Biochem Biophys Res Commun, 1984 Aug 30, 123(1), 186 - 93 Cytochrome P-450 inducibility by ethanol and 7-ethoxycoumarin O-deethylation in S . cerevisiae; Del Carratore R et al.; The level of cytochrome P-450 and some enzymatic activity cytochrome P-450 dependent in a diploid strain (D7) of S . cerevisiae are affected by the substrate supporting growth and its concentration and, in particular, by the growth phase of the culture . For these reasons we tested the hypothesis that the induction of the monooxygenase system in the D7 strain when grown in high concentration of glucose depended on one product of glycolysis, ethanol . There was a strict correlation between the level of cytochrome P-450 and the ethanol concentration . Moreover we developed a sensitive test measuring the ethoxycoumarin O-deethylation in order to detect the enzymatic activity cytochrome P-450 dependent in whole yeast cells, in different growth conditions. J Biol Chem, 1984 Aug 25, 259(16), 10499 - 506 On the nucleotide sequence recognized by a eukaryotic site-specific endonuclease, Endo.SceI from yeast; Shibata T et al.; Endo.SceI which is isolated from cells of Saccharomyces cerevisiae is a eukaryotic site-specific endonuclease active on double-stranded DNA . At each cleavage site, Endo.SceI cuts only a defined phosphodiester bond in each strand of the double helix . We compared nucleotide sequences around five cleavage sites for Endo.SceI using a computer . We could not find any common specific sequence consisting of five base pairs or more among them . However, we found a 26-base pair consensus sequence which included 15 conserved nucleotides, allowing any of the five sequences to include a few nucleotides deviated from the consensus sequence . The consensus sequence is 5'-CAn*PYnnAnnCYYGTTnnnPnYnnYA-3', where P, Y, n, and * denote purine, pyrimidine, any nucleotide, and the center of the cleavage site, respectively . The numbers of sites at which the consensus sequence appears in pBR322 DNA, phi X174 replicative form DNA, fd replicative form DNA, or SV40 DNA are close to those of the cleavage sites for Endo.SceI . We found that a 33-base pair fragment was efficiently cut at the defined phosphodiester bonds by Endo.SceI . This 33-base pair fragment included 25 base pairs out of the 26-base pair consensus sequence . The fragments in which a part of the consensus sequence was missing were not cut by Endo.SceI . These observations suggest that the consensus sequence described above is the major characteristic around the cleavage sites recognized by Endo.SceI and that the mode of recognition of cleavage sites by Endo.SceI is different from that by restriction endonucleases . We found homology between the consensus sequence for Endo.SceI and the sequences around the cleavage sites for two other site-specific endonucleases of S . cerevisiae: Endo.SceII and YZ-Endo which is involved in mating type switching. Mol Cell Biol, 1984 Aug, 4(8), 1515 - 20 Sequences responsible for transcription termination on a gene segment in Saccharomyces cerevisiae; Henikoff S et al.; We have mapped a signal sequence for mRNA 3'-end formation in Saccharomyces cerevisiae by using a Drosophila melanogaster DNA segment that complements a yeast adenine-8 mutation . That the 3' end of the transcript in S . cerevisiae nearly coincides with that in D . melanogaster is consistent with the possibility that mRNA termini are similarly determined in both organisms . Deletion analysis reveals that the complete signal is no more than 21 base pairs long . Part of the signal is the sequence TTTTTATA, which is seen in the termination region of several yeast genes . TTTTTATA appears to be able to act autonomously as a partial termination signal . The efficiency of the complete signal is affected by substitution of sequences downstream from it . This modulation of the effect of a signal is consistent with termination in S . cerevisiae, resembling rho-dependent termination in bacteria. EMBO J, 1984 Aug, 3(8), 1825 - 30 Polypeptide chain elongation factor 1 alpha (EF-1 alpha) from yeast: nucleotide sequence of one of the two genes for EF-1 alpha from Saccharomyces cerevisiae; Nagata S et al.; Messenger RNA for yeast cytosolic polypeptide chain elongation factor 1 alpha (EF-1 alpha) was partially purified from Saccharomyces cerevisiae . Double-stranded complementary DNA (cDNA) was synthesized and cloned in Escherichia coli with pBR327 as a vector . Recombinant plasmid carrying yEF-1 alpha cDNA was identified by cross-hybridization with the E . coli tufB gene and the yeast mitochondrial EF-Tu gene (tufM) under non-stringent conditions . A yeast gene library was then screened with the EF-1 alpha cDNA and several clones containing the chromosomal gene for EF-1 alpha were isolated . Restriction analysis of DNA fragments of these clones as well as the Southern hybridization of yeast genomic DNA with labelled EF-1 alpha cDNA indicated that there are two EF-1 alpha genes in S . cerevisiae . The nucleotide sequence of one of the two EF-1 alpha genes (designated as EF1 alpha A) was established together with its 5'- and 3'-flanking sequences . The sequence contained 1374 nucleotides coding for a protein of 458 amino acids with a calculated mol . wt . of 50 300 . The derived amino acid sequence showed homologies of 31% and 32% with yeast mitochondrial EF-Tu and E . coli EF-Tu, respectively. Virology, 1984 Aug, 137(1), 20 - 31 Genome structure and expression of a defective interfering mutant of the killer virus of yeast; Thiele DJ et al.; A large internal deletion in M1 double-stranded (ds) RNA from the killer virus of Saccharomyces cerevisiae generates a suppressive (S3) dsRNA molecule . Strains which harbor S3 dsRNA are defective in toxin production and immunity to the toxin . The biochemical defect in expression has been investigated and is apparently due to truncation of the protoxin polypeptide translation reading frame on S3 dsRNA . Transcription in vivo, and in isolated virions in vitro, results in the synthesis of a full-length positive polarity messenger RNA, denoted s . The s transcript contains no long poly(A) tracts as determined by its lack of affinity for oligo(dT)-cellulose, and as inferred by sequence analysis of approximately 87% of the S3 dsRNA genome . These data support a model for template coding of polyadenylate in transcripts derived from the wild-type M1 dsRNA . The orientation of the sequences conserved on S3 dsRNA with respect to M1 dsRNA has been determined . Some of the conserved sequences are likely to be required for the maintenance and replication of these viral dsRNA genomes in S . cerevisiae. Proc Natl Acad Sci U S A, 1984 Aug, 81(15), 4722 - 6 Identification of 17 beta-estradiol as the estrogenic substance in Saccharomyces cerevisiae; Feldman D et al.; Saccharomyces cerevisiae possesses a high-affinity estrogen binding protein and an endogenous ligand that displaces {3H}estradiol from both the yeast binding protein and mammalian estrogen receptors . Semipurified preparations of this ligand have been shown to exhibit estrogenic activity in mammalian systems . We now describe the purification procedure and ultimate identification of the estrogenic substance in extracts of S . cerevisiae as 17 beta-estradiol . Organic solvent extracts of commercially obtained dried yeast were sequentially chromatographed on silica gel columns and then subjected to a series of reversed phase HPLC fractionations . Active ligand was monitored by {3H}estradiol displacement in a rat uterine cytosol assay . After seven chromatography steps, the purified and highly active ligand exhibited a single peak with retention times identical to those of 17 beta-estradiol on both HPLC and GC . The yeast material was identified as 17 beta-estradiol by its UV absorbance and mass spectrometric fragmentation pattern . In addition, radioimmunoassay confirmed the presence of approximately the same mass of 17 beta-estradiol (approximately equal to 800 ng/1.5 kg of yeast) as estimated both by a competitive binding assay with estrogen receptor and by mass spectrometry . Extraneous contamination by estradiol was excluded by repeat experiments with different batches of starting material and demonstration of estradiol by RIA in conditioned medium and cell pellets of laboratory-grown S . cerevisiae whereas non-conditioned medium did not possess the steroid . We conclude that 17 beta-estradiol is a yeast product. Biochemistry, 1984 Jul 31, 23(16), 3582 - 9 Azasterol inhibition of delta 24-sterol methyltransferase in Saccharomyces cerevisiae; Oehlschlager AC et al.; The inhibition of the delta 24-sterol methyltransferase (24-SMT) of Saccharomyces cerevisiae by side-chain azasterols is related to their nuclear skeleton and side chain nitrogen position . Inhibitory power {I50 (microM)} was found to be in the order of 25-azacholesterol hydrochloride salt (0.05) greater than 25-aza-24,25-dihydrozymosterol (0.08) greater than 25-azacholesterol approximately equal to 25-azacholestanol (0.14) greater than (20R)- and (20S)-22,25-diazacholesterol (0.18) greater than 24-azacholesterol (0.22) greater than 25-aza-24,25-dihydrolanosterol (1.14) greater than 23-azacholesterol (4.8) . In the presence of azasterols, S . cerevisiae produces increased amounts of zymosterol, decreased amounts of ergosterol and ergostatetraenol, and the new metabolites cholesta-7,24-dienol, cholesta-5,7,24-trienol, and cholesta-5,7,22,24-tetraenol . Kinetic inhibition studies with partially purified 24-SMT and several azasterols suggest the azasterols act uncompetitively with respect to zymosterol and are competitive inhibitors with respect to S-adenosyl-L-methionine (SAM) . These results are consistent with at least two kinetic mechanisms . One excludes competition of azasterol and zymosterol for the same site, whereas a second could involve a ping-pong mechanism in which 24-SMT is methylated by SAM and the methylated enzyme reacts with sterol substrate. Biochim Biophys Acta, 1984 Jul 11, 774(1), 43 - 8 Effects of ethanol and other alkanols on passive proton influx in the yeast Saccharomyces cerevisiae; Leao C et al.; Ethanol, isopropanol, propanol and butanol enhanced the passive influx of protons into deenergized cells of Saccharomyces cerevisiae . The influx followed first-order kinetics with a rate constant that increased exponentially with the alkanol concentration . The exponential enhancement constants increased with the lipid solubility of the alkanols, which indicated hydrophobic membrane regions as the target sites . While the enhancement constants were independent of pH over the range tested (3.3-5.0), the rate constants decreased linearly with increasing extracellular proton concentration, indicating the presence of an additional surface barrier against proton penetration, the effectiveness of which increased with protonation . The alkanols affected the acidification curves of energized yeast suspensions in such a way that the final pH values were linear functions of the alkanol concentrations . These results were consistent with a balance between active and passive proton movements at the final pH, the exponential enhancement constants calculated from the slopes being nearly identical with those obtained with deenergized cells . It was concluded that passive proton influx contributes to the kinetics of acidification in S . cerevisiae and that uncoupling contributes to the overall kinetics of alkanol-inhibited secondary active transport across the yeast plasma membrane. Cell, 1984 Jul, 37(3), 1075 - 89 Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-alpha-factor; Julius D et al.; S . cerevisiae kex2 mutants are defective for the production of two biologically active secreted peptides: killer toxin and the mating pheromone, alpha-factor . Both molecules are excised from larger precursor polypeptides . In normal cells, the alpha-factor precursor is core-glycosylated and proteolytically processed intracellularly . In kex2 mutants, however, prepro-alpha-factor is not proteolytically cleaved and is secreted in a highly glycosylated form . All kex2 mutants examined (three independent alleles) lack a Zn++-sensitive membrane-associated endopeptidase with specificity for cleaving on the carboxyl side of a pair of basic residues . Absence of this activity cosegregates with the other phenotypes of a kex2 lesion in genetic crosses . The normal KEX2 gene was isolated by complementation of three of the phenotypes conferred by the kex2-1 mutation . The cloned DNA, either on a multicopy plasmid or integrated into the genome, restores both enzymatic activity in vitro and the normal pattern of proteolytic processing and glycosylation of prepro-alpha-factor in vivo . Gene dosage effects suggest that KEX2 is the structural gene for the endopeptidase. J Bacteriol, 1984 Jul, 159(1), 429 - 32 Two unlinked lysine genes (LYS9 and LYS14) are required for the synthesis of saccharopine reductase in Saccharomyces cerevisiae; Borell CW et al.; Three lysine auxotrophs, strains AU363, 7305d, and 8201-7A, were investigated genetically and biochemically to determine their gene loci, biochemical lesions, and roles in the lysine biosynthesis of Saccharomyces cerevisiae . These mutants were leaky and blocked after the alpha-aminoadipate step . Complementation studies placed these three mutations into a single, new complementation group, lys14 . Tetrad analysis from appropriate crosses provided evidence that the lys14 locus represented a single nuclear gene and that lys14 mutants were genetically distinct from the other mutants (lys1, lys2, lys5, and lys9) blocked after the alpha-aminoadipate step . The lys14 strains, like lys9 mutants, accumulated alpha-aminoadipate-semialdehyde and lacked significant amounts of saccharopine reductase activity . On the bases of these results, it was concluded, therefore, that LYS9 and LYS14, two distinct genes, were required for the biosynthesis of saccharopine