S. cerevisiae

Biochemistry, 1987 Aug 25, 26(17), 5377 - 82
Yeast DNA primase is encoded by a 59-kilodalton polypeptide: purification and immunochemical characterization; Biswas EE et al.; The DNA primase from the yeast Saccharomyces cerevisiae has been purified 9200-fold to homogeneity . The yeast DNA primase is a monomeric protein of molecular weight 59,000, and under conditions described in this report, it is stable at 4 or -80 degrees C . The primase does not bind to DEAE-cellulose, is not inhibited by a high concentration of alpha-amanitin (4 mg/mL), and is capable of synthesizing small (up to 15 nucleotides in length) ribo or ribo-deoxy mixed initiator RNA primers . The primer synthesis is stimulated by ATP; however, other ribonucleotides could be replaced by deoxynucleotides without any measurable effect on the overall DNA synthesis . Thus, the purified primase is distinct from the RNA polymerases of S . cerevisiae . Immunoblot analysis of the polypeptides in a crude cell extract using a mouse polyclonal antibody prepared against the highly purified primase indicates that the 59-kilodalton polypeptide is the native form and not a degraded form of a larger polypeptide; however, primase is degraded rapidly to smaller polypeptides by yeast proteases especially in the absence of protease inhibitors.

Nucleic Acids Res, 1987 Aug 25, 15(16), 6387 - 403
Analysis of transcripts of the major cluster of tRNA genes in the mitochondrial genome of S . cerevisiae; Francisci S et al.; The transcripts of a 6Kbp region of the mitochondrial genome of S . cerevisiae, localized in the 21S rRNA-OXI1 span and including 12 tRNA genes (from tRNA(thr) to tRNA(ala)) and several G+C clusters, have been studied by analysis of in vitro capped primary transcripts and by fine mapping of the 5' ends of transcripts . The study was performed in the w.t . strain D273-10B and in several rho- mutants retaining different, partially overlapping portions of the studied region; the mutants accumulate incompletely-processed precursors of tRNAs due to the absence of the tRNA synthesis locus . Results show the presence in the region of four sites at which initiation occurs at a consensus nonanucleotide ATTATAAGTA (or a minor variant of the same); however different initiation sites are used in different strains, and several differences as compared to initiation in vitro can also be observed . Termini arising by processing are often localized at AATATAA or AATATATTTT sequences localized immediately adjacent to a G+C cluster or a tRNA sequence.

Experientia, 1987 Aug 15, 43(8), 886 - 8
Protective effect of vitamins against trichothecene toxicity towards Saccharomyces cerevisiae; Yagen B et al.; Several trichothecene mycotoxins were shown to inhibit the growth of Saccharomyces cerevisiae . This effect was most pronounced with the macrocyclic trichothecenes, especially verrucarin A . Much less growth inhibition was observed with T-2 toxin . Verrucarol, diacetoxyscirpenol, acetyl T-2 toxin, HT-2 toxin, T-2 tetraol and neosolaniol were inactive at a concentration of 75 micrograms of toxin per disc . Incubation of S . cerevisiae with verrucarin A together with vitamins resulted in a decrease in toxicity . Pyridoxine-HCl, Ca-pantothenate, thiamine-HCl and alpha-tocopheryl acetate were amongst the most potent of the vitamins tested which reversed growth inhibition, overcoming the inhibitory potential of the toxins.

Biochem Biophys Res Commun, 1987 Aug 14, 146(3), 1093 - 101
Reversion of 7-methylguanosine 5'-phosphate inhibition of mRNA translation by polysomal and soluble factors isolated from Saccharomyces cerevisiae; Parets Suler A et al.; Protein fractions that overcome m7GMP inhibition of mRNA translation have been purified from the yeast S . cerevisiae . An active fraction isolated from polysomes contains two polypeptides of 220- and 190-kDa . The active fraction isolated from postribosomal supernatant contains a major polypeptide of 28-kDa and other species of 32-, 24-, 22- and 21-kDa, and sediments in sucrose gradients as a high molecular weight complex of about 200,000 . This fraction restored yeast mRNA translation in reticulocyte lysates under conditions of yeast and globin mRNA competition; however, this effect was not observed with the 220- and 190-kDa polypeptides from polysomes . Nevertheless, translation of yeast mRNA was stimulated by a partially purified fraction containing a 28-kDa polypeptide from polysomes.

Cell, 1987 Aug 14, 50(4), 593 - 602
S . cerevisiae U1 RNA is large and has limited primary sequence homology to metazoan U1 snRNA; Kretzner L et al.; We have cloned and sequenced the yeast SNR19 gene and show here that snR19 is the yeast homolog of metazoan U1 snRNA . sn R19 is 569 nucleotides long, strikingly larger than its metazoan counterpart . The two molecules resemble each other closely in the predicted secondary structure of their first 50 nucleotides . Primary sequence homology is restricted to some of their single-stranded regions, including 11 consecutive nucleotides at the 5' end of the two molecules, the region that interacts with pre-mRNA 5' splice junctions . snR19 is spliceosome-associated and required for in vitro pre-mRNA splicing . We also note that 8 sequences in snR19 have extensive complementarity to snR20, the large yeast U2 RNA, suggesting that yeast U1 may interact with yeast U2 by base-pairing.

Cell, 1987 Aug 14, 50(4), 585 - 92
An essential snRNA from S . cerevisiae has properties predicted for U4, including interaction with a U6-like snRNA; Siliciano PG et al.; Three yeast snRNAs (snR20, snR7, and snR14) have been implicated in pre-mRNA splicing . snR20 and snR7 contain domains of homology to U2 and U5, respectively, and each is required for viability . These RNAs are found associated with the spliceosome, as is snR14 . We show here that snR14 is also an essential gene product . Sequence analysis reveals that, like snR7 and snR20, snR14 contains a consensus binding site for the Sm antigen, a feature common to all mammalian snRNAs involved in splicing . Moreover, snR14 exhibits several blocks of sequence and structural homology to U4, which in metazoans is found in association with U6 . Native gel electrophoresis demonstrates that snR14 is in fact base-paired with another yeast snRNA, designated snR6, which has primary sequence homology to U6 . We conclude that snR14 is the yeast analog of U4.

J Biol Chem, 1987 Aug 5, 262(22), 10426 - 9
Identification of guanine nucleotides bound to ras-encoded proteins in growing yeast cells; Gibbs JB et al.; We have analyzed the guanine nucleotides bound to mammalian ras and yeast RAS proteins overexpressed in {32P}orthophosphate-labeled cultures of exponentially growing Saccharomyces cerevisiae cells . Whereas S . cerevisiae RAS1 and RAS2 proteins were immunoprecipitated bound entirely to GDP, mammalian Harvey ras was isolated with GTP and GDP bound in near-equimolar proportions . In a strain overexpressing a RAS2 variant where the RAS unique C-terminal domain was deleted, both GTP and GDP were detected in a ratio of 3:97 . Increased amounts of GTP (16-75% of total guanine nucleotide) were observed bound to all ras proteins containing mutations that inhibit GTP hydrolytic activity . Increasing proportions of GTP bound to the various ras proteins correlated with increasing biological potency to bypass cdc25 lethality in yeast.

Virology, 1987 Aug, 159(2), 450 - 3
Expression and glycosylation of the respiratory syncytial virus G protein in Saccharomyces cerevisiae; Ding MX et al.; A cDNA encoding the entire amino acid sequence of the G glycoprotein of respiratory syncytial virus (RSV) was inserted into a yeast-Escherichia coli shuttle vector such that expression of the virus gene was regulated by the yeast GAL1 promoter . Transformation of Saccharomyces cerevisiae with the vector led to the formation of the G protein when cells were grown in the presence of galactose . Under these conditions the RSV G appeared as a 60- to 65-kDa glycosylated protein . Expression of the G cDNA in secretory mutants of S . cerevisiae yielded a protein of 35 kDa in a mutant unable to glycosylate secreted proteins and a 65-kDa polypeptide in a mutant unable to transport proteins beyond the endoplasmic reticulum . The RSV protein formed in the latter mutant was converted to a 60-kDa protein by endoglycosidase H . Our results show that yeast can recognize the internal signal sequence of RSV G protein and add glycosyl groups to the polypeptide in the endoplasmic reticulum . Evidence is presented for both N- and O-linked glycosylation of the virus glycoprotein.

Mol Cell Biol, 1987 Aug, 7(8), 2914 - 24
Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression; Hoekema A et al.; The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets . The degree of this biased codon usage in each gene is positively correlated to its expression level . Highly expressed genes use these 25 major codons almost exclusively . As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene . The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome . Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level . The PGK protein levels dropped 10-fold . The steady-state mRNA levels also declined, but to a lesser extent (threefold) . Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons . By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically . We conclude that efficient mRNA translation is required for maintaining mRNA stability in S . cerevisiae . These findings have important implications for the study of the expression of heterologous genes in yeast cells.

Cell, 1987 Jul 17, 50(2), 277 - 87
Three different genes in S . cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase; Toda T et al.; We have isolated three genes (TPK1, TPK2, and TPK3) from the yeast S . cerevisiae that encode the catalytic subunits of the cAMP-dependent protein kinase . Gene disruption experiments demonstrated that no two of the three genes are essential by themselves but at least one TPK gene is required for a cell to grow normally . Comparison of the predicted amino acid sequences of the TPK genes indicates conserved and variable domains . The carboxy-terminal 320 amino acid residues have more than 75% homology to each other and more than 50% homology to the bovine catalytic subunit . The amino-terminal regions show no homology to each other and are heterogeneous in length . The TPK1 gene carried on a multicopy plasmid can suppress both a temperature-sensitive ras2 gene and adenylate cyclase gene.

J Biol Chem, 1987 Jul 5, 262(19), 9160 - 5
Construction of a synthetic Holliday junction analog and characterization of its interaction with a Saccharomyces cerevisiae endonuclease that cleaves Holliday junctions; Evans DH et al.; We describe the construction and characterization of an oligonucleotide Holliday junction analog and characterize its interaction with a Saccharomyces cerevisiae endonuclease that cleaves Holliday junctions . A Holliday junction analog containing four duplex arms and 54 base pairs was constructed by annealing four unique synthetic oligonucleotides . Mixing curve analysis showed that the complex contained a 1:1:1:1 mol ratio of the four unique sequence strands . In addition, a linear duplex with a sequence identical to two of the junction arms was also constructed for use as a control fragment . High resolution gel exclusion chromatography was used to purify and characterize the synthetic junction . The synthetic Holliday junction was found to be a specific inhibitor of a S . cerevisiae enzyme that catalyzes the cleavage of Holliday junctions . Under standard cleavage conditions, 50% inhibition was observed at a synthetic Holliday junction to substrate ratio of 7/1, whereas no inhibition by linear duplex was observed at molar ratios in excess of 150/1 . Kinetic analysis showed that Holliday junction was a competitive inhibitor of the reaction and had an apparent Ki = 2.5 nM, although the mode of inhibition was complex . The synthetic Holliday junction was not a substrate for the enzyme, but was found to form a specific complex with the enzyme as evidenced by polyacrylamide gel electrophoresis DNA binding assays.

Cell, 1987 Jul 3, 50(1), 17 - 29
Multiple exon-binding sites in class II self-splicing introns; Jacquier A et al.; Partial deletion of the exon 5' to S . cerevisiae intron a5, a self-splicing mitochondrial class II intron, reveals the existence of several sites of intron-exon interaction . We have identified two of the corresponding exon-binding sites in intron a5 by comparative sequence analysis and RNAase H digestion of the intron complexed to a DNA version of its 5' exon . Introduction of mutations in either the intronic sites or the complementary exonic sequences affects splicing in vitro, whereas double mutants in which intron-exon pairings have been restored show normal activity . Some of the mutants accumulate a product that was shown to be the intron-3' exon lariat, a postulated splicing intermediate . The possible role of one of the intronic sites in aligning exons for the ligation step is discussed.

Rev Argent Microbiol, 1987 Jul-Sep, 19(3), 109 - 19
{Saccharomyces cerevisiae: porphobilinogenase activity in a wild-type strain and its heme-deficient mutant}; Araujo LS et al.; Properties of Porphobilinogenase (PBGase), the enzyme complex converting porphobilinogen (PBG) into uroporphyrinogens, were comparatively studied in a wild strain D273-10B and its mutant B231 of Saccharomyces cerevisiae, Figure 1 shows the growth curves for both strains . The basic pattern of growth was observed but, although S . cerevisiae is a facultative aerobe and was grown on dextrose, a diauxic growth curve was not observed . The beginning of the exponential phase was slightly delayed for the mutant, so, its generation time (G = 3.20 h) was greater than that for the wild strain (G = 1.26 h) . Optimum conditions for extracting the enzyme from both strains were found to be sonication at 10 mu for 3 min (Table 1) . Table 2 shows the effect of centrifugation at 24,000 xg for 30 min on activity . For both strains the amount of porphyrins formed was the same either in the absence or presence of air . It was found (Figure 2) that urogen formation was linear with protein over a wide range of concentrations and with incubation time up to 2h in agreement with previous results for the enzyme of different sources . Figure 3 shows the effect of pH on PBGase activity . An optimum pH of 7.4 was found for both strains employing sodium phosphate buffer pH 8.0 . The shape of the pH curve as well as optimum pH were the same in both Tris-HCl and phosphate buffer, however PBGase was 15% less active in the former . When plots of velocity against PBG concentration were analyzed for PBGase, it was found that measuring the rate of the reaction on the basis of total urogen formation, saturation curves for wild and mutant strains harvested at the exponential phase, followed classical Michaelis-Menten kinetics . Saturation was reached at PBG concentration of about 70-90 microM . Therefore, double reciprocal plots (Figure 4) were linear and from these plots apparent Km's values of 20 and 14 microM were obtained for the wild and mutant strain respectively . It is known that in some organisms, the activity of the enzyme of heme synthesis is significantly influenced by the days of growing; therefore the effect of time growing on PBGase activity was studied (Figure 5) . A well defined maximum of enzyme activity was observed for the mutant strain after 20h of growing; while activity of wild strain did not significantly vary during growth.

