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 thre