reductase in wild-type S . cerevisiae. Mol Cell Biol, 1984 Jul, 4(7), 1191 - 7 Saccharomyces cerevisiae ribosomes recognize non-AUG initiation codons; Zitomer RS et al.; A series of Saccharomyces cerevisiae plasmids and mutant derivatives containing fusions of the Escherichia coli galactokinase gene, galK, to the yeast iso-1-cytochrome c CYC1 transcription unit were used to study the sequences affecting the initiation of translation in S . cerevisiae . When the CYC1 AUG initiation codon preceded the galK AUG codon and coding sequence and either the two AUGs were out of frame with each other or a nonsense codon was located between them, the expression of the galK gene was extremely low . Deletion of the CYC1 AUG and its surrounding sequences resulted in a 100-fold increase in galK expression . This dependence of galK expression on the elimination of the CYC1 AUG codon was used to select mutations in that codon . Then the ability of these altered initiation codons to serve in translational initiation was determined by reconstruction of the CYC1 gene 3' to and in frame with them . Initiation was found to occur at the codons UUG and AUA, but not at the codons AAA and AUC . Furthermore the codon UUG, when preceded by an A three nucleotides upstream, served as a better initiation codon than when a U was substituted for the A . The efficiency of translation from these non-AUG codons was quantitated by using a CYC1/galK protein-coding fusion and measuring cellular galactokinase levels . Initiation at the UUG codon was 6.9% as efficient as initiation at the wild-type AUG codon when preceded by an A three nucleotides upstream, but was over 10-fold less efficient when a U was substituted for that A . Initiation at AUA was 0.5% as efficient as at AUG . The effects of the sequences preceding the initiation codon are discussed in light of these results. J Bacteriol, 1984 Jul, 159(1), 413 - 7 Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation of the ADE1 gene; Crowley JC et al.; The ADE1 gene of Saccharomyces cerevisiae was isolated by complementation in S . cerevisiae from a yeast genomic DNA library carried on plasmid YEp13 . Electron microscopy of R-loop-containing DNA indicated the location of the ADE1 gene on the plasmid insert . Gene disruption and gene replacement were used to demonstrate that the ade1-complementing sequence was the actual ADE1 gene that maps on chromosome I . ade1 strains which normally form red colonies form white ones when transformed with the cloned ADE1 gene . This property should be very useful, since it enables detection of plasmids carrying this gene under nonselective conditions. Gene, 1984 Jul-Aug, 29(1-2), 103 - 12 A 189-bp fragment of Crithidia fasciculata maxicircle DNA confers autonomous replication in Saccharomyces cerevisiae; Kim R et al.; A 189-bp fragment capable of promoting high-frequency transformation in Saccharomyces cerevisiae has been isolated from the maxicircle of the insect trypanosomatid Crithidia fasciculata . Chimeric plasmids containing this autonomously replicating sequence (ars) are maintained as extrachromosomal elements in S . cerevisiae . The nucleotide sequence of the maxicircle fragment, termed ars189, has been determined, and its position has been mapped in the maxicircle . The ars189 fragment has an A + T content of 79.4% and shows a large asymmetry in the distribution of adenine and thymine residues between the two strands . In one strand (the T strand) thymine accounts for 118 out of 189 nucleotides while adenine accounts for only 32 nucleotides . The ars189 DNA does not hybridize with minicircles, and its sequence appears to be unique in the C . fasciculata maxicircle genome . This sequence also shows extensive homology to a sequence within a 2.6-kb ars fragment of the Leishmania tarentolae maxicircle . In addition, ars189 contains two copies of a yeast consensus ars sequence (A/T)TTTATPuTTT(T/A). J Biol Chem, 1984 Jun 25, 259(12), 7955 - 62 Correlations between transcription of a yeast tRNA gene and transcription factor-DNA interactions; Stillman DJ et al.; A partly purified fraction from Saccharomyces cerevisiae has a RNA polymerase III transcription factor activity and contains protein which binds specifically to two internal promoter regions of RNA polymerase III-transcribed genes . The influence of ionic strength and of dimethyl sulfoxide on specific binding to a S . cerevisiae tRNAleu3 gene has been analyzed by DNase I protection (footprinting) and by nitrocellulose filter binding . The effects of these agents on binding correlate with their effects on transcription and on the stability of transcription complexes . Dimethyl sulfoxide stabilizes binding and transcription complexes against dissociation by NaCl, with 10% dimethyl sulfoxide compensating for the addition of 50-60 mM NaCl . Binding of protein in the 5' proximal part of the S . cerevisiae tRNAleu3 gene is more NaCl-sensitive than binding in the 3' proximal part. Experientia, 1984 Jun 15, 40(6), 582 - 3 Reversal of pyrithiamine-induced growth inhibition of Saccharomyces cerevisiae by oxythiamine; Iwashima A et al.; Oxythiamine reversed the growth inhibition of Saccharomyces cerevisiae caused by pyrithiamine, although oxythiamine alone inhibited yeast cell growth . This phenomenon was explained by thiamine production from these 2 thiamine antagonists which was demonstrated using cell suspensions and the crude extract of S . cerevisiae. Nature, 1984 Jun 7-13, 309(5968), 556 - 8 A relationship between the yeast cell cycle genes CDC4 and CDC36 and the ets sequence of oncogenic virus E26; Peterson TA et al.; We report here significant primary sequence homology among the predicted translational products of three genes: CDC4 , CDC36 and ets . CDC4 and CDC36 are Saccharomyces cerevisiae cell division cycle genes, while ets is a transformation-specific sequence of avian erythroblastosis virus E26 . The deduced primary structures of the three gene products were compared by computer to a large data base of known and predicted protein sequences . The search revealed 22.0-25.5% identity over regions of 140-206 codons, respectively between the different pairwise combinations . For these particular sequences, these identity scores fall 3.4-4.0 standard deviations above the empirically-determined mean values of fortuitous similarity . S . cerevisiae calls require CDC36 and CDC4 in order to complete two early events in the cell cycle: execution of start ( CDC36 ) and spindle pole body separation ( CDC4 ) . In virus E26, the ets sequence is linked in frame with delta gag and mybE in the tripartite structure 5'-delta gag- mybE -ets-3', comprising the E26 transforming oncogene . The homologies described here suggest that the biochemical functions or regulation of the CDC4 , CDC36 and ets products may be related. Mutat Res, 1984 Jun, 136(3), 223 - 31 Apparent changes in structure-activity relationships for antimitochondrial effects of 9-anilinoacridines according to Saccharomyces cerevisiae strain and methodology; Ferguson LR; Sensitivity of detection of antimitochondrial effects in S . cerevisiae as measured by the induction of 'petite' mutants, has been investigated in a closely related series of 9-anilinoacridines, using a new microtitre test which has been compared to a range of other techniques . Drugs were chosen to span antimitochondrial activity between the inactive compounds 9-amino- or 3-amino-acridine and the moderately active proflavine, also between proflavine and the strong antimitochondrial agent, ethidium bromide . As previously reported using other techniques, no compound without an amino substituent caused antimitochondrial effects, whereas all 9 anilinoacridines with a 1'-substituted anilino group and 3,6-diamino-substituted acridine ring acted like ethidium in causing strong 'petite' mutagenesis . Compounds with a single acridine 3-amino group, together with proflavine, might or might not be scored as an antimitochondrial agent depending on the time and conditions of drug exposure and, more importantly, on the selection of yeast strain used in the screening . Measurement of 'petite' mutagenesis in strain 5178B, using the microtitre assay, provided the most sensitive and efficient means of detection of antimitochondrial effects for all physical DNA-binding agents . Detailed interpretation of structure-activity relationships and prediction of carcinogenic activity based upon induction of 'petite' mutagenesis would vary considerably if this procedure is not followed. J Bacteriol, 1984 Jun, 158(3), 860 - 5 Cystathionine accumulation in Saccharomyces cerevisiae; Ono B et al.; A cysteine-dependent strain of Saccharomyces cerevisiae and its prototrophic revertants accumulated cystathionine in cells . The cystathionine accumulation was caused by a single mutation having a high incidence of gene conversion . The mutation was designated cys3 and was shown to cause loss of gamma-cystathionase activity . Cysteine dependence of the initial strain was determined by two linked and interacting mutations, cys3 and cys1 . Since cys1 mutations cause a loss of serine acetyltransferase activity, our observation led to the conclusion that S . cerevisiae synthesizes cysteine by sulfhydrylation of serine with hydrogen sulfide and by cleavage of cystathionine which is synthesized from serine and homocysteine. Genetics, 1984 Jun, 107(2), 179 - 97 Mutations affecting Ty-mediated expression of the HIS4 gene of Saccharomyces cerevisiae; Winston F et al.; We have identified mutations in seven unlinked genes (SPT genes) that affect the phenotypes of Ty and delta insertion mutations in the 5' noncoding region of the HIS4 gene of S . cerevisiae . Spt mutants were selected for suppression of his4-912 delta, a solo delta derivative of Ty912 . Other Ty and delta insertions at HIS4 are suppressed by mutations in some but not all of the SPT genes . Only spt4 suppresses a non-Ty insertion at HIS4 . In addition to their effects on Ty and delta insertions, mutations in several SPT genes show defects in general cellular functions--mating, DNA repair and growth. Cell, 1984 Jun, 37(2), 437 - 45 Genetic analysis of yeast RAS1 and RAS2 genes; Kataoka T et al.; We present a genetic analysis of RAS1 and RAS2 of S . cerevisiae, two genes that are highly homologous to mammalian ras genes . By constructing in vitro ras genes disrupted by selectable genes and introducing these by gene replacement into the respective ras loci, we have determined that neither RAS1 nor RAS2 are by themselves essential genes . However, ras1 - ras2 - spores of doubly heterozygous diploids are incapable of resuming vegetative growth . We have determined that RAS1 is located on chromosome XV, 7 cM from ade2 and 63 cM from his3; and RAS2 is located on chromosome XIV, 2 cM from met4 . We have also constructed by site-directed mutagenesis a missense mutant, RAS2val19 , which encodes valine in place of glycine at the nineteenth amino acid position, the same sort of missense mutation that is found in some transforming alleles of mammalian ras genes . Diploid yeast cells that contain this mutation are incapable of sporulating efficiently, even when they contain wild-type alleles. J Biol Chem, 1984 May 25, 259(10), 6267 - 73 Coordinate regulation of phosphatidylserine decarboxylase activity and phospholipid N-methylation in yeast; Carson MA et al.; Membranes isolated from Saccharomyces cerevisiae, strain ATCC 26615, catalyze the decarboxylation of exogenous phosphatidylserine added as an aqueous dispersion in detergent . Active preparations of the decarboxylase can be obtained by extracting salt-washed membranes with 0.5% Cutscum . The properties of the phosphatidylserine decarboxylase activity associated with a particulate fraction and the detergent extracts have been characterized by assaying the enzymatic conversion of exogenous {14C}phosphatidylserine to {14C}phosphatidylethanolamine . The yeast decarboxylase does not require a divalent cation and is inhibited by hydroxylamine and p-hydroxymercuribenzoate . The rate of decarboxylation of exogenous phosphatidylserine catalyzed by membranes prepared from cells grown in the presence of choline is reduced by approximately 60% compared to membranes from cells grown in a choline-deficient medium . Relatively smaller reductions in phosphatidylserine decarboxylase activity are also seen in cells grown in the presence of mono- or dimethylethanolamine . In vitro incorporation studies with {14C}serine demonstrate that endogenous, prelabeled phosphatidylserine can be utilized for the biosynthesis of phosphatidylcholine by the coupled action of the hydroxylamine-sensitive decarboxylase and the phospholipid N-methyltransferases in the presence of 2 mM S-adenosylmethionine . A similar comparative enzymatic study shows that the rates of synthesis and decarboxylation of {14C}phosphatidylserine, as well as phospholipid N-methylation, are lower for membranes prepared from cells grown in the presence of choline relative to identical preparations from cells grown in the absence of choline . These studies describe the properties of particulate and detergent-solubilized phosphatidylserine decarboxylase activity in S . cerevisiae and provide evidence that its activity is regulated in coordination with other enzymes in the pathway for phosphatidylcholine biosynthesis involving N-methylation. Mutat Res, 1984 May-Jun, 131(5-6), 197 - 204 Introduction of the plasmid pKM101-associated muc genes into Saccharomyces cerevisiae; Potter AA et al.; Bacteria-yeast shuttle plasmids containing the pKM101-associated muc genes were constructed by cloning an ARS TRP fragment into the plasmid pGW270 in both possible orientations . The insertion of Saccharomyces cerevisiae DNA into pGW270 had no effect on the mutator and protective phenotypes associated with the plasmid in Escherichia coli . Two such recombinant plasmids, pAA90 and pAA91 , were capable of efficient