Mol Cell Biol, 1987 Jul, 7(7), 2397 - 405
Genetic manipulation of centromere function; Hill A et al.; A conditional centromere was constructed in Saccharomyces cerevisiae by placing the centromere of chromosome III immediately downstream from the inducible GAL1 promoter from S . cerevisiae . By utilizing growth conditions that favor either transcriptional induction (galactose-carbon source) or repression (glucose-carbon source) from the GAL1 promoter, centromere function can be switched off or on, respectively . With the conditional centromere we were able to radically alter the mitotic transmission pattern of both monocentric and dicentric plasmids . Moreover, it was possible to selectively induce the loss of a single chromosome from a mitotically dividing population of cells . We observed that the induction of chromosome III aneuploidy resulted in a dramatic change in cell morphology . The construction of a conditional centromere represents a novel way to create conditional mutations of cis-acting DNA elements and will be useful for further analysis of this important stabilizing element.

Mol Cell Biol, 1987 Jul, 7(7), 2329 - 34
Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae; Simon JR et al.; Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication . These constructs transform S . cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene . Single-stranded DNA was found to transform S . cerevisiae cells at efficiencies greater than that of double-stranded DNA . No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences . Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events . Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA.

J Biol Chem, 1987 Jun 15, 262(17), 8159 - 64
Carbon isotope effects on the pyruvate dehydrogenase reaction and their importance for relative carbon-13 depletion in lipids; Melzer E et al.; A method has been developed for the positional 13C isotope analysis of pyruvate and acetate by stepwise quantitative degradation . On its base, the kinetic isotope effects on the pyruvate dehydrogenase reaction (enzymes from Escherichia coli and Saccharomyces cerevisiae) for both of the carbon atoms involved in the bond scission (double isotope effect determination) and on C-3 of pyruvate have been determined . The experimental k12/k13 values with the enzyme from E . coli on C-1 and C-2 of pyruvate are 1.0093 +/- 0.0007 and 1.0213 +/- 0.0017, respectively, and, with the enzyme from S . cerevisiae, the values are 1.0238 +/- 0.0013 and 1.0254 +/- 0.0016, respectively . A secondary isotope effect of 1.0031 +/- 0.0009 on C-3 (CH3-group) was found with both enzymes . The size of the isotope on C-1 indicates that decarboxylation is more rate-determining with the yeast enzyme than with the enzyme from E . coli, although it is not the entirely rate-limiting step in the overall reaction sequence . Assuming appropriate values for the intrinsic isotope effect on the decarboxylation step (k3) and the equilibrium isotope effect on the reversible substrate binding (k1, k2), one can calculate values for the partitioning factor R (k3/k2: E . coli enzyme 4.67, S . cerevisiae enzyme 1.14) and the intrinsic isotope effects related to the carbonyl-C (k1/k'1 = 1.019; k3/k'3 = 1.033) . The isotope fractionation at C-2 of pyruvate gives strong evidence that the well known relative carbon-13 depletion in lipids from biological material is mainly caused by the isotope effect on the pyruvate dehydrogenase reaction . In addition, our results indicate an alternating 13C abundance in fatty acids, that has already been verified in some cases.

Yeast, 1987 Jun, 3(2), 77 - 84
Proliferation of microbodies in Saccharomyces cerevisiae; Veenhuis M et al.; The development of microbodies in the yeast Saccharomyces cerevisiae was studied in response to different conditions of growth . Various strains of S . cerevisiae were investigated, using cells from the exponential growth phase on glucose as an inoculum in all transfer experiments . Electron microscopy, including serial sectioning, revealed that these cells generally contained one to four small microbodies which were localized in the vicinity of the cell wall and characterized by the presence of catalase . Transfer of these glucose-grown cells into media supplemented with various compounds known to induce microbody proliferation in other yeasts--i.e . uric acid, alkylated amines, amino acids, C2-compounds such as ethanol or acetate, in the presence or absence of compounds that induce oxygen radical formation--did not result in a significant change in the number of microbody profiles observed . Marked microbody proliferation was, however, observed after a shift of cells into media containing oleic acid and was associated with the induction of activities of beta-oxidation enzymes . In addition, catalase and isocitrate lyase were present in enhanced levels . Kinetic experiments suggested that these microbodies developed from those originally present in the inoculum cells . In thin sections up to 14 microbody profiles were occasionally observed, often present in small clusters . Their ultimate volume fraction amounted to 8-10% of the cytoplasmic volume.

Yeast, 1987 Jun, 3(2), 63 - 70
Artifactual immunofluorescent labelling in yeast, demonstrated by affinity purification of antibody; Lillie SH et al.; In the course of making antibodies against various yeast (S . cerevisiae) proteins, we have noted that it is common to observe reactivity of rabbit sera with a number of extraneous bands on Western transfers of yeast proteins . The pattern of reactive bands can change within a period of weeks, even when the rabbit has not been injected with antigen . A simple method of affinity purification, using antigen bound to nitrocellulose, is employed to remove the reactivity with these extraneous bands from immune sera . The importance of affinity purification is demonstrated by our attempts to immunolocalize a 55 kd yeast protein (p55) . Immune serum stains yeast cells to give a striking pattern of spots and blotches not seen with preimmune serum . However, affinity purification of anti-p55 antibody shows that this pattern is not due to staining by anti-p55 antibody; rather the pattern is due to staining left in the serum depleted of anti-p55 antibody.

Yeast, 1987 Jun, 3(2), 131 - 7
Specificity of DNA uptake during whole cell transformation of S . cerevisiae; Bruschi CV et al.; We have studied the mechanism of DNA transformation of whole yeast cells in Saccharomyces cerevisiae with particular emphasis on the role of the cell wall complex in DNA uptake . Two new aspects of the process have been investigated in order to evaluate its specificity . Such aspects are: (i) effect of monovalent vs . divalent cations during incubation with the transforming DNA and (ii) timing of DNA adsorption and uptake . We found that the specificity for cation requirement is a strain-dependent characteristic influenced by the presence of transforming DNA in the cell suspension . This finding is supported by reports from several laboratories that some yeast strains show mutually exclusive transformability with monovalent vs . divalent cations . While irreversible adsorption of plasmid DNA molecules is induced by both heat shock and polyethylene-glycol (PEG), DNA uptake seems to occur only after the removal of PEG . In the course of this study we have developed a new, alternative method of whole cell DNA transformation with CaCl2 able to transform strains that do not respond to other methods.

J Gen Microbiol, 1987 Jun, 133 ( Pt 6), 1583 - 8
Novel genetic components controlling invertase production in Saccharomyces cerevisiae; del Castillo Agudo L et al.; Low levels of invertase (EC 3.2.1.26) activity were observed in most diploid strains of S . cerevisiae used in this work . There was no effect of mating type on invertase levels, and cell surface was not a limiting factor, because an increase in ploidy did not cause further decrease in specific invertase activity . Finally, some diploids showed the activity expected from the additive effects of different SUC genes, and haploid strains possessing two SUC genes expressed very variable invertase activities depending on the strain . This suggested the existence of one or more additional genes which control the levels of invertase . Genetic analysis of SUC5 strains provided evidence of the existence of a new gene, RPS5, which drastically reduced the specific invertase activity in strains possessing active SUC alleles . The recessive allele of this gene (rps5) allows expression of higher levels of invertase . We suggest that genes similar RPS5 are responsible for the low levels of invertase activity observed in diploid strains of S . cerevisiae.

Mol Gen Genet, 1987 Jun, 208(1-2), 159 - 67
Structure of the yeast HIS5 gene responsive to general control of amino acid biosynthesis; Nishiwaki K et al.; The nucleotide sequence of a 2.1 kb DNA fragment bearing the HIS5 gene of Saccharomyces cerevisiae, which encodes histidinol-phosphate aminotransferase (EC 2.6.1.9), has been determined . An open reading frame of 1,152 bp was found . S1 nuclease mapping indicated that the major transcription starts at position -37 from the ATG codon and the minor (approximately 20%) at -34 in both repressive and derepressive conditions . Northern analysis indicated that transcription of the HIS5 gene is under the general control of amino acid biosynthesis . The 5' noncoding region of the gene, thus far examined up to position -616, contains three copies of sequences homologous to the short repeats of the consensus sequence, 5'-AATGTGACTC-3', suggested for general amino acid control in the HIS1, HIS3, HIS4, and TRP5 at positions -336, -275 and -205 . The consensus sequence closest to the open reading frame was shown to be necessary but not sufficient for general amino acid control, by examination of beta-galactosidase appearance in S . cerevisiae cells carrying various mutant HIS5 promoter regions fused to the lac'Z gene and inserted at the leu2 locus of chromosome III.

Proc Natl Acad Sci U S A, 1987 Jun, 84(12), 4063 - 7
Import and processing of human ornithine transcarbamoylase precursor by mitochondria from Saccharomyces cerevisiae; Cheng MY et al.; Expression of the subunit precursor of the human mitochondrial matrix enzyme ornithine transcarbamoylase (OTCase; EC 2.1.3.3) was programmed in Saccharomyces cerevisiae from a 2-micron plasmid by using an inducible galactose operon promoter . In the presence of the inducing sugar (galactose), two polypeptides were specifically precipitable with anti-OTCase antiserum: the human OTCase precursor (40 kDa); and the mature OTCase subunit (36 kDa) . When yeast cells containing these species were lysed and fractionated, the OTCase precursor was found to be associated with mitochondrial membranes, while the mature subunit was found partly with mitochondrial membranes and partly in the soluble mitochondrial matrix-containing fraction . When OTCase enzymatic activity was assayed in fractions similarly derived from an S . cerevisiae strain devoid of yeast OTCase activity (an arg3 mutant) but expressing human OTCase, activity was detected specifically in the mitochondrial matrix fraction . A mutant human OTCase precursor containing an artificial mutation in the NH2-terminal leader peptide (arginine-23 to glycine) was similarly examined . As was previously observed with mammalian mitochondria, this precursor failed both to reach the matrix compartment and to be proteolytically processed; it also failed to exhibit OTCase enzymatic activity . Presence of OTCase enzymatic activity in an arg3 strain expressing wild-type precursor was utilized to obtain selective growth in a medium devoid of arginine but supplemented with the OTCase substrate ornithine . We conclude that, during evolution, the pathway of mitochondrial import utilized by the human OTCase precursor is conserved between yeast and humans, and that, by using selective growth conditions, it may be possible to examine genetically this pathway in S . cerevisiae.

Mol Cell Biol, 1987 Jun, 7(6), 2087 - 96
Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae; Sauer B; The procaryotic cre-lox site-specific recombination system of coliphage P1 was shown to function in an efficient manner in a eucaryote, the yeast Saccharomyces cerevisiae . The cre gene, which codes for a site-specific recombinase, was placed under control of the yeast GALI promoter . lox sites flanking the LEU2 gene were integrated into two different chromosomes in both orientations . Excisive recombination at the lox sites (as measured by loss of the LEU2 gene) was promoted efficiently and accurately by the Cre protein and was dependent upon induction by galactose . These results demonstrate that a procaryotic recombinase can enter a eucaryotic nucleus and, moreover, that the ability of the Cre recombinase to perform precise recombination events on the chromosomes of S . cerevisiae is unimpaired by chromatin structure.

Arch Biochem Biophys, 1987 May 1, 254(2), 568 - 78
In situ behavior of the pyrimidine pathway enzymes in Saccharomyces cerevisiae . 2 . Reaction mechanism of aspartate transcarbamylase dissociated from carbamylphosphate synthetase by genetic alteration; Belkaid M et al.; The reaction mechanism of Saccharomyces cerevisiae aspartate transcarbamylase was studied in permeabilized cells of a mutant in which this enzyme is not associated to carbamylphosphate synthetase . The results obtained indicate an ordered mechanism in which carbamylphosphate binds first, followed by aspartate, with dissociation of the products in the order phosphate then carbamylaspartate . Interestingly, this clear-cut mechanism differs from the more complex behavior shown by aspartate transcarbamylase when this enzyme is associated to carbamylphosphate synthetase in wild-type S . cerevisiae (B . Penverne and G . Herve, Arch . Biochem . Biophys . (1983) 225, 562-575) . This difference indicates that the association of the two enzymes within the multienzymatic complex alters the apparent kinetic properties of aspartate transcarbamylase . Such an enzyme-enzyme interaction might be related to the channeling of carbamylphosphate from one catalytic site to the other one.

J Bacteriol, 1987 May, 169(5), 2132 - 6
Regulation of dipeptide transport in Saccharomyces cerevisiae by micromolar amino acid concentrations; Island MD et al.; Prototrophic Saccharomyces cerevisiae X2180, when grown on unsupplemented minimal medium, displayed little sensitivity to ethionine- and m-fluorophenylalanine-containing toxic dipeptides . We examined the influence of the 20 naturally occurring amino acids on sensitivity to toxic dipeptides . A number of these amino acids, at concentrations as low as 1 microM (leucine and tryptophan), produced large increases in sensitivity to leucyl-ethionine, alanyl-ethionine, and leucyl-m-fluorophenylalanine . Sensitivity to ethionine and m-fluorophenylalanine remained high under either set of conditions . The addition of 0.15 mM tryptophan to a growing culture resulted in the induction of dipeptide transport, as indicated by a 25-fold increase in the initial rate of L-leucyl-L-{3H}leucine accumulation . This increase, which was prevented by the addition of cycloheximide, began within 30 min and peaked approximately 240 min after a shift to medium containing tryptophan . Comparable increases in peptidase activity were not apparent in crude cell extracts from tryptophan-induced cultures . We concluded that S . cerevisiae possesses a specific mechanism for the induction of dipeptide transport that can respond to very low concentrations of amino acids.

