, 35(3), 241 - 8
Synthesis of biologically active analogs of the dodecapeptide a-factor mating pheromone of Saccharomyces cerevisiae; Ewenson A et al.; A number of dodecapeptides with the sequence YIIKGVFWDPAC were synthesized using solid phase peptide synthesis . The purity of the crude cleavage product was found to be directly related to the cysteine protecting group and the conditions employed for cleavage of the peptide from the resin . When 4-methyl-benzyl cysteine was used, complete deprotection was only achieved with low-high HF conditions at temperatures of 10 degrees-25 degrees, whereas milder conditions could be used for dodecapeptides containing ethyl cysteine or acetamidomethyl cysteine . In several syntheses the biological activity of the crude cleavage product greatly exceeded the biological activity of a purified major peptide component . The high activity found in the crude cleavage peptide was probably due to minor peptide side products in which the cysteine sulfur was alkylated by hydrophobic species during HF treatment . Two dodecapeptides, YIIKGVFWDPAC and YIIKGFWDPAC(Ethyl), had significant a-factor activity against MAT alpha strains of Saccharomyces cerevisiae . These peptides represent the first synthetic analogs with a-factor activity.

Mol Cell Biol, 1990 Mar, 10(3), 947 - 57
Enhancer and promoter elements from simian virus 40 and polyomavirus can substitute for an upstream activation sequence in Saccharomyces cerevisiae; Axelrod NJ et al.; Ten fragments of higher eucaryotic DNA were tested for upstream activation sequence activity in Saccharomyces cerevisiae by inserting them upstream of a CYC1::lacZ promoter lacking an upstream activation sequence . Fragments containing the 21-base-pair repeat region, the enhancer of simian virus 40 or both strongly stimulated beta-galactosidase synthesis, and three fragments from the polyomavirus enhancer region stimulated moderate levels . Three of the four controls of random DNA sequences failed to stimulate significant levels, and the fourth stimulated moderate levels . The stimulation in all cases was independent of the orientation of the inserted fragment . Two series of clones were examined in which between one and six tandemly arranged copies of a fragment were inserted into the XhoI site of the vector . Very interestingly, we detected an apparent exponential relationship between the number of copies of a fragment and the amount of beta-galactosidase produced . Southern analysis showed that increases in enzyme activity were not a result of increased plasmid copy number . Rather, quantitative S1 nuclease analysis demonstrated that the increases were correlated with steady-state levels of lacZ-specific mRNA . We suggest that there may be an evolutionary relationship between some transcriptional activation sequences in yeast cells and the higher eucaryotic regulatory elements that we tested.

Mol Cell Biol, 1990 Mar, 10(3), 1297 - 300
Release of two Saccharomyces cerevisiae cytochrome genes, COX6 and CYC1, from glucose repression requires the SNF1 and SSN6 gene products; Wright RM et al.; We show here that SNF1 and SSN6 are required for derepression of the glucose-repressible yeast genes COX6 and CYC1, which encode the mitochondrial proteins cytochrome c oxidase subunit VI and iso-1-cytochrome c, respectively . In an snf1 mutant genetic background, the transcription of both COX6 and CYC1 continued to be repressed after cells were shifted into derepressing media . In an ssn6 mutant genetic background, both COX6 and CYC1 were expressed constitutively at high levels in repressing media . SSN6 acted epistatically to SNF1 in the regulation of both cytochrome genes . These findings are similar to previous findings on the effects of SNF1 and SSN6 on SUC2 expression in Saccharomyces cerevisiae and are consistent with a model proposing that SNF1 exerts its effect through SSN6 on COX6 and CYC1.

Mol Cell Biol, 1990 Mar, 10(3), 1049 - 55
A highly specific phosphatase from Saccharomyces cerevisiae implicated in tRNA splicing; McCraith SM et al.; We identified and partially purified a phosphatase from crude extracts of Saccharomyces cerevisiae cells that can catalyze the last step of tRNA splicing in vitro . This phosphatase can remove the 2'-phosphate left over at the splice junction after endonuclease has removed the intron and ligase has joined together the two half-molecules . We suggest that this phosphatase is responsible for the completion of tRNA splicing in vivo, based primarily on its specificity for the 2'-phosphate of spliced tRNA and on the resistance of the splice junction 2'-phosphate to a nonspecific phosphatase . Removal of the splice junction 2'-phosphate from the residue adjacent to the anticodon is likely necessary for efficient expression of spliced tRNA . The phosphatase appears to be composed of at least two components which, together with endonuclease and ligase, can be used to reconstitute the entire tRNA-splicing reaction.

J Biotechnol, 1990 Mar, 13(4), 267 - 78
Pilot scale production of a heterologous Trichoderma reesei cellulase by Saccharomyces cerevisiae; Zurbriggen B et al.; Cellobiohydrolase II of Trichoderma reesei was produced in laboratory and pilot scale using a transformant strain of Saccharomyces cerevisiae harbouring a multicopy expression plasmid . Different strategies were compared for concentration and partial purification of the enzyme produced in a 200 1 pilot cultivation . After efficient separation of biomass and sub-cellular particulate matter, a combination of ultrafiltration and adsorbent treatment for removal of protein impurities was used to provide a concentrate for chromatographic purification . Effective purification of the CBH II protein was obtained by passing the concentrate through a column of DEAE Sepharose, on which almost all the yeast proteins were adsorbed . The purified enzyme reacted with antibodies prepared against T . reesei CBH II and catalyzed partial solubilization of crystalline cellulose to soluble sugars.

J Biol Chem, 1990 Feb 25, 265(6), 3362 - 8
RAS2 protein of Saccharomyces cerevisiae undergoes removal of methionine at N terminus and removal of three amino acids at C terminus; Fujiyama A et al.; RAS2 protein of Saccharomyces cerevisiae undergoes post-translational modifications involving methyl esterification and palmitic acid addition, resulting in their association with the plasma membrane . In this paper, we provide evidence that two kinds of proteolytic events accompany the biosynthesis . This is shown by separating and characterizing three intracellular forms of RAS2 protein: precursor, intermediate, and mature (fatty acid-acylated) forms . N-Terminal sequencing has revealed that all three forms start with proline, which is the second amino acid expected from the RAS2 gene sequence . Thus, the first methionine is removed very early during the biosynthesis . Isolation and sequencing of C-terminal peptides indicate that three C-terminal amino acids present in the precursor form are removed in the intermediate and in the fatty acid acylated forms . C-Terminal proteolysis appears to accompany methyl esterification, since the methylation occurs with the intermediate and the fatty acid-acylated forms, but not with the precursor . Palmitic acid is identified as the major fatty acid attached to the fatty acid-acylated form.

J Biol Chem, 1990 Feb 25, 265(6), 3234 - 9
Duplicated NHP6 genes of Saccharomyces cerevisiae encode proteins homologous to bovine high mobility group protein 1; Kolodrubetz D et al.; The nonhistone chromosomal protein NHP6 from Saccharomyces cerevisiae has been previously isolated and its amino-terminal sequence determined . In this report, synthetic oligonucleotides, designed from the limited NHP6 amino acid sequence, were used as hybridization probes to clone the NHP6A gene from a yeast genomic library . Low stringency Southern blot analysis showed that there was a second gene homologous to NHP6A . This gene, NHP6B, was also cloned and sequenced . Nucleotide sequence analysis revealed that NHP6B has six extra amino acids at its amino terminus, but that NHP6A and NHP6B match at 87% of the rest of their sequences . S1 nuclease analysis was used to show that both genes are transcribed; the major transcription start sites lie 30 bases before the first ATG codon . Interestingly, the approximately 11-kDa NHP6A and NHP6B proteins are homologous to the middle segment of the 27-kDa chromatin-associated high mobility group protein 1 from calf; NHP6A and NHP6B each have over 40% identity with this part of high mobility group protein 1 . Possible functions for the NHP6 proteins are discussed in light of this homology.

Nucleic Acids Res, 1990 Feb 25, 18(4), 771 - 8
Expression of the Saccharomyces cerevisiae DNA repair gene RAD6 that encodes a ubiquitin conjugating enzyme, increases in response to DNA damage and in meiosis but remains constant during the mitotic cell cycle; Madura K et al.; The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating (E2) enzyme and is required for the repair of damaged DNA, mutagenesis, and sporulation . Here, we report our studies on the regulation of RAD6 gene expression after UV damage, during the mitotic cell cycle, in meiosis, and following heat shock and starvation . RAD6 mRNA levels became elevated in cells exposed to UV light, and at all UV doses the increase in mRNA levels was rapid and occurred within 30 min after exposure to UV . RAD6 mRNA levels also increased in sporulating MATa/MAT alpha cells, and the period of maximal accumulation of RAD6 mRNA during meiosis is coincident with the time during which recombination occurs . However, RAD6 mRNA levels showed no periodic fluctuation in the mitotic cell cycle, were not elevated upon heat shock, and fell in cells in the stationary phase of growth . These observations suggest that RAD6 activity is required throughout the cell cycle rather than being restricted to a specific stage, and that during meiosis, high levels of RAD6 activity may be needed at a stage coincident with genetic recombination . The observation that RAD6 transcription is not induced by heat and starvation, treatments that activate stress responses, suggests that the primary role of RAD6 is in the repair of damaged DNA rather than in adapting cells to stress situations.

J Biol Chem, 1990 Feb 25, 265(6), 3332 - 9
Pseudo-wild type revertants from inactive apocytochrome b mutants as a tool for the analysis of the structure/function relationships of the mitochondrial ubiquinol-cytochrome c reductase of Saccharomyces cerevisiae; di Rago JP et al.; We have analyzed the structure/function relationships of the yeast mitochondrial cytochrome b with a new methodology based upon the isolation of pseudo-wild type revertants from well-characterized cytochrome b respiratory deficient mutants . Our goal was to determine how cytochrome b function could be restored in such mutants, at least to some degree, by suppressor mutations within the protein . True wild type revertants were differentiated from pseudo-wild type revertants by the use of a simple and rapid screening technique based upon oligonucleotide hybridization . This can easily be used to analyze a large number of revertants . The suppressor mutations responsible for the restoration of respiratory competence were identified by sequencing the revertant's cytochrome b mRNA in crude mitochondrial RNA preparations . Using this new method we have analyzed 210 independent revertants . We report here nine novel cytochrome b structures conferring a variety of respiratory sufficient phenotypes, obtained from five respiratory deficient mutations affecting a short region of the protein (positions 131-138 of the polypeptide chain), presumably belonging to the ubiquinol oxidizing center of the bc1 complex.

J Biol Chem, 1990 Feb 15, 265(5), 2733 - 9
Differential stability of two apo-isocytochromes c in the yeast Saccharomyces cerevisiae; Dumont MD et al.; The yeast Saccharomyces cerevisiae contains two forms of cytochrome c, iso-1-cytochrome c and iso-2-cytochrome c, encoded by the genes CYC1 and CYC7, respectively . The amino acid sequences of these two isozymes are approximately 80% identical . Cyc3- mutants lack both holocytochromes c, because of a deficiency of cytochrome c heme lyase, the enzyme catalyzing covalent attachment of the heme group to apocytochrome c . A deficiency of heme lyase also prevents import into mitochondria . Surprisingly, apo-iso-1-cytochrome c is absent in cyc3- strains, although apo-iso-2-cytochrome c is present at approximately the same level at which holo-iso-2-cytochrome c is found in CYC3+ strains . The lack of apo-iso-1-cytochrome c is not due to a deficiency of either transcription or translation, but to rapid degradation of the protein . Apocytochromes c encoded by composite cytochrome c genes composed of the central portion of iso-2-cytochrome c flanked by amino and carboxyl regions of iso-1-cytochrome c exhibit increased stability compared with apo-iso-1-cytochrome c . A region encompassing no more than four amino acid differences between iso-1- and iso-2-cytochromes c is sufficient to partially stabilize the protein . In contrast to what is observed in vivo with the apo forms, the holo forms of the composite isocytochromes c are even less stable to thermal denaturation than iso-1-cytochrome c or iso-2-cytochrome c . Either a small region of the sequence of apo-iso-1-cytochrome c is involved in degradation of the protein, or the corresponding region in apo-iso-2-cytochrome c is preventing degradation . The differential stability of the two isocytochromes c may be part of a regulatory process that increases the proportion of iso-2-cytochrome c under certain physiological conditions.

Eur J Biochem, 1990 Feb 14, 187(3), 611 - 6
Induction of the 4-aminobutyrate and urea-catabolic pathways in Saccharomyces cerevisiae . Specific and common transcriptional regulators; Vissers S et al.; In the yeast Saccharomyces cerevisiae, induction of the 4-aminobutyrate-catabolic pathway by 4-aminobutyrate requires two positive regulatory factors, encoded by the UGA3 and the UGA35 genes respectively . In addition to this, expression of one gene of this pathway, namely the UGA4 gene encoding the 4-aminobutyrate-specific permease, is controlled negatively by the product of the UGA43 gene {Vissers, S., Andre, B., Muyldermans, F . & Grenson, M . (1989) Eur . J . Biochem . 181, 357-361} . We show here that the products of two of these regulatory genes, UGA35 and UGA43, also control the expression of the genes encoding the urea-catabolic pathway, although the 4-aminobutyrate and urea-catabolic pathways are synthesised under specific conditions and do not share any enzymatic step or metabolite: the UGA35 pathways are synthesised under specific conditions and do not share any enzymatic step or metabolite: the UGA35 gene is shown to be identical to the DURL gene which was previously identified as a positive regulatory factor of the urea-catabolic pathway; the UGA43 gene product is shown to behave like a negative regulatory factor of this pathway . In contrast to UGA35/DURL and UGA43, the positive regulatory factors encoded by the UGA3 gene and the previously identified DURM gene specifically control 4-aminobutyrate and urea catabolisms respectively . Northern hybridization experiments suggest that the UGA35/DURL and UGA43 common regulatory factors act at the transcriptional level . Our results show that the expression of two biochemically distinct nitrogenous catabolisms, as triggered by their respective inducers, seems to involve multiple regulatory factors, some of which are common to the two catabolic pathways.

Genetics, 1990 Feb, 124(2), 283 - 91
The CCR4 gene from Saccharomyces cerevisiae is required for both nonfermentative and spt-mediated gene expression; Denis CL et al.; Mutations in the yeast CCR4 gene inhibit expression of the glucose-repressible alcohol dehydrogenase (ADH2), as well as other nonfermentative genes, and suppress increased ADH2 expression caused by the cre1 and cre2 alleles . Both the cre1 and ccr4 alleles were shown to affect ADH II enzyme activity by altering the levels of ADH2 mRNA . Mutations in either CRE1 or CRE2 bypassed the inhibition of ADH2 expression caused by delta insertions at the ADH2 promoter which displace the ADH2 activation sequences 336 bp upstream of the TATA element . These cre1 and cre2 effects were suppressible by the ccr4 allele . The cre1 and ccr4 mutations also affected ADH2 expression when all the ADH2 regulatory sequences upstream of the TATA element were deleted . The relationship of the CRE genes to the SPT genes, which when mutated are capable of bypassing the inhibition of HIS4 expression caused by a delta promoter insertion (his4-912 delta allele), was examined . Both the cre1 and cre2 mutations allowed his4-912 delta expression . ccr4 mutations were able to suppress the ability of the cre alleles to increase his4-912 delta expression . CRE2 was shown to be allelic to the SPT6 gene, and CRE1 was found to be allelic to SPT10 . We suggest that the CRE genes comprise a general transcriptional control system in yeast that requires the function of the CCR4 gene.

Genetics, 1990 Feb, 124(2), 275 - 82
Identification and characterization of a mutation affecting the division arrest signaling of the pheromone response pathway in Saccharomyces cerevisiae; Fujimura H; Mating pheromones, a- and alpha-factors, arrest the division of cells of opposite mating types, alpha and a cells, respectively . I have isolated a sterile mutant of Saccharomyces cerevisiae that is defective in division arrest in response to alpha-factor but not defective in morphological changes and agglutinin induction . The mutation was designated dac2 for division arrest control by mating pheromones . The dac2 mutation was closely linked to gal1 and was different from the previously identified cell type nonspecific sterile mutations (ste4, ste5, ste7, ste11, ste12, ste18 and dac1) . Although dac2 cells had no phenotype in the absence of pheromones, they showed morphological alterations and divided continuously in the presence of pheromones . As a result, dac2 cells had a mating defect . The dac2 mutation could suppress the lethality caused by the disruption of the GPA1 gene (previously shown to encode a protein with similarity to the alpha subunit of mammalian G proteins) . In addition, dac2 cells formed prezygotes with wild-type cells of opposite mating types, although they could not undergo cell fusion . These results suggest that the DAC2 product may control the signal for G-protein-mediated cell-cycle arrest and indicate that the synchronization of haploid yeast cell cycles by mating pheromones is essential for cell fusion during conjugation.

Genetics, 1990 Feb, 124(2), 263 - 73
Distance-independence of mitotic intrachromosomal recombination in Saccharomyces cerevisiae; Yuan LW et al.; Many genetic studies have shown that the frequency of homologous recombination depends largely on the distance in which recombination can occur . We have studied the effect of varying the length of duplicated sequences on the frequency of mitotic intrachromosomal recombination in Saccharomyces cerevisiae . We find that the frequency of recombination resulting in the loss of one of the repeats and the intervening sequences reaches a plateau when the repeats are short . In addition, the frequency of recombination to correct a point mutation contained in one of these repeats is not proportional to the size of the duplication but rather depends dramatically on the location of the mutation within the repeated sequences . However, the frequency of mitotic interchromosomal reciprocal recombination is dependent on the distance separating the markers . The difference in the response of intrachromosomal and interchromosomal mitotic recombination to increasing lengths of homology may indicate there are different rate-limiting steps for recombination in these two cases . These findings have important implications for the maintenance and evolution of duplicated sequences.

Genetics, 1990 Feb, 124(2), 237 - 49
Mitotic chromosome transmission fidelity mutants in Saccharomyces cerevisiae; Spencer F et al.; We have isolated 136 independent mutations in haploid yeast strains that exhibit decreased chromosome transmission fidelity in mitosis . Eighty-five percent of the mutations are recessive and 15% are partially dominant . Complementation analysis between MATa and MAT alpha isolates identifies 11 chromosome transmission fidelity (CTF) complementation groups, the largest of which is identical to CHL1 . For 49 independent mutations, no corresponding allele has been recovered in the opposite mating type . The initial screen monitored the stability of a centromere-linked color marker on a nonessential yeast chromosome fragment; the mitotic inheritance of natural yeast chromosome III is also affected by the ctf mutations . Of the 136 isolates identified, seven were inviable at 37 degrees and five were inviable at 11 degrees . In all cases tested, these temperature conditional lethalities cosegregated with the chromosome instability phenotype . Five additional complementation groups (ctf12 through ctf16) have been defined by complementation analysis of the mutations causing inviability at 37 degrees . Twenty-three of the 136 isolates exhibited growth defects at concentrations of benomyl permissive for the parent strain, and nine appeared to be tolerant of inhibitory levels of benomyl . All of the mutant strains showed normal sensitivity to ultraviolet and gamma-irradiation . Further characterization of these mutant strains will describe the functions of gene products crucial to the successful execution of processes required for aspects of the chromosome cycle that are important for chromosome transmission fidelity in mitosis.

Mol Cell Biol, 1990 Feb, 10(2), 810 - 5
ARS binding factor I of the yeast Saccharomyces cerevisiae binds to sequences in telomeric and nontelomeric autonomously replicating sequences; Biswas SB et al.; We have analyzed various autonomously replicating sequences (ARSs) in yeast nuclear extract with ARS-specific synthetic oligonucleotides . The EI oligonucleotide sequence, which is derived from HMRE-ARS, and the F1 oligonucleotide sequence, which is derived from telomeric ARS120, appeared to bind to the same cellular factor with high specificity . In addition, each of these oligonucleotides was a competitive inhibitor of the binding of the other . Binding of the ARS binding factor (ABF) to either of these oligonucleotides was inhibited strongly by plasmids containing ARS1 and telomeric TF1-ARS . DNase I footprinting analyses with yeast nuclear extract showed that EI and F1 oligonucleotides eliminated protection of the binding site of ARS binding factor I (ABFI) in domain B of ARS1 . Sequence analyses of various telomeric (ARS120 and TF1-ARS) and nontelomeric ARSs (ARS1 and HMRE-ARS) showed the presence of consensus ABFI binding sites in the protein binding domains of all of these ARSs . Consequently, the ABFI and ABFI-like factors bind to these domain B-like sequences in a wide spectrum of ARSs, both telomeric and nontelomeric.

Mol Cell Biol, 1990 Feb, 10(2), 672 - 80
Genetic and biochemical evaluation of eucaryotic membrane protein topology: multiple transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl coenzyme A reductase; Sengstag C et al.; Both 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase isozymes of the yeast Saccharomyces cerevisiae are predicted to contain seven membrane-spanning domains . Previous work had established the utility of the histidinol dehydrogenase protein domain, encoded by HIS4C, as a topologically sensitive monitor that can be used to distinguish between the lumen of the endoplasmic reticulum and the cytoplasm . This study directly tested the structural predictions for HMG-CoA reductase by fusing the HIS4C domain to specific sites in the HMG-CoA reductase isozymes . Yeast cells containing the HMG-CoA reductase-histidinol dehydrogenase fusion proteins grew on histidinol-containing medium if the HIS4C domain was present on the cytoplasmic side of the endoplasmic reticulum membrane but not if the HIS4C domain was targeted to the endoplasmic reticulum lumen . Systematic exchanges of transmembrane domains between the isozymes confirmed that both isozymes had equivalent membrane topologies . In general, deletion of an even number of putative transmembrane domains did not interfere with the topology of the protein, but deletion or duplication of an odd number of transmembrane domains inverted the orientation of the protein . The data confirmed the earlier proposed topology for yeast HMG-CoA reductase, demonstrated that the yeast enzymes are core glycosylated, and provided in vivo evidence that the properties of transmembrane domains were, in part, dependent upon their context within the protein.

Curr Genet, 1990 Feb, 17(2), 105 - 11
Cloning and sequencing of URA10, a second gene encoding orotate phosphoribosyl transferase in Saccharomyces cerevisiae; de Montigny J et al.; Orotate phosphoribosyl transferase (OP-RTase) catalyses the transformation of orotate to OMP in the pyrimidine pathway . In the yeast Saccharomyces cerevisiae, the URA5 gene is known to encode this enzyme activity . In this paper we present the cloning and sequencing of a yeast gene, named URA10, encoding a second OPRTase enzyme . Comparison of the predicted amino acid sequences between URA5 and URA10 genes shows more than 75% similarity . These sequences have also been compared to those of Escherichia coli, Podospora anserina, Sordaria macrospora and Dictyostelium discoideum . Remarkable similarities in the primary structure of these proteins have been found . Gene disruption experiments revealed that URA10 gene expression is responsible for the leaky phenotype of a ura5 mutant . Assays of OPRTase activity in extracts from ura5 and ura10 mutants indicate that the URA10 product contributes only 20% of the total activity found in wild type cells.