transformation of S . cerevisiae and were stably maintained in this organism . Hybridization experiments suggest that muc-specific mRNA was present in transformed yeast cells and a small amount was polyadenylated . The RNAs were not of a discrete size, all being smaller than the muc genes . The presence of the plasmid pAA91 , and to a lesser extent, pAA90 , in yeast resulted in a detectable increase in the reversion frequencies of three markers and in ultraviolet protection . These results are discussed in terms of studying the relationship of error-prone repair in bacteria and yeast and of developing improved yeast tester strains. J Bacteriol, 1984 May, 158(2), 701 - 4 Buoyant density variation during the cell cycle of Saccharomyces cerevisiae; Baldwin WW et al.; Cell buoyant densities of the budding yeast Saccharomyces cerevisiae were determined for rapidly growing asynchronous and synchronous cultures by equilibrium sedimentation in Percoll gradients . The average cell density in exponentially growing cultures was 1.1126 g/ml, with a range of density variation of 0.010 g/ml . Densities were highest for cells with buds about one-fourth the diameter of their mother cells and lowest when bud diameters were about the same as their mother cells . In synchronous cultures inoculated from the least-dense cells, there was no observable perturbation of cell growth: cell numbers increased without lag, and the doubling time (66 min) was the same as that for the parent culture . Starting from a low value at the beginning of the cycle, cell buoyant density oscillated between a maximum density near midcycle (0.4 generations) and a minimum near the end of the cycle (0.9 generations) . The pattern of cyclic variation of buoyant density was quantitatively determined from density measurements for five cell classes, which were categorized by bud diameter . The observed variation in buoyant density during the cell cycle of S . cerevisiae contrasts sharply with the constancy in buoyant density observed for cells of Escherichia coli, Chinese hamster cells, and three murine cell lines. Cell, 1984 May, 37(1), 67 - 75 Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis; Schwartz DC et al.; A new type of gel electrophoresis separates DNA molecules up to 2000 kb with resolutions exceeding the logarithmic molecular weight dependence of conventional electrophoresis . The technique uses 1.5% agarose, 10 to 20 micrograms of DNA per well, and low ionic strength buffers . It employs alternately pulsed, perpendicularly oriented electrical fields, at least one of which is inhomogeneous . The duration of the applied electrical pulses is varied from 1 sec to 90 sec to achieve optimal separations for DNAs with sizes from 30 to 2000 kb . This pulsed field gradient gel electrophoresis fractionates intact S . cerevisiae chromosomal DNA, producing a molecular karyotype that greatly facilitates the assignment of genes to yeast chromosomes . Each yeast chromosome consists of a single piece of DNA; the chromosome sizes are consistent with the genetic linkage map . We also describe a general method for preparing spheroplasts, and cell lysates, without significant chromosomal DNA breakage. Mol Biochem Parasitol, 1984 May, 12(1), 61 - 7 Accurate transcription of a cloned gene from Babesia bovis in Saccharomyces cerevisiae; Kemp DJ et al.; Two cloned chromosomal segments of the hemoprotozoan Babesa bovis bearing genes from the polymorphic locus designated BabR were inserted into plasmid vector YCp19, cloned in Escherichia coli and then introduced into Saccharomyces cerevisiae . Both genes were transcribed producing poly(A+) RNAs . In at least one case, the transcript was very similar if not identical to that produced in B . bovis . It is therefore possible that S . cerevisiae can recognize at least some hemoprotozoan promoter and poly(A+)-addition signals. Nucleic Acids Res, 1984 Apr 25, 12(8), 3677 - 93 Primary and secondary structure of rat 28 S ribosomal RNA; Hadjiolov AA et al.; The primary structure of rat (Rattus norvegicus) 28 S rRNA is determined inferred from the sequence of cloned rDNA fragments . The rat 28 S rRNA contains 4802 nucleotides and has an estimated relative molecular mass (Mr, Na-salt) of 1.66 X 10(6) . Several regions of high sequence homology with S . cerevisiae 25 S rRNA are present . These regions can be folded in characteristic base-paired structures homologous to those proposed for Saccharomyces and E . coli . The excess of about 1400 nucleotides in the rat 28 S rRNA (as compared to Saccharomyces 25 S rRNA) is accounted for mainly by the presence of eight distinct G+C-rich segments of different length inserted within the regions of high sequence homology . The G+C content of the four insertions, containing more than 200 nucleotides, is in the range of 78 to 85 percent . All G+C-rich segments appear to form strongly base-paired structures . The two largest G+C-rich segments (about 760 and 560 nucleotides, respectively) are located near the 5'-end and in the middle of the 28 S rRNA molecule . These two segments can be folded into long base-paired structures, corresponding to the ones observed previously by electron microscopy of partly denatured 28 S rRNA molecules. Mutat Res, 1984 Apr, 139(4), 189 - 92 Studies of genetic effects in the D7 strain of Saccharomyces cerevisiae under different conditions of pH; Nanni N et al.; The genetic effects of variation in pH in culture media and in suspension tests were examined in a diploid strain (D7) of the yeast, Saccharomyces cerevisiae . Deviation from the normal pH of 6.24 in the liquid culture medium, has a significant effect on cellular growth and on mitotic gene conversion at the trp5 locus . Frequencies of reversion at the ilv I-92 locus and of mitotic crossing-over at the ade2 locus are not significantly influenced . Suspension tests, performed using phosphate buffer (pH 5.8), strongly confirm the original results . Our data suggest that the increase in mitotic gene conversion under various conditions of pH is due to a specific effect of pH itself on the cells of S . cerevisiae . In fact, increases were obtained using the same pH in both cellular growth and non-growth conditions . The maximum effect detected with both procedures was obtained at pH 5.8; in the growth test, at this pH, gene conversion frequency appeared to be most pronounced, being about 10 times higher than that of the control . These results suggest that pH exerts its specific action both on growing and non-growing yeast cells, and the difference in induction of genetic effect between these two conditions is probably due to a time factor. Mol Cell Biol, 1984 Apr, 4(4), 657 - 65 Effects of altered 5'-flanking sequences on the in vivo expression of a Saccharomyces cerevisiae tRNATyr gene; Shaw KJ et al.; Deletion mutations ending in the 5'-flanking sequences of the Saccharomyces cerevisiae SUP4-o gene have been analyzed for their effects on gene expression . This ochre-suppressing tRNATyr gene was cloned into a S . cerevisiae centromeric plasmid, and its level of in vivo expression was monitored by observing the suppressor phenotype of the gene after transformation into S . cerevisiae . A deletion mutant that retains only four base pairs of the 5'-flanking sequence is profoundly deficient in expression; deletion mutants extending to positions -18, -17, -16, or -15 are moderately deficient; deletion mutants extending to positions -36 or -27 are slightly defective; and mutants retaining more than 60 base pairs of the original 5'-flanking DNA are expressed normally . In some cases, the cloning procedure led to the introduction of multiple BamHI linkers at the SUP4-o-vector fusion site, and in one instance, the resulting structure dramatically affects gene function: the presence of three linkers abutting a -18 deletion completely inhibits the in vivo expression of SUP4-o . In contrast, three linkers that abut a -77 deletion have no effect on in vivo expression . The template properties of these plasmids in a homologous in vitro transcription system parallel the levels of in vivo expression, suggesting that the mutations predominantly affect transcription . The data demonstrate that there are significant functional constraints on the 5'-flanking sequences of this RNA polymerase III-transcribed gene . The dramatic effects of the multiple linker insertion at position -18 suggest that there may be extensive melting of the DNA in this region during normal transcription initiation. Mol Cell Biol, 1984 Apr, 4(4), 583 - 90 Saccharomyces cerevisiae CDC8 gene and its product; Birkenmeyer LG et al.; The product of the Saccharomyces cerevisiae CDC8 gene is essential for normal cellular DNA replication; the determination of the structure of the gene and the identification of its product would facilitate the examination of its role in this process . We have cloned a 1,000-base-pair fragment of the S . cerevisiae genome carrying the functional gene . The nucleotide sequence includes one long open reading frame; it is flanked by sequences typical of other S . cerevisiae genes . This sequence predicts a polypeptide chain product of 216 amino acids with a molecular weight of 24,600 . A polyadenylated RNA transcript of this sequence was identified by hybridization; in vitro translation of RNA samples enriched for this transcript produced a specific polypeptide chain of apparent molecular weight between 24,000 and 25,000 . Thus the reading frame identified represents the authentic CDC8 gene, and the amino acid sequence of its product has been deduced . Our observations differ from two previous reports of the identification of the putative CDC8 protein based upon in vitro complementation assays. Proc Natl Acad Sci U S A, 1984 Apr, 81(7), 2157 - 61 Identification, molecular cloning, and mutagenesis of Saccharomyces cerevisiae RNA polymerase genes; Ingles CJ et al.; Three different regions of Saccharomyces cerevisiae DNA were identified by using as hybridization probe a fragment of Drosophila melanogaster DNA that encodes an RNA polymerase II (EC 2.7.7.6) polypeptide . Two of these regions have been molecularly cloned . Each contains a sequence related not only to the D . melanogaster DNA fragment that was used as a probe in its isolation but also to the immediately adjacent DNA fragment of the D . melanogaster RNA polymerase II gene . The two cloned S . cerevisiae DNA sequences are each the template for single transcripts in vivo, one of 5.9 kilobases and the other of 4.6 kilobases . In vitro translation of hybrid-selected cellular RNA indicated that the former locus encodes a protein of Mr 220,000, equal in size to the largest polypeptide subunit of S . cerevisiae RNA polymerase II . Disruption of either gene by targeted integration of URA3+ DNA demonstrated that each is single-copy and essential in a haploid genome . We suggest that these S . cerevisiae loci are members of a family of related genes encoding the largest subunit polypeptides of RNA polymerases I, II, and III. Mol Cell Biol, 1984 Apr, 4(4), 703 - 11 Intrachromosomal movement of genetically marked Saccharomyces cerevisiae transposons by gene conversion; Roeder GS et al.; In this paper, we describe the movement of a genetically marked Saccharomyces cerevisiae transposon . Ty912(URA3), to new sites in the S . cerevisiae genome . Ty912 is an element present at the HIS4 locus in the his4-912 mutant . To detect movement of Ty912, this element has been genetically marked with the S . cerevisiae URA3 gene . Movement of Ty912(URA3) occurs by recombination between the marked element and homologous Ty elements elsewhere in the S . cerevisiae genome . Ty912(URA3) recombines most often with elements near the HIS4 locus on chromosome III, less often with Ty elements elsewhere on chromosome III, and least often with Ty elements on other chromosomes . These recombination events result in changes in the number of Ty elements present in the cell and in duplications and deletions of unique sequence DNA. Cell, 1984 Apr, 36(4), 1057 - 65 Targeting of E . coli beta-galactosidase to the nucleus in yeast; Hall MN et al.; In order to identify determinants governing nuclear protein localization, we constructed a set of hybrid genes by fusing the S . cerevisiae gene, MAT alpha 2, coding for a presumptive nuclear protein, and the E . coli gene, lacZ, coding for beta-galactosidase . The resultant hybrid proteins contain 3, 13, 25, 67, or all 210 amino acids of wild-type alpha 2 protein at the amino terminus and a constant, enzymatically active portion of beta-galactosidase at the carboxy terminus . Indirect immunofluorescence and subcellular fractionation studies with yeast cells containing the alpha 2-LacZ hybrid proteins indicate that the alpha 2 segment can direct localization of beta-galactosidase to the nucleus . A segment as small as 13 amino acids from alpha 2 is sufficient for this localization . Comparison of amino acid sequences of other nuclear proteins with this region of alpha 2 reveals a sequence that may be necessary for nuclear targeting . Production of some alpha 2-LacZ hybrid proteins causes cell death, perhaps as a result of improper or incomplete localization . These studies also indicate that the alpha 2 protein, argued on genetic grounds to be a negative regulator, acts in the yeast nucleus. EMBO J, 1984 Apr, 3(4), 847 - 53 Differential binding of a S . cerevisiae RNA polymerase III transcription factor to two promoter segments of a tRNA gene; Stillman DJ et al.; A Saccharomyces cerevisiae protein fraction which binds specifically to the internal promoter regions of genes that are transcribed by RNA polymerase III is shown to function as a transcription factor . We postulate that the stable DNA binding of the factor confers stability on polymerase III transcription complexes . Analysis of the binding by DNase 'foot-printing' distinguishes three segments of the S . cerevisiae tRNALeu3 gene: a region surrounding the so-called A block of the internal promoter, a region surrounding the B block and an intermediate segment . Binding to the A and B block regions is connected, but the B block region exerts a dominant effect. J Biol Chem, 1984 Mar 25, 259(6), 3450 - 6 Characterization of an estrogen-binding protein in the yeast Saccharomyces cerevisiae; Burshell A et al.; This paper further characterizes the estrogen-binding protein we have described in the cytosol of the yeast Saccharomyces cerevisiae . {3H}Estradiol was used as the radioprobe, and specific binding of cytosol fractions was measured by chromatography on Sephadex minicolumns . Other 3H-steroids did not exhibit specific binding . {3H}Estradiol binding was destroyed by treatment with trypsin, but not RNase, DNase, or phospholipase; N-ethylmaleimide substantially decreased the binding . The yeast did not metabolize estradiol added to the medium, and extraction and chromatography of the bound moiety showed it to be unmetabolized estradiol . Scatchard analysis of cytosol from both a and alpha mating types as well as the a/alpha diploid cell revealed similar binding properties: an apparent dissociation constant or Kd(25 degrees) for {3H}estradiol of 1.6-1.8 nM and a maximal binding capacity or Nmax of approximately 2000-2800 fmol/mg of cytosol protein . Gel exclusion chromatography on Sephacryl S-200 and high performance liquid chromatography suggested a Stokes radius of approximately 30 A . Sucrose gradient centrifugation showed a sedimentation coefficient of approximately 5 S, and the complex did not exhibit ionic dependent aggregation . The estrogen binder in S . cerevisiae differed in its steroidal specificities from classical mammalian estrogen receptors in rat uterus . 