Exp Cell Res, 1987 May, 170(1), 64 - 79
The cytoplasmic pH, ATP content and total protein synthesis rate during heat-shock protein inducing treatments in yeast; Weitzel G et al.; In S . cerevisiae the induction of heat-shock protein (HSP) synthesis is accompanied by a decrease in the cytoplasmic and vacuolar pH as determined by means of {31P}NMR spectroscopy . The relationship of HSP synthesis and acidification of the cytoplasmic pH is dose-dependent under a variety of treatments (temperature increases (23-32 degrees C), addition of 2,4-dinitrophenol (greater than 1 mM), sodium arsenite (greater than 3.75 X 10(-5) M) or sodium cyanide (greater than 10 mM} . Changes in the intracellular pH occur within 5 min after treatment, attain a maximum within 30 min and are subsequently stable . HSPs 98, 85 and 70 show maximum synthesis rates 1-2 h after a 40 degrees C heat shock . The synthesis rates then decline . HSPs 56, 44 and 33 reveal a smaller and slower increase and almost no decrease in the synthesis rate within 4 h at 40 degrees C . The similar dose dependencies of HSP synthesis and cytoplasmic pH . as well as the immediate response of the pH, can also be demonstrated in the mitochondrial mutant of S . cerevisiae (Q0) . This result indicates that the heat-shock response is mainly independent of intact oxidative phosphorylation . No correlation was observed between HSP synthesis rate and total intracellular ATP content.

Biochem Int, 1987 May, 14(5), 963 - 70
Status of calcium influx in cell cycle of S . cerevisiae; Anand S et al.; The transport of calcium was assayed in exponentially growing and G1 arrested temperature sensitive cdc mutants of Saccharomyces cerevisiae . There was no statistically significant difference in the rate of Ca2+ influx in cdc 28, cdc 37 and cdc 4 arrested cells, as well as in wild type cells arrested in G1 phase in comparison to exponentially growing cells . There was however a significant increase in Ca2+ uptake in cdc 7 and cdc 24 arrested cells . The former is known to arrest before bud emergence and initiation of DNA synthesis; arrest of the latter affects bud formation while DNA synthesis continues . The results suggest that Ca2+ may have a role in bud formation and growth.

Arch Microbiol, 1987 May, 147(4), 358 - 63
Glycerol production in relation to the ATP pool and heat production rate of the yeasts Debaryomyces hansenii and Saccharomyces cerevisiae during salt stress; Larsson C et al.; Changes in glycerol production and two parameters related to energy metabolism i.e . the heat production rate and the ATP pool, were assayed during growth of Saccharomyces cerevisiae and Debaryomyces hansenii in 4 mM and 1.35 M NaCl media . For both of the yeasts, the specific ATP pool changed during the growth cycle and reached maximum values around 10 nmol per mg dry weight in both types of media . The levels of glycerol were markedly enhanced by high salinity . In the presence of 1.35 M NaCl, D . hansenii retained most of its glycerol produced intracellularly, while S . cerevisiae extruded most of the glycerol to the environment . The intracellular glycerol level of S . cerevisiae equalled or exceeded that of D . hansenii, however, with values never lower than 3 mumol per mg dry weight at all phases of growth . When D . hansenii was grown at this high salinity the intracellular level of glycerol was found to correlate with the specific heat production rate . No such correlation was found for S . cerevisiae . We concluded that during salt stress, D . hansenii possesses the capacity to regulate the metabolism of glycerol to optimize growth, while S . cerevisiae may not be able to regulate when exposed to different demands on the glycerol metabolism.

Mol Gen Genet, 1987 May, 207(2-3), 342 - 8
Phleomycin resistance encoded by the ble gene from transposon Tn 5 as a dominant selectable marker in Saccharomyces cerevisiae; Gatignol A et al.; Phleomycin, a water-soluble antibiotic of the bleomycin family is as effective against Saccharomyces cerevisiae cells as against Escherichia coli cells . The ble gene of transposon Tn5, which confers resistance to phleomycin, was inserted in place of the iso-1-cytochrome C (CYC1) gene on an autonomously replicative multicopy E . coli-yeast shuttle plasmid . Higher resistance levels are obtained in S . cerevisiae when the region immediately upstream from the initiation codon conforms to the nucleotide sequence stringencies observed in almost every yeast gene . The expected regulation pattern of the whole CYC1 promoter confers different phleomycin resistance levels to the cell under varying physiological conditions . Partial deletions in the CYC1 promoter lead to changes in the resistance level of cells which are mostly accounted for by the removal of known positive and negative regulatory elements . Some of the vector constructions allow direct selection of phleomycin-resistant transformants on rich media.

Biochem Biophys Res Commun, 1987 Apr 29, 144(2), 613 - 9
A novel yeast secretion vector utilizing secretion signal of killer toxin encoded on the yeast linear DNA plasmid pGKL1; Tokunaga M et al.; The NH2-terminal signal region comprising of approximately 70% length of the prepro-sequence of the pGKL killer precursor protein was found to direct an efficient secretion of the mouse alpha-amylase into the culture medium of Saccharomyces cerevisiae . The alpha-amylase molecule secreted into the culture medium was identified by both immuno-blotting and assay of the enzyme activity . The amount of alpha-amylase secreted via the killer toxin signal was comparable to that directed by the leader sequence of mating factor alpha . The secretion of alpha-amylase using the killer toxin signal was blocked at 37C but not at 25C in sec18-1 host, indicating that alpha-amylase is exported through the normal secretion pathway of S . cerevisiae.

Biochim Biophys Acta, 1987 Apr 27, 906(1), 81 - 99
Protein glycosylation in yeast; Tanner W et al.; S . cerevisiae contains many mannose-rich glycoproteins that possess N- and O-linked carbohydrate chains, and both types may even occur on one and the same protein . The steps in the synthesis of asparagine-linked chains begin with assembly and transfer of the lipid-linked precursor to protein in a way common to all eucaryotes . Subsequent modifications lead to mannosyl extensions of various lengths, but complex type carbohydrate structures are not formed . Oligosaccharides O-linked to serine/threonine consist exclusively of mannose in S . cerevisiae . The mannose residue attached directly to the protein is transferred from Dol-P-Man in a unique way, which has been observed so far for fungal cells only . The cellular localization of the glycosylation reactions is summarized and the problem of transmembrane translocation of the sugar precursors at the ER and the Golgi is discussed . Some aspects of secretory (sec) and asparagine linked glycosylation (alg) mutants have been covered, and the various hypotheses related to the possible functions of this costly protein modification process are discussed . The article may also be helpful for those, who want to exploit the yeast's protein synthesizing machinery by genetically manipulating the cells.

FEBS Lett, 1987 Apr 6, 214(1), 158 - 62
Fatty acid synthesis in mitochondria from Saccharomyces cerevisiae; Bessoule JJ et al.; The ability of purified mitochondria isolated from S . cerevisiae to synthesize fatty acids and especially very long chain fatty acids (VLCFA) has been investigated . The VLCFA synthesis requires malonyl-CoA as the C2 unit donor and NADPH as the reducing agent . Moreover the yeast mitochondrial elongase is able to accept either exogenous long chain fatty acyl-CoAs as substrates or elongate endogenous substrates . In the latter case, ATP is required for full activity . Besides this important VLCFA formation, the mitochondria from S . cerevisiae were also able to synthesize C16 and C18.

Mol Cell Biol, 1987 Apr, 7(4), 1338 - 45
Phosphorylation of the Saccharomyces cerevisiae equivalent of ribosomal protein S6 has no detectable effect on growth; Johnson SP et al.; The phosphorylation of mammalian ribosomal protein S6 is affected by a variety of agents, including growth factors and tumor promoters, as well as by expressed oncogenes . Its potential role in the regulation of protein synthesis has been the object of much study . We have developed strains of Saccharomyces cerevisiae in which the phosphorylatable serines of the equivalent ribosomal protein (S10) were converted to alanines by site-directed mutagenesis . The S10 of such cells is not phosphorylated . Comparison of these cells with the parental cells, whose genomes differ by only six nucleotides, revealed no differences in the lag phase or logarithmic phase of a growth cycle, in growth on different carbon sources, in sporulation, or in sensitivity to heat shock . We conclude that in S . cerevisiae the phosphorylation of ribosomal protein S10 may play no role in regulating the synthesis of proteins . This conclusion leads one to ask whether certain protein phosphorylations are simply the adventitious, if easily observable, result of the imperfect specificity of one or another protein kinase.

Mol Cell Biol, 1987 Apr, 7(4), 1476 - 85
Signal processing, glycosylation, and secretion of mutant hemagglutinins of a human influenza virus by Saccharomyces cerevisiae; Abdul Jabbar M et al.; We investigated the nature of signal recognition, transport, and secretion of mutant hemagglutinins (HAs) of a human influenza virus by the yeast Saccharomyces cerevisiae . The cDNA sequences encoding variant forms of influenza HA were expressed in S . cerevisiae . The HA polypeptides (HA500 and HA325) that were synthesized with their N-terminal signal peptides were correctly targeted to the membrane compartment where they were glycosylated . In contrast, the HA polypeptides (HA484 and HA308) lacking the signal peptide were expressed in the cytoplasm and did not undergo any glycosidic modification, demonstrating the importance of the heterologous signal sequence in the early steps of translocation in S . cerevisiae . The analysis of the N-terminal amino acid sequence of HA500 and HA325 polypeptides demonstrated the correct cleavage of the signal peptide, indicating the structural compatibility of a heterologous signal peptide for efficient recognition and processing by the yeast translocation machinery . The membrane-sequestered and glycosylated HA polypeptides were relatively stable in S . cerevisiae compared with the signal-minus, nonglycosylated HA molecules . Although both the anchor-minus HA (HA500) and HA1 (HA325) polypeptides were targeted efficiently to the membrane, their glycosylation and transport patterns were shown to be different . During pulse-chase, the HA500 remained cell-associated with no detectable secretion into the extracellular medium, whereas the HA325 secreted into the medium . Furthermore, only the cell-associated and secreted forms of HA325 and not HA500 appeared to have undergone hyperglycosylation with the extensive addition of high-molecular-weight outer-chain mannans . Possible reasons for the observed phenotypic behavior of these two mutant HAs are discussed.

Mol Cell Biol, 1987 Apr, 7(4), 1371 - 7
Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae; Toda T et al.; We have cloned a gene (BCY1) from the yeast Saccharomyces cerevisiae that encodes a regulatory subunit of the cyclic AMP-dependent protein kinase . The encoded protein has a structural organization similar to that of the RI and RII regulatory subunits of the mammalian cyclic AMP-dependent protein kinase . Strains of S . cerevisiae with disrupted BCY1 genes do not display a cyclic AMP-dependent protein kinase in vitro, fail to grow on many carbon sources, and are exquisitely sensitive to heat shock and starvation.

J Biochem (Tokyo), 1987 Apr, 101(4), 949 - 56
Possible involvement of acetyl coenzyme A carboxylase as well as fatty acid synthetase in the temperature-controlled synthesis of fatty acids in Saccharomyces cerevisiae; Hori T et al.; Fatty acid synthetase (FAS) preparations from Saccharomyces cerevisiae cells grown at either 35 or 10 degrees C produced the same products at different temperatures and showed quite similar temperature-dependencies in Arrhenius plots, with break points at 25 degrees C . This break point does not appear to reflect a phase transition of phospholipids present in the purified FAS preparations but rather is associated with protein conformational changes . S . cerevisiae cells grown at 35 degrees C and then shifted to 10 degrees C produced fatty acids with a shorter average chain length than those fatty acids synthesized at 10 degrees C by cells already adapted to 10 degrees C (hyper response) . Acetyl-CoA carboxylase activity was relatively higher in the cells grown at 35 degrees C than in the cells grown at 10 degrees C; moreover, fatty acids with longer average chain lengths were synthesized in vitro at higher malonyl-CoA concentrations, which was consistent with the difference in the average chain lengths of newly synthesized fatty acids in cells grown at 35 and 10 degrees C . However, the activity levels of acetyl-CoA carboxylase and fatty acid synthetase alone did not account for the hyper response phenomena.

Cell, 1987 Mar 13, 48(5), 801 - 12
A genetic analysis of dicentric minichromosomes in Saccharomyces cerevisiae; Koshland D et al.; We have developed an assay in S . cerevisiae in which clones of cells that contain intact dicentric minichromosomes are visually distinct from those that have rearranged to monocentric minichromosomes . We find that the instability of dicentric minichromosomes is apparently due to mitotic nondisjunction accompanied by occasional structural rearrangements . Monocentric minichromosomes arising by rearrangement of the plasmid are rapidly selected in the population since dicentric minichromosomes depress the rate of cell division . We show that the ability of one centromere to compete with another in dicentric minichromosomes requires the presence of both of the conserved structural elements, CDE II and CDE III . Dicentric minichromosomes can be stabilized if one of the centromeres on the molecule is functionally hypomorphic because of mutations in CDE II even though these mutant centromeres are highly efficient in monocentric molecules . Stable dicentric molecules can also be produced by decreasing the space between two wild-type centromeres on the same molecule . These results suggest plausible pathways for changes in chromosome number that accompany evolution.

Cell, 1987 Mar 13, 48(5), 789 - 99
The S . cerevisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway; Broek D et al.; The gene corresponding to the S . cerevisiae cell division cycle mutant cdc25 has been cloned and sequenced, revealing an open reading frame encoding a protein of 1589 amino acids that contains no significant homologies with other known proteins . Cells lacking CDC25 have low levels of cyclic AMP and decreased levels of Mg2+-dependent adenylate cyclase activity . The lethality resulting from disruption of the CDC25 gene can be suppressed by the presence of the activated RAS2val19 gene, but not by high copy plasmids expressing a normal RAS2 or RAS1 gene . These results suggest that normal RAS is dependent on CDC25 function . Furthermore, mutationally activated alleles of CDC25 are capable of inducing a set of phenotypes similar to those observed in strains containing a genetically activated RAS/adenylate cyclase pathway, suggesting that CDC25 encodes a regulatory protein . We propose that CDC25 regulates adenylate cyclase by regulating the guanine nucleotide bound to RAS proteins.