Biochem Cell Biol, 1990 Feb, 68(2), 427 - 35
Regulation of proliferation by the budding yeast Saccharomyces cerevisiae; Johnston GC et al.; Mutations in the budding yeast Saccharomyces cerevisiae define regulatory activities both for the mitotic cell cycle and for resumption of proliferation from the quiescent stationary-phase state . In each case, the regulation of proliferation occurs in the prereplicative interval that precedes the initiation of DNA replication . This regulation is particularly responsive to the nutrient environment and the biosynthetic capacity of the cell . Mutations in components of the cAMP-mediated effector pathway and in components of the biosynthetic machinery itself affect regulation of proliferation within the mitotic cell cycle . In the extreme case of nutrient starvation, cells cease proliferation and enter stationary phase . Mutations in newly defined genes prevent stationary-phase cells from reentering the mitotic cell cycle, but have no effect on proliferating cells . Thus stationary phase represents a unique developmental state, with requirements to resume proliferation that differ from those for the maintenance of proliferation in the mitotic cell cycle.

Appl Biochem Biotechnol, 1990 Feb, 23(2), 181 - 6
Studies on Saccharomyces cerevisiae under carbon-limiting growth transformed with plasmid pCYG4 that carries the gene for NADP-GDH; Lima Filho JL et al.; The gene (GDH1) coding for the NADP-linked glutamate dehydrogenase system (NADP-GDH) has been cloned from Saccharomyces cerevisiae strain . Cells being transformed by the NADP-GDH gene on a 2 micron bared vector (pCYG4) plasmid confering 11-fold higher level on expressed GDH activity over the wild-type cells . The behavior of these cells was investigated under chemostatic growth with a carbon rate-limiting nutrient . Specific growth rates of cells carrying plasmid pCYG4 were found to be slightly slower than wild type cells . Furthermore, the NADP-GDH activity increases proportionally with the dilution rate . In addition, oscillations in the NADP-GDH activity, especially at a dilution rate up to 0.15/h, are probably consequential on the appearance of a changing mixed population (cells with and without plasmids).

J Bacteriol, 1990 Feb, 172(2), 1133 - 6
Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by cyclic AMP-dependent protein kinase; Kinney AJ et al.; The addition of cyclic AMP (cAMP) to Saccharomyces cerevisiae cyr1 mutant cells resulted in an increase in the rate of phosphatidylinositol synthesis at the expense of phosphatidylserine synthesis . The decrease in phosphatidylserine synthesis correlated with the down regulation of phosphatidylserine synthase activity by cAMP-dependent protein kinase phosphorylation . The increase in phosphatidylinositol synthesis was not due to the regulation of phosphatidylinositol synthase by cAMP-dependent protein kinase.

J Bacteriol, 1990 Feb, 172(2), 1014 - 8
The GLN3 gene product is required for transcriptional activation of allantoin system gene expression in Saccharomyces cerevisiae; Cooper TG et al.; We show that mutation at the GLN3 locus results in decreased steady-state levels of DAL7, DUR1,2, CAR1, and URA3 mRNAs derived from cultures grown in the presence of inducer . Basal levels of these RNA species, however, were not significantly affected by a gln3 mutation . The GLN3 product appears to affect gene expression in two ways . The pleiotropic requirement of GLN3 for induced gene expression probably derives from the need of the GLN3 product for inducer uptake into the cell and its loss in gln3 mutants . We also demonstrate that transcriptional activation, mediated by the DAL5 and DAL7 upstream activation sequences, requires a functional GLN3 gene product . This observation identified transcriptional activation as the most likely point of GLN3 participation in the expression of allantoin system genes.

Curr Genet, 1990 Feb, 17(2), 97 - 103
Biolistic nuclear transformation of Saccharomyces cerevisiae and other fungi; Armaleo D et al.; Tungsten microprojectiles coated with nucleic acid and accelerated to velocities of approximately 500 m/s, can penetrate living cells and tissues with consequent expression of the introduced genes (Klein et al . 1987) . Saccharomyces cerevisiae is used here as a model system to define the basic parameters governing the biolistic (biological-ballistic) delivery of DNA into cells . Among the physical factors affecting the efficiency of the process in yeast are the microprojectile's constitution, size, concentration and amount, and the procedure used for binding DNA to it . The biological parameters that affect the process include the cell's genotype, growth phase, plating density, and the osmotic composition of the medium during bombardment . By optimizing these physical and biological parameters, rates of transformation between 10(-5) and 10(-4) were achieved . Stable nuclear transformants result primarily from penetration of single particles of 0.5-0.65 micron in diameter, delivering on average 10-30 biologically active plasmids into the cell . The tungsten particles detectably increase the buoyant density of the transformants' progenitors.

Proc Natl Acad Sci U S A, 1990 Feb, 87(3), 1159 - 63
An essential member of the HSP70 gene family of Saccharomyces cerevisiae is homologous to immunoglobulin heavy chain binding protein; Nicholson RC et al.; Immunoglobulin heavy chain binding protein (BiP) is present in the lumen of the mammalian endoplasmic reticulum, where it associates transiently with a variety of newly synthesized secretory and membrane proteins or permanently with mutant proteins that are incorrectly folded . We describe a unique member of the Saccharomyces cerevisiae 70-kDa heat shock protein gene family (HSP70) that encodes a protein homologous to mammalian BiP . The DNA sequence contains a 2046-nucleotide open reading frame devoid of introns, and examination of the predicted amino acid sequence reveals features not found in most other yeast HSP70 proteins but which are present in BiP . Most notable are a 42-residue sequence at the N terminus that exhibits characteristics of a cleavable signal sequence and a C-terminal sequence, -His-Asp-Glu-Leu, that is involved in determining endoplasmic reticulum localization in yeast . The 5' flanking region of this gene contains two overlapping sequences between nucleotides -146 and -169 that closely resemble consensus heat shock elements . The yeast BiP gene is strongly heat shock-inducible, whereas the BiP genes in various other species are either weakly or non-heat-inducible . We demonstrate that a functional BiP gene is essential for vegetative growth . An evolutionary comparison of amino acid sequences of 34 HSP70 proteins from 17 species suggests that BiP genes share a common ancestor, which diverged from other HSP70 genes near the time when eukaryotes first appeared.

Mol Cell Biol, 1990 Feb, 10(2), 510 - 7
Stoichiometry of G protein subunits affects the Saccharomyces cerevisiae mating pheromone signal transduction pathway; Cole GM et al.; The Saccharomyces cerevisiae GPA1, STE4, and STE18 genes encode products homologous to mammalian G-protein alpha, beta, and gamma subunits, respectively . All three genes function in the transduction of the signal generated by mating pheromone in haploid cells . To characterize more completely the role of these genes in mating, we have conditionally overexpressed GPA1, STE4, and STE18, using the galactose-inducible GAL1 promoter . Overexpression of STE4 alone, or STE4 together with STE18, generated a response in haploid cells suggestive of pheromone signal transduction: arrest in G1 of the cell cycle, formation of cellular projections, and induction of the pheromone-inducible transcript FUS1 25- to 70-fold . High-level STE18 expression alone had none of these effects, nor did overexpression of STE4 in a MATa/alpha diploid . However, STE18 was essential for the response, since overexpression of STE4 was unable to activate a response in a ste18 null strain . GPA1 hyperexpression suppressed the phenotype of STE4 overexpression . In addition, cells that overexpressed GPA1 were more resistant to pheromone and recovered more quickly from pheromone than did wild-type cells, which suggests that GPA1 may function in an adaptation response to pheromone.

Proc Natl Acad Sci U S A, 1990 Feb, 87(3), 1076 - 80
Mutants of Saccharomyces cerevisiae that block intervacuole vesicular traffic and vacuole division and segregation; Weisman LS et al.; Intervacuole vesicular exchange and the segregation of parental vacuole material into the bud are strikingly impaired in a temperature-sensitive yeast mutant, vac1-1 . At the nonpermissive temperature, haploid vac1-1 cells show a pronounced delay in separation of mature buds from the mother cell and accumulate cells with multiple buds . At both the permissive and restrictive temperatures, daughter cells are produced that lack a detectable vacuole or contain a very small vacuole . In zygotes, vacuoles from a vac1-1 strain are defective as donors, or recipients, of the vesicles of intervacuole vesicular traffic . These defects are specific for the vacuole in that the segregation of nuclear DNA and of mitochondria into the bud appears normal . The isolation of the vac1-1 mutation is a first step in the genetic characterization of vacuole division and segregation.

Biochem Biophys Res Commun, 1990 Jan 30, 166(2), 681 - 6
Calmodulin-binding proteins of Saccharomyces cerevisiae; Liu YS et al.; The subcellular distribution of calmodulin-binding proteins in the soluble, plasma membrane, and nuclear fractions of Saccharomyces cerevisiae was analyzed with a gel binding assay using 125I-labeled calmodulin . Over 20 binding proteins were detected . The calmodulin-binding protein profiles were markedly different among the fractions . Calmodulin-binding proteins were most abundant in the nuclear fraction, followed by the membrane fraction and the soluble fraction in decreasing order . The amounts of certain calmodulin-binding proteins increased after treatment with alpha-mating factor.

Science, 1990 Jan 26, 247(4941), 464 - 7
Mutations of the adenylyl cyclase gene that block RAS function in Saccharomyces cerevisiae; Field J et al.; The interaction between RAS proteins and adenylyl cyclase was studied by using dominant interfering mutations of adenylyl cyclase from the yeast Saccharomyces cerevisiae . RAS proteins activate adenylyl cyclase in this organism . A plasmid expressing a catalytically inactive adenylyl cyclase was found to interfere dominantly with this activation . The interfering region mapped to the leucine-rich repeat region of adenylyl cyclase, which is homologous to domains present in several other proteins and is thought to participate in protein-protein interactions.

Eur J Biochem, 1990 Jan 26, 187(2), 315 - 20
Occurrence of S-(1,2-dicarboxyethyl)-cysteine at position 77 in mutant human lysozyme secreted by Saccharomyces cerevisiae; Kikuchi M et al.; A mutant human lysozyme P110, in which Val110 was replaced with Pro, was secreted by Saccharomyces cerevisiae; modification of the cysteine residue at position 77 was found in a purified mutant protein (P110-B) upon primary structure analysis . A peptide fragment containing 15 amino acid residues from Thr70 to Leu84 was obtained by proteolytic digestion of the protein and subsequently isolated by reverse-phase HPLC . This fragment was analyzed by high-resolution fast-atom-bombardment (FAB) mass spectrometry, which showed that 1,2-dicarboxyethyl group was attached to the thiol group of Cys77 . This modification was confirmed by comparing it with a sample of chemically synthesized S-(1,2-dicarboxyethyl)-L-cysteine . It was found that the modification caused a disruption of the disulfide bond Cys77-Cys95 in the mutant molecule . These observations, plus structural considerations, suggest that Cys77 and Cys95 either remain uncrosslinked or the disulfide bond Cys77-Cys95, once formed, is opened during the final step in the folding of human lysozyme in vivo.

J Biol Chem, 1990 Jan 25, 265(3), 1601 - 7
The purification and characterization of arginase from Saccharomyces cerevisiae; Green SM et al.; In Saccharomyces cerevisiae, ornithine transcarbamoylase and arginase form a regulatory multienzyme complex (Hensley, P . (1988) Curr . Top . Cell . Regul . 29, 35-75) . In this complex, arginase acts as a negative allosteric effector for ornithine transcarbamoylase . Before an analysis of the factors which promote and stabilize complex formation, arginase was purified in milligram quantities from a plasmid-containing, enzyme-overproducing, protease-deficient yeast strain and its physical characterization undertaken . The purified enzyme has a specific activity of 885 mumol urea min-1 mg-1 and a Km for arginine of 15.7 mM . The ultraviolet spectrum has a maximum absorbance at 279 nm, and the steady-state fluorescence emission spectrum has a maximum intensity at 337 nm, suggesting that the 3 tryptophans/polypeptide chain are in a relatively hydrophobic environment . Arginase has a weakly bound manganese responsible for the maintenance of the catalytic activity and is known to be heat activated in the presence of manganese . This effect is half-maximal at 12.1 microM manganese . In addition to a catalytic requirement for manganese, the presence of a more tightly bound metal is suggested from sedimentation studies . The native trimeric enzyme has a sedimentation coefficient of 5.95 S . Removal of the weakly associated metal results in no change in the sedimentation coefficient . However, dialysis with EDTA causes the s-value to decrease to 4.65 S, suggesting that under these conditions, the trimeric enzyme may partially dissociate . An analysis of CD spectra shows that significant spectral changes result from the removal of both the weakly bound metal and dialysis against EDTA.

J Biol Chem, 1990 Jan 25, 265(3), 1755 - 64
The sn-1,2-diacylglycerol cholinephosphotransferase of Saccharomyces cerevisiae . Nucleotide sequence, transcriptional mapping, and gene product analysis of the CPT1 gene; Hjelmstad RH et al.; The complete nucleotide sequence of the Saccharomyces cerevisiae CPT1 gene, a structural gene for the sn-1,2-diacylglycerol cholinephosphotransferase (Hjelmstad, R . H., and Bell, R . M . (1987) J . Biol . Chem . 262, 3909-3917), was determined . The 2,100-nucleotide extent of DNA sequenced contained an open reading frame encoding 407 amino acids interrupted by an intron near its 5'-end . Northern hybridization analysis detected the presence of 1.4- and 1.7-kilobase transcripts corresponding to the CPT1 gene . S1 nuclease mapping experiments indicated that the 1.4-kilobase transcript was initiated 80 nucleotides upstream from the translational start site near a poly(dA-dT) promoter element and established that the predicted intron was removed in vivo . The previously constructed cpt1::LEU2 insertional mutation was shown to involve disruption of the CPT1 open reading frame approximately in the middle; this construct did not support the production of a stable transcript . The CPT1 promoter region contained several elements homologous to the promoter regions of other phospholipid biosynthetic structural genes . A model for the membrane topography of the predicted 46,305-dalton cholinephosphotransferase was constructed on the basis of predictive methods . The presence of seven transmembrane helices and an asymmetric distribution of hydrophilic regions were predicted . Regional protein homologies to the acetylcholine receptor, phosphoglycerate kinase, and several cytidine diphosphate utilizing enzymes suggested a functional asymmetry which precisely correlated with the predicted topological asymmetry.

J Biol Chem, 1990 Jan 25, 265(3), 1594 - 600
Identification of the CBP1 polypeptide in mitochondrial extracts from Saccharomyces cerevisiae; Weber ER et al.; CBP1 is a nuclearly encoded yeast protein required for stability of mitochondrial cytochrome b pre-mRNA . Previous studies have shown that CBP1 stabilizes the cytochrome b transcripts via interaction with the 5'-end . For the present study, both rabbit polyclonal and mouse monoclonal antibodies against CBP1 were prepared using a trpE-CBP1 fusion polypeptide as a source of antigen . CBP1 was undetectable in a crude mitochondrial fraction from a wild-type strain by Western blot assay, but a 66-kDa immunoreactive protein was detected in a more purified fraction . The 66-kDa protein was absent in the equivalent fraction from a strain with a deletion in CBP1 . Assignment of Mr = 66,000 to the mature CBP1 polypeptide was verified by Western analysis of mitochondria from a strain which over-expresses CBP1 . Mitochondrial localization was verified by transcribing CBP1 in vitro with T3 polymerase, translating the artificial mRNA in a rabbit reticulocyte system and importing 35S-CBP1 precursor polypeptides into isolated mitochondria . The mature protein product was 66 kDa, whereas the precursor protein migrated as if it were 68 kDa rather than 76 kDa as predicted from the sequence . Analysis of polypeptides truncated at the carboxyl terminus showed that CBP1 polypeptides migrate anomalously fast in the Laemmli system due to a property of the carboxyl two-thirds of the primary sequence, several sections of which are extremely basic.

J Biol Chem, 1990 Jan 25, 265(3), 1563 - 8
Different kinetic properties of the two mutants, RAS2Ile152 and RAS2Val19, that suppress the CDC25 requirement in RAS/adenylate cyclase pathway in Saccharomyces cerevisiae; Crechet JB et al.; The properties of RAS2Gly19----Val and RAS2Thr152----Ile, two mutants suppressing the CDC25 requirement for the activation of adenylate cyclase in Saccharomyces cerevisiae, were compared with the properties of wild-type RAS2 . We examined (a) the guanine nucleotide interaction, (b) the intrinsic GTPase (EC 3.6.1-) activity, and (c) the ability to activate adenylate cyclase in vitro . The low GTPase of RAS2Val19 is associated with an increased stability of the GTP complex . By contrast, RAS2Ile152 shows a strong destabilization of the GDP complex (the dissociation rate constants of the RAS2Ile152.GDP complex is enhanced almost 50 times) and an increased GTPase activity . Remarkably, all the parameters of the interaction with GDP and GTP as well as the catalytic activity are modified by the two mutations in an opposite manner . Our kinetic results show that the functional modifications of RAS2 compensating for the CDC25 inactivation can not only be associated with the presence of a long-lived RAS2.GTP complex, but also with a rapid GDP to GTP exchange reaction . As a striking result, the functional modifications induced by Thr152----Ile activate the adenylate cyclase in vitro much more efficiently than those induced by Gly19----Val . This stresses the importance of a rapid regeneration of the RAS2.GTP complex for the activation of the adenylate cyclase pathway.

J Biol Chem, 1990 Jan 5, 265(1), 281 - 5
Intramitochondrial transfer of phospholipids in the yeast, Saccharomyces cerevisiae; Simbeni R et al.; Translocation of phosphatidylinositol, which is synthesized on the outer aspect of the outer membrane of isolated yeast mitochondria, to the inner membrane is linked to phosphatidylinositol synthesis and is therefore a vectorial process . Phosphatidylinositol once integrated into the inner mitochondrial membrane is not transferred back to the mitochondrial surface . Phosphatidylserine is also translocated from the outer to the inner mitochondrial membrane, where it is decarboxylated to phosphatidylethanolamine . We made use of this metabolic modification to characterize the intramitochondrial transfer of phosphatidylserine and phosphatidylethanolamine . Intramitochondrial phosphatidylserine transfer is insensitive to the uncoupler carbonyl cyanide m-chlorophenylhydrazone and to valinomycin and is thus independent of an electrochemical gradient across the inner membrane . Transfer of phosphatidylserine from the outer to the inner mitochondrial membrane occurs not only in intact mitochondria but also in mitoplasts which are devoid of intermembrane space proteins but have the outer membrane still adherent to the inner membrane . This result suggests that specific contact sites are involved in the intramitochondrial translocation of phospholipids . 3H-Labeled phosphatidylethanolamine synthesized from {3H}serine in isolated mitochondria is readily exported from the inner to the outer mitochondrial membrane without prior mixing with the pool of phosphatidylethanolamine of the inner membrane.

Genetics, 1990 Jan, 124(1), 81 - 90
Size selection identifies new genes that regulate Saccharomyces cerevisiae cell proliferation; Prendergast JA et al.; A centrifugation procedure to enrich for enlarged cells has been used to isolate temperature-sensitive cdc mutants of the yeast Saccharomyces cerevisiae . Among these mutants are strains containing mutations that arrest proliferation at the regulatory step start . These new start mutations define two previously unidentified genes, CDC67 and CDC68, and reveal that a previously identified gene, DNA33 (here termed CDC65), can harbour start mutations . Each new start mutation permits significant biosynthetic activity after transfer of mutant cells to the non-permissive temperature . The cdc68-1 start mutation causes arrest of cell proliferation without inhibition of mating ability, while the cdc65-1 and cdc67-1 mutations inhibit zygote formation and successful conjugation . The identification of new start genes by a novel selection procedure suggests that the catalog of genes that influence start is large.

Genetics, 1990 Jan, 124(1), 7 - 25
Mechanisms of gene conversion in Saccharomyces cerevisiae; Roman H et al.; In red-white sectored colonies of Saccharomyces cerevisiae, derived from mitotic cells grown to stationary phase and irradiated with a light dose of x-rays, all of the segregational products of gene conversion and crossing over can be ascertained . Approximately 80% of convertants are induced in G1, the remaining 20% in G2 . Crossing over, in the amount of 20%, is found among G1 convertants but most of the crossovers are delayed until G2 . About 20% of all sectored colonies had more than one genotype in one or the other sector, thus confirming the hypothesis that conversion also occurs in G2 . The principal primary event in G2 conversion is a single DNA heteroduplex . It is suggested that the close contact that this implies carries over to G2 when crossing over and a second round of conversion occurs.

Genetics, 1990 Jan, 124(1), 27 - 38
Genetic instability of clathrin-deficient strains of Saccharomyces cerevisiae; Lemmon SK et al.; Saccharomyces cerevisiae strains carrying a mutation in the clathrin heavy chain gene (CHC1) are genetically unstable and give rise to heterogeneous populations of cells . Manifestations of the instability include increases in genome copy number as well as compensatory genetic changes that allow better growing clathrin-deficient cells to take over the population . Increases in genome copy number appear to result from changes in ploidy as well as alterations in normal nuclear number . Genetic background influences the frequency at which cells with increased genome content are observed in different Chc- strains . We cannot distinguish whether genetic background affects the rate at which aberrant nuclear division events occur or a growth advantage of cells with increased nuclear and/or genome content . However, survival of chc1-delta cells does not require an increase in genome copy number . The clathrin heavy chain gene was mapped 1-2 cM distal to KEX1 on the left arm of chromosome VII by making use of integrated 2 mu plasmid sequences to destabilize distal chromosome segments and allow ordering of the genes.