17 beta-Estradiol was the best competitor, 17 alpha-estradiol had about 5% the activity, and diethylstilbestrol exhibited negligible binding affinity as did tamoxifen, nafoxidine, and the zearalenones . In summary, a high affinity, stereospecific, steroid-selective binding protein has been demonstrated in the cytosol of the simple yeast S . cerevisiae . We speculate that this molecule may represent a primitive hormone receptor system, possibly for an estrogen-like message molecule. J Biol Chem, 1984 Mar 25, 259(6), 3985 - 92 Nucleotide sequence of Saccharomyces cerevisiae genes TRP2 and TRP3 encoding bifunctional anthranilate synthase: indole-3-glycerol phosphate synthase; Zalkin H et al.; Saccharomyces cerevisiae anthranilate synthase:indole-3-glycerol phosphate synthase is a multifunctional hetero-oligomeric enzyme encoded by genes TRP2 and TRP3 . TRP2, encoding anthranilate synthase Component I, was cloned by complementation of a yeast trp2 mutant . The nucleotide sequence of TRP2 as well as that of TRP3 were determined . The deduced anthranilate synthase Component I primary structure from yeast exhibits only limited similarity to that of the corresponding Escherichia coli subunit encoded by trpE . On the other hand, yeast anthranilate synthase Component II and indole-3-glycerol phosphate synthase amino acid sequences from TRP3 are clearly homologous with the corresponding sequences of the E . coli trpG and trpC polypeptide segments and thereby establish the bifunctional structure of TRP3 protein . Based on comparisons of TRP3 amino acid sequence with homologous sequences from E . coli and Neurospora crassa, an 11-amino acid residue connecting segment was identified which fuses the trpG and trpC functions of the bifunctional TRP3 protein chain . These comparisons support the conclusion that the amino acid sequence of connectors in homologous multifunctional enzymes need not be conserved . Connector function is thus not dependent on a specific sequence . Nuclease S1 mapping was used to identify mRNA 5' termini . Heterogeneous 5' termini were found for both TRP2 and TRP3 mRNA . TRP2 and TRP3 5'-flanking regions were analyzed for sequences that might function in regulation of these genes by the S . cerevisiae general amino acid control system . The 9 base pair direct repeat (Hinnebusch, A.G., and Fink, G.R . (1983) J . Biol . Chem . 258, 5238-5247) and inverted repeats were identified in the 5'-flanking sequences of TRP2 and TRP3. Nucleic Acids Res, 1984 Mar 12, 12(5), 2303 - 15 Synthesis of Gp4N and Gp3N compounds by guanylyltransferase purified from yeast; Wang D et al.; Guanylyltransferase that catalyzes mRNA capping by the reaction, ppNpN + GTP----GpppNpN was purified from S . cerevisiae . The enzyme forms a nucleotidyl intermediate by phosphoamide linkage of GMP . Two guanylylated polypeptides of MR approximately 52,000 and 46,000 were obtained, the latter apparently by proteolysis of the larger component . Both forms transferred the covalently bound GMP to ppApG, yielding GpppApG . Dinucleoside tri- and tetraphosphates of the type Gp3N and Gp4N were also produced by using ribonucleoside 5'-di and triphosphates as acceptors . The purified yeast guanylyltransferase contained little or no RNA 5'-triphosphatase or methyltransferase. Mol Biol (Mosk), 1984 Mar-Apr, 18(2), 293 - 312 {Instability of the mitochondrial genome}; Nevzgliadova OV; A number of manifestations of mitochondrial DNA instability have been reviewed . Differences in organization of mitochondrial genomes of different origin have been regarded as well as variability concerning the genetic code . Examples of molecular heterogeneity of mtDNA and among them insertions and optional introns in Saccharomyces cerevisiae are given . Specific mutations in ascomycets and higher plants have been discussed as an aspect of instability since they cause the appearance of mitochondrial plasmids and episomes . One can regard the rate of mtDNA evolution particularly the high frequency of molecular rearrangement as connected with the fact that some of its regions behave as "egoistic" DNA . According to the Doolittle-Crick concept phenotypical selection always supports any useful function of that DNA, emerging by chance . Therefore we admit that some of the optional insertions into mt genes in S . cerevisiae have the adaptive function . It is also possible that in the course of evolution some higher plants "have learned" to use the DNA's ability to generate plasmids and episomes in order to create new means of gene activity regulation. Cell, 1984 Mar, 36(3), 607 - 12 Genes in S . cerevisiae encoding proteins with domains homologous to the mammalian ras proteins; Powers S et al.; The ras genes, which were first identified by their presence in RNA tumor viruses and which belong to a highly conserved gene family in vertebrates, have two close homologs in yeast, detectable by Southern blotting . We have cloned both genes (RAS1 and RAS2) from plasmid libraries and determined the complete nucleotide sequence of their coding regions . They encode proteins with nearly 90% homology to the first 80 positions of the mammalian ras proteins, and nearly 50% homology to the next 80 amino acids . Yeast RAS1 and RAS2 proteins are more homologous to each other, with about 90% homology for the first 180 positions . After this, at nearly the same position that the mammalian ras proteins begin to diverge from each other, the two yeast ras proteins diverge radically . The yeast ras proteins, like the proteins encoded by the mammalian genes, terminate with the sequence cysAAX, where A is an aliphatic amino acid . Thus the yeast ras proteins have the same overall structure and interrelationship as the family of mammalian ras proteins . The domains of divergence may correspond to functional domains of the ras proteins . Monoclonal antibody directed against mammalian ras proteins immunoprecipitates protein in yeast cells containing high copy numbers of the yeast RAS2 gene. Mol Cell Biol, 1984 Mar, 4(3), 407 - 14 Expression of plasmid R388-encoded type II dihydrofolate reductase as a dominant selective marker in Saccharomyces cerevisiae; Miyajima A et al.; The R388 plasmid-encoded drug-resistant type II dihydrofolate reductase gene (R . dhfr) was expressed in Saccharomyces cerevisiae by fusing the R . dhfr coding sequence to the yeast TRP5 promoter . Yeast cells harboring these recombinant plasmids grew in media with 10 micrograms of methotrexate per ml and 5 mg of sulfanilamide per ml, a condition which inhibits the growth of wild-type cells . Addition of a 390-base-pair fragment from the 3'-noncoding region of TRP5 downstream from R . dhfr increased expression . Presumably, the added segment promoted termination or polyadenylation or both of the R . dhfr transcript . The activity of the plasmid-encoded dihydrofolate reductase and the copy number of the R . dhfr plasmid in cells grown in drug-selective media were higher by one order of magnitude than those grown in nutrition-selective media . Plasmid copy number, as well as the plasmid-encoded enzyme level, decreased when cells were selected for prototrophy . In drug-selective media, the plasmid-encoded enzyme level and the content of R . dhfr transcripts were nearly constant in cells harboring R . dhfr plasmids containing different yeast promoters . In contrast, the plasmid copy number and beta-lactamase activity encoded in cis by plasmids were much higher when R . dhfr was associated with the weak TRP5 promoter than when it was fused to the strong ADC1 promoter . These results indicate that plasmid copy number, i.e., gene dosage of R . dhfr, correlates inversely with the strength of the promoter associated with R . dhfr, and cells with a higher plasmid copy number were enriched in drug-selective media . The transformation efficiency of R . dhfr fused to the ADC1 promoter was almost the same on drug-selective plates as on nutrition-selective plates, indicating that R . dhfr is suitable as a dominant selective transformation marker in S . cerevisiae. Genetics, 1984 Mar, 106(3), 365 - 85 Mutants of S . cerevisiae defective in the maintenance of minichromosomes; Maine GT et al.; We have isolated yeast mutants that are defective in the maintenance of circular minichromosomes . The minichromosomes are mitotically stable plasmids, each of which contains a different ARS (autonomously replicating sequence), a centrometeric sequence, CEN5, and two yeast genes, LEU2 and URA3 . Forty minichromosome maintenance-defective (Mcm-) mutants were characterized . They constitute 16 complementation groups . These mutants can be divided into two classes, specific and nonspecific, by their differential ability to maintain minichromosomes with different ARSs . The specific class of mutants is defective only in the maintenance of minichromosomes that carry a particular group of ARSs irrespective of the centromeric sequence present . The nonspecific class of mutants is defective in the maintenance of all minichromosomes tested irrespective of the ARS or centromeric sequence present . The specific class may include mutants that do not initiate DNA replication effectively at specific ARSs present on the minichromosomes; the nonspecific class may include mutants that are affected in the segregation and/or replication of circular plasmids in general. Biochem Biophys Res Commun, 1984 Feb 14, 118(3), 821 - 7 DNA sequences from a ligninolytic filamentous fungus Phanerochaete chrysosporium capable of autonomous replication in yeast; Rao TR et al.; Five different DNA sequences of Phanerochaete chrysosporium capable of supporting autonomous replication of yeast integration plasmid (YIp5) in Saccharomyces cerevisiae were isolated . These hybrid plasmids with the autonomous replication sequences from P . chrysosporium are maintained extra-chromosomally, are mitotically unstable and transform Ura3 deletion mutant of S . cerevisiae to Ura+ phenotype with high frequency . The autonomous replication sequence in pRR2, one of the recombinant plasmids, was further characterized and was shown to be homologous to P . chrysosporium genomic DNA . Restriction analyses showed that this plasmid has unique PvuII and SalI restriction sites for cloning. Nucleic Acids Res, 1984 Feb 10, 12(3), 1377 - 90 The in vivo and in vitro initiation site for transcription of the rRNA operon of Saccharomyces carlsbergensis; Klootwijk J et al.; We have performed a detailed analysis of the transcription initiation of the rRNA operon in the yeast Saccharomyces carlsbergensis . Electron microscopic analysis of R-looped pre-rRNA molecules together with a very sensitive S1-nuclease mapping showed the use of only a single transcription start at about 700 bp upstream of the 17S rRNA gene and not of the minor start sites proposed for the very closely related species S . cerevisiae by others {Bayev et al . (5), Swanson and Holland (6)} . The sequence of 730 bp of the initiating region is presented . In vitro transcription in concentrated lysates of yeast spheroplasts in the presence of (gamma-SH)ATP or (gamma-SH)GTP, followed by purification of the in vitro initiated RNA via Hg-agarose, revealed that on the endogenous template exactly the same site is used for transcription initiation as in vivo. Mol Cell Biol, 1984 Feb, 4(2), 329 - 39 Genetic mapping of Ty elements in Saccharomyces cerevisiae; Klein HL et al.; We used transformation to insert a selectable marker at various sites in the Saccharomyces cerevisiae genome occupied by the transposable element Ty . The vector CV9 contains the LEU2+ gene and a portion of the repeated element Ty1-17 . Transformation with this plasmid resulted in integration of the vector via a reciprocal exchange using homology at the LEU2 locus or at the various Ty elements that are dispersed throughout the S . cerevisiae genome . These transformants were used to map genetically sites of several Ty elements . The 24 transformants recovered at Ty sites define 19 distinct loci . Seven of these were placed on the genetic map . Two classes of Ty elements were identified in these experiments: a Ty1-17 class and Ty elements different from Ty1-17 . Statistical analysis of the number of transformants