Yeast, 1987 Mar, 3(1), 51 - 7
Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation of the MAK16 gene and analysis of an adjacent gene essential for growth at low temperatures; Wickner RB et al.; MAK16 is an essential gene on chromosome I defined by the thermosensitive lethal mak16-1 mutation . MAK16 is also necessary for M double-stranded RNA replication at the permissive temperature for cell growth . As part of an effort to clone all the DNA from chromosome I, plasmids that complemented both the temperature-sensitive growth defect, and the M1 replication defects of mak16-1 strains were isolated from a plasmid YCp50: Saccharomyces cerevisiae recombinant DNA library . The two plasmids analysed contained overlapping inserts that hybridized proportionally to strains carrying different dosages of chromosome I . Furthermore, integration of a fragment of one of these clones occurred at a site linked to ade 1, confirming that this clone was derived from the appropriate region of chromosome I . An open reading frame adjacent to MAK16 potentially coding for a 468 amino acid protein was defined by sequence analysis . 185 amino acids of this open reading frame were replaced with a 1.2 kb fragment carrying the S . cerevisiae URA3 gene by a one-step gene disruption . The resulting strains grew at a rate indistinguishable from the wild type at 20 degrees C, 30 degrees C, or 37 degrees C, but could not grow at 8 degrees C . The deleted region is thus essential only at 8 degrees C, and we name this gene LTE1 (low temperature essential).

Cell, 1987 Feb 13, 48(3), 507 - 15
The Saccharomyces and Drosophila heat shock transcription factors are identical in size and DNA binding properties; Wiederrecht G et al.; The heat shock transcription factor (HSTF) has been purified to apparent homogeneity from S . cerevisiae and D . melanogaster by sequence-specific DNA-affinity chromatography . A synthetic oligonucleotide containing an hsp83-like heat shock element (HSE) was prepared and ligated into concatamers and covalently coupled to Sepharose . This DNA-affinity resin allowed the rapid isolation of a yeast and a Drosophila protein with the same apparent molecular weight (70 kd) . The yeast HSTF will bind to both its own and the Drosophila HSEs . Similarly, the Drosophila HSTF will bind to both its own and the yeast HSEs . The yeast and Drosophila HSTFs were subjected to preparative SDS gel electrophoresis, and the 70 kd polypeptides were eluted, renatured, and observed to generate the identical footprint pattern as the native HSTFs . Affinity-purified Drosophila HSTF was further shown to stimulate specific HSE-dependent transcription from a Drosophila hsp70 gene in vitro.

J Bacteriol, 1987 Feb, 169(2), 779 - 84
Effect of cell cycle position on thermotolerance in Saccharomyces cerevisiae; Plesset J et al.; We showed that the heat killing curve for exponentially growing Saccharomyces cerevisiae was biphasic . This suggests two populations of cells with different thermal killing characteristics . When exponentially growing cells separated into cell cycle-specific fractions via centrifugal elutriation were heat shocked, the fractions enriched in small unbudded cells showed greater resistance to heat killing than did other cell cycle fractions . Cells arrested as unbudded cells fell into two groups on the basis of thermotolerance . Sulfur-starved cells and the temperature-sensitive mutants cdc25, cdc33, and cdc35 arrested as unbudded cells were in a thermotolerant state . Alpha-factor-treated cells arrested in a thermosensitive state, as did the temperature-sensitive mutant cdc36 when grown at the restrictive temperature . cdc7, which arrested at the G1-S boundary, arrested in a thermosensitive state . Our results suggest that there is a subpopulation of unbudded cells in exponentially growing cultures that is in G0 and not in G1 and that some but not all methods which cause arrest as unbudded cells lead to arrest in G0 as opposed to G1 . It has been shown previously that yeast cells acquire thermotolerance to a subsequent challenge at an otherwise lethal temperature during a preincubation at 36 degrees C . We showed that this acquisition of thermotolerance was corrected temporally with a transient increase in the percentage of unbudded cells during the preincubation at 36 degrees C . The results suggest a relationship between the heat shock phenomenon and the cell cycle in S . cerevisiae and relate thermotolerance to transient as well as to more prolonged residence in the G0 state.

Mol Cell Biol, 1987 Feb, 7(2), 632 - 8
Physical analysis of the COR region: a cluster of six genes in Saccharomyces cerevisiae; Barry K et al.; Six genes, CYC1, UTR1, UTR3, OSM1, tRNAGly, and RAD7, have been localized within an 8-kilobase region on chromosome X of the yeast Saccharomyces cerevisiae . The physical structures and the transcripts of these genes were identified by analyzing a normal strain and six deletion mutants by genomic blotting, transcriptional analysis, and gene disruption procedures . The well-studied CYC1 gene encodes iso-1-cytochrome c; the tRNAGly gene encodes a tRNA; deletion of OSM1 and RAD7 causes sensitivity to hypertonic medium and UV irradiation, respectively . There were no observable phenotypes in strains having deletions of the UTR1, UTR3, and tRNAGly gene . The high density of transcripts, with little or almost no intragenic regions, indicates that the chromosomal organization of S . cerevisiae resembles the chromosomal organization of procaryotes rather than higher eucaryotes.

Antonie Van Leeuwenhoek, 1987, 53(2), 77 - 84
Three newly delimited species of Saccharomyces sensu stricto; Martini AV et al.; Deoxyribonucleic acid reassociation studies of 24 different wine and beer-associated strains of Saccharomyces confirmed the presence of three separate species . S . cerevisiae and S . bayanus strains had only 22% of their genomes in common . S . pastorianus, with intermediate hybridization values between S . cerevisiae and S . bayanus (52 and 72%, respectively) could possibly be a natural hybrid of the two species . S . pastorianus replaces S . carlsbergensis with which it is homologous for 93% of its genome, since the former species was described first by Hansen in 1904 . These data do not agree with the results of traditional physiological tests.

Mol Cell Biol, 1987 Jan, 7(1), 68 - 75
Alterations in the adenine-plus-thymine-rich region of CEN3 affect centromere function in Saccharomyces cerevisiae; Gaudet A et al.; Centromere DNA from 11 of the 16 chromosomes of the yeast Saccharomyces cerevisiae have been analyzed and reveal three sequence elements common to each centromere, referred to as conserved centromere DNA elements (CDE) . The adenine-plus-thymine (A + T)-rich central core element, CDE II, is flanked by two short conserved sequences, CDE I (8 base pairs {bp}) and CDE III (25 bp) . Although no consensus sequence exists among the different CDE II regions, they do have three common features of sequence organization . First, the CDE II regions are similar in length, ranging from 78 to 86 bp measured from CDE I to the left boundary of CDE III . Second, the base composition is always greater than 90% A + T . Finally, the A and T residues in these segments are often arranged in runs of A and runs of T residues, sometimes with six or seven bases in a stretch . We constructed insertion, deletion, and replacement mutations in the CDE II region of the centromere from chromosome III, CEN3, designed to investigate the length and sequence requirements for function of the CDE II region of the centromere . We analyzed the effect of these altered centromeres on plasmid and chromosome segregation in S . cerevisiae . Our results show that increasing the length of CDE II from 84 to 154 bp causes a 100-fold increase in chromosome nondisjunction . Deletion mutations removing segments of the A + T-rich CDE II DNA also cause aberrant segregation . In some cases partial function could be restored by replacing the deleted DNA with fragments whose primary sequence or base composition is very different from that of the wild-type CDE II DNA . In addition, we found that identical mutations introduced into different positions in CDE II have very similar effects.

Mol Cell Biol, 1987 Jan, 7(1), 403 - 9
Isolation of a Saccharomyces cerevisiae centromere DNA-binding protein, its human homolog, and its possible role as a transcription factor; Bram RJ et al.; A protein that binds specifically to Saccharomyces cerevisiae centromere DNA element I was purified on the basis of a nitrocellulose filter-binding assay . This protein, termed centromere-binding protein 1 (CP1), was heat stable and renaturable from sodium dodecyl sulfate (SDS), and assays of eluates from SDS gels indicated a molecular weight of 57,000 to 64,000 . An activity with similar specificity and stability was detected in human lymphocyte extracts, and analysis in SDS gels revealed a molecular weight of 39,000 to 49,000 . CP1-binding sites occurred not only at centromeres but also near many transcription units, for example, adjacent to binding sites for the GAL4-positive regulatory protein upstream of the GAL2 gene in S . cerevisiae and adjacent to the TATA element of the adenovirus major late promoter . A factor (termed USF) that binds to the latter site and stimulates transcription has been isolated from HeLa cells by others.

Curr Genet, 1987, 11(6-7), 483 - 90
Gene-protein assignments within the yeast Yarrowia lipolytica dsRNA viral genome; el-Sherbeini M et al.; Some strains of the yeast Yarrowia lipolytica possess virus-like particles (VLPs) which encapsidate a double-stranded RNA (dsRNA) genome designated Ly . We report here that these VLPs have two associated polypeptides of molecular weights 83 kd (VLy-P1) and 77 kd (VLy-P2) . Denatured Ly-dsRNA was used to program a cell-free rabbit reticulocyte translation system, resulting in the appearance of four major products, viz . Ly-P1 (83 kd); Ly-P2 (77 kd); Ly-P3 (74 kd) and Ly-P4 (68 kd) . The in vivo viral-associated protein VLy-P1 co-migrated on SDS-polyacrylamide gels with the in vitro product Ly-P1 and, similarly, VLy-P2 co-migrated with Ly-P2 . Peptide mapping data confirm the identity of the in vivo products (VLy-P1 and VLy-P2) and their in vitro counterparts . The conclusion made is that VLy-P1 and VLy-P2 are almost identical primary translation products of the Ly genome, derived from a single or multiple species of Ly-dsRNA . RNA blot hybridizations using L1A M1 and separately, L2A M2 probes prepared from appropriate K1 and K2 Saccharomyces cerevisiae killer strains, failed to show any detectable homology to Ly-dsRNA, substantiating the uniqueness of the Ly genome with respect to the K1 and K2 S . cerevisiae dsRNA killer systems.

Curr Genet, 1987, 11(6-7), 445 - 50
Genetic mapping of two pairs of linked ribosomal protein genes in Saccharomyces cerevisiae; Papciak SM et al.; We have used the 2 mu mapping method described by Falco and Botstein (1983) and tetrad analysis to map four ribosomal protein genes (two linked pairs) in S . cerevisiae . One pair (rp28-rp55 copy 1) is on chromosome XV, 14 cM proximal to ARG8 . The other pair (rp55-rp28 copy 2) is 19 cM from the centromere on the left arm of chromosome XIV . To map copy 1 we used the E . coli beta-galactosidase gene rather than a yeast gene to mark the ribosomal protein chromosomal locus . This provided a more sensitive color screening assay for chromosome loss in the 2 mu method . It also removed the restriction that the mapping tester strains must be mutant for the plasmid marker.

Curr Genet, 1987, 11(6-7), 429 - 34
Mutations suppressing the effects of a deletion of the phosphoglucose isomerase gene PGI1 in Saccharomyces cerevisiae; Aguilera A; A mutant with a deletion covering the phosphoglucose isomerase gene PGI1, allele pgil delta, can only grow on a medium containing fructose and low concentrations of glucose whereas growth is completely inhibited by glucose concentrations higher than 0.4% . This was used to select suppressor mutants restoring growth on synthetic media with 2% glucose as the sole carbon source . One complementation group, SPG1, was defined by recessive mutations . The ability to grow on glucose media was strictly dependent on functional mitochondria . The generation time of the selected mutants on YEP glucose was 6-8 h . No ethanol was formed from glucose and the levels of respiration were very high . These phenotypes were also observed in single pgil delta mutants when growing on fructose media supplemented with 0.4% glucose . The other glycolytic enzymes, the enzymes of the glucose-6-phosphate oxidation pathway as well as catabolite repression were normal in suppressed pgil delta mutants . The suppressor mutation alone caused no abnormal phenotype . The results suggest that the spg1 suppressor mutations allow S . cerevisiae pgil delta mutant strains to grow on glucose by using the Pentose-P cycle in combination with unusual strong respiration.

Curr Genet, 1987, 11(4), 295 - 301
A mitochondrial frameshift-suppressor (+1) {corrected} of the yeast S . cerevisiae maps in the mitochondrial 15S rRNA locus; Weiss-Brummer B et al.; The first case of a +1 "extrageneic" frameshift suppressor (MF1), mapping in the yeast mitochondrial 15S rRNA gene is reported . The suppressor was identified by genetic analyses in a leaky mitochondrial oxil frameshift mutant and the respective wild-type strain 777-3A of the yeast S . cerevisiae . This is in accordance with the finding that all mitochondrial frameshift mutants isolated from this strain tend to be leaky to a variable degree . MF1 does not suppress known nonsense mutations created by a direct basepair exchange in strain 777-3A . These mutants exhibit a non-leaky phenotype (Weiss-Brummer et al . 1984).

Gene, 1987, 55(2-3), 303 - 17
Expression of a wheat alpha-gliadin gene in Saccharomyces cerevisiae; Neill JD et al.; A vector was constructed that directs the expression of foreign genes in the yeast Saccharomyces cerevisiae . This vector contains an expression site that was constructed by in vitro modification of the iso-1-cytochrome c (CYC1) gene of S . cerevisiae . The expression of heterologous sequences can be experimentally controlled by catabolite control sequences, promoter and transcription initiation sequences and termination sequence derived from the CYC1 gene . A portion of a genomic wheat alpha-gliadin gene consisting of the entire 861 bp of protein-coding sequence, 18 bp of 5' leader sequence and 54 bp of 3'-noncoding sequence was inserted into the expression site . A CYC1::alpha-gliadin transcript of approx . 1050 nucleotides was synthesized in transformed yeast under the control of the CYC1 regulatory region . The transcripts terminated within the alpha-gliadin 3'-noncoding region, near a nucleotide sequence similar to the yeast transcription termination consensus sequence . The alpha-gliadin was immunochemically detected in total protein extracts from transformed cells and accounted for approx . 0.1% of the total cellular protein . The size of alpha-gliadin synthesized in yeast is the same as that of mature wheat alpha-gliadin . This is consistent with recognition and cleavage of the signal peptide by yeast . Due to the amino acid composition of alpha-gliadin, the availability of glutamine tRNA is a potential translational limitation to high-level synthesis in yeast.