Mutat Res, 1990 Jan, 235(1), 1 - 7
Photobiological effects in Saccharomyces cerevisiae induced by the monofunctional furocoumarin 4,4',6-trimethylangelicin (TMA) and the bifunctional furocoumarin 8-methoxypsoralen (8-MOP); Bianchi L et al.; Recently, the monofunctional furocoumarin 4,4',6-trimethylangelicin (TMA) has been proposed for photochemotherapeutic use . In order to assess its genotoxic potential, the photobiological (genetic) effects of TMA were studied in a diploid strain of Saccharomyces cerevisiae (D7) and compared to those of the bifunctional furocoumarin 8-methoxypsoralen (8-MOP) . At equimolar concentrations, the induction of lethal effects by TMA in the presence of equal 365-nm radiation was higher than that exerted by 8-MOP . TMA was also more active than 8-MOP in inducing nuclear events such as nuclear reverse mutation and mitotic recombination (crossing-overs and gene conversion) per unit dose of 365-nm radiation . At equal survival, however, TMA was less efficient in inducing reverse mutation and crossing-over, showing the same activity as 8-MOP in the induction of gene conversion . TMA was more active than 8-MOP in the induction of cytoplasmic 'petite' mutations per unit dose of 365-nm radiation and per viable cell . The high photobiological activity of this monofunctional furocoumarin is mainly related to its strong DNA photobinding but also to the type of monoaddition induced, to the sequential distribution in DNA and to the generation of active forms of oxygen.

EMBO J, 1990 Jan, 9(1), 271 - 7
The U6 gene of Saccharomyces cerevisiae is transcribed by RNA polymerase C (III) in vivo and in vitro; Moenne A et al.; Unlike the majority of genes encoding small nuclear RNAs, which are transcribed by RNA polymerase B, the U6 gene contains features found in both class B and class C genes, indicating the involvement of a combination of transcription factors normally specific to each class of genes . We present direct genetic and biochemical evidence that the U6 gene of Saccharomyces cerevisiae is transcribed by RNA polymerase C in vivo as well as in vitro . A mutant strain with a temperature-sensitive defect in the large subunit of RNA polymerase C that results in defective transcription of tRNA and 5S RNA genes shows a corresponding defect in U6 RNA levels . Also, purified RNA polymerase C transcribes the U6 gene when supplemented with partially purified TFIIIB . The other class C transcription factors, TFIIIA and Tau (TFIIIC), are not required in this system.

Mol Cell Biol, 1990 Jan, 10(1), 409 - 12
The sum1-1 mutation affects silent mating-type gene transcription in Saccharomyces cerevisiae; Livi GP et al.; The silent mating-type genes (HML and HMR) of Saccharomyces cerevisiae are kept under negative transcriptional control by the trans-acting products of the four MAR/SIR loci . MAR/SIR gene mutations result in the simultaneous derepression of HML and HMR gene expression . The sum1-1 mutation was previously identified as an extragenic suppressor of mutations in MAR1 (SIR2) and MAR2 (SIR3) . As assayed genetically, sum1-1 is capable of restoring repression of silent mating-type information in cells containing mar1 or mar2 null mutations . We show here that the mating-type phenotype associated with sum1-1 results from a dramatic reduction in the steady-state level of HML and HMR gene transcripts . At the same time, the sum1-1 mutation has no significant effect on the level of each of the four MAR/SIR mRNAs.

Mol Cell Biol, 1990 Jan, 10(1), 265 - 72
Reversion of autonomously replicating sequence mutations in Saccharomyces cerevisiae: creation of a eucaryotic replication origin within procaryotic vector DNA; Kipling D et al.; To investigate how a defective replicon might acquire replication competence, we have studied the reversion of autonomously replicating sequence (ARS) mutations . By mutagenesis of a Saccharomyces cerevisiae plasmid lacking a functional origin of replication, we have obtained a series of cis-acting mutations which confer ARS activity on the plasmid . The original plasmid contained an ARS element inactivated by point mutation, but surprisingly only 1 of the 10 independent Ars+ revertants obtained shows a back mutation in this element . In the remainder of the revertants, sequence changes in the M13 vector DNA generate new ARSs . In two cases, a single nucleotide change results in an improved match to the ARS consensus, while six other cases show small duplications of vector sequence creating additional matches to the ARS consensus . These results suggest that changes in replication origin distribution may arise de novo by point mutation rather than by transposition of preexisting origin sequences.

Mol Cell Biol, 1990 Jan, 10(1), 223 - 34
Chromosome instability mutants of Saccharomyces cerevisiae that are defective in microtubule-mediated processes; Hoyt MA et al.; By using a multiply marked supernumerary chromosome III as an indicator, we isolated mutants of Saccharomyces cerevisiae that display increased rates of chromosome loss . In addition to mutations in the tubulin-encoding TUB genes, we found mutations in the CIN1, CIN2, and CIN4 genes . These genes have been defined independently by mutations causing benomyl supersensitivity and are distinct from other known yeast genes that affect chromosome segregation . Detailed phenotypic characterization of cin mutants revealed several other phenotypes similar to those of tub mutants . Null alleles of these genes caused cold sensitivity for viability . At 11 degrees C, cin mutants arrest at the mitosis stage of their cell cycle because of loss of most microtubule structure . cin1, cin2, and cin4 mutations also cause defects in two other microtubule-mediated processes, nuclear migration and nuclear fusion (karyogamy) . Overproduction of the CIN1 gene product was found to cause the same phenotype as loss of function, supersensitivity to benomyl . Our findings suggest that the CIN1, CIN2, and CIN4 proteins contribute to microtubule stability either by regulating the activity of a yeast microtubule component or as structural components of microtubules.

J Bacteriol, 1990 Jan, 172(1), 317 - 25
Cu,Zn superoxide dismutase and copper deprivation and toxicity in Saccharomyces cerevisiae; Greco MA et al.; A wild-type strain of the yeast Saccharomyces cerevisiae grown at a medium {Cu} of less than or equal to 50 nM contained less Cu,Zn superoxide dismutase (SOD) mRNA (60%), protein (50%), and activity (50%) in comparison with control cultures grown in normal synthetic dextrose medium ({Cu} approximately 150 nM) . A compensating increase in the activity of MnSOD was observed, as well as a smaller increase in MnSOD mRNA . These medium {Cu}-dependent differences were observed in cultures under N2 as well . Addition of Cu2+ (100 microM) to Cu-depleted cultures resulted in a rapid (30 min) increase in Cu,ZnSOD mRNA (2.5-fold), protein (3.5-fold), and activity (4-fold) . Ethidium bromide (200 micrograms/ml of culture) inhibited by 50% the increase in Cu,ZnSOD mRNA, while cycloheximide (100 micrograms/ml of culture) inhibited completely the increase in protein and activity . Addition of Cu2+ to greater than or equal to 100 microM caused no further increase in these parameters but did result in a loss of total cellular RNA and translatable RNA, a decline in the population of specific mRNAs, a decrease in total soluble protein and the activity of specific enzymes, and an inhibition of incorporation of {3H}uracil and {3H}leucine into trichloroacetic acid-insoluble material . Cu,ZnSOD mRNA, protein, and activity appeared relatively more resistant to these effects of Cu toxicity than did the other cellular constituents examined . When evaluated in cultures under N2, the cellular response to {Cu} of greater than or equal to 100 microM was limited to the inhibition of radiolabel incorporation into trichloroacetic acid-insoluble material . All other effects were absent in the absence of O2 . The data indicated that medium (cellular) Cu alters the steady-state level of Cu, ZnSOD . This regulation may be at the level of transcription . In addition, Cu,ZnSOD exhibits the characteristics of Cu-stress protein in that it and its mRNA are enhanced relative to other cellular species under conditions of Cu excess . This observation and the O2-dependence of some of the manifestations of Cu excess suggest that one mechanism of Cu toxicity involves the superoxide radical anion O2-.

Radiat Environ Biophys, 1990, 29(4), 293 - 301
Molecular analysis of the REV2 gene of Saccharomyces cerevisiae--a review; Ahne F et al.; The REV2 gene controls DNA repair, induced mutagenesis and, probably, some fidelity mechanism of replication . Of particular interest is the notion that it is inducible by DNA-damaging agents . We wanted to find molecular evidence for these results derived from numerous biological experiments . We cloned the REV2 gene from a yeast genomic DNA library based on the YCp50 centromere vector, sequenced it and studied its regulation on the transcriptional level . The coding region of the REV2 gene consists of a 1425 pb reading frame with a coding capacity for a polypeptide of 52 kD; no significant homology to any gene filed in available data bases was found . Examination of a hydrophobicity plot of the putative Rev2 protein predicts the existence of transmembrane helices . Quantitative Northern analysis confirmed the working hypothesis that DNA-damaging agents increase the level of REV2 gene expression in stationary cells . Thus, the REV2 gene seems to code for a membrane protein which is inducible by DNA-damaging agents and which controls processes of repair and mutagenesis in yeast.

Int J Biochem, 1990, 22(8), 841 - 6
Identification of peptides from autolysates of Saccharomyces cerevisiae that exhibit glucose tolerance factor activity in a yeast assay; O'Donoghue EM et al.; 1 . Cationic fractions were isolated from a low chromium (less than 0.2 ppm) commercial yeast extract in an attempt to purify the material responsible for glucose tolerance factor (GTF) activity observed in a standard yeast assay system . 2 . Following previously described procedures a fraction with GTF activity but containing negligible chromium was isolated, which on further purification was found to be composed of many separate small basic peptides . 3 . Much of the activity of the yeast GTF material in the yeast assay could be attributed to the presence of basic peptides and free amino acids acting as nitrogen sources for the yeast . 4 . Additional activity was present in the yeast GTF sample, which was not due to a synergistic effect of the mixed amino acids and peptides although the component of the yeast extract responsible for this activity was not identified . 5 . The results show that the GTF fractions isolated according to most previously published procedures are highly impure, and conclusions drawn about the nature of GTF based on these isolates must remain open to question . 6 . The activity due to the presence of peptides and amino acids is a major cause of lack of specificity of the yeast systems as an assay for GTF.

Folia Biol (Praha), 1990, 36(6), 312 - 8
Microtubular dynamics of Saccharomyces cerevisiae temperature-sensitive secretory mutants: the sec 1 product and polymerization of microtubules; Briestanska J; The relation of cytoplasmic microtubules to intracellular transport was studied in temperature-sensitive (ts) secretory mutants of Saccharomyces cerevisiae at permissive and nonpermissive temperature using indirect immunofluorescence with monoclonal antibody TU-01 against alpha-tubulin . It was found that in the sec 1 mutant, which at 37 degrees C accumulated secretory vesicles and in which therefore transport of secretory material from secretory vesicles to cytoplasmic membrane was inhibited, cytoplasmic and in some cases nuclear microtubules were impaired . After 4 h of postcultivation at 24 degrees C the altered phenotype reverted to the original state, the cells began to divide, and were comparable with control . Use of the sec 1 mutant protoplasts suggested that the product, whose gene is mutated, is probably involved in microtubular polymerization . The sec 7 mutant, which accumulates the Golgi complex under nonpermissive conditions and in which the transfer of secretory material from the Golgi complex to the secretory vesicles is thus inhibited, showed no significant changes in the length or number of cytoplasmic microtubules . As a result of secretory product accumulation, the cytoplasmic microtubules were displaced towards the periphery in some cells.

Arch Microbiol, 1990, 154(6), 544 - 9
Genetic mapping and biochemical analysis of mutants in the maltose regulatory gene of the MAL1 locus of Saccharomyces cerevisiae; Goldenthal MJ et al.; The MAL1 locus of Saccharomyces cerevisiae comprises three genes necessary for maltose utilization: a regulatory (MALR), a maltose transport (MALT) and a maltase gene (MALS) . A fine structure genetic map of the MAL1R gene was constructed and the order of mutations was confirmed by plasmid-mediated chromosomal recombination . The mutations cluster non-randomly within the 5' half of the gene, where the putative DNA binding domain of the encoded protein is located . Only mutations mal1R-22 and MAL1R-72 map in the 3' terminal half of the gene; these mutations cause a different pattern of transcriptional regulation of plasmid-borne MAL6T genes . Experiments supporting a direct involvement of the MALR-encoded protein in carbon catabolite repression of MAL gene expression are reported.

Rev Argent Microbiol, 1990 Jan-Mar, 22(1), 7 - 16
{Effect of ammonium ions on the uptake of L-leucine in Saccharomyces cerevisiae . Repression and inhibition of transport systems}; Kotliar N et al.; L-leucine entrance into Saccharomyces cerevisiae is mediated by the general amino acid permease, GAP and two transport systems, S1 and S2, kinetically characterized . S1 is a high-affinity, low-velocity transport system, operating at lower L-leucine external concentration (0.05-0.1 mM), while S2 is a low-affinity, high-velocity transport system, operating at higher L-leucine external concentration (1.0 mM) . In cells grown in minimal medium containing ammonium as sole nitrogen source the values of L-leucine entrance and uptake are smaller than those in cells grown in L-proline containing medium . When GAP is repressed by ammonium, L-leucine entrance is mediate by systems S1 and S2 . Both systems are inhibited by ammonium . When GAP is derepressed, in cells grown in L-proline medium, L-leucine is transported by systems S1 and GAP (lower L-leucine external concentration), and mainly by S2 (higher L-leucine external concentration) . GAP is the largest system inhibited by ammonium.

J Basic Microbiol, 1990, 30(7), 535 - 40
Protein extracts for nutritional purposes from fragile strains of Saccharomyces cerevisiae: construction of strains and conditions for lysis; Stateva L et al.; A superfragile strain, Saccharomyces cerevisiae 211, carrying three nonallelic nuclear determinants of cell lysis by osmotic shock was isolated from the haploid progeny of a cross between two laboratory fragile strains VY 1160 and SY 15 . The strain Sacch . cerevisiae 211 is prototrophic, grows well in both laboratory and industrial media, only when supplemented with osmotic stabilizers . The average protein content of its biomass after growth in a molasses-based nutritional medium is 48.7% . In laboratory conditions this strain releases about 60% of its cellular proteins by spontaneous lysis in water . However, on a pilot scale the protein yield decreases to about 25% . After a single disintegration step at 650 atm, practically all of the cellular protein--91.5%, is extracted without any additional chemical treatment . This result cannot be reached in any of the wild type strains tested even after triple treatment at 650 atm.

Cell Mol Biol, 1990, 36(3), 337 - 44
Influence of the structure of alkanols on their inhibition of protein synthesis in Saccharomyces cerevisiae var . ellipsoideus; Ferreras JM et al.; The effects of several short chain alcohols on protein synthesis by intact Saccharomyces cerevisiae var . ellipsoideus cells were studied . The results show that the relative inhibitory potencies correlate well with the size of the carbon backbone, thus suggesting that a hydrophobicity-related effect is involved in the inhibitory action of these alcohols . Additionally, the branching nature of the carbon backbone contributes to determining the action of the alcohol, as proved by the relative potencies of the four-carbon alcohol series.

J Basic Microbiol, 1990, 30(4), 301 - 4
DNA sequences from Saccharomycopsis fibuligera capable of autonomous replication in Saccharomyces cerevisiae; Jursky F et al.; A Sau3AI digest of total DNA from Saccharomycopsis fibuligera was cloned with the yeast-integrating plasmid YEp24 delta EcoRI and the capacity for autonomous replication (ARS) was assayed in yeast . From eighty clones, five mitoticaly unstable yeast transformants were picked up, recombinant plasmids from these clones were recovered with Escherichia coli, mapped, hybridized with total DNA of S . fibuligera and tested to mitotical stability in Saccharomyces cerevisiae . Experiments suggested the existence of DNA sequences from dimorphic Saccharomycopsis fibuligera with ARS activity in Saccharomyces cerevisiae.

Folia Microbiol (Praha), 1990, 35(2), 168 - 71
Dominant resistance to oxythiamin in Saccharomyces cerevisiae and its mapping; Ruml T et al.; A dominant mutation, responsible for the resistance to oxythiamin in Saccharomyces cerevisiae was mapped on the right arm of chromosome IV, 4.6 cM centromere-distally to trp1 . The corresponding gene is not involved in the control of intracellular content of total thiamin during growth on a minimal medium without thiamin.

Genetika, 1990 Jan, 26(1), 30 - 6
{The effect of mutation him1 characterized by enhanced induced mutagenesis on the genetic effects of 6-N-hydroxylaminopurine in Saccharomyces cerevisiae}; Ivanov EL et al.; The him1 mutation has been shown to influence the genetic effects of the mutagenic purine base analog 6-hydroxylaminopurine, i . e . inactivation of haploid cells, mutation induction, and inhibition of DNA synthesis in vivo . The influence observed is well consistent with the idea that the him1 mutation affects mismatch correction . We present evidence that during in vivo DNA replication 6-hydroxylaminopurine incorporates into the yeast DNA.

Chem Pharm Bull (Tokyo), 1990 Jan, 38(1), 246 - 8
Presence of a dipeptidyl aminopeptidase III in Saccharomyces cerevisiae; Watanabe Y et al.; The soluble fraction of a cell extract of Saccharomyces cerevisiae was found to contain hydrolytic activity toward arginyl-arginyl-beta-naphthylamide (Arg-Arg-beta NA) . Most of the contaminating Arg-beta NA hydrolysis activity was removed by diethylaminoethyl (DEAE)-cellulose column chromatography and the enzyme was partially purified . It is very similar in various properties to mammalian dipeptidyl aminopeptidase III (DAP III), although it differs slightly in some respects.

Arch Microbiol, 1990, 153(4), 384 - 91
Effects of ethanol on Saccharomyces cerevisiae as monitored by in vivo 31P and 13C nuclear magnetic resonance; Loureiro-Dias MC et al.; Cell suspensions of a respiratory deficient mutant of Saccharomyces cerevisiae were monitored by in vivo 31P and 13C Nuclear Magnetic Resonance in order to evaluate the effect of ethanol in intracellular pH and metabolism . In the absence of an added energy source, ethanol caused acidification of the cytoplasm, as indicated by the shift to higher field of the resonance assigned to the cytoplasmic orthophosphate . Under the experimental conditions used this acidification was not a consequence of an increase in the passive influx of H+ . With cells energized with glucose, a lower value for the cytoplasmic pH was also observed, when ethanol was added . Furthermore, lower levels of phosphomonoesters were detected in the presence of ethanol, indicating that an early event in glycolysis is an important target of the ethanol action . Acetic acid was identified as responsible for the acidification of the cytoplasm, in experiments where {13C}ethanol was added and formation of labeled acetic acid was detected . The intracellular and the extracellular concentrations of acetic acid were respectively, 30 mM and 2 mM when 0.5% (120 mM) {13C}ethanol was added.

Can J Microbiol, 1990 Jan, 36(1), 61 - 4
A biometric study of higher alcohol production in Saccharomyces cerevisiae; Giudici P et al.; A hundred strains of Saccharomyces cerevisiae were examined for the ability to produce higher alcohols . In the strains tested the production of higher alcohols was found to be an individual strain characteristic and, as such, was statistically significant . The characteristics of the strains used (flocculation ability, foaming ability, killer character, and non-H2S production) were found to be uncorrelated to isobutanol and isoamyl alcohol production, whereas the production of high levels of n-propanol was found to be related to inability to produce H2S . This, in turn, suggests a link to methionine biosynthesis.

Mutagenesis, 1990 Jan, 5(1), 39 - 44
Genetic and biochemical analysis of glutathione-deficient mutants of Saccharomyces cerevisiae; Kistler M et al.; Five independently isolated glutathione-deficient (gsh-) mutants of Saccharomyces cerevisae with maximally 6% residual glutathione content have been analysed genetically . Complementation as well as tetrad analysis of the homo- and heterozygous diploids constructed by suitable crosses of the five mutants indicated that all isolates belong to one complementation group and hence represent different alleles of one gene, GSH1 . In order to determine the Gsh1 gene product an assay suitable for yeast was developed to determine the activity of gamma-glutamyl-cysteine synthetase catalysing the first step of glutathione biosynthesis . All mutants are severely deficient in gamma-glutamyl-cysteine synthetase (less than 6.5% of the activity of the glutathione competent parental strain) which is in good accordance with the genetic data.

Mol Gen Genet, 1990 Jan, 220(2), 186 - 90
Molecular analysis of the mitochondrial transcription factor mtf2 of Saccharomyces cerevisiae; Lisowsky T; The nuclear gene for a new mitochondrial transcription factor (mtf2) was isolated by transformation of mutant pet-ts3504 . It was localized on a 6.4 kb fragment of yeast genomic DNA by subcloning and complementation tests . Sequencing of a 1.7 kb DNA fragment revealed an open reading frame of 1320 bp . A transcript of 1400 nucleotides can be assigned to this region . Gene disruption of this reading frame in a wild-type yeast strain created a stable pet phenotype . Further analysis of this insertion mutation showed that it is allelic to the mutated gene of pet-ts3504 . Comparison of the 5' upstream regions of MTF2 and a previously characterized mitochondrial transcription factor (MTF1) revealed common sequence motifs which may be important for coordinated regulation of gene expression.

Curr Genet, 1990 Jan, 17(1), 7 - 12
Conditional hyporecombination mutants of three REC genes of Saccharomyces cerevisiae; Esposito MS et al.; We have isolated and characterized three conditional hyporecombination mutants, rec1-1, rec3-1 and rec4-1, that define three REC genes of Saccharomyces cerevisiae required for spontaneous general mitotic interchromosomal recombination . Each MATa/MAT alpha rec/rec diploid is deficient in mitotic single site gene conversion, intragenic recombination, intergenic recombination and sporulation at the restrictive temperature (36 degrees C) . The rec1-1 mutation also confers conditional enhanced sensitivity to the killing effects of X-rays . The rec1-1 and rec3-1 mutations have been mapped to chromosome VII . The rec1-1, rec3-1 and rec4-1 mutations exhibit complementation at 36 degrees C for both mitotic recombination and sporulation.

Curr Genet, 1990 Jan, 17(1), 1 - 6
Heterologous expression of human 5-aminolevulinate dehydratase in Saccharomyces cerevisiae; Schauer WE et al.; A cDNA coding for human 5-amino-levulinate dehydratase was placed in a yeast expression vector under the control of the GAL10 promoter . The resulting multicopy plasmid was then used to transform a yeast mutant which contains a defective hem2 gene coding for 5-aminolevulinate dehydratase . Expression of the human cDNA was shown in four ways: (1) restoration of normal growth on glycerol/galactose as primary carbon source, (2) decrease in intracellular 5-aminolevulinic acid concentration, (3) restoration of cytochrome biosynthesis and (4) direct, in situ assay of 5-aminolevulinic acid dehydratase . Curing transformed cells of plasmid restored the hem2 mutant phenotype . This heterologous system could be used to produce large quantities of human 5-aminolevulinic acid dehydratase for physical and biochemical studies.