at each class of Ty elements shows that there is preferential integration of the CV9 plasmid into the Ty1-17 class. Mol Cell Biol, 1984 Feb, 4(2), 290 - 5 Saccharomyces cerevisiae RAD2 gene: isolation, subcloning, and partial characterization; Naumovski L et al.; A plasmid (pNF2000) containing a 9.7-kilobase pair DNA insert that complements the UV sensitivity of rad2-1, rad2-2, and rad2-4 mutants of Saccharomyces cerevisiae has been isolated from a yeast genomic library . Genetic analysis of strains derived by transformation of rad2 mutants with an integrating plasmid containing a 9.3-kilobase pair fragment from pNF2000 shows that the fragment integrates exclusively at the chromosomal rad2 gene . We therefore conclude that this plasmid contains the RAD2 gene . The 9.3-kilobase pair fragment was partially digested with Sau3A and cloned into a multicopy yeast vector designed for easy retrieval of Sau3A inserts . The smallest subclone that retains the RAD2 gene is 4.5 kilobase pairs . This fragment was partially digested with Sau3A and cloned into an integrating plasmid . These plasmids were isolated and integrated into a heterozygous rad2/RAD2 strain . Plasmids containing internal fragments of the RAD2 gene were identified because they yielded UV-sensitive transformants due to disruption of the RAD2 gene . Sporulation of diploids transformed with integrating plasmids containing internal fragments of RAD2 gave rise to four viable haploids per tetrad, indicating that unlike the RAD3 gene of S . cerevisiae, the RAD2 gene is not essential for the viability of haploid cells under normal growth conditions . Measurements of the RNA transcript by RNA-DNA hybridization with the internal fragment as the probe indicate a size of approximately 3.2 kilobases. Mol Cell Biol, 1984 Feb, 4(2), 268 - 75 Identification of two proteins encoded by the Saccharomyces cerevisiae GAL4 gene; Laughon A et al.; We placed the Saccharomyces cerevisiae GAL4 gene under control of the galactose regulatory system by fusing it to the S . cerevisiae GAL1 promoter . After induction with galactose, GAL4 is now transcribed at about 1,000-fold higher levels than in wild-type S . cerevisiae . This regulated high-level expression has enabled us to tentatively identify two GAL4-encoded proteins. Exp Cell Res, 1984 Feb, 150(2), 309 - 13 A temperature-sensitive N-glycosylation mutant of S . cerevisiae that behaves like a cell-cycle mutant; Klebl F et al.; The temperature-sensitive S . cerevisiae mutant alg1-1, defective in the N-glycosylation of proteins, shows a first cycle arrest at the non-permissive temperature of 36 degrees C . The cell number increases by 50% and the absorbance approximately doubles . The budding index of 0.4 at 26 degrees C drops to 0.15 and DNA synthesis quickly comes to a halt at 36 degrees C . When the temperature is lowered again, budding and DNA synthesis start after a lag of 2-3 h; alpha-factor prevents both these processes in cells of mating type a . In addition, cells arrested at 26 degrees C in G1 with alpha-factor also do not start budding at the non-permissive temperature after removal of alpha-factor . The results support recent findings obtained with tunicamycin and suggest that at least one glycoprotein is required for G1-S phase transition in yeast. Cell, 1984 Feb, 36(2), 503 - 11 Distinctly regulated tandem upstream activation sites mediate catabolite repression of the CYC1 gene of S . cerevisiae; Guarente L et al.; The upstream activation site (UAS) of the yeast CYC1 gene is shown to contain two homologous subsites, UAS1 and UAS2 . Each site, when placed upstream of the transcriptional initiation region of the yeast LEU2 gene, activates LEU2 transcription which is regulated by catabolite repression . UAS1 is responsible for most of the transcription under glucose repressed conditions, while UAS1 and UAS2 contribute equally to lactate derepressed transcription . A single point mutation in UAS2 increases its activity in glucose 10- to 20-fold . Several experiments indicate that UAS1 and UAS2 are regulated distinctly at the molecular level . First, UAS1 but not UAS2 is fully depressed in glucose by increasing the levels of intracellular heme . Second, trans-acting regulatory mutations, hap1-1 and hap2-1, selectively abolish the activity of UAS1 or UAS2 . HAP1 appears to encode a protein that mediates catabolite repression of UAS1 by responding to intracellular heme levels. Immun Infekt, 1984 Feb, 12(1), 69 - 72 {Phagocytosis of neutrophilic granulocytes of intensive care patients: effect of immunoglobulin preparations}; Stubner G et al.; Phagocytic capacity and the amount of PMN-granulocytes which take part in phagocytosis was investigated in patients with septicemia and polytrauma patients in comparison to PMN-granulocytes withdrawn from healthy volunteers by use of a standardized test-system in which autoclavated S.cerevisiae is the phagocytic agent . Influence of polyvalent immunoglobulin preparations together with patients' own serum incubation was compared with serum preincubation only in the test system mentioned above . It became evident that phagocytic capacity and the number of PMN-granulocytes which take part in phagocytosis in patients with septicemia are primary on a significant higher rate than those of other groups . PMN-granulocytes from polytraumated patients showed a significant higher rate on ingestion of S.cerevisiae pretreated with patients' own serum - especially after preopsonisation with immunoglobulins - than granulocytes from patients with septicemia or healthy volunteers. Nature, 1984 Jan 12-18, 307(5947), 183 - 5 Primary structure homology between the product of yeast cell division control gene CDC28 and vertebrate oncogenes; Lorincz AT et al.; In the budding yeast, Saccharomyces cerevisiae, division is controlled in response to nutrient limitation and in preparation for conjugation . Cells deprived of an essential nutrient or responding to mating pheromones cease division and become synchronous in the G1 interval, apparently constrained from completing a critical event . This event has been given the operational designation of 'start' . We have isolated a large number of start mutations which confer on S . cerevisiae cells a conditional inability to complete start (Fig . 1) presumably because they define genes which must be expressed for the start event to be successfully completed . We have described the isolation on plasmids of one of the start genes, CDC28, by genetic complementation and initial characterization of its product . We now describe the DNA sequence of the gene CDC28. J Biol Chem, 1984 Jan 10, 259(1), 504 - 11 Cytochrome b gene of Neurospora crassa mitochondria . Partial sequence and location of introns at sites different from those in Saccharomyces cerevisiae and Aspergillus nidulans; Burke JM et al.; We have sequenced a 2614-base pair fragment of the Neurospora crassa mitochondrial DNA which contains part of the structural gene for apocytochrome b . This gene is split by at least two introns . The sequence reported here begins within one intron, extends through the next exon, another intron 1276 base pairs long, and the last exon which encodes the COOH terminus of cytochrome b . Within the 254 amino acids encoded by the two exons, there is a high degree of sequence conservation, 81%, with cytochrome b of Aspergillus nidulans . Surprisingly, both introns in the N . crassa cytochrome b gene are located at positions different from introns in the corresponding genes in Saccharomyces cerevisiae or A . nidulans . The upstream intron is located 22 nucleotides before the first intron in the long form of the S . cerevisiae cytochrome b gene . The downstream intron is located 16 nucleotides before the third intron in the long form of the S . cerevisiae gene and the only intron in the A . nidulans cytochrome b gene . The 1276-base pair downstream intron contains a 314 amino acid long open reading frame, which is in-phase with the preceding exon . The protein product of this reading frame has some resemblance to intron-encoded proteins, known as "mRNA maturases," which are thought to participate in RNA splicing in the mitochondria of S . cerevisiae . Another feature shared by the downstream intron and most other mitochondrial introns is the presence of the Box 9 and Box 2 consensus sequences, which may also be important for RNA splicing. Mol Cell Biol, 1984 Jan, 4(1), 23 - 9 Saccharomyces cerevisiae synthesizes proteins related to the p21 gene product of ras genes found in mammals; Papageorge AG et al.; A family of normal vertebrate genes and oncogenes has been called the ras gene family . The name ras was assigned to this gene family based on the species of origin of the viral oncogenes of the rat-derived Harvey and Kirsten murine sarcoma viruses . There are now three known functional members of the ras gene family, and genes homologous to ras genes have been detected in the DNA of a wide variety of mammals and in Drosophila melanogaster . Prior experiments have detected proteins coded for by ras genes in a large number of normal cells, cell lines, and tumors . We report here the detection of ras-related proteins in D . melanogaster, a result predicted by the earlier detection of ras-related genes in the Drosophila genome . We also report for the first time the detection of ras-related proteins in a single-cell eucaryocyte, Saccharomyces cerevisiae . These proteins, approximately 30K in size, are recognized by both a monoclonal antibody which binds to the p21 coded for by mammalian ras genes and a polyclonal rat serum made by transplanting a v-Ha-ras-induced tumor in Osborne-Mendel rats . The p21 of v-Ha-ras and the 30K proteins from S . cerevisiae share methionine-labeled peptides as detected by two-dimensional tryptic peptide maps . The results indicate that S . cerevisiae synthesizes ras-related proteins . A genetic analysis of the function of these proteins for yeast cells may now be possible. Microbiol Immunol, 1984, 28(12), 1283 - 92 Immunochemical properties of mannan-protein complex isolated from viable cells of Saccharomyces cerevisiae 4484-24D-1 mutant strain by the action of zymolyase; Shibata N et al.; Viable cells of Saccharomyces cerevisiae 4484-24D-1 mutant strain were treated with an Arthrobacter sp . beta-1,3-glucanase, Zymolyase-60,000, in the presence of a serine protease inhibitor, phenylmethylsulfonyl fluoride . Fractionation of the solubilized materials with Cetavlon (cetyltrimethylammonium bromide) yielded a purified mannan-protein complex, which had a molecular weight of ca . 150,000, approximately three times higher than that of the mannan isolated from the same cells by the hot-water extraction method at 135 C . The amino acid composition of the mannan-protein complex was found to be very similar to that of the mannan-protein complexes of S . cerevisiae X2180-1A wild and S . cerevisiae X2180-1A-5 mutant strains, indicating the presence of large amounts of serine and threonine . It was unexpected that the antibody-precipitating activity of this complex against the homologous anti-whole cell serum was about twice as great as that of the mannan isolated by hot-water extraction . Treatment of this complex with 100 mM NaOH, hot water at 135 C, and pronase, respectively, gave degradation products having the same molecular weight and antibody-precipitating activity as those of the hot-water extracted mannan, allowing the assumption that the protein moiety participated in a large part of this activity. Mol Gen Genet, 1984, 198(1), 69 - 74 Modification of nuclear gene expression by inhibition of mitochondrial translation during sporulation in MAT alpha/MATa diploids of Saccharomyces cerevisiae; Marmiroli N et al.; Sporulation of S . cerevisiae MAT alpha-/MATa was accompanied by a novel pattern of protein synthesis as shown by the disappearance of some "mitotic" polypeptides and by the appearance of a new set of "meiotic" polypeptides . Inhibition of mitochondrial protein synthesis by erythromycin within the 1st h caused the disappearance of several "meiotic" polypeptides . These meiotic polypeptides were also sensitive to cycloheximide and were localized in the cytosol, demonstrating that they were not mitochondrial translational products . Since erythromycin affected neither protein synthesis nor sporulation in a mitochondrially inherited eryr mutant, we conclude that mitochondrial protein synthesis is needed for the expression of some nuclear genes during sporulation. Sabouraudia, 1984, 22(5), 403 - 7 Saccharomyces cerevisiae infections in man; Eng RH et al.; Saccharomyces cerevisiae is a yeast commonly used to make food products such as bread, beer and wine, or ingested whole as a 'health' food . We report five cases of infections involving S . cerevisiae, including one in which S . cerevisiae alone was implicated, and review the literature on its pathogenicity. Mol Gen Genet, 1984, 195(1-2), 361 - 3 Effect of caffeine on ozone-sensitivity in Saccharomyces cerevisiae; Dubeau H et al.; The addition of 0.1% caffeine to the plating medium markedly reduced the ozone-survival of the wild-type and the rad1 and rad6 mutants of Saccharomyces cerevisiae, whereas no effect was observed in the rad52 mutant . Since, in S . cerevisiae, caffeine has been reported to interfere with the recombinational repair pathway under the control of the RAD52 gene, these results support previous observations suggesting that this pathway is involved in the repair of ozone-induced DNA damage. Mol Cell Biol, 1984 Jan, 4(1), 86 - 91 Isolation and characterization of the centromere from chromosome V (CEN5) of Saccharomyces cerevisiae; Maine GT et al.; We have cloned a functional centromeric DNA sequence from Saccharomyces cerevisiae . Using the 2 mu chromosome-loss mapping technique and meiotic tetrad analysis, we have identified this DNA sequence as the centromere of chromosome V (CEN5) . The CEN5 sequence has been localized on an 1,100-base-pair BamHI-BglII restriction fragment . Plasmids containing CEN5 and an autonomously replicating sequence are mitotically stable in S . cerevisiae and segregate in a Mendelian fashion during meiosis. Mol Cell Biol, 1984 Jan, 4(1), 54 - 60 Structure and expression of the SNF1 gene of Saccharomyces cerevisiae; Celenza JL et al.; The SNF1 gene of Saccharomyces cerevisiae is essential for normal regulation of gene expression by glucose repression . A functional SNF1 gene product is required to derepress many glucose-repressible genes in response to conditions of low external glucose . In the case of the SUC2 structural gene for invertase, SNF1 acts at the RNA level . We have reported the isolation of a cloned gene that complements the snf1 defect in S . cerevisiae and that is homologous to DNA at the SNF1 locus (J . L . Celenza and M . Carlson, Mol . Cell . Biol . 