Microbios, 1987, 51(206), 37 - 42
Analysis of fatty acid composition of Candida species by gas-liquid chromatography using a polar column; Kobayashi K et al.; The fatty acid composition of representative Candida species was examined by gas-liquid chromatography (GLC) using a polar column . The major fatty acids were C14:0, C16:0, C18:0 saturated, C16:1 and C18:1 monoenoic series, with or without C18 polyunsaturated acids (C18:2 and C18:3) . In Torulopsis glabrata and Saccharomyces cerevisiae the C18:2 and C18:3 acids were not found, but the C10:0 and C12:0 acids were detected in S . cerevisiae . These results indicated that the Candida genus could be distinguished from Torulopsis and Saccharomyces genera by GLC analysis of fatty acids . Quantitative differences in the fatty acid composition between cells grown at high temperature (37 degrees C) and low temperature (25 degrees C) were found generally in Candida species, and the amounts of C18 polyunsaturated acids (C18:2 and C18:3) increased in the cells grown at 25 degrees C . Each Candida species showed a characteristic profile in fatty acid composition . Determination of the cellular fatty acid composition in Candida species is likely to be useful for the grouping or chemotaxonomy of newer isolates of Candida species.

Gene, 1987, 54(1), 1 - 22
The AT spacers and the var1 genes from the mitochondrial genomes of Saccharomyces cerevisiae and Torulopsis glabrata: evolutionary origin and mechanism of formation; de Zamaroczy M et al.; Intergenic sequences represent 63% of the mitochondrial 'long' (85 kb) genome of Saccharomyces cerevisiae . They comprise 170-200 AT spacers that correspond to 47% of the genome and are separated from each other by GC clusters, ORFs, ori sequences, as well as by protein-coding genes . Intergenic AT spacers have an average size of 190 bp, and a GC level of 5%; they are formed by short (20-30 nt on the average) A/T stretches separated by C/G mono- to trinucleotides . An analysis of the primary structures of all intergenic AT spacers already sequenced (32 kb; 80% of the total) has shown that they are characterized by an extremely high level of short sequence repetitiveness and by a characteristic sequence pattern; the frequencies of A/T isostichs conspicuously deviate from statistical expectations, and exponentially decrease when their (AT + TA)/(AA + TT) ratio, R, decreases . A situation basically identical was found in the AT spacers of the mitochondrial genome (19 kb) of Torulopsis glabrata . The sequence features of the AT spacers indicate that they were built in evolution by an expansion process mainly involving rounds of duplication, inversion and translocation events which affected an initial oligodeoxynucleotide (endowed with a particular R ratio) and the sequences derived from it . In turn, the initial oligodeoxynucleotide appears to have arisen from an ancestral promoter-replicator sequence which was at the origin of the nonanucleotide promoters present in the mitochondrial genomes of several yeasts . Common sequence patterns indicate that the AT spacers so formed gave rise to the var1 gene (by linking and phasing of short ORFs), to the DNA stretches corresponding to the untranslated mRNA sequences and to the central stretches of ori sequences from S . cerevisiae.

J Mol Evol, 1987, 24(3), 252 - 9
Molecular evolution of the Saccharomyces cerevisiae histone gene loci; Smith MM; The core histone genes of Saccharomyces cerevisiae are arranged as duplicate nonallelic sets of specifically paired genes . The identity of structural organization between the duplicated gene pairs would have its simplest evolutionary origin in the duplication of a complete locus in a single event . In such a case, the time since the duplication of one of the genes should be identical to that since duplication of the gene adjacent to it on the chromosome . A calculation of the evolutionary distances between the coding DNA sequences of the histone genes leads to a duplication paradox: The extents of sequence divergence in the silent component of third-base positions for adjacent pairs of genes are not identical . Estimates of the evolutionary distance between the two H3-H4 noncoding intergene DNA sequences are large; the divergence between the two separate sequences is indistinguishable from the divergence between either of the regions and a randomly generated permutation of itself . These results suggest that the duplication event may have occurred much earlier than previously estimated . The potential age of the duplication, and the attractive simplicity of the duplication of both the H3-H4 and the H2A-H2B gene pairs having taken place in a single event, leads to the hypothesis that modern haploid S . cerevisiae may have evolved by diploidization or fusion of two ancient fungi.

Gene, 1987, 54(1), 113 - 23
Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus; Schultz LD et al.; The major envelope glycoprotein (gp350) of Epstein-Barr virus has been expressed and secreted in the yeast Saccharomyces cerevisiae as a 400-kDa glycoprotein . This is the first example of the secretion of such a large, heavily glycosylated heterologous protein in yeast . Since gp350 proved highly toxic to S . cerevisiae, initial cellular growth required repression of the expression of gp350 . Using temperature- or galactose-inducible promoters, cells could be grown and the expression of gp350 then induced . After induction, the glycoprotein accumulated both intracellularly as well as in the culture medium . Only the most heavily glycosylated form was secreted, suggesting a role for N-linked glycans in directing secretion . The extent of O-linked glycosylation of the yeast-derived protein was similar to that of the mature viral gp350 . N-linked glycosylation varied slightly depending upon culture conditions and host strain used and was more extensive than that associated with the mature viral gp350 . Although there is no evidence that more than a single mRNA for the glycoprotein was expressed from the recombinant plasmid, variously sized glycoproteins accumulated in yeast at early stages after induction, probably reflecting intermediates in glycosylation . The yeast-derived glycoproteins reacted with animal and human polyclonal antibodies to gp350 as well as with a neutralizing murine monoclonal antibody to gp350, suggesting that this glycoprotein retains several epitopes of the native glycoprotein.

Mol Cell Biol, 1987 Jan, 7(1), 410 - 9
Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and analysis of the CEN1-ADE1-CDC15 region; Steensma HY et al.; To continue the systematic examination of the physical and genetic organization of an entire Saccharomyces cerevisiae chromosome, the DNA from the CEN1-ADE1-CDC15 region from chromosome I was isolated and characterized . Starting with the previously cloned ADE1 gene (J . C . Crowley and D . B . Kaback, J . Bacteriol . 159:413-417, 1984), a series of recombinant lambda bacteriophages containing 82 kilobases of contiguous DNA from chromosome I were obtained by overlap hybridization . The cloned sequences were mapped with restriction endonucleases and oriented with respect to the genetic map by determining the physical positions of the CDC15 gene and the centromeric DNA (CEN1) . The CDC15 gene was located by isolating plasmids from a YCp50 S . cerevisiae genomic library that complemented the cdc15-1 mutation . S . cerevisiae sequences from these plasmids were found to be represented among those already obtained by overlap hybridization . The cdc15-1-complementing plasmids all shared only one intact transcribed region that was shown to contain the bona fide CDC15 gene by in vitro gene disruption and one-step replacement to delete the chromosomal copy of this gene . This deletion produced a recessive lethal phenotype that was also recessive to cdc15-1 . CEN1 was located by finding a sequence from the appropriate part of the cloned region that stabilized the inheritance of autonomously replicating S . cerevisiae plasmid vectors . Finally, RNA blot hybridization and electron microscopy of R-loop-containing DNA were used to map transcribed regions in the 23 kilobases of DNA that went from CEN1 to CDC15 . In addition to the transcribed regions corresponding to the ADE1 and ADC15 genes, this DNA contained five regions that gave rise to polyadenylated RNA, at least two regions complementary to 4S RNA species, and a Ty1 transposable element . Notably, a higher than average proportion of the DNA examined was transcribed into RNA.

Mol Cell Biol, 1987 Jan, 7(1), 258 - 65
Ty1 sequence with enhancer and mating-type-dependent regulatory activities; Errede B et al.; Some insertion mutations in Saccharomyces cerevisiae activate the expression of adjacent structural genes . The CYC7-H2 mutation is a Ty1 insertion 5' to the iso-2-cytochrome c coding region of CYC7 . The Ty1 insertion causes a 20-fold increase in CYC7 expression in a and alpha haploid cell types of S . cerevisiae . This activation is repressed in the a/alpha diploid cell type . Previous computer analysis of the CYC7-H2 Ty1 activator region identified two related sequences with homology both to mammalian enhancers and to a yeast a/alpha control site . A 112-base-pair (bp) DNA fragment encompassing one of these blocks of homology functioned as one component of the Ty1 activator . A 28-bp synthetic oligonucleotide with the wild-type homology block sequence was also functional . A single base pair mutation within the enhancer core of the synthetic 28-bp regulatory element reduced its activation ability to near background amounts . In addition, the 112-bp Ty1 fragment by itself functioned as a target for repression of adjacent gene expression in a/alpha diploid cells.

Mol Cell Biol, 1987 Jan, 7(1), 121 - 8
Signal peptide specificity in posttranslational processing of the plant protein phaseolin in Saccharomyces cerevisiae; Cramer JH et al.; We linked the cDNA coding region for the bean storage protein phaseolin to the promoter and regulatory region of the Saccharomyces cerevisiae repressible acid phosphatase gene (PHO5) in multicopy expression plasmids . Yeast transformants containing these plasmids expressed phaseolin at levels up to 3% of the total soluble cellular protein . Phaseolin polypeptides in S . cerevisiae were glycosylated, and their molecular weights suggested that the signal peptide had been processed . We also constructed a series of plasmids in which the phaseolin signal-peptide-coding region was either removed or replaced with increasing amounts of the amino-terminal coding region for acid phosphatase . Phaseolin polypeptides with no signal peptide were not posttranslationally modified in S . cerevisiae . Partial or complete substitution of the phaseolin signal peptide with that from acid phosphatase dramatically inhibited both signal peptide processing and glycosylation, suggesting that some specific feature of the phaseolin signal amino acid sequence was required for these modifications to occur . Larger hybrid proteins that included approximately one-half of the acid phosphatase sequence linked to the amino terminus of the mature phaseolin polypeptide did undergo proteolytic processing and glycosylation . However, these polypeptides were cleaved at several sites that are not normally used in the unaltered acid phosphatase protein.

Biochimie, 1987 Jan, 69(1), 53 - 62
Sensitivity of yeast cells to reactive oxygen species generated in the extracellular space; Chaput M et al.; Even when cytoplasmic scavenging activities are plentiful, yeast cells (S . cerevisiae) remain particularly sensitive towards reactive oxygen species generated in the extracellular space (either by the xanthine/xanthine oxidase reaction or by the redox cycling of menadione) . A sharp reduction of the extent of cellular alterations when SOD and/or catalase were supplemented in the incubation buffer, points to a contribution of both O-.2 and H2O2 in the toxic process . Although oxygen metabolites as well as t-butylhydroperoxide (tBH), a highly toxic organic peroxide, may be directly responsible for cellular damage, their toxicity is largely reduced in the presence of Desferal . A role of metal ions in potentiating the toxicity points to the involvement of OH . radicals, actually produced in the medium . With tBH, metal cations would be rather active in promoting peroxidative chain reactions . In the case of an extracellular oxidative attack, it may be foreseen that the plasma membrane will form a preferential target . An increased permeability of the plasma membrane towards ionized molecules and uncharged polycarboxylic acids is indeed observed after an oxidative treatment . The loss of selective permeability is, as a rule, correlated with a drop in viability . Early alterations, disrupting the functional organization of the plasma membrane have been sought . The permease involved in the active transport of purine(s) has appeared to be an appropriate marker for checking its functional integrity . This transport function appears to be very sensitive to damage induced by O-.2 generators, particularly under conditions in which the resulting lethality is still kept low and in which the energization of active transport processes remains unimpaired.

Biol Cell, 1987, 61(3), 171 - 5
The energetic growth yields of the yeast Candida parapsilosis; Camougrand N et al.; The energetic growth yields of Candida parapsilosis were compared with those of Saccharomyces cerevisiae as a function of the energy source in the presence or absence of antimycin A, an inhibitor of the second phosphorylation site . When glycerol was used as energy source, the energetic growth yields were quite similar in C . parapsilosis and S . cerevisiae . On the other hand, when experiments were carried out with glucose as energy source, although three phosphorylation sites were available, glucose was found to be a poor energy source for C . parapsilosis . When C . parapsilosis was grown in the presence of antimycin A, on glucose: YGluS = 3YGlu + AS and on glycerol: YGlyS = 2 YGly + AS . It was concluded that growth in the presence of antimycin A could occur due to the functioning of the third phosphorylation site . This result agrees with previous works indicating that in C . parapsilosis the alternative pathway merges into the main respiratory chain at the cytochrome c level . Although the doubling time of C . parapsilosis was much less temperature-sensitive than that of S . cerevisiae, the energetic growth yield was the same at 13 degrees C and 28 degrees C, and consequently, the secondary pathway did not seem to be thermogenic.