J Basic Microbiol, 1990, 30(6), 443 - 50
{Substrate-induced cAMP signals in wild-type and mutant strains of Saccharomyces cerevisiae}; Sachse O; Addition of glucose or other substrates to starved Saccharomyces cerevisiae cells triggers a cyclic AMP signal which induces the protein phosphorylating cascade . Before the addition of various substrates the wild-type and mutant yeast strains were arrested at the G1 phase of the cell division cycle by transferring the cells, grown at 26 degrees C to 36 degrees C in a synthetic medium without any substrate . After the temperature shift back to 26 degrees C different substrates were added and the cAMP levels were measured . The highest cAMP levels were observed immediately after the addition of the substrates . A relationship between the maximum growth rate of the individual strains or mutants at a given substrate and the intracellular cAMP level is discussed.

Braz J Med Biol Res, 1990, 23(2), 105 - 11
Effect of dimethylsulfoxide on signal transduction in mutants of Saccharomyces cerevisiae; Panek AC et al.; 1 . As the first part of a study of pesticide toxicity we report the effects of the solvent dimethylsulfoxide (DMSO) on signal transduction in mutants of Saccharomyces cerevisiae . 2 . The enzymes of trehalose metabolism, which are activated and deactivated by a "glucose signal" and by heat shock treatment, were chosen as targets for this study . 3 . DMSO was shown to be able to permeate glucose and cAMP . The effects of glucose and cAMP were enhanced by pre-incubating the cells in the presence of DMSO . 4 . No effects were observed during the heat shock, suggesting that the solvent acts on the cell membrane . 5 . The results suggest that DMSO may be used as a vehicle for small molecules which do not easily penetrate yeast cell membranes, thus providing a new tool for biochemical and toxicological studies.

Arch Microbiol, 1990, 154(2), 199 - 205
Absence of glucose-induced cAMP signaling in the Saccharomyces cerevisiae mutants cat1 and cat3 which are deficient in derepression of glucose-repressible proteins; Arguelles JC et al.; Addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae induces a transient, specific cAMP signal . Intracellular acidification in these cells, as caused by addition of protonophores like 2,4-dinitrophenol (DNP) causes a large, lasting increase in the cAMP level . The effect of glucose and DNP was investigated in glucose-repressed wild type cells and in cells of two mutants which are deficient in derepression of glucose-repressible proteins, cat1 and cat3 . Addition of glucose to cells of the cat3 mutant caused a transient increase in the cAMP level whereas cells of the cat1 mutant and in most cases also repressed wild type cells did not respond to glucose addition with a cAMP increase . The glucose-induced cAMP increase in cat3 cells and the cAMP increase occasionally present in repressed wild type cells however could be prevented completely by addition of a very low level of glucose in advance . In derepressed wild type cells this does not prevent the specific glucose-induced cAMP signal at all . These results indicate that repressed cells do not show a true glucose-induced cAMP signal . When DNP was added to glucose-repressed wild type cells or to cells of the cat1 and cat3 mutants no cAMP increase was observed . Addition of a very low level of glucose before the DNP restored the cAMP increase which points to lack of ATP as the cause for the absence of the DNP effect . These data show that intracellular acidification is able to enhance the cAMP level in repressed cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochimie, 1990 Jan, 72(1), 77 - 9
Regulation of trehalose metabolism in Saccharomyces cerevisiae mutants during temperature shifts; Panek AC et al.; Temperature shifts from 23 degrees C to 36 degrees C resulted in trehalose accumulation in Saccharomyces independently of genetic lesions in the cAMP-protein kinase cascade . In parallel, trehalose 6-phosphate synthase activity increased about 3-fold in all strains; the increase could be inhibited by cycloheximide, suggesting that protein synthesis was required . Heat shock treatment after the temperature shift led to a drastic increase in trehalose activity, and deactivation of the biosynthetic enzyme with a consequent drop in trehalose . Up to now no definite correlation between acquisition of thermotolerance and trehalose accumulation has been made.

Mol Gen Genet, 1990 Jan, 220(2), 213 - 21
Ty RNA levels determine the spectrum of retrotransposition events that activate gene expression in Saccharomyces cerevisiae; Curcio MJ et al.; To learn more about the variety of Ty elements capable of activating gene expression, we characterized 206 spontaneous Ty transpositions that activate the promoterless gene his3 delta 4 . Most of the Ty elements appear to be full-length, although a few deleted elements were recovered . Over 95% of the insertions belong to the Ty1 family, and the rest are Ty2 elements . The excessive number of Ty1 transpositions was unexpected because there are only 2-fold more Ty1 than Ty2 elements in the yeast strains used in the selection . However, there is 20-fold more Ty1 than Ty2 RNA present in these yeast strains . This difference in RNA level explains the greater number of Ty1 verses Ty2 transpositions at his3 delta 4, because Ty elements transpose through an RNA intermediate . A similar association between the Ty transcript level and transpositional activation of his3 delta 4 is obtained in cells expressing GAL1-promoted Ty2-H556 or Ty2-917 elements, but only if the element does not contain a marker . Genetically marked Ty2-H556NEO and -917NEO elements transpose into and activate his3 delta 4 with the same efficiency as the previously characterized Ty1-H3NEO element, but are underrepresented relative to the levels of TyNEO transcript . We also found that chromosomal Ty transcripts are even more abundant than previously estimated and comprise about 1% of total cellular RNA.

Yeast, 1990 Jan-Feb, 6(1), 53 - 60
Intracellular and extracellular levels of cyclic AMP during the cell cycle of Saccharomyces cerevisiae; Smith ME et al.; Using the technique of centrifugal elutriation it was demonstrated that during the cell division cycle of the budding yeast Saccharomyces cerevisiae there are stage-specific fluctuations in the intracellular concentration of adenosine 3',5'-cyclic monophosphate (cAMP) . Results shown here indicate that the intracellular concentration of cAMP is at its highest during the division cycle, and at its lowest immediately prior to and just after cell separation . Results also show the extrusion of extracellular cAMP into the medium by Saccharomyces cerevisiae, extracellular cAMP levels being ten to one hundred times higher than intracellular levels . During the cell cycle of Saccharomyces cerevisiae the extracellular level of cAMP does not fluctuate.

Mol Cell Biol, 1990 Jan, 10(1), 10 - 5
RPO41-independent maintenance of {rho-} mitochondrial DNA in Saccharomyces cerevisiae; Fangman WL et al.; A subset of promoters in the mitochondrial DNA (mtDNA) of the yeast Saccharomyces cerevisiae has been proposed to participate in replication initiation, giving rise to a primer through site-specific cleavage of an RNA transcript . To test whether transcription is essential for mtDNA maintenance, we examined two simple mtDNA deletion ({rho-}) genomes in yeast cells . One genome (HS3324) contains a consensus promoter (ATATAAGTA) for the mitochondrial RNA polymerase encoded by the nuclear gene RPO41, and the other genome (4a) does not . As anticipated, in RPO41 cells transcripts from the HS3324 genome were more abundant than were transcripts from the 4a genome . When the RPO41 gene was disrupted, both {rho-} genomes were efficiently maintained . The level of transcripts from HS3324 mtDNA was decreased greater than 400-fold in cells carrying the RPO41 disrupted gene; however, the low-level transcripts from 4a mtDNA were undiminished . These results indicate that replication of {rho-} genomes can be initiated in the absence of wild-type levels of the RPO41-encoded RNA polymerase.

J Bacteriol, 1990 Jan, 172(1), 102 - 9
The functional domain of adenylate cyclase associated with entry into meiosis in Saccharomyces cerevisiae; Uno I et al.; Diploid yeast cells that carry a part of the CYR1 gene deficient in a region coding for the N-terminal domain of adenylate cyclase were growth arrested and accumulated unbudded cells after inoculation into complete medium or nitrogen-free medium, but produced many cells which had one or more buds after incubation in sporulation medium . The cells incubated in sporulation medium had abnormal spindles which were free from the spindle pole bodies, larger in size, or frequently distributed in cytoplasm . The levels of cyclic AMP in these cells did not decrease to the wild-type level after transfer to the sporulation medium and remained at a constant level . The results suggest that the N-terminal domain of adenylate cyclase is associated with the regulatory function for sporulation . The environmental signals for sporulation may be transferred to the adenylate cyclase system through a factor that negatively interacts with the N-terminal domain of this enzyme.

J Basic Microbiol, 1990, 30(7), 523 - 8
Protein extracts for nutritional purposes from fragile strains of Saccharomyces cerevisiae: reduction of the nucleic acid content and applicability of the protein extracts; Lyutskanov N et al.; 92% of the cellular proteins from the osmotically fragile strain Sacch . cerevisiae 211 (MATa srbl srb2 tsl) were extracted by one passage in a high pressure disintegrator at 650 atm . After isoelectric precipitation, 60.1% of the proteins were isolated . The protein extract was rich in proteins (60-70%) but also contained 12-13% nucleic acids and 13-14% carbohydrates . Mild conditions were created for simultaneous reduction of nucleic acids and carbohydrates in the lysate prior to precipitation of proteins . Under these conditions the amount of nucleic acids in the protein extract was reduced to 2%, and that of the carbohydrates to 5.7% . As a result of the whole procedure on a pilot scale, 11 culture of Sacch . cerevisiae 211 grown in molasses based nutritional medium yielded 1 g protein extract . The applicability of this protein extract for nutritional purposes was demonstrated by determination of its amino acid composition, acute toxicity tests with Tetrahymena pyriformis and short-term animal nutrition tests . The results showed that the protein extract is a suitable source of SCP for nutritional purposes.

Mol Gen Genet, 1990 Jan, 220(2), 269 - 76
The UGA3 gene regulating the GABA catabolic pathway in Saccharomyces cerevisiae codes for a putative zinc-finger protein acting on RNA amount; Andre B; The UGA3 gene of Saccharomyces cerevisiae is required for 4-aminobutyric acid (GABA)-dependent induction of the UGA1, UGA2 and UGA4 genes which encode the two GABA catabolic enzymes and a GABA-specific permease, respectively . Measurements of UGA1-specific transcripts show that induction of UGA1 correlates with accumulation of its RNA and requires a functional UGA3 gene . A 2 kb DNA fragment complementing the uga3 mutation was isolated and shown to contain the UGA3 gene . The primary structure of the UGA3 encoded protein was deduced from the DNA sequence, and contains an N-terminal, cysteine-rich motif similar in sequence to regions found in other fungal regulatory proteins and which are supposed to form zinc finger structures involved in DNA binding . Mutations were identified in the UGA3 genes isolated from uninducible and constitutive uga3 alleles . One case of intragenic complementation between two uninducible uga3 mutants is reported, indicating a possible oligomeric structure for UGAe . The role of UGA3 is discussed in relation to its genetic properties and its predicted structure.

Mol Cell Biol, 1990 Jan, 10(1), 217 - 22
Overexpression of the STE4 gene leads to mating response in haploid Saccharomyces cerevisiae; Whiteway M et al.; The STE4 gene of Saccharomyces cerevisiae encodes the beta subunit of the yeast pheromone receptor-coupled G protein . Overexpression of the STE4 protein led to cell cycle arrest of haploid cells . This arrest was like the arrest mediated by mating pheromones in that it led to similar morphological changes in the arrested cells . The arrest occurred in haploid cells of either mating type but not in MATa/MAT alpha diploids, and it was suppressed by defects in genes such as STE12 that are needed for pheromone response . Overexpression of the STE4 gene product also suppressed the sterility of cells defective in the mating pheromone receptors encoded by the STE2 and STE3 genes . Cell cycle arrest mediated by STE4 overexpression was prevented in cells that either were overexpressing the SCG1 gene product (the alpha subunit of the G protein) or lacked the STE18 gene product (the gamma subunit of the G protein) . This finding suggests that in yeast cells, the beta subunit is the limiting component of the active beta gamma element and that a proper balance in the levels of the G-protein subunits is critical to a normal mating pheromone response.

Chin J Biotechnol, 1990, 6(3), 207 - 13
Study on the protoplast fusion between a thermotolerant yeast and Saccharomyces cerevisiae; Fang AQ et al.; Intergenetic protoplast fusion between Saccharomyces cerevisiae 396 and thermotolerant yeast Candida sp . C6 has been studied . 396 is a molasses distiller's yeast, and C6 which was isolated from the hot spring in Yunnan, grows well at 45 degrees C . The auxotrophic mutants of 396 (Arg.) and C6 (Lys.) were obtained by treatment of NTG . The fusion frequency was 0.91 per 10(5) protoplasts . Six fusants were studied, the average size of their cells is 1.3 times the size of their parents, while the DNA content is 1.6 times . Other characteristics of the fusants including colonial and cell morphology, physiology, especially in carbon sources assimilation appeared different . The possibility of heterokaryon formation was excluded except for F2 and F14 . The growth curves, efficiency of sugar utilization and ethanol yield of original parents 396 and C6, immediate parents 396 (Arg.) and C6 (Lys.), fusants F1, F7, F12 and F13 have been compared at 28 degrees C, 40 degrees C and 45 degrees C . The intergenetic fusant F13 with efficiency of substrate utilization 94.3% and the final ethanol concentration 59.7 g/l at 40 degrees C has been selected.

Chin J Biotechnol, 1990, 6(2), 103 - 10
A rapid and efficient procedure for transformation of Saccharomyces cerevisiae intact cells with plasmid DNA; Mao XH et al.; A rapid and efficient yeast transformation procedure has been developed through investigation of factors affecting transformation efficiency . The manipulation of the entire procedure can be done within one and one half hours . High yield of transformants is obtained by: adding calf thymus DNA as carrier DNA; adding PEG4000, carrier DNA and plasmid DNA to cell suspension simultaneously; prolonging heat shock at 42 degrees C from 5 min to 25 min and spreading the transformation mixture directly onto agar plates after heat shock . The pretreatment of yeast intact cells with LiAc can be omitted in this procedure . The transformation rates of four types of plasmid DNA were as follows: pCN60: 3.5-7.2 x 10(4) (for linear pCN60/BamHI: 1.6 x 10(5)); YEp13: 1.7-2.6 x 10(4) (for linear YEp13/BamHI: 8.0 x 10(4)); RC4: 3.7 x 10(4); YIp5/StuI: 7.6 x 10(3) . Seven recipient strains transformed by using this procedure all reached the yields of over 10(4) transformants per microgramme of DNA.

J Basic Microbiol, 1990, 30(8), 569 - 75
A cytoplasmic factor involved in the expression of resistance to C8-ATC in Saccharomyces cerevisiae; Misiewicz MH et al.; A dozen mutants of Saccharomyces cerevisiae, resistant to C8-ATC have been characterized . C8-ATC was previously established as a biologically toxic compound . Frequency of mutants (10(-7)) was typical for spontaneous mutations . One very stable mutant was characterized extensively . The genetical analysis revealed that resistance in this mutant was determined by single-gene mutation . The rho 0 cells, obtained by ethidium bromide (EB) mutagenesis of the resistant strain, were completely devoid of resistance . A large percentage of rho- cells, obtained by a moderate EB treatment of resistant cells were still able to express resistance to C8-ATC . Therefore we hypothesized that, in our particular strain, a cytoplasmic factor is involved in nuclear determination of resistance.

Cell Mol Biol, 1990, 36(5), 581 - 7
Differential D-glucose requirements of the general amino-acid permease and protein synthesis in Saccharomyces cerevisiae var . ellipsoideus; Iglesias R et al.; The dependence of the general aminoacid permease and protein synthesis on the availability of D-glucose as energy source was studied . Stimulation by the sugar was immediate once added to the cell suspensions and seems to be mediated by energy derived directly from glycolysis . The general aminoacid permease was saturated linearly with D-glucose whereas protein synthesis was saturated sigmoidealy requiring much higher concentration of the sugar than the general aminoacid permease.

Acta Microbiol Pol, 1990, 39(3-4), 157 - 62
Amino acid auxotrophy increases sensitivity of Saccharomyces cerevisiae to a quaternary ammonium salt IM; Lachowicz TM et al.; Relationships between amino acid auxotrophy and N-dodecyloxy-carboxy-methyl-N-N-N-trimethyl ammonium chloride (IM) sensitivity have been investigated in isogenic yeast strains Saccharomyces cerevisiae and their meiotic segregants . It has been found, that auxotrophy increases the level of sensitivity to this salt markedly . A gene conferring resistance to that drug cancels the auxotrophy-dependent sensitivity.

Gene, 1989 Dec 28, 85(2), 511 - 6
Expression and secretion of biologically active echistatin in Saccharomyces cerevisiae; Jacobson MA et al.; A synthetic gene coding for a platelet aggregation inhibitor, echistatin (ECS), was inserted into a Saccharomyces cerevisiae expression vector utilizing the alpha-mating factor pre-pro leader sequence and galactose-inducible promoter, GAL10 . Cleavage of the pre-pro leader sequence in vivo results in the secretion of a properly processed recombinant ECS with the native N-terminal glutamic acid residue . Recombinant ECS was recovered from yeast supernatants and purified by reverse phase high performance liquid chromatography . Recombinant ECS expressed and purified from yeast was identical to native ECS in its ability to inhibit platelet aggregation.

Gene, 1989 Dec 28, 85(2), 303 - 11
Characterization of the prephenate dehydrogenase-encoding gene, TYR1, from Saccharomyces cerevisiae; Mannhaupt G et al.; TYR1, the gene from Saccharomyces cerevisiae, which encodes prephenate dehydrogenase, one of the tyrosine biosynthetic enzymes, has been cloned by complementing a yeast tyr1 mutant strain . The DNA fragment containing the gene is part of a 45-kb cosmid clone which represents a region of chromosome II covering the genetically mapped tyr1 locus . The nucleotide sequence of a 3.1-kb region carrying the TYR1 gene and adjacent regions has been determined . The open reading frame contains 441 codons, corresponding to about 52.2 kDa for the encoded protein . The canonical NAD-binding domain is located within the first 45 amino acids of the protein . By primer extension, we show that there is one transcription start point . Presumably, the expression of TYR1 is not under the general GCN4 control . Instead, we find a dependence on the presence or absence of phenylalanine . These data were obtained by analysing CAT activity in constructs containing promoter fragments of TYR1 and the cat reporter gene.

Gene, 1989 Dec 28, 85(2), 545 - 51
Production and secretion of porcine urokinase in Saccharomyces cerevisiae: characterization of the secreted gene product; Zaworski PG et al.; The properties of porcine urokinase plasminogen activator (u-PA), produced and secreted by Saccharomyces cerevisiae, were studied to evaluate processing of the enzyme by yeast . Porcine u-PA cDNA was positioned behind the triosephosphate isomerase promoter and the yeast alpha-mating factor secretion signal sequences in a yeast expression vector, pZV125 . Greater than 99% of the secreted PA activity was found to be single chain (pro-urokinase) . The secreted gene product could be converted to two-chain (tc) with plasmin and then purified to homogeneity on benzamidine sepharose . Plasmin cleavage resulted in the formation of high Mr (HMW) and low Mr moieties representing HMW tc and free catalytic domain, respectively, as detected by N-terminal amino acid sequence analysis . Approximately 60-70% of the secreted activity was found to be associated with hyperglycosylated fractions from G-75 sizing columns . Approximately 30% of the total activity was secreted into the culture medium, where levels of activity approached 200 I.U./ml.

J Biol Chem, 1989 Dec 25, 264(36), 21613 - 8
Expression of functional chicken oviduct progesterone receptors in yeast (Saccharomyces cerevisiae); Mak P et al.; The cDNAs encoding full-length chicken oviduct progesterone receptor B (PRB) and a truncated receptor (C1C2) lacking the amino-terminal domain were expressed in yeast (Saccharomyces cerevisiae) using a ubiquitin fusion system . The expression of the fusion protein is under the control of a copper-responsive yeast metallothionein promoter, and the fusion protein is subsequently cleaved by the yeast host enzyme to produce receptor protein . Western immunoblot analyses of yeast extracts containing full-length PRB revealed a polypeptide co-migrating with authentic chicken oviduct PRB . Using a polyclonal antibody (907) directed against the "hinge" region of the authentic chicken progesterone receptor, a 42-kDa polypeptide was detected by Western analysis in yeast extracts containing C1C2 receptors . Standard hormone binding assays indicated that these receptors produced in yeast cells exhibited steroid binding affinity and specificity characteristic of the authentic chicken progesterone receptor . To test for progesterone receptor-mediated activation of transcription in yeast, reporter plasmids were constructed to transform yeast cells expressing PRB or C1C2 receptors . The reporter gene contained two copies of a progesterone response element upstream of the yeast proximal CYC1 promoter fused to the beta-galactosidase gene of Escherichia coli . The induction of beta-galactosidase activity by PRB and C1C2 was strictly dependent on specific ligand and the presence of a progesterone response element . However, overproduced C1C2 receptors had an adverse effect on the transcription of the lacZ gene . It was found that when overproduced C1C2 was activated by progesterone, an inhibitory effect on normal yeast cell growth was evident . These observations suggest that C1C2 is a potent trans-acting factor in yeast and that the amino-terminal domain of the chicken progesterone receptor may play a role in selective modulation of target gene activation.

EMBO J, 1989 Dec 20, 8(13), 4265 - 72
Sequence, expression and mutational analysis of BAF1, a transcriptional activator and ARS1-binding protein of the yeast Saccharomyces cerevisiae; Halfter H et al.; We report the cloning and sequence analysis of the yeast BAF1 gene which encodes an abundant protein previously shown to act as a transcription activator in the YPT1-TUB2 intergene region . As predicted from the DNA sequence, the highly hydrophilic BAf1 protein is 731 amino acids long and has a molecular mass of 81 748 daltons . The protein product of the cloned BAF1 gene produced in Escherichia coli is able to form specific complexes with DNA fragments containing the conserved element TCN7ACG . The protein binds also to the ABF1-binding site of the B-domain of ARS1, entertaining the possibility that BAF1 and ABF1 are identical proteins . Extensive deletion studies identified the N-terminal two thirds of the Baf1 protein to be required for specific DNA binding . Amino acid substitutions point to the N-terminal sequence CysX7HisX3HisX4CysX4Cys to form an atypical metal-binding 'finger' structure . Disruption of the BAF1 gene is lethal . The existence of five potential Baf1-protein binding sites in the 5' region of the gene suggests the involvement of the Baf1 protein in transcription regulation of its own gene.