4:49-53, 1984) . In this work we identified a 2.4-kilobase polyadenylate-containing RNA encoded by the SNF1 gene and showed that its level is neither regulated by glucose repression nor dependent on a functional SNF1 product . The position of the SNF1 RNA relative to the cloned DNA was mapped, and the direction of transcription was determined . The cloned DNA was used to disrupt the SNF1 gene at its chromosomal locus . Gene disruption resulted in A Snf1- phenotype, thereby proving that the cloned gene is the SNF1 gene and showing that the phenotype of a true null mutation is indistinguishable from that of previously isolated snf1 mutations. Mol Cell Biol, 1984 Jan, 4(1), 49 - 53 Cloning and genetic mapping of SNF1, a gene required for expression of glucose-repressible genes in Saccharomyces cerevisiae; Celenza JL et al.; A functional SNF1 gene product is required to derepress expression of many glucose-repressible genes in Saccharomyces cerevisiae . Strains carrying a snf1 mutation are unable to grow on sucrose, galactose, maltose, melibiose, or nonfermentable carbon sources; utilization of these carbon sources is regulated by glucose repression . The inability of snf1 mutants to utilize sucrose results from failure to derepress expression of the structural gene for invertase at the RNA level . We isolated recombinant plasmids carrying the SNF1 gene by complementation of the snf1 defect in S . cerevisiae . A 3.5-kilobase region is common to the DNA segments cloned in five different plasmids . Transformation of S . cerevisiae with an integrating vector carrying a segment of the cloned DNA resulted in integration of the plasmid at the SNF1 locus . This result indicates that the cloned DNA is homologous to sequences at the SNF1 locus . By mapping a plasmid marker linked to SNF1 in this transformant, we showed that the SNF1 gene is located on chromosome IV . We then mapped snf1 to a position 5.6 centimorgans distal to rna3 on the right arm; snf1 is not extremely closely linked to any previously mapped mutation. Mol Cell Biol, 1984 Jan, 4(1), 195 - 8 Recombinational instability of a chimeric plasmid in Saccharomyces cerevisiae; Whiteway MS et al.; Wild-type strains of Saccharomyces cerevisiae exhibit mitotic recombination between the chimeric plasmid TLC-1 and the endogenous 2mu circle that involves sequence homologies between the two plasmids that are not acted on by the 2mu circle site-specific recombination system . This generalized recombination can be detected because it separates the LEU2 and CAN1 markers of TLC-1 from each other through the formation of a plasmid containing only the S . cerevisiae LEU2 region and the 2mu circle . This derivative plasmid is maintained more stably during vegetative growth than TLC-1, and strains which carry it frequently lose the endogenous 2mu circle . Therefore, TLC-1 can provide a convenient selection for {cir0} cells . Formation of this new plasmid is greatly reduced, but not eliminated, in strains containing the rad52-1 mutation . This indicates that generalized mitotic recombination between plasmid sequences utilizes functions required for chromosomal recombination in S . cerevisiae. Mol Gen Genet, 1984, 193(2), 275 - 9 Mitochondrial ribosomal RNA genes of yeast: their mutations and a common nuclear suppressor; Julou C et al.; Due to the absence of repetition of the rRNA genes in S . cerevisiae mitochondria, isolation of ribosomal mutants at the level of the rRNA genes is relatively easy in this system . We describe here a novel thermosensitive mutation, ts1297, localized by rho- deletion mapping in (or very close to) the sequence corresponding to the small ribosomal RNA (15S) gene . Defective mutations of the small rRNA have not been reported so far . In the mutant, the amount of 15S rRNA and of the small ribosomal subunit, 37S, is reduced . The quantity of the large ribosomal RNA (21S), directly extracted from mitochondria, appears normal . However, the large ribosomal subunit, 50S, seems to be fragile and could be recovered only in the presence of Ca2+ in place of Mg2+ . The 50S particles seem to be completely degraded under normal conditions of extraction with Mg2+ . The thermosensitive phenotype of the ts1297 mutant is suppressed by a nuclear mutation SU101 . The SU101 mutation had been originally isolated as a suppressor of another mitochondrial mutation, ts902, which is located within the 21S rRNA gene . These results suggest that the mitochondrial mutations ts1297 and ts902 are both involved in the interaction of the large and small ribosomal subunits. Mol Gen Genet, 1984, 194(1-2), 31 - 41 Control of Herpes simplex virus thymidine kinase gene expression in Saccharomyces cerevisiae by a yeast promoter sequence; Zhu XL et al.; This study presents the first evidence that the 5' promoter region of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene (G-3-PD) promoter will permit expression of an adjacent foreign gene . The S . cerevisiae G-3-PD promoter was linked to the herpes simplex virus--thymidine kinase (HSV-TK) gene in a shuttle plasmid capable of autonomous replication in both yeast and Escherichia coli . Since the HSV-TK gene promoter is not functional in yeast, yeast cells containing these plasmids will express the HSV-TK gene and synthesize thymidine kinase only if the yeast promoter fragment is fused to the HSV-TK gene in the proper orientation . The 5' flanking sequences necessary for the expression of heterologous eukaryotic genes in S . cerevisiae are discussed. Mol Biol (Mosk), 1984 Jan-Feb, 18(1), 21 - 9 {Study of the stability of hybrid plasmids replicating in Saccharomyces cerevisiae due to DNA fragments from polyoma virus}; Oganesian NA et al.; Hybrid plasmid pSP97 carrying the entire genome of polyoma virus (PY), inserted into bacterial vector psV3, transforms yeast cells with the frequency 1 x 10(-2) . Plasmid pSP97 is capable of autonomous replication in S . cerevisiae, while its structure remains unaltered, the stability of hybrid plasmid in transformants is 44%--100% . Plasmid pSP155 consisting of Ori-containing DNA segment from polyoma, pBR322 and yeast gene arg4, transforms yeast cells with the frequency 5 x 10(-3), the stability of plasmid in transformants is 23%--29% . Two types of plasmids were isolated from transformants: one was identical to SP155, while the another differed structurally and phenotypically from SP155 . Plasmids pSP113 and pSP114, in addition to pBR322 and yeast gene arg4, contain a viral DNA segment that encodes genes from small and middle T-antigens . These plasmids transform yeast cells with low frequency (2 x 10(-4), 3 x 10(-5)), the stability of plasmids in yeast transformants is 100% . However, hybrid plasmids identical to pSP113 were isolated from transformants . Structural rearrangements have been observed in pSP114, which carries the arg4 gene in reversed orientation compared to pSP113. Mol Gen Genet, 1984, 193(3), 557 - 60 Identification of a Ty insertion within the coding sequence of the S . cerevisiae URA3 gene; Rose M et al.; Insertion mutations in yeast caused by the transposable element Ty have been identified at several genetic loci . In all cases so far, the site of Ty insertion has been in the 5' non-coding region of the affected gene . Experiments presented here demonstrate that the ura3-52 mutation, a non-reverting ura3 mutation, is caused by a Ty insertion mutation within the coding region of the URA3 gene . This Ty insertion within a gene has a similar structure to those in non-coding regions. Biochimie, 1984 Jan, 66(1), 71 - 4 Glutathione metabolism in yeast Saccharomyces cerevisiae . Evidence that gamma-glutamyltranspeptidase is a vacuolar enzyme; Jaspers CJ et al.; In a first experiment we have shown that S . cerevisiae beta-glutamyltranspeptidase is associated with a particulate fraction obtained by differential centrifugation . We have subsequently shown that this enzyme activity followed accurately the distribution of vacuolar markers . Liberation of vacuoles was carried out by mechanical disruption of spheroplast under isotonic conditions and the vacuoles were purified by centrifugation of Ficoll gradients . Yeast beta-glutamyltranspeptidase could be implicated in the exchanges of amino acids between the cytoplasm and the vacuolar sap. Cold Spring Harb Symp Quant Biol, 1984, 49, 77 - 88 Involvement of double-strand chromosomal breaks for mating-type switching in Saccharomyces cerevisiae; Klar AJ et al.; The yeast S . cerevisiae switches a and alpha cell types by a transposition mechanism that replaces genetic information residing at the mating-type locus (MAT) with information copied from either of the two donor loci, HML and HMR . The donor HML and HMR loci contain the same genetic information as the MATa and MAT alpha alleles, yet they do not switch . Additionally, Strathern et al . (1982) have described an in vivo double-strand DNA break found at subgenomic levels (approximately 2% of MAT DNA) within the MAT locus but not within HML and HMR . We have examined the role of this double-strand DNA break in the switching process . Cell lineage studies show that strains containing deletions of the donor HML and HMR loci produce lethal progeny in the exact pattern described for MAT switching in standard strains . Our interpretation is that the double-strand MAT break in the deletion strains cannot be repaired because of the lack of the donor loci, resulting in cell death . We suggest that the double-strand DNA break is an initiating event for switching and that this event is lethal in the absence of the donor loci . MAT mutants isolated as survivors from this "pedigree of death" define a site required for switching where the double-strand break occurs . We have also examined marl mutant strains in which the donor loci are expressed and observed to switch (Klar et al . 1981a) . The double-strand DNA cut appears at the HM loci in these strains . Thus, there is a strong correlation between the presence or absence of the double-strand break at each cassette and its ability or inability to switch as observed at the single cell level. Nature, 1983 Dec 15-21, 306(5944), 704 - 7 A yeast gene encoding a protein homologous to the human c-has/bas proto-oncogene product; Gallwitz D et al.; Organisms amenable to easy genetic analysis should prove helpful in assessing the function of at least those proto-oncogene products which are highly conserved in different eukaryotic cells . One obvious possibility is to pursue the matter in Drosophila melanogaster DNA, which has sequences homologous to several vertebrate oncogenes . Another is to turn to the yeast Saccharomyces cerevisiae, if it contains proto-oncogene sequences . Here we report the identification of a gene in S . cerevisiae which codes for a 206 amino acid protein (YP2) that exhibits striking homology to the p21 products of the human c-has/bas proto-oncogenes and the transforming p21 proteins of the Harvey (v-rasH) and Kirsten (v-rasK) murine sarcoma viral oncogenes . The YP2 gene is located between the actin and the tubulin gene on chromosome VI and is expressed in growing cells . The protein it encodes might share the nucleotide-binding capacity of p21 proteins. Biochem Int, 1983 Dec, 7(6), 707 - 17 Absence of derepression of amino acids transport in Candida; Verma RS et al.; The transport of glycine, L-alanine, L-proline, L-leucine, L-lysine, L-phenylalanine and L-glutamic acid did not enhance in various strains of Candida cells, when they were grown in proline containing medium or preincubated with proline . However, under similar conditions, a significant enhancement in the level of accumulation of amino acids (derepression) was observed in Saccharomyces cerevisiae X-2180-A2 (GAP+) cells, which was sensitive to ammonium ions (NH4+) . As expected, the derepression was absent in GAP- cells of S . cerevisiae X-2180 (GAP- mutant) . In contrast to S . cerevisiae (GAP+) cells, the increase in few amino acids uptake in different Candida strains, grown in proline or preincubated in proline, could not be inhibited by cycloheximide, NH4+ or their D-stereoisomers . It appears that derepression of amino acids transport, a well known phenomenon in S . cerevisiae, may not exist in Candida species. J Bacteriol, 1983 Dec, 156(3), 1204 - 13 Separation and characterization of six (1 leads to 3)-beta-glucanases from Saccharomyces cerevisiae; Hien NH et al.; Using a system of chromatography through columns of DEAE-Bio-Gel, HTP-Bio-Gel, and CM-Bio-Gel, we isolated and characterized six different (1 leads to 3)-beta-glucanases from cell wall autolysates and cell extracts of Saccharomyces cerevisiae haploid strain 2180B . These enzymes were designated glucanases I, II, IIIA, IIIB, IV, and V . The haploid mating type S . cerevisiae strain 2180A and the diploid strains S . cerevisiae 2180D and S . cerevisiae 595 contained the same complex of glucanases . Glucanases II and IIIA were exoenzymes, and glucanases I, IIIB, IV, and V were endoenzymes . The enzymes exhibited different molecular weights, kinetic