Experientia Suppl, 1987, 52, 431 - 7
Synaptic relations in meiotic gene conversion at the iterated CUP1r locus of S . cerevisiae; Welch JW et al.; This study concerns a comparative molecular analysis of copy number changes in two hybrids that differ in the extent of homologies at the CUP1r locus . Hybrid JW1020 is a diploid wherein each parent contributed nine identical, tandemly arrayed 2.0 kb repeat units . Genomic DNA was isolated from each of the spore colonies in a sample of 200 unselected tetrads . About 15% displayed copy number changes, i.e., increases or decreases of one or more complete units . Changes on a per tetrad basis occurred as often in a single spore colony as changes in each of two spores . Such double changes are rarely reciprocal in character . To account for the observed qualitative and quantitative copy number shifts, we propose a molecular recombination model that posits partial, incomplete synaptic pairing and gene conversion of the unpaired regions with or without associated crossing over . A second contrasting study centers on the copy number alterations and recombinational events uncovered in a molecular analysis of 50 unselected tetrads generated by hybrid EB8 . Unlike the hybrid JW1020, the EB8 diploid strain carried a six copy tandem array of 1.1 kb units at the CUP1r locus in one parental homologue and a five copy array of 1.6 kb units at the corresponding chromosome VIII locus . These natural alleles were recovered from industrial yeast strains by conventional genetic procedures and characterized by restrictional analysis . Twelve tetrads exhibit evidence for several different types of recombination events . However,ordinary crossover exchanges are conspicuously absent . We suppose that the repetitious nonhomologies generate DNA configurations sufficient to disrupt the effective synapsis over the entire locus.(ABSTRACT TRUNCATED AT 250 WORDS)

Curr Genet, 1987, 11(6-7), 451 - 7
Nucleotide sequencing analysis of a LEU gene of Candida maltosa which complements leuB mutation of Escherichia coli and leu2 mutation of Saccharomyces cerevisiae; Takagi M et al.; The expression of a LEU gene from Candida maltosa (designated as C-LEU2) isolated previously (Kawamura et al . 1983) was shown to be regulated, when transferred into Saccharomyces cerevisiae, by leucine and threonine in the medium, as in the case of LEU2 gene of S . cerevisiae . The coding region together with the regulatory region was subcloned and the nucleotide sequence was determined . When the sequence of the coding region was compared with that of LEU2, the homology was 72% for base pairs and 76% for deduced amino acids . Comparison of the regulatory region of C-LEU2 with those of LEU1 and LEU2 suggested a few short consensus sequences which are involved in regulation of gene expression by leucine and threonine in the medium.

Curr Genet, 1987, 11(5), 377 - 83
The Yarrowia lipolytica LEU2 gene; Davidow LS et al.; A 2810 bp DNA fragment containing the beta-isopropylmalate dehydrogenase gene of the dimorphic yeast Yarrowia lipolytica has been sequenced . The sequence contains an open reading frame of 405 codons, predicting a protein of 43,366 molecular weight . Protein sequence homology with the polypeptide encoded by the LEU2 gene of Saccharomyces cerevisiae is 64%, whereas DNA sequence homology is 61% . The 5'- and 3'-flanking regions of the Y . lipolytica LEU2 gene share only some general structural features common to genes of S . cerevisiae such as the presence and location of TATA boxes, CAAT boxes, CACACA repeats, the lack of G residues in the 5'-untranslated region and 3'-transcription terminators . Transcription of a 1.4 kb mRNA begins at a small cluster of sites approximately 40 base pairs before the initial ATG.

Curr Genet, 1987, 12(5), 337 - 48
Efficient integrative transformation of Cephalosporium acremonium; Skatrud PL et al.; A hybrid gene, IPNSp/HPTorf, was constructed by placing an 850 bp sequence of Cephalosporium acremonium DNA next to the 5' end of a bacterial open reading frame, HPTorf . The sequence was obtained as an 850 bp NcoI restriction fragment from the 5' non-coding region of the C . acremonium isopenicillin N synthetase (IPNS) gene . The HPTorf was obtained from a bacterial gene that coded for a hygromycin B phosphotransferase (HPT) . Plasmids that contained IPNSp/HPTorf transformed C . acremonium to a stably maintained hygromycin B resistant phenotype . Southern analysis of total DNA from transformants demonstrated multiple integrations of the transforming DNA in the high molecular weight DNA of most transformants, but single integrations were observed in a few transformants . The number of transformants per microgram of DNA was about 100 times greater than for plasmids that contained the HPTorf without any juxtaposed eucaryotic promoter sequence . Plasmids with the promoterless HPTorf and plasmids with a truncated S . cerevisiae phosphoglycerate kinase promoter juxtaposed to the HPTorf transformed C . acremonium at equivalent low frequencies . Transformation of C . acremonium with linearized plasmid DNA produced at least 2-3 fold more transformants than the corresponding circular molecule . Several observations were made concerning protoplast formation and handling which made the transformation procedure more efficient and allowed a greater proportion of protoplasts to regenerate to viable walled cells . Plasmids were constructed that contained both the IPNSp/HPTorf and additional elements: fragments of C . acremonium ribosomal DNA (rDNA), or a fragment of C . acremonium mitochondrial DNA possessing activity as an autonomous replication sequence (ARS) in S . cerevisiae, or putative transcriptional termination/polyadenylation signals from the IPNS gene . These plasmids transformed C . acremonium at frequencies experimentally equivalent to those containing IPNSp/HPTorf without any of these additional elements.

Gene, 1987, 58(2-3), 189 - 99
Complete nucleotide sequence of the Rhodosporidium toruloides gene coding for phenylalanine ammonia-lyase; Anson JG et al.; The complete nucleotide sequence of the Rhodosporidium toruloides gene coding for the enzyme phenylalanine ammonia-lyase (PAL) has been determined . The primary structure of PAL was deduced from the nucleotide sequence of the two cDNA clones, pPAL1 and pPAL2, which covered the entire amino acid-coding sequence . Comparison of cDNA and genomic sequences of pal revealed the presence of six introns . The nucleotide sequences of these introns were compared to those from other fungi . The primary amino acid sequence of the enzyme exhibits only 30.8% identity with the determined primary sequence of PAL from Phaseolus vulgaris . Upstream from the structural gene there is a stretch of C + T-rich DNA similar to that found upstream from a number of Neurospora and Saccharomyces cerevisiae genes . In the case of S . cerevisiae, these C + T-rich sequences are thought to be involved in the transcription of highly expressed genes.

Curr Genet, 1987, 12(8), 561 - 7
The secretion and post translational modification of interferons from Saccharomyces cerevisiae; Piggott JR et al.; Studies with three interferon molecules, IFN-alpha 2, IFN-beta 1, and a "hybrid" interferon, IFNX-430 are described which illustrate that both the expression and secretion characteristics of heterologous proteins in yeast cells reflect properties of the proteins themselves . Recombinant DNA techniques have also been used to demonstrate that the efficient processing of mature heterologous proteins from the yeast alpha factor secretion leader can be affected by sequences on the carboxyl side of the initial cleavage site . Secretion studies with heterologous proteins in S . cerevisiae are aimed at maximising yield, the percentage of extracellular product and correct amino terminus sequence . The results presented here show that all three factors are susceptible to currently unpredictable properties of the foreign sequence . This situation, in turn, means that heterologous proteins can be used as tools in the biochemical dissection of the yeast secretion process.

Dev Biol Stand, 1987, 67, 185 - 99
Construction of expression vectors for the production of interferons in yeast; Devenish RJ et al.; Expression vectors designed for the production of foreign proteins in yeast are constructed as a composite of functional DNA segments brought together in a single plasmid DNA molecule . Such a composite DNA molecule constitutes a complex entity in terms of its replication and selection functions (in both E . coli and S . cerevisiae) and its gene expression properties . The latter depend critically upon transcriptional control signals (the promoter and terminator regions from a highly expressed yeast gene) that are used to direct the expression of foreign genes in yeast cells . This paper describes the construction of such an expression vector based on the transcription control signals of the yeast phosphoglycerate kinase gene and its application to the production of human interferon-alpha's in yeast cells.

J Biol Chem, 1986 Dec 25, 261(36), 17183 - 91
Characterization of COX9, the nuclear gene encoding the yeast mitochondrial protein cytochrome c oxidase subunit VIIa . Subunit VIIa lacks a leader peptide and is an essential component of the holoenzyme; Wright RM et al.; The gene COX9 for subunit VIIa of cytochrome c oxidase from Saccharomyces cerevisiae has been cloned with the aid of an oligonucleotide probe . From the nucleotide sequence of COX9, we deduce that subunit VIIa is derived from a precursor that is 59 amino acids in length (Mr = 6963) . This precursor is longer than mature subunit VIIa by one amino acid at its NH2 terminus and four amino acids at its COOH terminus . COX9 exists as a single copy in the haploid genome of S . cerevisiae and produces one major transcript . When the genomic copy of COX9 is removed, cells lack a functional cytochrome c oxidase holoenzyme . From the predicted secondary structure of subunit VIIa, previous data concerning its relationship to the lipid bilayer of the inner membrane and the location of its hydrophobic domains (Power, S.D., Lochrie, M.A., and Poyton, R.O . (1986) J . Biol . Chem . 261, 9206-9209) and the finding that it is essential for the holoenzyme, we propose a model for subunit VIIa which suggests that this small integral protein plays a role in holoenzyme assembly or stability.

Mol Cell Biol, 1986 Dec, 6(12), 4516 - 25
Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome; Coleman KG et al.; Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae . A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector . Plasmids containing pyk1-complementing DNA were obtained from other investigators . Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes . These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector . A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined . Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3) . Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome . The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans . As previous studies with S . cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.

J Bacteriol, 1986 Dec, 168(3), 1468 - 71
Biological activity of the Asn-5,Arg-7 tridecapeptide encoded by MF alpha 2 of Saccharomyces cerevisiae; Raths S et al.; The precursor predicted by the nucleotide sequence of the MF alpha 2 gene of Saccharomyces cerevisiae contains one copy of the tridecapeptide alpha-factor previously characterized (H2N-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-COOH) and one copy of a peptide that contains two conservative amino acid substitutions (H2N-Trp-His-Trp-Leu-Asn-Leu-Arg-Pro-Gly-Gln-Pro-Met-Tyr-COOH) . To determine whether the novel molecule possesses biological activity, the Asn-5,Arg-7 tridecapeptide was prepared chemically by solid-phase peptide synthesis . Growth arrest and morphogenesis assays gave identical activity profiles for the Asn-5,Arg-7 peptide and the other gene product, the Gln-5,Lys-7 peptide . The activities of the two peptides were additive and indistinguishable for S . cerevisiae X2180-1A . When present in fourfold molar excess, the biologically inactive desTrp-1,Ala-3 dodecapeptide reversed activity of the Asn-5,Arg-7 and Gln-5,Lys-7 tridecapeptides . Furthermore, neither peptide caused growth arrest of a MATa ste2(Ts) mutant when assayed at the restrictive temperature . These studies suggest that both pheromones interact with the alpha-factor receptor in a similar manner.

Mol Cell Biol, 1986 Dec, 6(12), 4763 - 6
DNA binding is not sufficient for nuclear localization of regulatory proteins in Saccharomyces cerevisiae; Silver PA et al.; We showed by immunofluorescence that the procaryotic DNA-binding protein LexA and a chimeric protein that contains the DNA-binding portion of LexA (amino acids 1 to 87) and a large portion (amino acids 74 to 881) of the Saccharomyces cerevisiae positive regulatory GAL4 protein (GAL4 gene product) are not preferentially localized in the nucleus in S . cerevisiae.

Mol Cell Biol, 1986 Dec, 6(12), 4281 - 94
Structure of the Saccharomyces cerevisiae HO gene and analysis of its upstream regulatory region; Russell DW et al.; The HO gene product of Saccharomyces cerevisiae is a site-specific endonuclease that initiates mating type interconversion . We have determined the nucleotide sequence of a 3,129-base-pair (bp) segment containing HO . The segment contains a single long open reading frame encoding a polypeptide of 586 amino acids, which has unusual (unbiased) codon usage and is preceded by 762 bp of upstream region . The predicted HO protein is basic (16% lysine and arginine) and is calculated to have a secondary structure that is 30% helical . The corresponding transcript is initiated approximately 50 nucleotides prior to the presumed initiation codon . Insertion of an Escherichia coli lacZ gene fragment into the putative HO coding segment inactivated HO and formed a hybrid HO-lacZ gene whose beta-galactosidase activity was regulated by the mating type locus in the same manner as HO (repressed by a 1-alpha 2) . Upstream regions of 1,360 and 762 bp conferred strong repression; 436 bp led to partial constitutivity and 301 bp to full constitutivity . Thus, DNA sequences that confer repression of HO by a1-alpha 2 are at least 250 nucleotides upstream of the transcription start point and are within 436 nucleotides of the HO initiation codon . The progressive loss of repression suggests that both the -762 to -436 and the -436 to -301 intervals contain sites for regulation by a1-alpha 2 . The HO gene contains two distinct regions that promote autonomous replication of plasmids in S . cerevisiae . These regions contain sequences that are homologous to the two conserved sequences that are associated with ARS activity.

Mol Cell Biol, 1986 Dec, 6(12), 4425 - 32
Highly mutable sites for ICR-170-induced frameshift mutations are associated with potential DNA hairpin structures: studies with SUP4 and other Saccharomyces cerevisiae genes; Hampsey DM et al.; The majority of the mutations induced by ICR-170 in both the CYC1 gene (J . F . Ernst et al . Genetics 111:233-241, 1985) and the HIS4 gene (L . Mathison and M . R . Culbertson, Mol . Cell . Biol . 5:2247-2256, 1985) of the yeast Saccharomyces cerevisiae were recently shown to be single G . C base-pair insertions at monotonous runs of two or more G . C base pairs . However, not all sites were equally mutable; in both the CYC1 and HIS4 genes there is a single highly mutable site where a G . C base pair is preferentially inserted at a {sequence in text} . Here we report the ICR-170 mutagen specificity at the SUP4-o tyrosine tRNA gene of yeast . Genetic fine structure analysis and representative DNA sequence determination of ICR-170-induced mutations revealed that there is also a single highly mutable site in SUP4-o and that the mutation is a G . C base-pair insertion at a monotonous run of G . C base pairs . Analysis of DNA sequences encompassing the regions of highly mutable sites for all three genes indicated that the mutable sites are at the bases of potential hairpin structures; this type of structure could not be found at any of the other, less mutable G . C runs in SUP4, CYC1, and HIS4 . Based on these results and recent information regarding novel DNA structural conformations, we present a mechanism for ICR-170-induced mutagenesis . (i) ICR-170 preferentially binds to DNA in the beta conformation; factors that increase the temporal stability of this structure, such as adjacent stem-and-loop formation, increase the frequency of ICR-170 binding; (ii) the observed mutagen specificity reflects formation of a preferred ICR-170 intercalative geometry at {sequence in text} sites; (iii) during replication or repair, ICR-170 remains associated with the single-stranded template; (iv) stuttering or strand slippage by the polymerization complex as it encounters the mutagen results in nucleotide duplication; (v) subsequent replication or mismatch repair fixes the insertion into the genome . This mechanism accounts for both the IRC-170 mutagenic specificity and the molecular basis of the highly mutable sites in S . cerevisiae.