EMBO J, 1989 Dec 20, 8(13), 4325 - 34
Cruciform cutting endonucleases from Saccharomyces cerevisiae and phage T4 show conserved reactions with branched DNAs; Jensch F et al.; We have purified a cruciform DNA resolving endonuclease (Endo X3) greater than 1000-fold from crude extracts of mitotically growing Saccharomyces cerevisiae . The enzyme shows high specificity for DNAs with secondary structures and introduces characteristic patterns of staggered 'nicks' in the immediate vicinity of the structure . The following substrates were analyzed in detail: (i) naturally occurring four-way X junctions in cruciform DNA of a supercoiled plasmid; (ii) synthetic four-way X junctions with arms of 9 bp; (iii) synthetic three-way Y junctions with arms of 10 bp; and (iv) heteroduplex loops with 19 nucleotides in the loop . Cleavages were always found in the double stranded portion of the DNA, located immediately adjacent to the junction of the respective structure . The Endo X3 induced cleavage patterns are identical or very similar to the cleavage patterns induced in the same substrates by endonuclease VII (Endo VII) from phage T4 . Furthermore, the activity of Endo X3 is completely inhibited in the presence of anti-Endo VII antiserum . Endo X3 has an apparent mol . wt of 43,000 daltons, determined by gel filtration and of approximately 18,000 daltons in SDS--polyacrylamide gels . Maximum activity of the enzyme was obtained in the presence of 10 mM MgCl2 at 31 degrees C in Tris-HCl buffer over a broad pH range with a maximum approximately 8.0 . About 70% of maximal activity was obtained when Mg2+ was replaced by equimolar amounts of Mn2+ or Ca2+.

J Biol Chem, 1989 Dec 15, 264(35), 21403 - 12
Purification and characterization of a DNA-pairing and strand transfer activity from mitotic Saccharomyces cerevisiae; Halbrook J et al.; An enzyme catalyzing homologous pairing of DNA chains has been extensively purified from mitotic yeast . The most highly purified fractions are enriched for a polypeptide with a molecular mass of approximately 120 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Protein-dependent pairing of single-stranded DNAs requires a divalent cation (Mg2+ or Ca2+) but proceeds rapidly in the absence of any nucleoside triphosphates . The kinetics of reassociation are extremely rapid, with more than 60% of the single-stranded DNA becoming resistant to S1 nuclease within 1 min at a ratio of 1 protein monomer/50 nucleotides . The results of enzyme titration and DNA challenge experiments suggest that this protein does not act catalytically during renaturation but is required stoichiometrically . The protein promotes formation of joint molecules between linear M13 replicative form DNA (form III) containing short single-stranded tails and homologous single-stranded M13 viral DNA . Removal of approximately 50 nucleotides from the ends of the linear duplex using either exonuclease III (5' ends) or T7 gene 6 exonuclease (3' ends) activates the duplex for extensive strand exchange . Electron microscopic analysis of product molecules suggests that the homologous circular DNA initially associates with the single-stranded tails of the duplexes, and the heteroduplex region is extended with displacement of the noncomplementary strand . The ability of this protein to pair and to promote strand transfer using either exonuclease III or T7 gene 6 exonuclease-treated duplex substrates suggests that this activity promotes heteroduplex extension in a nonpolar fashion . The biochemical properties of the transferase are consistent with a role for this protein in heteroduplex joint formation during mitotic recombination in Saccharomyces cerevisiae.

Biochem Biophys Res Commun, 1989 Dec 15, 165(2), 667 - 74
Saccharomyces cerevisiae chromatin associated protein Yp20 is a guanine nucleotide binding protein; Valansi C et al.; Yp20 is a 20kD protein whose role is still obscure which copurifies with yeast histones . Yeast histones were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose . Incubation of the nitrocellulose blots with {gamma-35S} GTP gamma S demonstrated that Yp20 is a GTP binding protein . A polyclonal antiserum raised against purified Yp20 cross reacted with bacterially expressed cHras and T24 Hras genuine ras products . The results obtained suggest that Yp20 is a yeast chromatin associated ras-related antigen.

Cancer Res, 1989 Dec 15, 49(24 Pt 1), 6935 - 40
Cleavage of cellular and extracellular Saccharomyces cerevisiae DNA by bleomycin and phleomycin; Moore CW; Low-molecular-weight phleomycin (Mr approximately 1500-1600) is considerably less active on a per mol basis than structurally related bleomycin in degrading purified Saccharomyces cerevisiae DNA . Phleomycin also exhibits a substantially higher requirement than bleomycin for ferrous ions . However, phleomycin (0.13 to 3.3 x 10(-6) M) produced 7 to 350 times more breaks than bleomycin in prelabeled intracellular {2-14C}DNA and {6-3H}DNA and is considerably more cytotoxic than bleomycin . Phleomycin and bleomycin produced equivalent numbers of DNA breaks at equivalent, physiologically meaningful levels of survival, indicating that DNA breaks are related to lethal properties of the anticancer glycopeptides . Phleomycin degradation of extracellular DNA was only detectable at greater than or equal to 1.7 x 10(-4) M, approximately two orders of magnitude higher than the concentrations of phleomycin which yielded equivalent fragmentation of intracellular DNA, indicating that phleomycin causes substantially more degradation of intracellular DNA than extracellular DNA . In contrast, bleomycin (greater than or equal to 1.7 x 10(-5) M) degradation of purified DNA is quite extensive and considerably greater than the degradation of DNA in cells incubated with the same or higher concentrations of bleomycin . Neither phleomycin nor bleomycin cleaved extracellular DNA in the absence of ferrous ions, although both chemical analogues cleaved intracellular DNA without adding iron . Therefore, the requirement for metal ion in stimulating DNA degradation by the two structural families of glycopeptidic antibiotics is met by the cell itself.

Eur J Biochem, 1989 Dec 8, 186(1-2), 361 - 6
Purification and properties of the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (phenylalanine-inhibitable) of Saccharomyces cerevisiae; Paravicini G et al.; The phenylalanine-inhibitable 3-deoxy-D-arabino-heptulosonate-7-phosphate (dHp1P) synthase from Saccharomyces cerevisiae has been purified to apparent homogeneity by a 1250-fold enrichment of the enzyme activity present in wild-type crude extracts, employing an overproducing strain . The estimated molecular mass of 42 kDa corresponds to the calculated molecular mass of 42.13 kDa deduced from the previously determined primary sequence . Gel filtration indicates that the active enzyme is a monomer . The enzyme is an Fe protein and is inactivated by EDTA in a reaction which is reversible by several bivalent metal ions . The Michaelis constant of the enzyme is 18 microM for phosphoenolpyruvate (P-pyruvate) and 130 microM for erythrose 4-phosphate (Ery4P) and the rate constant was calculated as 10 s-1 . Inhibition by phenylalanine is competitive with respect to erythrose 4-phosphate and non-competitive to phosphoenolpyruvate, with a Ki of 10 microM.

Gene, 1989 Dec 7, 84(1), 207 - 8
U3 small nuclear RNA (snR17A RNA) and DFR1 genes are very closely linked in Saccharomyces cerevisiae; Bachellerie JP; SNR17A and DFR1 genes of Saccharomyces cerevisiae are only 313 bp apart and in the same orientation.

J Biol Chem, 1989 Dec 5, 264(34), 20532 - 9
Purification and characterization of Rad3 ATPase/DNA helicase from Saccharomyces cerevisiae; Harosh I et al.; The Rad3 ATPase/DNA helicase was purified to physical homogeneity from extracts of yeast cells containing overexpressed Rad3 protein . The DNA helicase can unwind duplex regions as short as 11 base pairs in a partially duplex circular DNA substrate and does so by a strictly processive mechanism . On partially duplex linear substrates, the enzyme has a strict 5'----3' polarity with respect to the single strand to which it binds . Nicked circular DNA is not utilized as a substrate, and the enzyme requires single-stranded gaps between 5 and 21 nucleotides long to unwind oligonucleotide fragments from partially duplex linear molecules . The enzyme also requires duplex regions at least 11 base pairs long when these are present at the ends of linear molecules . Rad3 DNA helicase activity is inhibited by the presence of ultraviolet-induced photoproducts in duplex regions of partially duplex circular molecules.

J Biol Chem, 1989 Dec 5, 264(34), 20181 - 4
Phosphatidylinositol 3-kinase and its novel product, phosphatidylinositol 3-phosphate, are present in Saccharomyces cerevisiae; Auger KR et al.; The metabolism of polyphosphoinositides has been shown to be an important factor in controlling the proliferation of Saccharomyces cerevisiae . The monophosphate form of phosphatidylinositol has been assumed to be phosphatidylinositol 4-phosphate (PI-4-P) . Recent evidence from our laboratory has established that a phosphatidylinositol (PI) kinase, which phosphorylates the D-3 position of the inositol ring (PI 3-kinase), is associated with many activated protein-tyrosine kinases and may play an important role in the signaling of cell proliferation (Auger, K . R., Serunian, L . A., Soltoff, S . P., Libby, P., and Cantley, L . C . (1989) Cell 57, 167-175) . To determine the evolutionary conservation of this enzymatic activity, we investigated its presence in yeast . In vitro PI kinase assays of yeast cell homogenates demonstrated that PI 3-kinase activity was present . Preliminary biochemical characterization of the activity suggested that it was quite different from the mammalian enzyme yet catalyzed the same reaction, i.e . phosphorylating the D-3 hydroxyl position of the inositol ring of phosphatidyl-myo-inositol . {3H}Inositol labeling of intact yeast cells with the subsequent extraction, deacylation, and high performance liquid chromatography analysis of the lipids demonstrated that PI-3-P was as abundant as the PI-4-P isomer . The conservation of the enzymatic activity from yeast to man suggests that it has an important functional role in the cell cycle.

Genetika, 1989 Dec, 25(12), 2111 - 20
{The effect of him mutations characterized by enhanced induced mutagenesis on the spontaneous mitotic recombination in the ADE2 gene in Saccharomyces cerevisiae}; Koval'tsova SV et al.; We have studied the influence of him1, him2, him3 and himX mutations on the frequency of spontaneous mitotic gene conversion in the yeast Saccharomyces cerevisiae using the set of heteroallelic combinations in the ADE2 gene . Data obtained on the HIM/HIM, him/him homozygotes and HIM/him heterozygotes indicate that the him1 mutation is recessive with respect to conversion, whereas the him2, him3 and himX mutations are semidominant . Gene conversion was increased in the majority of heteroalleles of mutant diploids him1/him1 . On the contrary, the him2, him3 and himX mutants have hypo-rec phenotypes on mitotic conversion . The him mutations do not affect some heteroalleles, moreover, for some heteroalleles, the effects of the him mutations was opposite . On the basis of the sum of genetical data and, particularly, of conversion event pattern in the him mutants, we suggest that him mutations analysed affect the repair pathway for mismatch correction.

Mol Microbiol, 1989 Dec, 3(12), 1697 - 707
Regulation of the RAD2 gene of Saccharomyces cerevisiae; Siede W et al.; Regulation of the DNA damage-inducible RAD2 gene was investigated in yeast cells transformed with centromeric plasmids containing RAD2-lacZ fusion constructs . Deletion analysis defined several regions in the 350bp region upstream of the translational start codon which are required for induction of beta-galactosidase activity . No deletions resulted in constitutively enhanced expression . We therefore conclude that induction of RAD2 by DNA-damaging agents is positively regulated . Two domains required for induction have a similar sequence and are located approximately 70 and approximately 140bp upstream of the major transcriptional start site . Four other sequence domains required for induction contain uninterrupted poly(dA) poly(dT) stretches 9-13bp long . Deletion of some of these AT-rich domains also affects constitutive expression of RAD2 . Expression of RAD2 is not cell-cycle-regulated in mitotic cells . However, meiosis is accompanied by increased steady-state levels of RAD2 mRNA in the absence of DNA damage . This enhanced transcription is not dependent on the presence of upstream sequences required for regulation of induction by DNA damage . Increased steady-state levels of RAD2 mRNA are induced by cycloheximide in asynchronously dividing populations of cells, but not in non-replicating cells arrested in G1 phase of the cell cycle . Following exposure to u.v . irradiation induction is also dramatically reduced in non-replicating cells.

Antimicrob Agents Chemother, 1989 Dec, 33(12), 2092 - 5
Antibodies to nystatin demonstrate polyene sterol specificity and allow immunolabeling of sterols in Saccharomyces cerevisiae; Walker-Caprioglio HM et al.; Polyclonal antibodies elicited by injection into rabbits of a nystatin-bovine serum albumin conjugate were reactive with both nystatin and amphotericin B . Upon labeling of polyene-treated Saccharomyces cerevisiae sterol auxotrophs grown on various sterols, nystatin reacted specifically with ergosterol, while amphotericin B did not react preferentially with ergosterol, cholesterol, or cholestanol . Time course labeling experiments demonstrated the rate of ergosterol transport into cholesterol-grown cells.

Lipids, 1989 Dec, 24(12), 1020 - 3
Regulation of squalene synthetase and squalene epoxidase activities in Saccharomyces cerevisiae; M'Baya B et al.; Squalene synthetase (EC 2.5.1.21) and squalene epoxidase (EC 1.14 99.7) activities have been measured in cell-free extracts of wild type yeast grown in aerobic and semi-anaerobic conditions as well as in sterol-auxotrophic mutant strains grown aerobically . The results show that both enzymes are induced resulting in an almost two- to five-fold increase in enzymatic activities in mutant strains containing limited sterol amounts and are repressed in the wild type strain cultured in anaerobiosis in excess of sterol . The results show also that squalene epoxidase is repressed by lanosterol, and that the mevalonic acid pool may regulate squalene synthetase levels . The large change in the activities of the two enzymes, depending on the sterol needs of the cells, as well as their low specific activities in comparison with those of the enzymes involved in the early stages of sterol synthesis strongly suggests that squalene synthetase and squalene epoxidase are of importance in regulating the amount of sterol synthesized by yeast.

Genetics, 1989 Dec, 123(4), 715 - 24
Intragenic and extragenic suppressors of mutations in the heptapeptide repeat domain of Saccharomyces cerevisiae RNA polymerase II; Nonet ML et al.; The largest subunit of RNA polymerase II contains a repeated heptapeptide sequence at its carboxy terminus . Yeast mutants with certain partial deletions of the carboxy-terminal repeat (CTR) domain are temperature-sensitive, cold-sensitive and are inositol auxotrophs . Intragenic and extragenic suppressors of the cold-sensitive phenotype of CTR domain deletion mutants were isolated and studied to investigate the function of this domain . Two types of intragenic suppressing mutations suppress the temperature-sensitivity, cold-sensitivity and inositol auxotrophy of CTR domain deletion mutants . Most intragenic mutations enlarge the repeat domain by duplicating various portions of the repeat coding sequence . Other intragenic suppressing mutations are point mutations in a conserved segment of the large subunit . An extragenic suppressing mutation (SRB2-1) was isolated that strongly suppresses the conditional and auxotrophic phenotypes of CTR domain mutations . The SRB2 gene was isolated and mapped, and an SRB2 partial deletion mutation (srb2 delta 10) was constructed . The srb2 delta 10 mutants are temperature-sensitive, cold-sensitive and are inositol auxotrophs . These phenotypes are characteristic of mutations in genes encoding components of the transcription apparatus . We propose that the SRB2 gene encodes a factor that is involved in RNA synthesis and may interact with the CTR domain of the large subunit of RNA polymerase II.

Curr Genet, 1989 Dec, 16(5-6), 331 - 8
Formation and stability of interstrand cross-links induced by cis- and trans-diamminedichloroplatinum (II) in the DNA of Saccharomyces cerevisiae strains differing in repair capacity; Wilborn F et al.; Four haploid yeast strains differing in proficiency for DNA repair were treated with cis- or transDDP . The wild type was least sensitive while the excision-deficient mutants rad1, rad2 and snm1 exhibited higher sensitivities to either platinum compound . In all four strains tested cisDDP showed a two- to five-fold higher cytotoxicity than equimolar concentrations of transDDP . DNA interstrand cross-linking was caused by both agents in all strains . However, transDDP introduced more DNA cross-links at exposure times up to 6 h while cisDDP was the more active cross-linking agent at longer times . There was no clear-cut correlation of the number of DNA interstrand cross-links with survival . Formaldehyde-treated cells showed DNA with lower buoyant density due to proteinase K sensitive DNA-protein cross-linking; this effect was not observed after treatment with either platinum compound . Post-treatment incubation of wild-type cells exposed to cisDDP led to degradation of DNA by single and double-strand breaks, parallel with further increase of DNA interstrand cross-linking . DNA from transDDP-treated cells did not show extensive degradation although interstrand cross-links were lost during liquid holding.

J Cell Biol, 1989 Dec, 109(6 Pt 2), 3355 - 66
The dynamics of chromosome movement in the budding yeast Saccharomyces cerevisiae; Palmer RE et al.; Nuclear DNA movement in the yeast, Saccharomyces cerevisiae, was analyzed in live cells using digital imaging microscopy and corroborated by the analysis of nuclear DNA position in fixed cells . During anaphase, the replicated nuclear genomes initially separated at a rate of 1 micron/min . As the genomes separated, the rate of movement became discontinuous . In addition, the axis defined by the segregating genomes rotated relative to the cell surface . The similarity between these results and those previously obtained in higher eukaryotes suggest that the mechanism of anaphase movement may be highly conserved . Before chromosome separation, novel nuclear DNA movements were observed in cdc13, cdc16, and cdc23 cells but not in wild-type or cdc20 cells . These novel nuclear DNA movements correlated with variability in spindle position and length in cdc16 cells . Models for the mechanism of these movements and their induction by certain cdc mutants are discussed.

Experientia, 1989 Dec 1, 45(11-12), 1018 - 23
Kinetic analysis and simulation of glucose transport in plasma membrane vesicles of glucose-repressed and derepressed Saccharomyces cerevisiae cells; Fuhrmann GF et al.; In this study experimental data on the kinetic parameters investigated by other authors 1-5, 11 together with own data on plasma membrane vesicles, have been subjected to a computer simulation based on the equations describing facilitated diffusion . The simulation led to an ideal fit describing the above data . From this it can be concluded that glucose is transported by facilitated diffusion, and not by active transport as was postulated by Van Steveninck 14,15 . The simulation method also demonstrates that the fast sampling technique used by these authors 1-5, 11 underestimated the fluxes . Thus, the parameters given do not contribute to the understand of glucose transport under different metabolic conditions . The K value of plasma membrane vesicles prepared from glucose-repressed cells is around 7 mM . Derepression, particularly by galactose, causes a highly significant increase in affinity as shown by a decrease in the K value to 2 mM . The highest affinity was measured in a triple kinaseless mutant grown on glycerol with a K value of 1 mM . It seems, therefore, that the kinetic parameters derived from initial uptake rates of glucose in intact cells 1-5, 11 using single flux analysis, such as Eadie-Hofstee- or Lineweaver-Burk-plots, are in error.

Mutat Res, 1989 Dec, 224(4), 471 - 8
Observations on chromosome loss detection by multiple recessive marker expression in strain D61.M of Saccharomyces cerevisiae; Mayer VW et al.; Since chromosomes of fungi are difficult to observe directly, strains have been developed in which chromosome loss can be detected by the use of genetic markers . In the diploid D61.M strain of Saccharomyces cerevisiae, the loss of a copy of chromosome VII that carries 3 dominant wild-type alleles is measured by expression of 3 recessive mutant alleles carried on the other remaining copy of chromosome VII . We have tested the hypothesis that expression of the 3 recessive alleles might be due to 3 simultaneous independent genetic events other than chromosome loss, such as mutation or recombination . We have measured, when possible, the frequencies of expression for each of these recessive alleles, independently and in combination one with another, under both selective and non-selective conditions . Our results show that simultaneous expression of these 3 recessive alleles is attributable to chromosome loss (greater than 98%) . Similarly, at least 99% of the nocodazole-induced events are attributable to chromosome loss . In contrast, most if not all of the apparent chromosome loss induced by ethyl methanesulfonate is due to multiple events of mutation or recombination.

Mol Cell Biol, 1989 Dec, 9(12), 5702 - 12
Positive and negative transcriptional control by heme of genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae; Thorsness M et al.; Responses of the yeast genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, HMG1 and HMG2, to in vivo changes in heme concentrations were investigated . Expression of the genes was determined by direct measurement of the mRNA transcribed from each gene, by direct assay of the enzyme activity encoded by each gene, and by measurement of the expression of lacZ fusions to the control regions of each gene . These studies indicated that expression of HMG1 was stimulated by heme, whereas expression of HMG2 was repressed by heme . The effect of heme on HMG1 expression was mediated by the HAP1 transcriptional regulator and was independent of HAP2 . Thus, the genes encoding the 3-hydroxy-3-methylglutaryl coenzyme A reductase isozymes join a growing list of gene pairs that are regulated by heme in opposite ways.

Mol Cell Biol, 1989 Dec, 9(12), 5643 - 9
The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae; Ma H et al.; Saccharomyces cerevisiae mutants containing different point mutations in the HXK2 gene were used to study the relationship between phosphorylation by hexokinase II and glucose repression in yeast cells . Mutants showing different levels of hexokinase activity were examined for the degree of glucose repression as indicated by the levels of invertase activity . The levels of hexokinase activity and invertase activity showed a strong inverse correlation, with a few exceptions attributable to very unstable hexokinase II proteins . The in vivo hexokinase II activity was determined by measuring growth rates, using fructose as a carbon source . This in vivo hexokinase II activity was similarly inversely correlated with invertase activity . Several hxk2 alleles were transferred to multicopy plasmids to study the effects of increasing the amounts of mutant proteins . The cells that contained the multicopy plasmids exhibited less invertase and more hexokinase activity, further strengthening the correlation . These results strongly support the hypothesis that the phosphorylation activity of hexokinase II is correlated with glucose repression.

Mol Cell Biol, 1989 Dec, 9(12), 5630 - 42
Isolation and characterization of mutations in the HXK2 gene of Saccharomyces cerevisiae; Ma H et al.; Several hundred new mutations in the gene (HXK2) encoding hexokinase II of Saccharomyces cerevisiae were isolated, and a subset of them was mapped, resulting in a fine-structure genetic map . Among the mutations that were sequenced, 35 were independent missense mutations . The mutations were obtained by mutagenesis of cloned HXK2 DNA carried on a low-copy-number plasmid vector and screened for a number of different phenotypes in yeast strains bearing chromosomal hxk1 and hxk2 null mutations . Some of these mutants were characterized both in vivo and in vitro; they displayed a wide spectrum of residual hexokinase activities, as indicated by three assays: in vitro enzyme activity, ability to grow on glucose and fructose, and ability to repress invertase production when growing on glucose . Of those that failed to support growth on fructose, only a small minority made normal-size, stable, and inactive protein . Analysis of the amino acid changes in these mutants in light of the crystallographically determined three-dimensional structure of hexokinase II suggests important roles in structure or catalysis for six amino acid residues, only two of which are near the active site.