properties, and activities on isolated yeast cell walls . The products of substrate (laminarin) hydrolysis were quantified by using high-pressure liquid chromatography and were significantly different for the four endoglucanases. Cell, 1983 Dec, 35(3 Pt 2), 733 - 42 An mRNA maturase is encoded by the first intron of the mitochondrial gene for the subunit I of cytochrome oxidase in S . cerevisiae; Carignani G et al.; We have localized ten oxi3- mutations in the first, al1, intron of the coxl gene . All are splicing deficient, being unable to excise the intron . Complementation experiments disclose several domains in the intron al1: the 5'-proximal and 3'-proximal domains harbor cis-dominant mutations, while trans-recessive ones are located in the intron's open reading frame . Comprehensive analyses of allele-specific polypeptides accumulating in mutants show that they result from the translation of the intron's ORF . We conclude that a specific mRNA maturase involved in splicing of oxidase mRNA is encoded by the intron al1 in a manner similar to the cytochrome b mRNA maturase. Mikrobiologiia, 1983 Nov-Dec, 52(6), 909 - 16 {Effect of cyclic changes in culture conditions on the growth kinetics and physiological characteristics of yeasts}; Sokolov DP et al.; Candida utilis, Saccharomyces cerevisiae and Candida scottii were used to study the effect of cyclic changes in the pH and pO2 within a range of 1 to 60 min on their growth kinetics and physiological properties . These changes were shown to increase the specific growth rate from 0.33 to 0.5-0.6 h-1 without decreasing the economic coefficient and the quantity of budding cells during 2-3 generations of the exponentially growing batch culture of C . utilis . Optimal conditions of cyclic changes in the pO2 (minutes) were found to increase the specific growth rate of C . scottii and S . cerevisiae . The authors discuss a hypothesis for the formation of intermediate products in the substrate oxidation in the course of pulse aeration by the yeasts during the aerobic stage and the utilization of the products at the anaerobic stage of cyclic regimes . The paper describes a mathematical model for the yeast growth under the nonsteady-state conditions of pH and pO2, which accounts for the formation and utilization of possible intermediate biosynthetic products within the studied time intervals. Mol Cell Biol, 1983 Nov, 3(11), 1949 - 57 Comparison of the levels of the 21S mitochondrial rRNA in derepressed and glucose-repressed Saccharomyces cerevisiae; Kelly R et al.; A cDNA preparation, synthesized by using Saccharomyces cerevisiae mitochondrial RNA as template and oligodeoxythymidylic acid as primer, was found to specifically hybridize to the mitochondrial 21S rRNA by the following criteria: (i) it hybridizes only to the 21S RNA species in mitochondrial RNA and not to RNA from a {rho0} mutant, and (ii) it hybridizes to fragments in restriction digests of mitochondrial DNA that contain the 21S rRNA gene but not to nuclear DNA . This cDNA was used as a probe to demonstrate that a 2.6-fold decrease in the cellular level of the mitochondrial large rRNA is associated with glucose repression of mitochondrial function in S . cerevisiae . A corresponding decrease in the level of mitochondrial DNA was not observed. Genetics, 1983 Nov, 105(3), 501 - 15 Genetics of yeast glucokinase; Maitra PK et al.; Mutants of Saccharomyces cerevisiae lacking glucokinase (EC 2.7.1.2) have no discernible phenotypic difference from the wild-type strain; in a hexokinaseless background, however, they are unable to grow on any sugar except galactose . Reversion studies with glucokinase mutants indicate that the yeast S . cerevisiae has no other enzyme for phosphorylating glucose except the two hexokinases, P1 and P2, and glucokinase . Spontaneous revertants of hxk1 hxk2 glk1 strains collected on glucose regain any one of these three enzymes . The majority of glucokinase revertants synthesize species of enzyme activity that are kinetically or otherwise indistinguishable from the wild-type enzyme . In a few cases the reverted enzyme is very perceptibly altered in properties with a Km for glucose two orders of magnitude higher than that of the enzyme from the wild-type parent . These recessive, noncomplementing mutants, thus, define a single structural gene GLK1 of glucokinase . Yeast diploids lacking all of the three enzymes for glucose phosphorylation fail to sporulate . Heterozygosity of either of the hexokinase genes HXK1 or HXK2, but not GLK1, restores sporulation . The location of GLK1 on chromosome III was indicated by loss of this chromosome when hexokinaseless diploids heterozygous for glk1 were selected for resistance to 2-deoxyglucose; the homologue of chromosome III carrying GLK1, the mating-type allele and other nutritional markers on this chromosome was lost . Meiotic mapping of glucokinase executed with heterozygosity of one of the hexokinases indicated that the gene GLK1 defining the structure of glucokinase protein is located on the left arm of chromosome III 24 cM to the left of his4 in the order: leu2--his4--glk1 . --Only two of 206 independent glucokinase mutants are nonsense ochre, both of which map at one end of the gene . In hxk1 only one of 130 isolates is a nonsense mutation, whereas in hxk2 none has been found among 220 independent mutants . These results raise the possibility that the protein products of these genes have some other essential function . --An earlier mapping result for hxk2 has been corrected . The new location is on the left arm of chromosome VII, 17 cM distal to ade5 in the order: lys5--ade5--hxk2. J Bacteriol, 1983 Nov, 156(2), 552 - 8 Isolation of mannan-protein complexes from viable cells of Saccharomyces cerevisiae X2180-1A wild type and Saccharomyces cerevisiae X2180-1 A-5 mutant strains by the action of Zymolyase-60,000; Shibata N et al.; The viable whole cells of Saccharomyces cerevisiae X2180-1A wild type and its mannan mutant strain S . cerevisiae X2180-1A-5, were treated with an Arthrobacter sp . beta-1,3-glucanase in the presence of a serine protease inhibitor, phenyl-methylsulfonyl fluoride . Fractionation of the solubilized materials of each strain with Cetavlon (cetyltrimethylammonium bromide) yielded one mannan-protein complex . Molecular weights of these complexes were almost the same as that of the mannoprotein of the mutant strain prepared by Nakajima and Ballou, which had a molecular weight of 133,000 and were approximately three times larger than those of the mannans isolated from the same cells by hot-water extraction . Each mannan-protein complex contained up to 2% glucose residue, which was not removed by specific precipitation with anti-mannan sera or by affinity chromatography on a column of concanavalin A-Sepharose . Treatment of these complexes with alkaline NaBH4 produced peptide-free mannan containing small amounts of glucose nearly identical to those of the parent complexes . The above findings provide evidence that the glucose residues exist in a covalently linked form to the mannan moiety . Fractionation of the mannan-protein complex of the S . cerevisiae wild-type strain by DEAE-Sephadex chromatography yielded five subfractions of different phosphate content, indicating that these highly intact mannan-protein complexes were of heterogeneous material consisting of many molecular species of different phosphate content. Cell, 1983 Nov, 35(1), 167 - 74 A site-specific endonuclease essential for mating-type switching in Saccharomyces cerevisiae; Kostriken R et al.; We have detected two site-specific endonucleases in strains of Saccharomyces cerevisiae . One endonuclease, which we call YZ endo, is present only in yeast strains that are undergoing mating-type interconversion . The site at which YZ endo cleaves corresponds to the in vivo double-strand break occurring at the mating-type locus in yeast undergoing mating-type interconversion . YZ endo generates a site-specific double-strand break having 4-base 3' extensions terminating in 3' hydroxyl groups . The site of cleavage occurs in the Z1 region near the YZ junction of the mating-type locus . Mutant mating-type loci known to decrease the frequency of mating-type interconversion are correspondingly poor substrates for YZ endo in vitro . In vitro analysis of a number of such altered recognition sites has delimited the sequences required for cleavage . The molecular genetics of mating-type interconversion is discussed in the context of this endonucleolytic activity . The second endonuclease, which we refer to as Sce II, is present in all strains of S . cerevisiae we have examined . The cleavage site of Sce II has been determined and proves to be unrelated to the cleavage site of YZ endo. Biochim Biophys Acta, 1983 Oct 25, 763(3), 251 - 7 Study of the interaction of Saccharomyces cerevisiae with glucose by particle microelectrophoresis; Beezer AE et al.; Saccharomyces cerevisiae NCYC 239 in the presence of glucose at temperatures under 303 K shows a time-dependent lowering of electrophoreric mobility v . At temperatures above 303 K, this time-dependent change in v is in the direction of increased mobilities . Cells suspended in buffer indicate a surface pKa of less than 4, whereas for cells suspended in buffered glucose it is impossible to derive a surface pKa . A kinetic study of the interaction of S . cerevisiae with glucose as a function of temperature allows calculation of an activation energy of 140 kJ X mol-1 for the combined processes of (i) uptake of glucose onto the cell wall, (ii) transfer through the cell wall and membrane, and (iii) the establishment of a steady glucose flux through the wall and membrane. Mol Cell Biol, 1983 Oct, 3(10), 1846 - 56 Proline utilization in Saccharomyces cerevisiae: analysis of the cloned PUT2 gene; Brandriss MC; The PUT2 gene was isolated on a 6.5-kilobase insert of a recombinant DNA plasmid by functional complementation of a put2 (delta 1-pyrroline-5-carboxylate dehydrogenase-deficient) mutation in Saccharomyces cerevisiae . Its identity was confirmed by a gene disruption technique in which the chromosomal PUT2+ gene was replaced by plasmid DNA carrying the put2 gene into which the S . cerevisiae HIS3+ gene had been inserted . The cloned PUT2 gene was used to probe specific mRNA levels: full induction of the PUT2 gene resulted in a 15-fold increase over the uninduced level . The PUT2-specific mRNA was approximately 2 kilobases in length and was used in S1 nuclease protection experiments to locate the gene to a 3-kilobase HindIII fragment . When delta 1-pyrroline-5-carboxylate dehydrogenase activity levels were measured in strains carrying the original plasmid, as well as in subclones, similar induction ratios were found as compared with enzyme levels in haploid yeast strains . Effects due to increased copy number or position were also seen . The cloned gene on a 2 mu-containing vector was used to map the PUT2 gene to chromosome VIII. Proc Natl Acad Sci U S A, 1983 Oct, 80(20), 6192 - 6 Molecular cloning and sequence determination of the nuclear gene coding for mitochondrial elongation factor Tu of Saccharomyces cerevisiae; Nagata S et al.; A 3.1-kilobase Bgl II fragment of Saccharomyces cerevisiae carrying the nuclear gene encoding the mitochondrial polypeptide chain elongation factor (EF) Tu has been cloned on pBR327 to yield a chimeric plasmid pYYB . The identification of the gene designated as tufM was based on the cross-hybridization with the Escherichia coli tufB gene, under low stringency conditions . The complete nucleotide sequence of the yeast tufM gene was established together with its 5'- and 3'-flanking regions . The sequence contained 1,311 nucleotides coding for a protein of 437 amino acids with a calculated Mr of 47,980 . The nucleotide sequence and the deduced amino acid sequence of tufM were 60% and 66% homologous, respectively, to the corresponding sequences of E . coli tufA, when aligned to obtain the maximal homology . Plasmid YRpYB was then constructed by cloning the 2.5-kilobase EcoRI fragment of pYYB carrying tufM into a yeast cloning vector YRp-7 . A mRNA hybridizable with tufM was isolated from the total mRNA of S . cerevisiae D13-1A transformed with YRpYB and translated in the reticulocyte lysate . The mRNA could direct the synthesis of a protein with Mr 48,000, which was immunoprecipitated with an anti-E . coli EF-Tu antibody but not with an antibody against yeast cytoplasmic EF-1 alpha . The results indicate that the tufM gene is a nuclear gene coding for the yeast mitochondrial EF-Tu. J Bacteriol, 1983 Oct, 156(1), 141 - 7 In situ assay for 5-aminolevulinate dehydratase and application to the study of a catabolite repression-resistant Saccharomyces cerevisiae mutant; Borralho LM et al.; To facilitate the study of the effects of carbon catabolite repression and mutations on 5-aminolevulinate dehydratase (EC 4.2.1.24) from Saccharomyces cerevisiae, a sensitive in situ assay was developed, using cells permeabilized by five cycles of freezing and thawing . Enzymatic activity was measured by colorimetric determination of porphobilinogen with a modified Ehrlich reagent . For normal strains, porphobilinogen production was linear for 15 min, and the reaction rate was directly proportional to the permeabilized cell concentration up to 20 mg (dry weight) per ml . The reaction exhibited Michaelis-Menten-type kinetics, and an apparent Km of 2.6 mM was obtained for 5-aminolevulinic acid . This value is only slightly higher than the value of 1.8 mM obtained for the enzyme assayed in cell extracts . The in situ assay was used to assess catabolite repression-dependent changes in 5-aminolevulinate dehydratase during batch culture on glucose medium . In normal S . cerevisiae cells, the enzyme is strongly repressed as long as glucose is present in the medium . In contrast, a strain bearing the hex2-3 mutation exhibits derepressed levels of enzyme activity