J Biol Chem, 1986 Nov 25, 261(33), 15572 - 6
Reconstitution of Saccharomyces cerevisiae phosphatidylserine synthase into phospholipid vesicles . Modulation of activity by phospholipids; Hromy JM et al.; Membrane-associated phosphatidylserine synthase was purified from Saccharomyces cerevisiae (Bae-Lee, M., and Carman, G . M . (1984) J . Biol . Chem . 259, 10857-10862) and reconstituted into phospholipid vesicles containing phosphatidylcholine/phosphatidylethanolamine/ phosphatidylinositol/phosphatidylserine . Reconstitution was performed by removing detergent from an octyl glucoside/phospholipid/Triton X-100/enzyme mixed micelle by Sephadex G-50 super-fine chromatography . The average diameter of the vesicles was 90 nm, and the enzyme was reconstituted asymmetrically with the active site facing outward . The enzymological properties of reconstituted phosphatidylserine synthase were determined in the absence of detergent . The enzyme was reconstituted into vesicles with phospholipid compositions approximating those of wild type and mutant strains of S . cerevisiae . Reconstituted activity was modulated by the phosphatidylinositol/phosphatidylserine ratio in the vesicles . The modulation of activity observed in the vesicles is enough to account for some of the fluctuations in the phosphatidylserine content in vivo.

Nucleic Acids Res, 1986 Nov 11, 14(21), 8347 - 60
The relationship between mRNA stability and length in Saccharomyces cerevisiae; Santiago TC et al.; A rapid and convenient procedure has been developed for the measurement of mRNA half-life in S.cerevisiae using the transcriptional inhibitor, 1,10-phenanthroline . A range of half-lives from 6.6 +/- 0.67 minutes to over 100 minutes, relative to the stability of the 18S rRNA control, has been obtained for fifteen mRNAs . They include the pyruvate kinase and actin mRNAs, as well as 13 randomly picked mRNAs of unknown function . The mRNAs clearly fall into two populations when their lengths and half-lives are analysed; one population is considerably more stable than the other when mRNAs of similar length are compared . Also, within each population, there is an inverse relationship between mRNA length and half-life . These results suggest that mRNA length and at least one additional factor strongly influence mRNA stability in yeast.

Cell, 1986 Nov 7, 47(3), 413 - 22
RAM, a gene of yeast required for a functional modification of RAS proteins and for production of mating pheromone a-factor; Powers S et al.; We have identified a gene (SUPH) of S . cerevisiae that is required for both RAS function and mating by cells of a mating type . supH is allelic to ste16, a gene required for the production of the mating pheromone a-factor . Both RAS and a-factor coding sequences terminate with the potential acyltransferase recognition sequence Cys-A-A-X, where A is an aliphatic amino acid . Mutations in SUPH-STE16 prevent the membrane localization and maturation of RAS protein, as well as the fatty acid acylation of it and other membrane proteins . We propose the designation RAM (RAS protein and a-factor maturation function) for SUPH and STE16 . RAM may encode an enzyme responsible for the modification and membrane localization of proteins with this C-terminal sequence.

Cell, 1986 Nov 7, 47(3), 401 - 12
The ras-related YPT1 gene product in yeast: a GTP-binding protein that might be involved in microtubule organization; Schmitt HD et al.; The 23.5 kd protein product of the ras-related YPT1 gene of S . cerevisiae was found to be essential for cell growth . The loss of YPT1 function, studied in cells with the YPT1 gene on chromosome VI regulated by the galactose-inducible GAL10 promoter, led to arrested cells that were multibudded and exhibited a complete disorganization of microtubules and an apparent loss of nuclear integrity . The YPT protein binds GTP specifically . GTP binding of the protein is essential for its intracellular function . The Asn121----IIe substitution, generated by site-directed mutagenesis, had a dominant lethal phenotype, the expression of the mutant protein led to binucleated cells and abnormal spindles . In contrast to the S . cerevisiae RAS1 and RAS2 gene products, the YPT protein seems to be involved, directly or indirectly, in microtubule organization and function.

Environ Health Perspect, 1986 Nov, 69, 183 - 202
Toxicology of haloacetonitriles; Hayes JR et al.; Haloacetonitriles are by-products of water chlorination and may form in vivo from the reaction of residual chlorine with endogenous compounds such as amino acids . Dibromoacetonitrile (DBAN) was negative in selected mutagenic assays; dichloroacetonitrile (DCAN) was mutagenic in S . typhimurium, but not in S . cerevisiae . Both DBAN and DCAN may be carcinogenic . There is a paucity of basic toxicological data for these compounds . The studies described were conducted to determine the acute, subacute, and subchronic toxicity of DBAN and DCAN . The acute oral LD50 values (mg/kg) in mice and rats are: DBAN, mice: 289 (M), 303 (F); DBAN, rats: 245 (M), 361 (F); DCAN, mice: 270 (M), 279 (F); DCAN, rats: 339 (M), 330 (F) . Death was preceded by slowed respiration, depressed activity, prostration, and coma . There were no apparent compound-related gross pathological effects . DBAN (in corn oil) was administered by gavage to male and female CD rats for 14 or 90 days at levels of 23, 45, 90, and 180 mg/kg/day or 6, 23, and 45 mg/kg/day, respectively . Mortality was 100% at 180 mg/kg and 40% (M) and 20% (F) at 90 mg/kg/day . Compound-related mortality was 10% (M) and 5% (F) at 45 mg/kg and 0% (M) and 10% (F) at 23 mg/kg during the 90-day study . No consistent, significant, adverse compound-related effects on any of the parameters evaluated were evident . Possible target organs might be spleen, thymus, and liver . The no-observed adverse-effect level (NOAEL) for 14 days was 45 mg/kg/day and for 90 days was 23 mg/kg/day . DCAN (in corn oil) was administered by gavage to male and female CD rats for 14 or 90 days at levels of 12, 23, 45, and 90 mg/kg/day or 8, 33, and 65 mg/kg/day, respectively . There were no deaths during the 14-day study . Compound-related mortality was 50% (M) and 25% (F) at 65 mg/kg, 10% (M) and 5% (F) at 33 mg/kg, and 5% (M) and 0% (F) at 8 mg/kg during the 90-day study . Body weights were significantly lower at 90 and 65 mg/kg/day; weight and ratios of spleen and gonads and cholesterol levels were significantly lower at 90 mg/kg/day . No consistent, significant adverse compound-related effects on any of the parameters evaluated were evident . The NOAEL for 14 days was 45 mg/kg/day and for 90 days was 8 mg/kg/day.

Mol Cell Biol, 1986 Nov, 6(11), 3847 - 53
Constitutive and inducible Saccharomyces cerevisiae promoters: evidence for two distinct molecular mechanisms; Struhl K; his3 and pet56 are adjacent Saccharomyces cerevisiae genes that are transcribed in opposite directions from initiation sites that are separated by 200 base pairs . Under normal growth conditions, in which his3 and pet56 are transcribed at similar basal levels, a poly(dA-dT) sequence located between the genes serves as the upstream promoter element for both . In contrast, his3 but not pet56 transcription is induced during conditions of amino acid starvation, even though the critical regulatory site is located upstream of both respective TATA regions . Moreover, only one of the two normal his3 initiation sites is subject to induction . From genetic and biochemical evidence, I suggest that the his3-pet56 intergenic region contains constitutive and inducible promoters with different properties . In particular, two classes of TATA elements, constitutive (Tc) and regulatory (Tr), can be distinguished by their ability to respond to upstream regulatory elements, by their effects on the selection of initiation sites, and by their physical structure in nuclear chromatin . Constitutive and inducible his3 transcription is mediated by distinct promoters representing each class, whereas pet56 transcription is mediated by a constitutive promoter . Molecular mechanisms for these different kinds of S . cerevisiae promoters are proposed.

Mol Cell Biol, 1986 Nov, 6(11), 3774 - 84
Glycolytic gene expression in Saccharomyces cerevisiae: nucleotide sequence of GCR1, null mutants, and evidence for expression; Baker HV; In Saccharomyces cerevisiae, the gcr mutation is known to have a profound effect on the levels of most glycolytic enzymes, reducing them to 5% of normal or less in growth on noncarbohydrates . Here I report the preparation of chromosomal gcr insertion and deletion mutations . The null mutations were recessive, were not lethal, and caused a pattern of glycolytic enzyme deficiency similar to that seen earlier for the gcr1-1 allele, including the partial inducibility by glucose of the residual enzyme activities . DNA sequence analysis showed that GCR1 encoded a protein of molecular weight 94,414, with a very low codon bias index, characteristic of several S . cerevisiae regulatory genes; adjacent 5' and 3' sequences contained elements suggesting that it was transcribed, polyadenylated, and translated . RNA gel transfer hybridization experiments with purified polyadenylated RNA and a probe complementary to the 5' portion of the open reading frame showed that Ger was expressed as a polyadenylated transcript . Together with previous work, the present results suggest that the Gcr product may be a transcriptional factor necessary specifically for the high-level transcription of a limited set of genes whose products, the enzymes of glycolysis, constitute a substantial fraction of cell proteins and are responsible for the primary metabolic flux in many cells.

Genetics, 1986 Nov, 114(3), 753 - 67
Thymidine utilization by tut mutants and facile cloning of mutant alleles by plasmid conversion in S . cerevisiae; Sclafani RA et al.; Plasmid pJM81 contains a Herpes simplex virus thymidine kinase (TK) gene that is expressed in yeast . Cells containing the plasmid utilize thymidine (TdR) and the analogue 5-bromodeoxyuridine (BUdR) for specific incorporation into DNA . TdR auxotrophs, harboring plasmid pJM81 and a mutation in the yeast gene TMP1 require high concentrations of TdR (300 micrograms/ml) to support normal growth rates and the wild-type mitochondrial genome (rho+) cannot be maintained . We have identified a yeast gene, TUT1, in which recessive mutations allow efficient utilization of lower concentrations of TdR . Strains containing the mutations tmp1 and tut1, as well as plasmid pJM81, form colonies at 2 micrograms/ml TdR, grow at nearly normal rates and maintain the rho+ genome at 50 micrograms/ml TdR . These strains can be used to radiolabel DNA specifically and to synchronize DNA replication by TdR starvation . In addition, the substitution of BUdR for TdR allows the selective killing of DNA-synthesizing cells by 310-nm irradiation and allows the separation of replicated and unreplicated forms of DNA by CsCl equilibrium density banding . We also describe a unique, generally applicable system for cloning mutant alleles that exploits the fact that Tk+ yeast cells are sensitive to 5-fluorodeoxyuridine (FUdR) and that gene conversions can occur between a yeast chromosome and a TK-containing plasmid.

J Bacteriol, 1986 Oct, 168(1), 467 - 9
Mediation, by Saccharomyces cerevisiae translocation signals, of beta-lactamase transport through the Escherichia coli inner membrane and sensitive method for detection of signal sequences; Roggenkamp R et al.; Signal sequences of Saccharomyces cerevisiae invertase and alpha-factor pheromone were tested for the ability to mediate protein transport through the inner membrane of Escherichia coli by fusion to bacterial beta-lactamase lacking the signal sequence (blaS0) . Both types of transformants exhibited ampicillin resistance in accordance with the transport of the fused protein to the periplasmic compartment . This compartment contained most of the beta-lactamase activity present in the cell . Therefore, the tested yeast signal sequences, which conferred translocation of their proteins across the membrane of the endoplasmic reticulum in S . cerevisiae, can provide the same function in E . coli . The screening for ampicillin resistance among blaS0 fusions provides a convenient method for the isolation of functional yeast and possibly higher eucaryotic signal sequences.

Mol Cell Biol, 1986 Oct, 6(10), 3502 - 12
Amino-terminal fragments of delta 1-pyrroline-5-carboxylate dehydrogenase direct beta-galactosidase to the mitochondrial matrix in Saccharomyces cerevisiae; Brandriss MC et al.; delta 1-Pyrroline-5-carboxylate (P5C) dehydrogenase, the second enzyme in the proline utilization (Put) pathway of Saccharomyces cerevisiae and the product of the PUT2 gene, was localized to the matrix compartment by a mitochondrial fractionation procedure . This result was confirmed by demonstrating that the enzyme had limited activity toward an externally added substrate that could not penetrate the inner mitochondrial membrane (latency) . To learn more about the nature of the import of this enzyme, three gene fusions were constructed that carried 5'-regulatory sequences through codons 14, 124, or 366 of the PUT2 gene ligated to the lacZ gene of Escherichia coli . When these fusions were introduced into S . cerevisiae either on multicopy plasmids or stably integrated into the genome, proline-inducible beta-galactosidase was made . The shortest gene fusion, PUT2-lacZ14, caused the production of a high level of beta-galactosidase that was found exclusively in the cytoplasm . The PUT2-lacZ124 and PUT2-lacZ366 fusions made lower levels of beta-galactosidases that were mitochondrially localized . Mitochondrial fractionation and protease-protection experiments showed that the PUT2-lacZ124 hybrid protein was located exclusively in the matrix, while the PUT2-lacZ366 hybrid was found in the matrix as well as the inner membrane . Thus, the amino-terminal 124 amino acids of P5C dehydrogenase carries sufficient information to target and deliver beta-galactosidase to the matrix compartment . The expression of the longer hybrids had deleterious effects on cell growth; PUT2-lacZ366-containing strains failed to grow on proline as the sole source of nitrogen . In the presence of the longest hybrid beta-galactosidase, the wild-type P5C dehydrogenase was still properly localized in the matrix compartment, but its activity was reduced . The nature of the effects of these hybrid proteins on cell growth is discussed.