Mol Cell Biol, 1989 Dec, 9(12), 5602 - 9
The Saccharomyces cerevisiae SPT13/GAL11 gene has both positive and negative regulatory roles in transcription; Fassler JS et al.; To understand the function of SPT13, a gene encoding a trans-acting factor involved in regulation of Ty-mediated gene expression, we have cloned and sequenced the gene . Our analysis revealed that SPT13 is the same gene as GAL11, a gene in which mutations cause reduced expression of some GAL4-regulated genes . Further analysis of spt13/gal11 mutants suggested that the SPT13 gene also affects transcription of genes involved in mating-type specialization . We show here that SPT13 has both positive and negative regulatory roles in transcription.

Mol Cell Biol, 1989 Dec, 9(12), 5585 - 93
Purification of a protein binding to the CDEI subregion of Saccharomyces cerevisiae centromere DNA; Jiang WD et al.; The DNA subregions CDEI and CDEIII of Saccharomyces cerevisiae centromeres are highly conserved, and both are binding sites for proteins . We describe here the purfication of a CDEI-specific binding protein using biotin-labeled synthetic CDEI DNA coupled to streptavidin agarose . The binding properties of this 64-kilodalton (kDa) protein were characterized by competition assays and by methylation interference assays . DNA fragments with single base-pair changes at positions 7 and 8 of CDEI were less efficient competitors than fragments with nonmutated CDEI . Mutations at these positions have previously been shown to decrease centromere activity in vivo . Methylation of guanosines at either side of the 8-base-pair CDEI sequence did not interfere with binding, whereas methylation of any of the four guanosines within CDEI prevented binding . A smaller CDEI-specific binding protein of 37 kDa was also purified and characterized . It is most likely a degradation product of the 64-kDa protein.

Genetics, 1989 Dec, 123(4), 739 - 48
Isolation and characterization of temperature-sensitive mutations in the RAS2 and CYR1 genes of Saccharomyces cerevisiae; Mitsuzawa H et al.; The yeast Saccharomyces cerevisiae contains two ras homologues, RAS1 and RAS2, whose products have been shown to modulate the activity of adenylate cyclase encoded by the CYR1 gene . To isolate temperature-sensitive mutations in the RAS2 gene, we constructed a plasmid carrying a RAS2 gene whose expression is under the control of the galactose-inducible GAL1 promoter . A ras1 strain transformed with this plasmid was subjected to ethyl methanesulfonate mutagenesis and nystatin enrichment . Screening of approximately 13,000 mutagenized colonies for galactose-dependent growth at a high temperature (37 degrees) yielded six temperature-sensitive ras2 (ras2ts) mutations and one temperature-sensitive cyr1 (cyr1ts) mutation that can be suppressed by overexpression or increased dosage of RAS2 . Some ras2ts mutations were shown to be suppressed by an extra copy of CYR1 . Therefore increased dosage of either RAS2 or CYR1 can suppress the temperature sensitivity caused by a mutation in the other . ras1 ras2ts and ras1 cyr1ts mutants arrested in the G1 phase of the cell cycle at the restrictive temperature, and showed pleiotropic phenotypes to varying degrees even at a temperature permissive for growth (25 degrees), including slow growth, sporulation on rich media, increased accumulation of glycogen, impaired growth on nonfermentable carbon sources, heat-shock resistance, impaired growth on low concentrations of glucose, and lithium sensitivity . Of these, impaired growth on low concentrations of glucose and sensitivity to lithium are new phenotypes, which have not been reported for mutants defective in the cAMP pathway.

Mol Gen Genet, 1989 Dec, 220(1), 33 - 42
Analysis of the HO-cleaved MAT DNA intermediate generated during the mating type switch in the yeast Saccharomyces cerevisiae; Raveh D et al.; A galactose-inducible HO gene was used to induce mating type switching in heterothallic Saccharomyces cerevisiae cells arrested in G1, in rad52 mutants defective in DNA damage repair, and in cells lacking the donor cassettes . The HO-cleaved MAT intermediate is stable over significant lengths of time, i.e . HO cleavage is not coupled to the subsequent gene conversion event . The in vivo cleavage site was mapped to single base resolution by primer extension experiments on total genomic DNA . Cells arrested in G1 with alpha-factor switched mating type thus demonstrating that switches can occur in the absence of replication of the genome . rad52 mutants did not produce MAT DNA of the opposite mating type indicating that the block is prior to the gene duplication stage of the switch . In strains in which the HM donor cassettes are deleted the cut MAT DNA was degraded after induction of the HO gene.

Mol Gen Genet, 1989 Dec, 220(1), 133 - 9
Molecular characterization of a specific p-nitrophenylphosphatase gene, PHO13, and its mapping by chromosome fragmentation in Saccharomyces cerevisiae; Kaneko Y et al.; The structural gene, PHO13, for the specific p-nitrophenyl phosphatase of Saccharomyces cerevisiae was cloned and its nucleotide sequence determined . The deduced PHO13 protein consists of 312 amino acids and its molecular weight is 34635 . The disruption of the PHO13 gene produced no effect on cell growth, sporulation, or viability of ascospores . The PHO13 locus was mapped at 1.9 centimorgans from the HO locus on the left arm of chromosome IV . By chromosome fragmentation, the PHO13 locus was found to be located about 72 kb from the left-hand telomere of chromosome IV and distal to the HO locus.

Mol Cell Biol, 1989 Dec, 9(12), 5350 - 8
Identification of an upstream activation sequence and other cis-acting elements required for transcription of COX6 from Saccharomyces cerevisiae; Trawick JD et al.; Transcription of Saccharomyces cerevisiae COX6, the nuclear gene for subunit VI of cytochrome c oxidase, is activated in heme-proficient cells, requires the HAP2 gene, and is subject to glucose repression . In this study, by deletion mutagenesis of the COX6 promoter, we identified two regions that are important for transcription . The first was an upstream activation site, UAS6 . It was found to be contained within an 84-base-pair (bp) sequence, between bp -256 and -340 of the COX6 translational initiation codon, and to contain sequences required for activation by heme and HAP2 and for release from glucose repression . When located upstream of a CYC1-lacZ fusion gene, deleted for both of its UASs, this segment functioned as a UAS element . Although UAS6 could promote expression in either orientation, it showed a marked orientation dependence in its response to HAP2 and the carbon source . The second region lay between bp -255 and -91 . It contained two of the three major 5' termini of COX6 mRNAs and a putative TATA box . Deletion analysis of this region demonstrated that the putative TATA box is not required for transcription and that this region is separable into two redundant domains.

Mol Cell Biol, 1989 Dec, 9(12), 5440 - 4
Requirement of upstream activation sequences for nitrogen catabolite repression of the allantoin system genes in Saccharomyces cerevisiae; Cooper TG et al.; Synthesis of the transport systems and enzymes mediating uptake and catabolism of nitrogenous compounds is sensitive to nitrogen catabolite repression . In spite of the widespread occurrence of the control process, little is known about its mechanism . We have previously demonstrated that growth of cells on repressive nitrogen sources results in a dramatic decrease in the steady-state levels of mRNA encoded by the allantoin and arginine catabolic pathway genes and of the transport systems associated with allantoin metabolism . The present study identified the upstream activation sequences in the 5'-flanking regions of the allantoin system genes as the cis-acting sites through which nitrogen catabolite repression is exerted.

Gene, 1989 Nov 30, 83(2), 271 - 9
The suppressor gene scl1+ of Saccharomyces cerevisiae is essential for growth; Balzi E et al.; In Saccharomyces cerevisiae, the SCL-1 mutation is a dominant suppressor of the cycloheximide-resistant, temperature-sensitive (ts) lethal mutation, crl3 {McCusker and Haber, Genetics 119 (1988a) 303-315} . The wild-type scl1+ gene was isolated by screening subclones of the 35-kb region between TRP5 and LEU1 for restoration of the ts phenotype in an SCL1-1 crl3-2 strain . The scl1+ mRNA is about 900 nt long and encodes an open reading frame of 810 bp . The polypeptide deduced from scl1+ possesses a putative secretory signal peptide . The 5'-noncoding region may be under multiple controls, since it contains significant homology to the consensus sequences for the DNA-binding proteins, GCN4, GFI and, possibly, TUF . Gene disruption of scl1+ demonstrates that it is an essential gene.

Biochim Biophys Acta, 1989 Nov 27, 986(2), 301 - 9
Membrane properties modulate the activity of a phosphatidylinositol transfer protein from the yeast, Saccharomyces cerevisiae; Szolderits G et al.; A phospholipid transfer protein from yeast (Daum, G . and Paltauf, F . (1984) Biochim . Biophys . Acta 794, 385-391) was 2800-fold enriched by an improved procedure . The specificity of this transfer protein and the influence of membrane properties of acceptor vesicles (lipid composition, charge, fluidity) on the transfer activity were determined in vitro using pyrene-labeled phospholipids . The yeast transfer protein forms a complex with phosphatidylinositol or phosphatidylcholine, respectively, and transfers these two phospholipids between biological and/or artificial membranes . The transfer rate for phosphatidylinositol is 19-fold higher than for phosphatidylcholine as determined with 1:8 mixtures of phosphatidylinositol and phosphatidylcholine in donor and acceptor membrane vesicles . If acceptor membranes consist only of non-transferable phospholipids, e.g., phosphatidylethanolamine, a moderate but significant net transfer of phosphatidylcholine occurs . Phosphatidylcholine transfer is inhibited to a variable extent by negatively charged phospholipids and by fatty acids . Differences in the accessibility of the charged groups of lipids to the transfer protein might account for the different inhibitory effects, which occur in the order phosphatidylserine which is greater than phosphatidylglycerol which is greater than phosphatidylinositol which is greater than cardiolipin which is greater than phosphatidic acid which is greater than fatty acids . Although mitochondrial membranes contain high amounts of negatively charged phospholipids, they serve effectively as acceptor membranes, whereas transfer to vesicles prepared from total mitochondrial lipids is essentially zero . Ergosterol reduces the transfer rate, probably by decreasing membrane fluidity . This notion is supported by data obtained with dipalmitoyl phosphatidylcholine as acceptor vesicle component; in this case the transfer rate is significantly reduced below the phase transition temperature of the phospholipid.

J Biol Chem, 1989 Nov 25, 264(33), 20060 - 7
Transcription of the 5 S rRNA gene of Saccharomyces cerevisiae requires a promoter element at +1 and a 14-base pair internal control region; Challice JM et al.; In vitro transcription of templates containing deletion-substitution mutations has localized two essential promoter elements of the 5 S rRNA gene of Saccharomyces cerevisiae . A promoter element spanning the start site of transcription extends from -14 to +8, and a short internal control region (ICR) extends from +81 to +94 . Changes in spacing between these elements by more than a few base pairs significantly reduce transcription . The site of RNA polymerase III transcription factor A (TFIIIA) binding, mapped by determination of the G residues that are protected from methylation on exposure of the TFIIIA.5 S DNA complex to dimethyl sulfate, is coincident with the ICR . Incorporation of TFIIIC into the TFIIIA.5 S rRNA gene complex protects additional G residues 5' and 3' of the ICR from methylation.

Biochim Biophys Acta, 1989 Nov 20, 1014(2), 153 - 61
Studies on the degradative mechanism of phosphoenolpyruvate carboxykinase from yeast Saccharomyces cerevisiae; Burlini N et al.; Previous work carried out in our laboratory (Burlini, N., Lamponi S., Radrizzani, M., Monti, E . and Tortora P . (1987) Biochim . Biophys . Acta 930, 220-229) led to the immunological identification of a yeast 65-kDa phosphoprotein as a modified form of phosphoenolpyruvate carboxykinase; moreover the appearance of this phospho form was proven to be independent of cAMP, whereas the glucose-induced inactivation of the native enzyme is cAMP-dependent . Here, we report further investigations on the mechanism of the glucose-triggered degradation of the enzyme which led to the following results: (a) the aforementioned phospho form displayed a binding pattern to 5 AMP-Sepharose 4B quite similar to that of native enzyme, although it did not retain its oligomeric structure, nor was it catalytically active; (b) its phosphate content was of about two residues per monomer; (c) its isoelectric point was slightly higher than that of native enzyme, this shows that the enzyme undergoes additional modifications besides phosphorylation; (d) it represented about 4% of the native enzyme in glucose-depressed cells; (e) other forms immunologically cross-reactive with the native enzyme were also isolated, whose molecular mass was in the range of 60-62 kDa, and they are probable candidates as degradation products of the phospho form; (f) time courses of the native and phospho forms in the presence and the absence of glucose provided data consistent with a kinetic model involving a strong stimulation of the decay of both forms effected by the sugar; (g) in the mutant ABYS1 (Achstetter, T., Emter, O., Ehmann, C . and Wolf, D.H . (1984) J . Biol . Chem . 259, 13334-13343) which is devoid of the four major vacuolar proteinases, the decay pattern was essentially the same as in wild-type; (h) effectors lowering intracellular ATP also retarded the first step of enzyme degradation; this points to an ATP-dependence of this step . Based on these results we propose a degradation mechanism consisting of an initial cAMP- and ATP-dependent modification of the enzyme, followed by a cAMP-independent phosphorylation, which leads to the appearance of the aforementioned monomeric phospho form; this in turn seems to undergo limited proteolysis . These data strongly suggest the occurrence of an intermediate form arising from the native one and whose phosphorylation gives rise to the 65-kDa phosphoprotein described here.

Gene, 1989 Nov 15, 83(1), 65 - 74
Synthesis and secretion of human nerve growth factor by Saccharomyces cerevisiae; Kanaya E et al.; The DNA coding for human nerve growth factor (hNGF) was chemically synthesized and introduced into Saccharomyces cerevisiae . Expression and secretion of hNGF was obtained by use of the yeast phosphoglycerate kinase-encoding gene promoter and the pre-pro sequence of the yeast alpha-mating factor . Immunoblotting with antiserum raised against a protein A-hNGF fusion protein, allowed the detection of an immunoreactive material secreted into the culture medium . A preparation from the culture medium, partially purified by ion-exchange column chromatography, stimulated neurite outgrowth from rat pheochromocytoma PC12h cells.

Biochem Biophys Res Commun, 1989 Nov 15, 164(3), 1157 - 64
A rapid and efficient procedure for transformation of intact Saccharomyces cerevisiae by electroporation; Simon JR et al.; A rapid and efficient procedure is described for transforming Saccharomyces cerevisiae using electroporation to render intact cells permeable to DNA . The technique uses relatively low voltages and is particularly sensitive to low concentrations of plasmid DNA . At the highest voltage used (400 volts), the frequency of transformation increased with the amount of plasmid DNA between 25 ng and 100 ng . At higher concentrations of DNA (1-1.5 micrograms) electroporation yielded one-third to one-half the number of transformants obtained with a standard lithium acetate pretreatment . Because this method requires neither pretreatment of cells nor addition of polyethylene glycol (PEG), it has several advantages over currently used transformation procedures.

J Biol Chem, 1989 Nov 15, 264(32), 19176 - 84
Farnesyl diphosphate synthetase . Molecular cloning, sequence, and expression of an essential gene from Saccharomyces cerevisiae; Anderson MS et al.; Farnesyl diphosphate (FPP) synthetase is a key enzyme in isoprenoid biosynthesis which supplies C15 precursors for several classes of essential metabolites including sterols, dolichols, and ubiquinones . The structural gene for FPP synthetase was isolated on a 4.5-kilobase EcoRI genomic restriction fragment from the yeast Saccharomyces cerevisiae . The clone encodes a 40,483-dalton polypeptide of 342 amino acids with a high degree of similarity to the protein encoded by a putative rat liver clone of FPP synthetase (Clarke, C . F., Tanaka, R . D., Svenson, K., Wamsley, M., Fogelman, A . M., and Edwards, P . A . (1987) Mol . Cell Biol . 7, 3138-3146) and to an active site protein fragment from avian liver FPP synthetase (Brems, D . N., Bruenger, E., and Rilling, H . C . (1981) Biochemistry 20, 3711-3718) . When cloned into the yeast shuttle vector YRp17, the 4.5-kilobase EcoRI fragment directed a 2-3-fold over-expression of FPP synthetase activity in transformed yeast cells . The levels of expression were independent of culture growth phase and orientation of the insert, indicative of a functional promoter in the clone . Disruption of the FPP synthetase gene from a diploid yeast strain, followed by dissection and analysis of tetrads, demonstrates that the gene is an essential, single copy number gene in yeast . The gene for FPP synthetase resides on chromosome XI as judged from Southern blots of separated yeast chromosomes.

J Biol Chem, 1989 Nov 15, 264(32), 19169 - 75
Isopentenyl diphosphate:dimethylallyl diphosphate isomerase . An improved purification of the enzyme and isolation of the gene from Saccharomyces cerevisiae; Anderson MS et al.; Isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IPP isomerase) is an enzyme in the isoprenoid biosynthetic pathway which catalyzes the interconversion of the primary five-carbon homoallylic and allylic diphosphate building blocks . We report a substantially improved procedure for purification of this enzyme from Saccharomyces cerevisiae . An amino-terminal sequence (35 amino acids) was obtained from a highly purified preparation of IPP isomerase . Oligonucleotide probes based on the protein sequence were used to isolate the structural gene encoding IPP isomerase from a yeast lambda library . The cloned gene encodes a 33,350-dalton polypeptide of 288 amino acids . A 3.5-kilobase EcoRI fragment containing the gene was subcloned into the yeast shuttle vector YRp17 . Upon transformation with plasmids containing the insert, a 5-6-fold increase in IPP isomerase activity was seen in transformed cells relative to YRp17 controls, confirming the identity of the cloned gene . This is the first reported isolation of the gene for IPP isomerase.

J Biol Chem, 1989 Nov 15, 264(32), 18874 - 7
Isolation and characterization of a Saccharomyces cerevisiae mutant with a disrupted gene for the IP subunit of succinate dehydrogenase; Lombardo A et al.; A partial cDNA clone corresponding to the iron-sulfur protein of succinate dehydrogenase (EC 1.3.99.1) has been isolated by an application of the polymerase chain reaction (Gould, S . J., Subramani, S., and Scheffler, I . E . (1989) Proc . Natl . Acad . Sci . U . S . A . 86, 1934-1938) . We used this clone for targeted gene disruption to isolate clones of Saccharomyces cerevisiae which were totally defective in this enzyme . The disruption of the gene was verified by Southern analysis . A Northern analysis revealed the existence of a new transcript which includes the 5' end of the mRNA for the IP subunit, but extends into the vector sequence disrupting the gene . A Western analysis suggests that the large flavoprotein subunit (FP) is still made in the mutant and imported into the mitochondria, but at significantly lower levels.

Gene, 1989 Nov 15, 83(1), 25 - 38
The differential overamplification of short sequences in the mitochondrial DNA of rho- petites in Saccharomyces cerevisiae stimulates recombination; Netter P et al.; It is well known that the yeast Saccharomyces cerevisiae can be affected by mutations, called 'petite colonie', which correspond to the loss of the major part of the mitochondrial DNA and the concomitant amplification of the remaining sequence, the basic repeat unit (BRU) . We describe here a new phenomenon, the internal overamplification (IOA), due to the differential amplification (up to 20-fold) of short sequences within the BRU . These IOAs are very stable and stimulate the recombination . We discuss here the possible mechanisms giving rise to the appearance and maintenance of the IOAs within the BRU and their effect on the recombination process.

Gene, 1989 Nov 15, 83(1), 1 - 14
Expression and activity of a gene encoding rat cytochrome c in the yeast Saccharomyces cerevisiae; Clements JM et al.; A rat-processed pseudogene, which encodes normal rat cytochrome c, has been expressed in the yeast, Saccharomyces cerevisiae . The translated region of the chromosomal CYC1+ locus, which encodes yeast iso-1-cytochrome c, was replaced by the translated region of the gene encoding rat cytochrome c (CYC1-RAT), thus preserving the proper CYC1 transcription initiation and termination signals . Although the levels of transcription of the normal CYC1+ gene and the CYC1-RAT gene in yeast were equivalent, rat cytochrome c was produced at approx . 40% of the level of iso-1-cytochrome c . In addition, the specific activity in vivo was estimated to be approx . 60% that of the yeast iso-1-cytochrome c . N-terminal processing of indigenous rat cytochrome c, in which the N-terminal methionine residue is cleaved and the penultimate glycine residue is acetylated, also occurred in yeast . Methionine cleavage was complete, while acetylation proceeded to only 70% completion . Lys-72 was trimethylated to 66% completion in the rat cytochrome c produced in yeast . The near normal expression (40%) and specific activity (60%) in vivo indicates that the 40% difference in amino acid sequence is not critical for mitochondrial import, heme attachment and interactions with redox partners.

Nucleic Acids Res, 1989 Nov 11, 17(21), 8595 - 609
Structure and transcription of the mitochondrial genome in heteroplasmic strains of Saccharomyces cerevisiae; Kang YW et al.; Saccharomyces cerevisiae strain FF1210-6C/170 is respiratory deficient due to a mutation of the penultimate base of the mitochondrial tRNA(Asp) gene . We have identified a number of progeny from this strain which have reverted to respiratory sufficiency by the excision and tandem amplification of a small region of the mitochondrial (mt) DNA carrying the tRNA(Asp) gene, while also maintaining the full-length mtDNA . We have studied the structure of the mtDNA and mitochondrial transcription in a number of these heteroplasmic strains . The exact site of the recombination involved in the excision of the repeating unit of the amplified mtDNA has been determined for five of the revertants . Recombination occurs between identical sequences 4-13 base pairs in length . Each of the different repeating units of the amplified DNA retains an active promoter which has been moved to a site just upstream of the tRNA(Asp) gene by the excision/amplification . Transcripts from the heteroplasmic strains have been characterized to determine the sites of mitochondrial RNA termini . We find that in addition to the 5' and 3' processing of the tRNAs, many of the transcripts terminate at a position about 300 base pairs downstream of the gene for tRNA(Asp) . We also find that 3' processing of tRNA(Asp) precursors is absent in petite strains which lack 5' processing indicating that 5' processing of tRNA(Asp) may be a prerequisite for 3' processing in this mutant.

Genetika, 1989 Nov, 25(11), 1952 - 9
{Spontaneous mutation of leu2 in Saccharomyces cerevisiae}; Chepurnoi AI et al.; The data obtained indicate that spontaneous mutations in Saccharomyces cerevisiae are formed during DNA replication . With no DNA replication in the lag-period, in the stationary growth phase, spontaneous mutations are not formed in cell culture during the G1 phase of cell cycle . Experimental data show the absence of primary spontaneously occurring DNA lesion accumulation in the cell G1 phase . Spontaneous mutations of yeasts are formed in the S phase of cell cycle, apparently as DNA replication errors . It is established that the frequency of spontaneous reversions of the leu2 gene in Saccharomyces cerevisiae strain NA3-24 increases when the cells are cultivated on the culture medium with different concentrations of leucine.