during growth on glucose . Synthesis of cytochromes by this strain is also resistant to catabolite repression . Similar studies employing a strain containing the glc1 mutation, which enhances porphyrin accumulation, did not reveal any significant phenotypic change in catabolite regulation of 5-aminolevulinate dehydratase. Gene, 1983 Oct, 24(2-3), 157 - 62 Cloning of a LEU gene and an ARS site of Candida maltosa; Kawamura M et al.; Gene libraries of DNA from an n-alkane-assimilating yeast strain, Candida maltosa IAM12247, were constructed, using Escherichia coli plasmid vector pBR322 . A LEU gene from C . maltosa was cloned, and found to complement leu- mutations in E . coli and Saccharomyces cerevisiae . In E . coli, the LEU gene in the cloned yeast DNA fragment was efficiently expressed when inserted into the vector in one orientation, while in the other orientation, it was expressed only weakly . In S . cerevisiae, the Candida LEU gene was efficiently expressed when inserted into a shuttle vector pRC3 in both orientations, suggesting that the isolated Candida DNA fragment contains a promoter sequence of Candida in front of the LEU gene, which is operative in S . cerevisiae but not in E . coli . In addition, our data suggest that the cloned LEU fragment also contains an ARS (autonomously replicating sequence) site of C . maltosa. Biochim Biophys Acta, 1983 Sep 28, 747(3), 209 - 14 Measurement of the amount of ornithine decarboxylase in Saccharomyces cerevisiae and Saccharomyces uvarum by using alpha-{5-14C}difluoromethylornithine; Poso H et al.; Ornithine decarboxylase (EC 4.1.1.17) activity was about 3-times higher in Saccharomyces uvarum than in Saccharomyces cerevisiae in the middle of logarithmic growth . The enzyme from both sources was inactivated by alpha-difluoromethylornithine . When the binding of {5-14 C}difluoromethylornithine to yeast ornithine decarboxylase was studied it was shown that S . uvarum extracts contained about 40 ng of active ornithine decarboxylase per mg of cellular protein, and that of S . cerevisiae 10-12 ng of active enzyme . It appeared that S . uvarum ornithine decarboxylase could be highly purified by affinity chromatography, but S . cerevisiae enzyme did not bind to the same column . The purified preparation from S . uvarum had an Mr of 73 000 and a 100-fold purification (purified by conventional methods) of ornithine decarboxylase from S . cerevisiae had an Mr of 69 000 on a gel filtration column . When the purified S . ovarum ornithine decarboxylase was labelled with difluoromethylornithine, it co-eluted with native enzyme on a gel filtration column and it ran as a single band on polyacrylamide gel electrophoresis under denaturing conditions at a position corresponding to an Mr of 72 000, indicating that the active enzyme is a monomer . The loss of ornithine decarboxylase activity after addition of cycloheximide and spermidine to culture correlated with the decrease of the binding of difluoromethylornithine to protein. Mol Cell Biol, 1983 Sep, 3(9), 1625 - 33 Heat shock-regulated production of Escherichia coli beta-galactosidase in Saccharomyces cerevisiae; Finkelstein DB et al.; The HSP90 gene of the yeast Saccharomyces cerevisiae encodes a heat shock-inducible protein with an Mr of 90,000 (hsp90) and unknown function . We fused DNA fragments of a known sequence (namely, either end of a 1.4-kilobase EcoRI fragment which contains the S . cerevisiae TRP1 gene) to an EcoRI site within the coding sequence of the HSP90 gene . When these fusions are introduced into S . cerevisiae they direct the synthesis of unique truncated hsp90 proteins . By determining the size and charge of these proteins we were able to deduce the translational reading frame at the (EcoRI) fusion site . This information allowed us to design and construct a well-defined in-frame fusion between the S . cerevisiae HSP90 gene and the Escherichia coli lacZ gene . When this fused gene is introduced into S . cerevisiae on a multicopy plasmid vector, it directs the heat shock-inducible synthesis of a fused protein, which is an enzymatically active beta-galactosidase . Thus, for the first time, it is possible to quantitate the heat shock response in a eucaryotic organism with a simple enzyme assay. Mol Cell Biol, 1983 Sep, 3(9), 1609 - 14 Isolation of the ARO1 cluster gene of Saccharomyces cerevisiae; Larimer FW et al.; The AROl cluster gene was isolated by complementation in Saccharomyces cerevisiae after transformation with a comprehensive yeast DNA library of BamHI restriction fragments inserted into the shuttle vector YEp13 . Most of the transformants exhibited the expected episomal inheritance of the ARO+ phenotype; however, one stable transformant has been shown to be an integration of the AROl fragment and the vector YEp13 at the arol locus . The insert containing AROl is a 17.2-kilobase pair (kbp) BamHI fragment which complements both nonsense and missense alleles of arol . Subcloning by Sau3AI partial digestion further locates the AROl segment to a 6.2-kbp region . An autonomously replicating sequence (ars) was found on the 17.2-kbp fragment . Yeast arol mutants transformed with the AROl episome express 5 to 12 times the normal level of the five AROl enzyme activities and possess elevated amounts of the AROl protein . The yeast AROl fragment also complemented aroA, aroB, aroD, and aroE mutants of Escherichia coli . The expression of AROl in both S . cerevisiae and E . coli was independent of the orientation of the fragment with respect to the vector. Mol Cell Biol, 1983 Sep, 3(9), 1545 - 51 Isolation of the catalase T structural gene of Saccharomyces cerevisiae by functional complementation; Spevak W et al.; The catalase T structural gene of Saccharomyces cerevisiae was cloned by functional complementation of a mutation causing specific lack of the enzyme (cttl) . Catalase T-deficient mutants were obtained by UV mutagenesis of an S . cerevisiae strain bearing the cas1 mutation, which causes insensitivity of catalase T to glucose repression . Since the second catalase protein of S . cerevisiae, catalase A, is completely repressed on 10% glucose, catalase T-deficient mutant colonies could be detected under such conditions . A cttl mutant was transformed with an S . cerevisiae gene library in plasmid YEp13 . Among the catalase T-positive clones, four contained overlapping DNA fragments according to restriction analysis . Hybridization selection of yeast mRNA binding specifically to one of the cloned DNAs, translation of this mRNA in cell-free protein synthesis systems, and demonstration of catalase T protein formation by specific immunoadsorption showed that the catalase T structural gene had been cloned . By subcloning, the gene was located within a 3.5-kilobase S . cerevisiae DNA fragment . As in wild-type cells, catalase T synthesis in cttl mutant cells transformed with plasmids containing this fragment is sensitive to glucose repression . By DNA-RNA hybridization, catalase T transcripts were shown to be present in oxygen-adapting cells but absent from heme-deficient cells. Biochem J, 1983 Sep 1, 213(3), 687 - 700 The conformation of eukaryotic cytochrome c around residues 39, 57, 59 and 74; Robinson MN et al.; 1H-n.m.r . studies of horse, tuna, Candida krusei and Saccharomyces cerevisiae cytochromes c showed that each of the proteins contains a similar cluster of residues at the bottom of the protein that assists in shielding the haem from the solvent . The relative positions of the residues forming these clusters vary continuously with temperature, and they change with the change in protein redox state . This conformational heterogeneity is discussed with reference to the conformational flexibility of cytochrome c around residues 57, 59 and 74 . Spectroscopic measurements of pKa values for Lys-55 (horse and tuna cytochromes c) and His-33 and His-39 (C . krusei and S . cerevisiae cytochromes c) are in excellent agreement with expectations based on chemical-modification studies of horse cytochrome c . {Bosshard & Zurrer (1980) J . Biol . Chem . 255, 6694-6699} and on the X-ray-crystallographic structure of tuna cytochrome c {Takano & Dickerson (1981) J . Mol . Biol . 153, 79-94, 95-115}. Mol Cell Biol, 1983 Aug, 3(8), 1440 - 50 A functional prepro-alpha-factor gene in Saccharomyces yeasts can contain three, four, or five repeats of the mature pheromone sequence; Brake AJ et al.; The chromosomal region containing a structural gene for the mating pheromone precursor prepro-alpha-factor was examined in a variety of Saccharomyces yeasts by using a cloned putative prepro-alpha-factor gene of Saccharomyces cerevisiae as the probe . Analysis by restriction endonuclease digestion and Southern blot hybridization indicated that the physical arrangement of this region is highly conserved in all the Saccharomyces species analyzed, but displays length polymorphisms of limited size (50 to 60 base pairs) . The observed polymorphisms were shown to be due solely to differences in the number of tandemly arranged spacer peptide/pheromone units within the coding sequence of these genes . Analysis of polyadenylated RNA indicated that these genes specified RNA transcripts and that these RNA molecules could be translated in vitro into prepro-alpha-factor polypeptides immunoprecipitable with anti-alpha-factor antibodies . The sizes of both the mRNAs and the proteins synthesized from them reflected exactly the differences observed in the lengths of the genes . These findings demonstrate conclusively that the putative prepro-alpha-factor DNA cloned from S . cerevisiae, as well as the sequences detected in the other Saccharomyces species, are indeed expressed and functional genes, and suggest that proper proteolytic processing of prepro-alpha-factor is unaffected by the number of pheromone repeats encoded within this precursor protein. FEBS Lett, 1983 Jul 25, 158(2), 335 - 8 O-glycosylation in Saccharomyces cerevisiae is initiated at the endoplasmic reticulum; Haselbeck A et al.; The first mannose of O-linked oligomannose chains in S . cerevisiae is transferred to Ser/Thr residues via dolichylphosphate mannose . Only this reaction (and not the subsequent reactions requiring GDP-Man) proceeds at the endoplasmic reticulum. J Biol Chem, 1983 Jul 10, 258(13), 7911 - 4 A guanine nucleotide-sensitive adenylate cyclase in the yeast Saccharomyces cerevisiae; Casperson GF et al.; Adenylate cyclase in particulate extracts of Saccharomyces cerevisiae utilized either MnATP or MgATP as substrate . A mutation in the CYR1 gene, which codes for the catalytic unit of yeast adenylate cyclase (Matsumoto, K., Uno, I., and Ishikawa, T . (1983) Cell 32, 417-423), eliminated utilization of both MgATP and MnATP, indicating that a single enzyme was responsible for both activities . GTP and guanylyl-5'-imidodiphosphate stimulated yeast adenylate cyclase, while a GDP analog, guanosine-5'-O-(2-thiodiphosphate), competitively inhibited this stimulation . Thermal inactivation studies distinguished putative guanine-nucleotide regulatory protein (N) from the catalytic unit (C) of yeast adenylate cyclase . Yeast N, which conferred guanine nucleotide regulation and the ability to utilize MgATP on yeast C, was quickly inactivated by incubation of particulate extracts at 30 degrees C . In contrast, yeast C, which apparently utilized MnATP as substrate in the absence of a functional N protein, resisted inactivation at 30 degrees C . These observations suggested that physically distinct protein components mediated the catalytic activity of yeast adenylate cyclase and its regulation by guanine nucleotides . These findings indicate a striking homology between the adenylate cyclase systems of S . cerevisiae and those of vertebrate cells. Mol Cell Biol, 1983 Jul, 3(7), 1204 - 11 Recombination of plasmids into the Saccharomyces cerevisiae chromosome is reduced by small amounts of sequence heterogeneity; Smolik-Utlaut S et al.; As a model system for studying the properties of mitotic recombination in the yeast Saccharomyces cerevisiae, we have examined recombination between a recombinant plasmid (introduced into the S . cerevisiae cell by transformation) and homologous chromosomal loci . The recombinant plasmids used in these experiments contained S . cerevisiae rRNA genes . We found that the frequency of integrative recombination is sensitive to small amounts of sequence heterogeneity . In addition, the frequency and specificity of these recombination events are affected by the lengths of the interacting homologous DNA sequences. Biochem Pharmacol, 1983 Jun 15, 32(12), 1825 - 30 Effects of econazole nitrate on yeast cells and mitochondria; Wilm K et al.; The inhibitory effect of econazole nitrate on the growth of yeast Saccharomyces cerevisiae is proportional to the concentration of the product . It depends on the phase of culture and on the number of cells present at the moment of econazole addition into the medium . The most important inhibition is obtained in the exponential phase of growth with a low concentration of cells . It is enhanced with cells which were previously in contact with the product . There is no adaptation of the yeast toward increased concentrations of econazole . The product penetrates the cells and attaches first to particular fractions, later to soluble fractions . The highest concentration of econazole nitrate in cells lies in the mitochondria . No product of econazole metabolism by S . cerevisiae was uncovered . Econazole nitrate does not slow down the in vivo activities of mitochondrial enzymes (cytochrome c oxidase, succinate dehydrogenase and phenylalanyl-tRNA synthetase), but inhibits the biosynthesis of mitochondrial membrane enzymes without affecting that of the synthetase, a matrix enzyme. Cell, 1983 Jun, 33(2), 563 - 73 Organization of DNA sequences and replication origins at yeast telomeres; Chan CS et al.; We have shown that the DNA sequences adjacent t