Mol Gen Genet, 1989 Nov, 219(3), 439 - 44
Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor; Steden M et al.; Nine independent mutants which are supersensitive (ssl-) to G1 arrest by the mating hormone a-factor were isolated by screening mutagenized Saccharomyces cerevisiae MAT alpha cells on solid medium for increased growth inhibition with a-factor . These mutants carried lesions in two complementation groups, ssl1 and ssl2 . Mutations at the ssl1 locus were mating type specific: MAT alpha ssl1- cells were supersensitive to a-factor but MATa ssl1- were not supersensitive to alpha-factor . In contrast, mutations at the ssl2 locus conferred supersensitivity to the mating hormone of the opposite mating type on both MAT alpha and MATa cells . The alpha-cell specific capacity to inactivate externally added a-factor was shown to be lacking in MAT alpha ssl1- mutants whereas MAT alpha ssl2- cells were able to inactivate a-factor . Complementation analysis showed that ssl2 and sst2, a mutation originally isolated as conferring supersensitivity to alpha-factor to MATa cells, are lesions in the same gene . The ssl1 gene was mapped 30.5 centiMorgans distal to ilv5 on chromosome XII.

Yeast, 1989 Nov-Dec, 5(6), 497 - 507
High level of expression of a protective antigen of schistosomes in Saccharomyces cerevisiae; Loison G et al.; Strains of Saccharomyces cerevisiae expressing P28-I, an antigen inducing protection against schistosomiasis, have been constructed . Transformants containing a very high copy number of a P28-I expression vector were selected by genetic complementation involving deficient LEU2 or URA3 alleles carried by plasmids . Using the ura3 fur1 auto-selection system, constitutive and stable expression of P28-I could be obtained in cultures grown in rich medium . The accumulation of the foreign protein exceeds 25% of total yeast proteins when estimated by Coomassie Brilliant Blue staining of SDS-PAGE . Moreover, P28-I which was located intracellularly was soluble and biologically active.

FEMS Microbiol Lett, 1989 Nov, 53(1-2), 233 - 7
Curing of the killer character of Saccharomyces cerevisiae with acridine orange; Cansado J et al.; Acridine orange, an intercalating dye usually employed in the curing of bacterial plasmids, was tested for its ability to cure K1 and K2 killer strains (laboratory and wine strains) . The results showed a high curing percentage of the killer character . This was demonstrated by the loss of M1 or M2 dsRNAs (responsible for toxin production and resistance to it) and because the meiotic products exhibited non-Mendelian segregation . The curing percentages varied, depending on the strain but not on the killer type, and showed similar efficiency as compared with other known curing agents.

Mol Cell Biol, 1989 Nov, 9(11), 5265 - 71
hsp26 of Saccharomyces cerevisiae is related to the superfamily of small heat shock proteins but is without a demonstrable function; Susek RE et al.; Analysis of the cloned gene confirms that hsp26 of Saccharomyces cerevisiae is a member of the small heat shock protein superfamily . Previous mutational analysis failed to demonstrate any function for the protein . Further experiments presented here demonstrate that hsp26 has no obvious regulatory role and no major effect on thermotolerance . It is possible that the small heat shock protein genes originated as primitive viral or selfish DNA elements.

Mol Cell Biol, 1989 Nov, 9(11), 4882 - 8
Arginine restriction induced by delta-N-(phosphonacetyl)-L-ornithine signals increased expression of HIS3, TRP5, CPA1, and CPA2 in Saccharomyces cerevisiae; Kinney DM et al.; delta-N-(Phosphonacetyl)-L-ornithine (PALO), a transition state analog inhibitor of ornithine transcarbamylase, induced arginine limitation in vivo in Saccharomyces cerevisiae . Arginine restriction caused increased expression of HIS3 and TRP5, measured by the beta-galactosidase activity in strains carrying chromosomally integrated fusions of the promoter regions of each gene with the lacZ gene of Escherichia coli . The increase in beta-galactosidase activity induced by PALO was reversed by the addition of arginine and was dependent on GCN4 protein . These results indicate that PALO, like 3-amino-1,2,4-triazole DL-5-methyltryptophan, can be used to study the effect of limitation of a single amino acid, arginine, on the expression of genes under the general amino acid control regulatory system . Arginine deprivation imposed by PALO also caused increased expression of CPA1 and CPA2, coding respectively for the small and large subunits of arginine-specific carbamyl-phosphate synthetase . The observed increase was GCN4 dependent and was genetically separable from arginine-specific repression of CPA1 mRNA translation . The 5'-flanking regions of CPA1 (reported previously) and CPA2 determined in this study each contained at least two copies of the sequence TGACTC, shown to bind GCN4 protein . The beta-galactosidase activities expressed from CPA1- and CPA2-lacZ fusions integrated into the nuclear DNA of gcn4 mutant strains were five to six times less than in the wild type, when both strains were grown under depressed conditions . The gcn4 mutation reduced basal expression of both CPA1 and CPA2 . The addition of arginine to strains containing the CPA1-lacZ fusion further reduced beta-galactosidase activity of the gcn4 mutant, indicating independent regulation of the CPA1 gene by the general amino acid control and by arginine-specific repression . In strains overproducing GCN4 protein, the translational control completely overrode transcriptional activation of CPA1 by general amino acid control.

Mol Cell Biol, 1989 Nov, 9(11), 4706 - 12
The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences; Siddiqui AH et al.; The PUT1 and PUT2 genes encoding the enzymes of the proline utilization pathway of Saccharomyces cerevisiae are induced by proline and activated by the product of the PUT3 gene . Two upstream activation sequences (UASs) in the PUT1 promoter were identified by homology to the PUT2 UAS . Deletion analysis of the two PUT1 UASs showed that they were functionally independent and additive in producing maximal levels of gene expression . The consensus PUT UAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes . The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor . In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of the PUT3 gene or the cloned PUT3 gene on a 2 microns plasmid, and the binding was dosage dependent . DNA-binding activity was not observed in extracts of strains carrying either a put3 mutation that caused a noninducible (Put-) phenotype or a deletion of the gene . Wild-type levels of complex formation were observed in an extract of a strain carrying an allele of PUT3 that resulted in a constitutive (Put+) phenotype . Extracts from a strain carrying a PUT3-lacZ gene fusion formed two complexes of slower mobility than the wild-type complex . We conclude that the PUT3 product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of both PUT1 and PUT2 . Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction.

Mol Cell Biol, 1989 Nov, 9(11), 4621 - 30
The HML mating-type cassette of Saccharomyces cerevisiae is regulated by two separate but functionally equivalent silencers; Mahoney DJ et al.; Mating-type genes resident in the silent cassette HML at the left arm of chromosome III are repressed by the action of four SIR gene products, most likely mediated through two cis-acting sites located on opposite sides of the locus . We showed that deletion of either of these two cis-acting sites from the chromosome did not yield any detectable derepression of HML, while deletion of both sites yielded full expression of the locus . In addition, each of these sites was capable of exerting repression of heterologous genes inserted in their vicinity . Thus, HML expression is regulated by two independent silencers, each fully competent for maintaining repression . This situation was distinct from the organization of the other silent locus, HMR, at which a single silencer served as the predominant repressor of expression . Examination of identifiable domains and binding sites within the HML silencers suggested that silencing activity can be achieved by a variety of combinations of various functional domains.

Proc Natl Acad Sci U S A, 1989 Nov, 86(22), 8732 - 6
Cloning and nucleotide sequence of the gene for protein X from Saccharomyces cerevisiae; Behal RH et al.; The gene encoding the protein X component of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae has been cloned and sequenced . A 487-base fragment of yeast genomic DNA encoding the amino-terminal region of protein X was amplified by the polymerase chain reaction using synthetic oligonucleotide primers based on amino-terminal and internal amino acid sequences . This DNA fragment was used as a probe to select two genomic DNA restriction fragments, which were cloned and sequenced . A 2.1-kilobase insert contains the complete sequence of the protein X gene . This insert has an open reading frame of 1230 nucleotides encoding a presequence of 30 amino acid residues and a mature protein of 380 amino acid residues (Mr, 42,052) . Hybridization analysis showed that there is a single copy of the protein X gene and that the size of the mRNA is approximately 1.5 kilobases . Comparison of the deduced amino acid sequences of yeast protein X and dihydrolipoamide acetyltransferase indicates that the two proteins evolved from a common ancestor . The amino-terminal part of protein X (residues 1-195) resembles the acetyltransferase, but the remainder is quite different . There is strong homology between protein X and the acetyltransferase in the amino-terminal region (residues 1-84) that corresponds to the putative lipoyl domain . Protein X lacks the highly conserved sequence His-Xaa-Xaa-Xaa-Asp-Gly near the carboxyl terminus, which is thought to be part of the active site of all dihydrolipoamide acyltransferases.

J Bacteriol, 1989 Nov, 171(11), 6169 - 73
Structural discrimination in the sparking function of sterols in the yeast Saccharomyces cerevisiae; Lorenz RT et al.; A Saccharomyces cerevisiae sterol auxotroph, SPK14 (a hem1 erg6 erg7 ura), was constructed to test the ability of selected C-5,6 unsaturated sterols at growth-limiting concentrations to spark growth on bulk cholestanol . The native sterol, ergosterol, initiated growth faster and allowed a greater cell yield than did other sterols selectively altered in one or more features of the sterol . Although the C-5,6 unsaturation is required for the sparking function, the presence of the C-22 unsaturation was found to facilitate sparking far better than did the C-7 unsaturation, whereas the C-24 methyl was the least important group . The addition of delta-aminolevulinic acid to the medium allowed the sparking of FY3 (hem1 erg7 ura) on bulk cholestanol due to the derepression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and the production of endogenous ergosterol . The optimal concentration of delta-aminolevulinic acid to spark growth was 800 ng/ml, whereas higher concentrations caused a growth inhibition . The growth yield of FY3 reached a plateau maximum at about 5 micrograms/ml when the bulk cholestanol was varied in the presence of 10 ng of sparking erogosterol per ml.

J Bacteriol, 1989 Nov, 171(11), 5795 - 802
N alpha acetylation is required for normal growth and mating of Saccharomyces cerevisiae; Lee FJ et al.; Acetylation is the most frequently occurring chemical modification of the alpha-NH2 group of eucaryotic proteins and is catalyzed by N alpha-acetyltransferase . The yeast enzyme is encoded by the AAA1 (amino-terminal alpha-amino acetyltransferase) gene . A null mutation (aaa1-1) created by gene replacement, while not lethal, slows cell growth and results in heterogeneous colony morphology . In comparison with wild-type cells, aaa1-1/aaa1-1 diploids cannot enter stationary phase, are sporulation defective, and are sensitive to heat shock . In addition, the aaa1-1 mutation specifically reduces mating functions of MATa cells . These results indicate that N alpha acetylation plays a crucial role in yeast cell growth and mating.

Carcinogenesis, 1989 Nov, 10(11), 2139 - 43
The expression of cytochrome P450IIB1 in Saccharomyces cerevisiae results in an increased mutation frequency when exposed to cyclophosphamide; Black SM et al.; A recombinant plasmid containing a full length cDNA encoding the rat cytochrome P450IIB1 under the control of the Saccharomyces cerevisiae ADC1 promoter was constructed and transformed into the yeast strain KY118 . The encoded P450IIB1 protein was produced at a level of between 0.1 and 0.2% of total yeast cellular protein (0.068 nmol/mg total cellular protein) . This protein was localized in the microsomal fraction and had activity towards the substrate benzyloxyresorufin, the activity being 0.16 nmol resorufin produced/min/mg microsomal protein . When exposed to the anticancer drug cyclophosphamide the mutation frequency, as determined by the development of resistance to the arginine analogue canavanine, increased in a dose-dependent manner over a control strain and was up to 16-fold higher at the highest doses used.

Mol Cell Biol, 1989 Nov, 9(11), 4613 - 20
Role of intron splicing in the function of the MATa1 gene of Saccharomyces cerevisiae; Ner SS et al.; The MATa1 gene of Saccharomyces cerevisiae is unique in yeast cells in that it contains two short intervening sequences (IVS1 and IVS2) 54 and 51 nucleotides long . The 3' intron is inefficiently spliced and results in the accumulation of transcript with only the first intron removed, leading to the speculation that the gene may produce different protein products by alternative splicing patterns . We have used in vitro mutagenic techniques to construct intronless MATa1 genes and have introduced point substitutions in the 5'-TACTAAC-3' internal conserved sequence of each intron to identify the protein product that is required for repression of haploid-specific genes . Analysis of these constructs for the ability to repress expression of an HO::lacZ fusion and for the ability to allow diploid cells to undergo sporulation during conditions of starvation revealed that the gene is functional with two, one, or no introns and that the only functional protein is the one produced when both introns are spliced from the mRNA.

Mol Cell Biol, 1989 Nov, 9(11), 5034 - 44
Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with the SNF4 protein; Celenza JL et al.; The SNF1 gene of Saccharomyces cerevisiae encodes a protein-serine/threonine kinase that is required for derepression of gene expression in response to glucose limitation . We present evidence that the protein kinase activity is essential for SNF1 function: substitution of Arg for Lys in the putative ATP-binding site results in a mutant phenotype . A polyhistidine tract near the N terminus was found to be dispensable . Deletion of the large region C terminal to the kinase domain only partially impaired SNF1 function, causing expression of invertase to be somewhat reduced but still glucose repressible . The function of the SNF4 gene, another component of the regulatory system, was required for maximal in vitro activity of the SNF1 protein kinase . Increased SNF1 gene dosage partially alleviated the requirement for SNF4 . C-terminal deletions of SNF1 also reduced dependence on SNF4 . Our findings suggest that SNF4 acts as a positive effector of the kinase but does not serve a regulatory function in signaling glucose availability.

Proc Natl Acad Sci U S A, 1989 Nov, 86(21), 8348 - 52
Transcription terminates near the poly(A) site in the CYC1 gene of the yeast Saccharomyces cerevisiae; Russo P et al.; A 38-base-pair region required for normal CYC1 mRNA 3' end formation in Saccharomyces cerevisiae was shown to be necessary for the termination of transcription in vivo by examining the stability of CEN3 plasmids . CEN3 plasmids were stably maintained during vegetative growth, unless a GAL1 transcript impinged on the CEN3 region . Transcription from the GAL1 promoter was terminated, and plasmid stability was restored by the insertion of a fragment containing the 38-base-pair region of CYC1 . In contrast, a similar fragment lacking the 38-base-pair region had no such stabilizing effect . Furthermore, CYC1 mRNA transcription terminated in a region less than 100 nucleotides downstream from the normal poly(A) site, thus establishing that CYC1 mRNA 3' end formation does not involve overly extended precursors as are observed in higher eukaryotes.

Biochimie, 1989 Nov-Dec, 71(11-12), 1201 - 4
Identification of proteins crosslinked to RNA in 40S ribosomal subunits of Saccharomyces cerevisiae; Xiang RH et al.; RNA-protein crosslinks were introduced into the 40S ribosomal subunits from Saccharomyces cerevisiae by mild UV treatment . Proteins crosslinked to the 18S rRNA molecule were separated from free proteins by repeated extraction of the treated subunits and centrifugation in glycerol gradients . After digestion with RNase to remove the RNA molecules, proteins were radio-labeled with 125I and identified by electrophoresis on two-dimensional polyacrylamide gels with carrier total 40S ribosomal proteins and autoradiography . Proteins S2, S7, S13, S14, S17/22/27, and S18 were linked to the 18S rRNA . A shorter period of irradiation resulted in crosslinking of S2 and S17/22/27 only . Several of these proteins were previously demonstrated to be present in ribosomal core particles or early assembled proteins.

J Bacteriol, 1989 Nov, 171(11), 6259 - 64
Molecular cloning of a cell wall exo-beta-1,3-glucanase from Saccharomyces cerevisiae; Klebl F et al.; A major protein of Saccharomyces cerevisiae cell walls is a 29-kilodalton glycoprotein which shows lectinlike binding to beta-1,3-glucan and chitin . It was solubilized by heating isolated cell walls at 90 degrees C and purified to homogeneity by running two high-pressure liquid chromatography columns . With the sequence information of the N terminus and seven peptides, two oligonucleotides were synthesized and the gene was cloned . Its sequence is similar to those of two plant beta-glucanases, and the protein was shown to possess beta-1,3-exoglucanase activity with laminarin as substrate . Haploid yeast cells contained one copy of the gene (BGL2) . Gene disruption did not result in a phenotype.

Mol Cell Biol, 1989 Nov, 9(11), 5045 - 54
Molecular analysis of the SNF4 gene of Saccharomyces cerevisiae: evidence for physical association of the SNF4 protein with the SNF1 protein kinase; Celenza JL et al.; The SNF4 gene is required for expression of glucose-repressible genes in response to glucose deprivation in Saccharomyces cerevisiae . Previous evidence suggested that SNF4 is functionally related to SNF1, another essential gene in this global regulatory system that encodes a protein kinase . Increased SNF1 gene dosage partially compensates for a mutation in SNF4, and the SNF4 function is required for maximal SNF1 protein kinase activity in vitro . We have cloned SNF4 and identified its 1.2-kilobase RNA, which is not regulated by glucose repression . A 36-kilodalton SNF4 protein is predicted from the nucleotide sequence . Disruption of the chromosomal SNF4 locus revealed that the requirement for SNF4 function is less stringent at low temperature (23 degrees C) . A bifunctional SNF4-lacZ gene fusion that includes almost the entire SNF4 coding sequence was constructed . The fusion protein was shown by immunofluorescence microscopy to be distributed throughout the cell, with partial localization to the nucleus . The SNF4-beta-galactosidase protein coimmunoprecipitated with the SNF1 protein kinase, thus providing evidence for the physical association of the two proteins.

Mol Cell Biol, 1989 Nov, 9(11), 4824 - 34
Enhancer and silencerlike sites within the transcribed portion of a Ty2 transposable element of Saccharomyces cerevisiae; Farabaugh P et al.; The Ty2-917 element is a member of the Ty2 class of retroviruslike transposable elements of Saccharomyces cerevisiae . We showed that regions downstream of the Ty2-917 transcription start site modulate its transcription . One region was located downstream of the transcription initiation site (position 240) and within the first 559 base pairs of the element . This region had a dramatic effect, causing an approximately 1,000-fold increase in steady-state levels of RNA . The region stimulated transcription when placed in either orientation upstream of a heterologous gene, HIS4, lacking its own upstream activation sequence (UAS) . We termed this positively acting region an enhancer, by analogy to sites described in higher cells, to distinguish it from yeast UASs which do not function when placed within the transcribed portion of the gene . Though, like some higher eucaryotic enhancers, the Ty2-917 enhancer is located within the transcribed region, it is unlike them in that it occurs within a coding region rather than in an intron . The Ty2-917 enhancer and the Ty2-917 UAS had a synergistic effect on transcription, together stimulating transcription 15-fold over the predicted additive effect . We also identified a site which decreases RNA accumulation, located about 750 base pairs into the element . This site functioned in only one orientation when inserted upstream of the UAS-less heterologous gene . The site was similar to silencers, or negative enhancers, in that it acted to repress transcription from outside the transcribed region, but was distinct in that the function of a canonical silencer was independent of orientation.

J Biol Chem, 1989 Oct 25, 264(30), 18110 - 8
Regulation of Saccharomyces cerevisiae ornithine decarboxylase expression in response to polyamine; Fonzi WA; The mechanism of yeast ornithine decarboxylase (ODC) regulation in response to polyamines was examined . ODC catalyzes the first step of polyamine biosynthesis, the conversion of ornithine to putrescine . ODC activity was modulated approximately 200-fold by varying the availability of polyamines . Variations in ODC activity were associated with parallel changes in the amount of active enzyme molecules . Regulation of ODC activity did not appear to be mediated by changes in the stability of the enzyme . The half-life of ODC was approximately 75 min and was unaffected by the availability of polyamines . Polyamines had no demonstrable effect on the transcriptional or translational stages of ODC expression . The amount of ODC mRNA and the number of ribosomes associated with the mRNA were constant despite the variations in ODC activity . Unlike translationally controlled genes, the regulation of ODC expression did not require either the 5'- or the 3'-untranslated regions of the ODC gene transcript . While gene dosage experiments indicated that negative trans-acting factors are involved in control of ODC expression, these implied factors did not appear to affect translation of ODC mRNA . The results are interpreted to suggest that polyamines regulate ODC expression at a post-translational step prior to assembly of the active form of the enzyme.

Biochim Biophys Acta, 1989 Oct 16, 985(2), 127 - 32
Modifications of the relative proteolipid composition in the ATP synthase of a respiratory competent mutant of Saccharomyces cerevisiae; Manon S et al.; A comparative study of the proteolipid composition of the F0-sector of the ATP synthase of wild-type strain of Saccharomyces cerevisiae and of nuclear mutants, modified at the level of the oxidative phosphorylation due to an enhanced proton permeability of the inner membrane, was carried out . Analysis of the crude proteolipid extract by electrophoresis and high liquid performance chromatography showed some differences at the level of mitochondrial DNA encoded proteolipids . Subunit 6 and in particular subunit 8 were present in reduced amounts, whereas subunit 9 was present in equal amounts in both types of strain . However, the phosphate binding affinity of subunit 8 was the same in wild-type and mutant strains . The fact that subunit 6 and subunit 8 are cotranscripted on a single mRNA led to the problem of the regulation of the mitochondrial synthesis of these two proteins by a nuclear gene.

FEMS Microbiol Lett, 1989 Oct 15, 52(3), 297 - 300
Ultrastructural alterations in Saccharomyces cerevisiae cells in association with elevated temperature-induced autolysis; Takeo K et al.; By means of the freeze-etching technique ultrastructural alterations in Saccharomyces cerevisiae cells undergoing autolysis at elevated temperature were studied . Wall surfaces of intact cells were smooth . During autolysis wall surfaces became rough with granules of 20-40 nm diameter . This alteration occurred after extensive disintegration of cytoplasmic organelles and after functional and ultrastructural impairments of the plasma membrane, but well before the rupture of the plasma membrane.

J Biol Chem, 1989 Oct 15, 264(29), 17126 - 30
Deletion analysis in the amino-terminal extension of methionyl-tRNA synthetase from Saccharomyces cerevisiae shows that a small region is important for the activity and stability of the enzyme; Walter P et al.; MESI, the structural gene for methionyl-tRNA synthetase from Saccharomyces cerevisiae encodes an amino-terminal extension of 193 amino acids, based on the comparison of the encoded protein with the Escherichia coli methionyl-tRNA synthetase . We examined the contribution of this polypeptide region to the activity of the enzyme by creating several internal deletions in MESI which preserve the correct reading frame . The results show that 185 amino acids are dispensable for activity and stability . Removal of the next 5 residues affects the activity of the enzyme . The effect is more pronounced on the tRNA aminoacylation step than on the adenylate formation step . The Km for ATP and methionine are unaltered indicating that the global structure of the enzyme is maintained . The Km for tRNA increased slightly by a factor of 3 which indicates that the positioning of the tRNA on the surface of the molecule is not affected . There is, however, a great effect on the Vmax of the enzyme . Examination of the three-dimension structure of the homologous E . coli methionyl-tRNA synthetase indicates that the amino acid region preceding the mononucleotide-binding fold does not participate directly in the catalytic cleft . It could, however, act at a distance by propagating a mutational alteration to the catalytic residues.

Biochim Biophys Acta, 1989 Oct 13, 993(1), 51 - 5
Cadmium-binding protein in a cadmium-resistant strain of Saccharomyces cerevisiae; Inouhe M et al.; A Cd-binding protein in the Cd2+-resistant strain 301N of Saccharomyces cerevisiae was induced by administration to 0.5 mM CdSO4 . The protein was purified by a gel-permeation and subsequent ion-exchange column chromatographies . The purified Cd-binding protein had the characteristics of metallothioneins: (1) low molecular weight (9.0 kDa), (2) high Cd content (63 micrograms/mg protein), (3) amino-acid composition rich in cysteine (18%), basic and acidic amino acids and free from aromatic amino acids, and (4) an absorption shoulder at near 250 nm . Acid pH or EDTA treatments abolished 250 nm absorption of the Cd-binding protein, and the formed apoprotein was capable of binding Cd2+, Cu2+ and Zn2+, respectively . Heat treatment (75 degrees C) little affected the ultraviolet absorption or sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of Cd-binding protein . These results suggest that metallothionein generally found in animals also occurs in Cd-adapted yeast cells and thus has a role in its Cd-resistance.

FEBS Lett, 1989 Oct 9, 256(1-2), 139 - 42
N alpha-acetyltransferase deficiency alters protein synthesis in Saccharomyces cerevisiae; Lee FJ et al.; Acetylation is the most frequently occurring chemical modification of the alpha-NH2 group of eukaryotic proteins and is catalyzed by a N alpha-acetyltransferase . Two-dimensional gel electrophoresis was used to compare the soluble proteins synthesized in wild type and a mutant (aaa1) yeast cells lacking N alpha-acetyltransferase . Among 855 soluble proteins identified in wild type and mutant, approximately 20% of the proteins in the mutant either disappeared or were shifted to higher pI without a change of molecular mass, and 27 proteins were observed only in the mutant . In addition, the synthesis of another 12% of the proteins in the mutant was either diminished or enhanced, suggesting that the acetylation of certain regulatory proteins may affect their expression . This is the first demonstration of the broad-based functional role of N alpha-acetylation in eukaryotic protein synthesis.

J Gen Microbiol, 1989 Oct, 135 ( Pt 10), 2693 - 7
Role of glutamine aminotransferase in glutamine catabolism by Saccharomyces cerevisiae under microaerophilic conditions; Soberon M et al.; The involvement of glutamine aminotransferase activity in glutamine catabolism by Saccharomyces cerevisiae under microaerophilic conditions was studied . We were able to show that there are at least two different glutamine aminotransferase activities that are differentiated genetically, by their substrate specificity (pyruvate and glyoxylate dependence), and their different modes of regulation . The pyruvate-dependent glutamine aminotransferase activity plays a major role in glutamine catabolism under microaerophilic conditions since the wild-type strain S288C showed a 10-fold higher activity in static cultures than in agitated ones . The same strain also had 3-fold higher glutaminase B activity in agitated cultures than in static ones . Pyruvate-dependent glutamine aminotransferase activity is not regulated directly by O2 itself since a rho- strain showed a high activity regardless of the extent of aeration of cultures . Finally, we were able to isolate a mutant, strain CN20, derived from the rho- strain and unable to utilize glutamine as the sole nitrogen source, which was severely affected in pyruvate-dependent but not in glyoxylate-dependent aminotransferase activity.

J Cell Sci, 1989 Oct, 94 ( Pt 2), 207 - 16
Three-dimensional analysis of morphogenesis induced by mating pheromone alpha factor in Saccharomyces cerevisiae; Baba M et al.; Ultrastructural analyses of cytoplasmic changes in Saccharomyces cerevisiae X2180-1A (MATa) that had been treated with alpha factor were performed by using the freeze-substitution fixation method . After alpha factor treatment, cells exhibited a pointed projection, which is a unique pattern of oriented cell surface growth . The relationship between projection formation and intracellular organelles was examined using serial thin sections and computer-aided three-dimensional reconstructions . Using these analyses membrane vesicles and other organelles were detected, and studies on their dynamic structural reorganization became feasible . Production of membrane vesicles (average 65 nm in diameter) was induced upon exposure of the cells to alpha factor before projection emergence . The total number of membrane vesicles increased at the early stage and decreased at the late stage of projection formation . Three-dimensional analysis indicated that the vesicles were at first dispersed throughout the cell, then accumulated at the site where the projection formed . Morphological changes and multiplication of the Golgi body were seen during the process of projection formation . Other intracellular organelles (nucleus, vacuole, rough endoplasmic reticulum and mitochondria) were also rearranged, showing a polar organization of the cytoplasm during projection formation.

Genetika, 1989 Oct, 25(10), 1725 - 39
{Genetic control of the number of copies of type K2 killer plasmids in Saccharomyces cerevisiae}; Nesterova GF et al.; 13 non-linked chromosomal mutations derepress the negative genetic control of copy number of K2 yeast killer plasmids and lead to 1.5-2-fold elevating of copy number of that type plasmids -L2A and M2 virus-like dsRNA . The content of both plasmids is increased 3-5-fold in cells with chromosomal ski5 mutation, as compared to the strains of wild type . Expression of ski5 allele is recessive . The dose effect of this allele is observed on haploid and diploid levels . Dominant ochre nonsense suppressors suppress the action of ski5 and the ski6 allele epistates that of ski5.

Mol Gen Genet, 1989 Oct, 219(1-2), 75 - 80
The PSO3 gene is involved in error-prone intragenic recombinational DNA repair in Saccharomyces cerevisiae; de Andrade HH et al.; The induction of gene conversion and mitotic crossing-over by photoaddition of psoralens, 254 nm ultraviolet radiation, and nitrogen mustards was determined in diploid cells homozygous for the pso3-1 mutation and in the corresponding wild type of Saccharomyces cerevisiae . For these different agents, the frequency of non-reciprocal events (conversion) is reduced in the pso3-1 mutant compared to the wild type . In contrast, the frequency of reciprocal events (crossing-over) is increased at a range of doses . These observations, together with the block in induced mutagenesis for both reverse and forward mutations previously reported for the pso3-1 mutant, suggest that the PSO3 gene product plays a role in mismatch repair of short patch regions . The block in gene conversion in the pso3 homozygous diploid leads, in the case of nitrogen mustards, to specific repair intermediates which are lethal to the cells.

Mol Gen Genet, 1989 Oct, 219(1-2), 33 - 8
A full length cDNA clone for the alkaline protease from Aspergillus oryzae: structural analysis and expression in Saccharomyces cerevisiae; Tatsumi H et al.; We have cloned and determined the nucleotide sequence of a cDNA fragment for the entire coding region of the alkaline protease (Alp) from a filamentous ascomycete Aspergillus oryzae . According to the deduced amino acid sequence, Alp has a putative prepro region of 121 amino acids preceding the mature region, which consists of 282 amino acids . A consensus sequence of a signal peptide consisting of 21 amino acids is found at the N-terminus of the prepro region . The primary structure of the mature region shares extensive homology (29%-44%) with those of subtilisin families, and the three residues (Asp 32, His 64 and Ser 221 in subtilisin BPN') composing the active site are preserved . The entire cDNA, coding for prepro Alp, when introduced into the yeast Saccharomyces cerevisiae, directed the secretion of enzymatically active Alp into the culture medium, with its N-terminus and specific activity identical to native Aspergillus Alp.

Mol Gen Genet, 1989 Oct, 219(1-2), 161 - 7
Identification of a gene conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae; Kamizono A et al.; A DNA fragment conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae was isolated from a library of yeast genomic DNA . Its nucleotide sequence revealed the presence of a single open reading frame (ORF; 1326 bp) having the potential to encode a protein of 442 amino acid residues (molecular mass of 48.3 kDa) . A frameshift mutation introduced within the ORF abolished resistance to heavy metal ions, indicating the ORF is required for resistance . Therefore, we termed it the ZRC1 (zinc resistance conferring) gene . The deduced amino acid sequence of the gene product predicts a rather hydrophobic protein with six possible membrane-spanning regions . While multiple copies of the ZRC1 gene enable yeast cells to grow in the presence of 40 mM Zn2+, a level at which wild-type cells cannot survive, the disruption of the chromosomal ZRC1 locus, though not a lethal event, makes cells more sensitive to zinc ions than are wild-type cells.

Mol Gen Genet, 1989 Oct, 219(1-2), 125 - 8
Mutations in the genes for mitochondrial RNA polymerase and a second mitochondrial transcription factor of Saccharomyces cerevisiae; Lisowsky T et al.; In our previous work (Lisowsky et al . 1987; Lisowsky and Michaelis 1988) we have identified two nuclear pet genes of yeast that are required for mitochondrial transcription . In this report we show that one of these pet mutations, pet-ts798, maps in the RP041 gene encoding mitochondrial RNA polymerase . The temperature-sensitive lesion of mutant pet-ts798 can be suppressed by a second nuclear gene RF1023 (mtf1) when inserted into a high copy number plasmid . Our assumption that mtf1 codes for a 40 kDa mitochondrial transciription factor is supported by the fact that the cloned gene acts as an intergenic suppressor of a temperature-sensitive RNA polymerase mutant . A third nuclear gene (mtf2) for mitochondrial transcription was identified by analysing mutant pet-ts3504 . The in vitro transcriptional activity of isolated mutant mitochondria is temperature sensitive suggesting the presence of an altered component of transcription inside mitochondria . The defect was confirmed by studies with a transcriptionally active DNA-protein complex and by testing the DNA-binding ability of mitochondrial proteins.

Mol Microbiol, 1989 Oct, 3(10), 1319 - 27
Cloning and characterization of NSP1, a locus encoding a component of a CDC25-dependent, nutrient-responsive pathway in Saccharomyces cerevisiae; Tripp ML et al.; The NSP1 gene in Saccharomyces cerevisiae has been identified by its ability, when expressed at high levels, to bypass the CDC25 requirement for growth . Sequence analysis of the cloned NSP1 locus suggests that the NSP1 product contains 269 amino acids and has a membrane-spanning domain at its carboxyl terminus . The NSP1 protein does not have sequence similarity to other known proteins, and is not related to the CDC25 protein, or to any of the previously described suppressors of CDC25 mutants . Phosphoprotein analysis of NSP1-suppressed cells indicates that the NSP1 product controls the phosphorylation of two 31 kD proteins whose phosphorylation and dephosphorylation are strongly correlated with cell-cycle arrest and proliferation, respectively, and suggests that the NSP1 product is an important downstream element of a CDC25-dependent, nutrient-responsive, phosphorylation pathway.

FEMS Microbiol Lett, 1989 Oct 1, 52(1-2), 189 - 94
Characterization of non-flocculent cells isolated from a culture of flocculent Saccharomyces cerevisiae NCYC 1001; Bielecki S et al.; During cultivation of a flocculent yeast, Saccharomyces cerevisiae 1001, two cell fractions, flocs and free cells, appeared in the medium . Free cells contained cells with a normal ability to flocculate, less flocculent cells and not-flocculent cells . When the non-flocculent cells and not-flocculent cells . When the non-flocculent cell fraction from the postexponential phase of growth was collected and used as an inoculum, the culture showed synchronous growth . The floc forming ability of the yeast cells from this culture increased gradually with the number of divisions.

Mol Cell Biol, 1989 Oct, 9(10), 4571 - 5
Elongation factor EF-1 alpha gene dosage alters translational fidelity in Saccharomyces cerevisiae; Song JM et al.; Changes in the dosage of genes encoding elongation factor EF-1 alpha were shown to cause parallel changes in the misreading of nonsense codons . Higher amounts of EF-1 alpha were correlated with increased nonsense suppression, suggesting that the level of EF-1 alpha is critically involved in translational fidelity.

Mol Cell Biol, 1989 Oct, 9(10), 4523 - 30
Mating-type control in Saccharomyces cerevisiae: isolation and characterization of mutants defective in repression by a1-alpha 2; Harashima S et al.; The alpha 2 protein, the product of the MAT alpha 2 cistron, represses various genes specific to the a mating type (alpha 2 repression), and when combined with the MATa1 gene product, it represses MAT alpha 1 and various haploid-specific genes (a1-alpha 2 repression) . One target of a1-alpha 2 repression is RME1, which is a negative regulator of a/alpha-specific genes . We have isolated 13 recessive mutants whose a1-alpha 2 repression is defective but which retain alpha 2 repression in a genetic background of ho MATa HML alpha HMRa sir3 or ho MAT alpha HMRa HMRa sir3 . These mutations can be divided into three different classes . One class contains a missense mutation, designated hml alpha 2-102, in the alpha 2 cistron of HML, and another class contains two mat alpha 2-202, in the MAT alpha locus . These three mutants each have an amino acid substitution of tyrosine or acid substitution of tyrosine or phenylalanine for cysteine at the 33rd codon from the translation initiation codon in the alpha 2 cistron of HML alpha or MAT alpha . The remaining 10 mutants make up the third class and form a single complementation group, having mutations designated aar1 (a1-alpha 2 repression), at a gene other than MAT, HML, HMR, RME1, or the four SIR genes . Although a diploid cell homozygous for the aarl and sir3 mutations and for the MATa, HML alpha, and HMRa alleles showed alpha mating type, it could sporulate and gave rise to asci containing four alpha mating-type spores . These facts indicate that the domain for alpha2 repression is separable from that for a1-alpha2 protein interaction or complex formation in the alpha2 protein and that an additional regulation gene, AAR1, is associated with the a1-alpha2 repression of the alpha1 cistron and haploid-specific genes.

Mol Cell Biol, 1989 Oct, 9(10), 4507 - 14
Regulation of alpha-factor production in Saccharomyces cerevisiae: a-factor pheromone-induced expression of the MF alpha 1 and STE13 genes; Achstetter T; Production of the mating pheromone alpha-factor was examined in Saccharomyces cerevisiae MAT alpha cells that had been exposed to the mating pheromone a-factor . A 2-h treatment with a-factor caused a significant increase in alpha-factor concentration in the medium as demonstrated by a halo assay . MF alpha 1 is one of the two genes coding for a precursor of alpha-factor . A Northern (RNA) analysis of total RNA from a-factor-treated MAT alpha cells revealed a rapid two- to threefold increase in MF alpha 1 transcript levels, reaching maximum within 60 min of exposure to the pheromone . Pheromone induction did not require ongoing protein synthesis . a-Factor-induced MF alpha 1 expression was quantitated by analysis of an MF alpha 1::SUC2 fusion gene whose product was assayed for invertase activity . Expression of the MF alpha 1::SUC2 gene in MAT alpha cells responded to the a-factor signal like the chromosomal version of MF alpha 1 . Maturation of the alpha-factor precursor involves three proteolytic activities which are encoded by the KEX1, KEX2, and STE13 genes, respectively . Two of these genes, namely, KEX2 and STE13, were examined for pheromone-induced expression . Only the STE13 gene exhibited pheromone induction at the transcriptional level.

Mol Cell Biol, 1989 Oct, 9(10), 4220 - 8
Mutations in the anticodon stem affect removal of introns from pre-tRNA in Saccharomyces cerevisiae; Mathison L et al.; To evaluate the role of exon domains in tRNA splicing, the anti-codon stem of proline pre-tRNAUGG from Saccharomyces cerevisiae was altered by site-directed mutagenesis of the suf8 gene . Sixteen alleles were constructed that encode mutant pre-tRNAs containing all possible base combinations in the last base pair of the anticodon stem adjacent to the anticodon loop (positions 31 and 39) . The altered pre-tRNAs were screened by using an in vitro endonucleolytic cleavage assay to determine whether perturbations in secondary structure affect the intron excision reaction . The pre-tRNAs were cleaved efficiently whenever secondary structure in the anticodon stem was maintained through standard base pairing or G.U interactions . However, most of the pre-tRNAs with disrupted secondary structure were poor substrates for intron excision . We also determined the extent to which the suf8 alleles produce functional products in vivo . Each allele was integrated in one to three copies into a yeast chromosome or introduced on a high-copy-number plasmid by transformation . The formation of a functional product was assayed by the ability of each allele to suppress the +1 frameshift mutation his4-713 through four-base codon reading, as shown previously for the SUF8-1 suppressor allele . We found that alleles containing any standard base pair or G.U pair at position 31/39 in the anticodon stem failed to suppress his4-713 . We could not assess in vivo splicing with these alleles because the tRNA products, even if they are made, would be expected to read a normal triplet rather than a quadruplet codon . However, all of the alleles that contained a disrupted base pair at position 31/ 39 in the anticodon stem altered the structure of the tRNA in a manner that caused frameshift suppression . Suppression indicated that splicing must have occurred to some extent in vivo even though most of the suppression alleles produced pre-tRNAs that were cleaved with low efficiency or not at all in vitro . These results have important implications for the interpretation of in vitro cleavage assays in general and for the potential use of suppressors to select mutations that affects tRNA splicing.

Genetics, 1989 Oct, 123(2), 261 - 8
Position effects in ectopic and allelic mitotic recombination in Saccharomyces cerevisiae; Lichten M et al.; We have examined the role that genomic location plays in mitotic intragenic recombination . Mutant alleles of the LEU2 gene were inserted at five locations in the yeast genome . Diploid and haploid strains containing various combinations of these inserts were used to examine both allelic recombination (between sequences at the same position on parental homologs) and ectopic recombination (between sequences at nonallelic locations) . Chromosomal location had little effect on mitotic allelic recombination . The rate of recombination to LEU2 at five different loci varied less than threefold . This finding contrasts with previous observations of strong position effects in meiosis; frequencies of meiotic recombination at the same five loci differ by about a factor of forty . Mitotic recombination between dispersed copies of leu2 displayed strong position effects . Copies of leu2 located approximately 20 kb apart on the same chromosome recombined at rates 6-13-fold higher than those observed for allelic copies of leu2 . leu2 sequences located on nonhomologous chromosomes or at distant loci on the same chromosome recombined at rates similar to those observed for allelic copies . We suggest that, during mitosis, parental homologs interact with each other no more frequently than do nonhomologous chromosomes.

EMBO J, 1989 Oct, 8(10), 3039 - 44
Mutants of H-ras that interfere with RAS effector function in Saccharomyces cerevisiae; Michaeli T et al.; We report a class of interfering mutants of the human H-ras gene capable of inhibiting phenotypes arising from the expression of the activated RAS2 gene, RAS2val19, in the yeast Saccharomyces cerevisiae . All these mutants encode unprocessed H-ras proteins that remain in the cytoplasm . One of the mutants, H-rasarg186, was examined in detail . H-rasarg186 protein is a competitive inhibitor of RAS2val19 protein . It does not interfere with processing and membrane localization of RAS2val19, nor does it appear to compete with RAS protein for its proposed regulator, the CDC25 protein . By several criteria the RAS2val19 adenylate cyclase interaction is unaffected by H-rasarg186 . We infer from our results that H-rasarg186 protein interferes with an alternative function of RAS2val19.

Proc Natl Acad Sci U S A, 1989 Oct, 86(19), 7515 - 9
Mutations in the structural genes for eukaryotic initiation factors 2 alpha and 2 beta of Saccharomyces cerevisiae disrupt translational control of GCN4 mRNA; Williams NP et al.; The SUI2 and SUI3 genes of Saccharomyces cerevisiae encode the alpha and beta subunits, respectively, of translation initiation factor eIF-2 (eukaryotic initiation factor 2) . Previously isolated mutations in these genes restore expression from his4 mutant alleles lacking an ATG initiation codon . The SUI mutations also lead to increased levels of HIS4 mRNA . We show that the latter phenotype exists because the SUI mutations elevate expression of GCN4, an activator of HIS4 transcription . Increased GCN4 expression in the SUI mutants occurs independently of the GCN2 and GCN3 gene products that are normally required to stimulate translation of GCN4 mRNA under conditions of amino acid starvation . Derepression of GCN4 expression in the SUI mutants requires the multiple AUG codons in the leader of the GCN4 transcript that normally mediate its translational control by amino acid availability . In these respects, the SUI mutations resemble mutations in GCD genes whose products function as translational repressors of GCN4 . Thus, in addition to its general role in AUG start codon selection, eIF-2 appears to be an important factor in GCN4 translational control . We also show that deletion of GCN3 in sui2-1 strains is lethal, suggesting that GCN3 contributes to eIF-2 alpha function in addition to its role as a translational activator of GCN4.

J Cell Biol, 1989 Oct, 109(4 Pt 1), 1411 - 9
N2,N2-dimethylguanosine-specific tRNA methyltransferase contains both nuclear and mitochondrial targeting signals in Saccharomyces cerevisiae; Li JM et al.; The TRM1 gene of Saccharomyces cerevisiae encodes a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase, which modifies both mitochondrial and cytoplasmic tRNAs . The enzyme is targeted to mitochondria for the modification of mitochondrial tRNAs . Cellular fractionation and indirect immunofluorescence studies reported here demonstrate that this enzyme is also localized to the nucleus . Further, immunofluorescence experiments using strains that overproduce the enzyme show a staining at the periphery of the nucleus suggesting that the enzyme is found in a subnuclear destination near or at the nuclear membrane . There is no obvious cytoplasmic staining in these overproducing strains . Fusion protein technology was used to begin to localize sequences involved in the nuclear targeting of this enzyme . Indirect immunofluorescence studies indicate that sequences between the first 70 and 213 NH2-terminal amino acids of the methyltransferase are sufficient to target Escherichia coli beta-galactosidase to nuclei.

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