Mol Cell Biol, 1986 Oct, 6(10), 3401 - 9
Repair of heteroduplex plasmid DNA after transformation into Saccharomyces cerevisiae; Bishop DK et al.; Purified heteroduplex plasmid DNAs containing 8- or 12-base-pair insertion mismatches or AC or CT substitution mismatches were used to transform Saccharomyces cerevisiae . Two insertion mismatches, separated by 943 base pairs, were repaired independently of each other at least 55% of the time . This suggested that repair tracts were frequently shorter than 1 kilobase . The two insertion mismatches were repaired with different efficiencies . Comparison of the repair efficiency of one mismatched site with or without an adjacent mismatch suggests that mismatches promote their own repair and can influence the repair of neighboring mismatches . When two different plasmids containing single-insertion mismatches were transformed into S . cerevisiae cells, a slight preference towards insertion was detected among repair products of one of the two plasmids, while no repair preference was detected among transformants with the second plasmid.

Biochemistry, 1986 Sep 9, 25(18), 5117 - 25
Identification and assignment of base pairs in three helical stems of Torulopsis utilis ribosomal 5S RNA and its RNase T1 and RNase T2 cleaved fragments via 500-MHz proton homonuclear overhauser enhancements; Chen SM et al.; Imino proton resonances in the downfield region (10-14 ppm) of the 500-MHz 1H NMR spectrum of Torulopsis utilis 5S RNA are identified (A X U, G X C, or G X U) and assigned to base pairs in helices I, IV, and V via analysis of homonuclear Overhauser enhancements (NOE) from intact T . utilis 5S RNA, its RNase T1 and RNase T2 digested fragments, and a second yeast (Saccharomyces cerevisiae) 5S RNA whose nucleotide sequence differs at only six residues from that of T . utilis 5S RNA . The near-identical chemical shifts and NOE behavior of most of the common peaks from these four RNAs strongly suggest that helices I, IV, and V retain the same conformation after RNase digestion and that both T . utilis and S . cerevisiae 5S RNAs share a common secondary and tertiary structure . Of the four G X U base pairs identified in the intact 5S RNA, two are assigned to the terminal stem (helix I) and the other two to helices IV and V . Seven of the nine base pairs of the terminal stem have been assigned . Our experimental demonstration of a G X U base pair in helix V supports the 5S RNA secondary structural model of Luehrsen and Fox {Luehrsen, K . R., & Fox, G.E . (1981) Proc . Natl . Acad . Sci . U.S.A . 78, 2150-2154} . Finally, the base-pair proton peak assigned to the terminal G X U in helix V of the RNase T2 cleaved fragment is shifted downfield from that in the intact 5S RNA, suggesting that helices I and V may be coaxial in intact T . utilis 5S RNA.

Eur J Biochem, 1986 Sep 1, 159(2), 239 - 45
Isolation and characterization of a lectin from tulip bulbs, Tulipa gesneriana; Oda Y et al.; A lectin, which agglutinated specifically the yeast cells of the Saccharomyces genus, was isolated from tulip bulbs (Tulipa gesneriana) using affinity chromatography on mannan-Sepharose 4B . Its relative molecular mass was determined by gel filtration to be approximately 67,000 . On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, a relative molecular mass of 17,000 was obtained, suggesting that the lectin is a tetramer . Binding studies performed with iodinated lectin indicated that Saccharomyces cerevisiae cells contained approximately 5.7 X 10(6) binding sites per cell, whereas little binding was observed with yeasts other than the Saccharomyces genus, bacteria and animal erythrocytes . D-Mannose, D-mannose 6-phosphate, L-fucose and L-fucosylamine were potent inhibitors of the lectin binding to S . cerevisiae cells, while, D-glucose, D-galactose and D-mannosamine were inactive, indicating that hydroxyl group at C-2 of D-mannose was essential for the lectin binding . Furthermore, inhibition experiments, using various manno-oligosaccharides, suggested that the lectin recognized (1----6)-linked manno-oligosaccharide units larger than mannobiose.

J Bacteriol, 1986 Sep, 167(3), 981 - 5
Characteristics of sterol uptake in Saccharomyces cerevisiae; Lorenz RT et al.; A Saccharomyces cerevisiae sterol auxotroph, FY3 (alpha hem1 erg7 ura), was used to probe the characteristics of sterol uptake in S . cerevisiae . The steady-state cellular concentration of free sterol at the late exponential phase of growth could be adjusted within a 10-fold range by varying the concentration of exogenously supplied sterol . When cultured on 1 microgram of sterol ml-1, the cells contained a minimal cellular free-cholesterol concentration of 0.85 nmol/mg (dry weight) and were termed sterol depleted . When cultured on 11 micrograms of sterol ml-1 or more, the cells contained a maximal cellular free-cholesterol concentration of 6.8 nmol/mg (dry weight) and were termed free sterol saturated . Cells with free-sterol concentrations below the maximal level were capable of accumulating free sterol from the medium . The capacity of the cells for cholesterol uptake was inversely proportional to the initial intracellular concentration . The uptake of sterol was shown to be a nonactive process that is independent of cellular energy sources or viability . The intracellular transport of sterol for esterification is not sensitive to anti-microtubule agents.

Endocrinology, 1986 Sep, 119(3), 1362 - 9
Unexpected presence of estrogens in culture medium supplements: subsequent metabolism by the yeast Sacchromyces cerevisiae; Miller SC et al.; We have previously shown the presence of 17 beta-estradiol in extracts of commercially prepared Saccharomyces cerevisiae ss well as the production of estradiol by yeast grown in the laboratory . In our current study, yeast grown in a chemically defined medium synthesized estradiol in only small amounts, (less than 500 pg/liter) . We have analyzed a variety of media commonly used for growing yeast and found that substantial estradiol production (greater than 5 ng/liter) was obtained when yeast were grown in medium supplemented with Bacto-peptone . The peptone was shown to contain significant amounts of estrone, and the results of the experiments establish a precursor-product relationship where estrone from the medium is metabolized to estradiol by S . cerevisiae . Studies with added {3H}estrone demonstrated rapid conversion into {3H}estradiol and a 3H-labeled nonpolar estrogen derivative . The commercially obtained yeast used previously had been grown in a molasses medium . We demonstrate here that the molasses medium contains substantial amounts of estrone and estradiol . We conclude that the conversion of estrone in a culture medium to estradiol in laboratory grown yeast and estrone and estradiol present in the commercially grown yeast medium account for the majority of estradiol found in yeast.

Mol Biol (Mosk), 1986 Sep-Oct, 20(5), 1273 - 80
{Isolation of recipient yeast strains for the stable reproduction of 2-micron DNA vectors}; Nevzgliadova OV et al.; Most Leu- clones of yeast transformants (cir0, pJDB219) can stabilize the replication of 2 micron-vectors with REP3, the stability obtained being comparable to the one for the standard cir0 strain . One of the Leu- clones was used to isolate a plasmid with Rep 1.2 functions ("Rep-helper plasmid") . The plasmid was shown to carry a partially active LEU2 gene by transforming both E . coli and S . cerevisiae to Leu+ phenotype . A restriction analysis performed demonstrated that the Rep-helper plasmid has lost approximately 1.9 kb compared to the parent pJDB219, deletion and rearrangement having taken place at the bacterial and 2 mem components boundary . The Rep-helper plasmid carrying host strains allows to quantify the REP3 function on different 2 microns vectors . Some but not all cir+ stabilized vectors show greater stability in Rep-helper strains compared to the standard cir0 ones . Manipulating the Rep-helper plasmid level, by selecting for Leu+ phenotype, stabilized REP3 +/- plasmid p3030, but mostly destabilizes REP3+ plasmid YEp13HIS3.

Mol Gen Genet, 1986 Sep, 204(3), 397 - 403
Yeast LEU5 is a PET-like gene that is not essential for leucine biosynthesis; Drain P et al.; Alpha-IPM synthase catalyzes the first committed step in leucine biosynthesis in the yeast S . cerevisiae . LEU4 is known to encode this enzyme activity . A second gene, LEU5, has been proposed to encode a second enzyme with this activity . We cloned LEU5 and genetically defined the locus . LEU5 maps to chromosome VIII and is tightly linked to CEN8 . Five different mutations in LEU5 were analyzed: a site-directed deletion and a disruption, as well as three distinct mutations produced by chemical mutagenesis . In a leu4 background, each leu5 mutation causes a Leu--phenotype; in a LEU4 background, none of the mutations alters the Leu+ phenotype . This shows that LEU5 is not essential for leucine biosynthesis . In either a leu4 or LEU4 background, each leu5 mutation causes a glycerol--phenotype . This operationally defines LEU5 as a PET gene . Two distinct suppressors of the Pet--phenotype of leu5 strains have been isolated . These suppressors revert the Pet--phenotype of each of four mutant leu5 alleles that were tested . Suppression occurs regardless of the allele at LEU4 . Moreover, the suppressors co-revert the Leu--phenotype for each of the four leu5 mutations that is combined with a leu4 allele . This establishes the presence of a gene other than LEU5 that encodes a second alpha-IPM synthase . Further analysis provided no evidence for synthase activity that is encoded by LEU5.

J Steroid Biochem, 1986 Sep, 25(3), 299 - 304
Yeast alpha-factor and somatostatin enhance binding of {3H}estradiol to proteins in rat pancreas and Saccharomyces cerevisiae; Grossman A et al.; Pancreatic tissue contains an {3H}estradiol-binding protein that requires a coligand in the steroid-binding reaction . The endogenous coligand appears to be the tetradecapeptide somatostatin . Yeast alpha-factor, a tridecapeptide pheromone that induces conjugation between haploid cells of opposite mating type, was found to be as effective as somatostatin in enhancing specific binding of {3H}estradiol to partially purified pancreatic protein . Supernatant fractions from yeast cells also contain an {3H}estradiol-binding protein . alpha-Factor can enhance specific binding of {3H}estradiol to such yeast fractions . Somatostatin, somatostatin analogues, and an analogue of alpha-factor enhanced binding of {3H}estradiol but did not inhibit cell growth or induce morphological changes in S . cerevisiae . Thus, it appears that coligand-requiring {3H}estradiol-binding activity and mating in yeast are not directly related.

Cell, 1986 Aug 29, 46(5), 733 - 40
Double-strand breaks can initiate meiotic recombination in S . cerevisiae; Kolodkin AL et al.; We investigated the effects of double-strand breaks on meiotic recombination in yeast . A double-strand break was introduced at the MATa locus by sporulation of a MAT alpha inc/MATa diploid under inducing conditions for the HO-encoded endonuclease; 14% of the resulting tetrads had undergone 4 alpha:0a conversion . Conversion at MAT was associated with co-conversion of a closely linked marker and an increased recombination frequency for flanking markers . We also studied the sporulation products of a diploid heterozygous at the HIS4 locus for an insertion of a 100 bp fragment of MATa containing the HO endonuclease cut site . Under inducing conditions, a significant number of tetrads were formed that had undergone gene conversions in favor of the HIS4+ allele . Although double-strand breaks can initiate meiotic recombination in yeast, the data suggest that they do not normally do so.

J Mol Biol, 1986 Aug 20, 190(4), 519 - 28
The Saccharomyces cerevisiae ADE5,7 protein is homologous to overlapping Drosophila melanogaster Gart polypeptides; Henikoff S; The Drosophila melanogaster Gart locus encodes two polypeptides specified by overlapping alternative transcripts . One transcript encodes only glycinamide ribotide synthetase (GARSase) on a 45,000 Mr polypeptide, while the other encodes GARSase aminoimidazole ribotide synthetase (AIRSase), and glycinamide ribotide transformylase (GARTase) on a 145,000 Mr polypeptide . In Saccharomyces cerevisiae, glycinamide ribotide synthetase and aminoimidazole ribotide synthetase are encoded at the ADE5,7 locus . Here I report the cloning, sequencing, and determination of the transcriptional organization of the yeast ADE5,7 gene . There is sufficient homology to align the predicted 802 amino acid ADE5,7 polypeptide with its Drosophila counterpart . These results, together with the sequence of the S . cerevisiae ADE8 gene encoding glycinamide ribotide transformylase, show that the entire Drosophila large polypeptide can be accounted for by the three enzymatic activities . A novel finding is that successive Drosophila domains are each homologous to the aminoimidazole ribotide synthetase portion of the yeast ADE5,7 gene, such that regions of homology with yeast aminoimidazole ribotide synthetase alternate from one of the Drosophila repeats to the other . Such a relationship suggests that the two Drosophila aminoimidazole ribotide synthetase domains together participate in catalysis . This model is consistent with a 20-year-old explanation of complex interallelic complementation such as that characterizing the gene segment encoding yeast aminoimidazole ribotide synthetase.

Cell, 1986 Aug 15, 46(4), 541 - 50
Site-specific recombination promotes plasmid amplification in yeast; Volkert FC et al.; All stable, naturally occurring circular yeast DNA plasmids contain a pair of long, nontandem inverted repeats that undergo frequent reciprocal recombination . This yields two plasmid inversion isomers that exist in the cell in equal numbers . In the 2 mu circle plasmid of S . cerevisiae such inversion is catalyzed by a plasmid-encoded site-specific recombinase, FLP . We show that the site-specific recombination system of 2 mu circle enables the plasmid to increase its mean intracellular copy number in yeast cells growing under nonselective conditions . This apparently occurs by a FLP-induced transient shift in the mode of replication from theta to double rolling circle as initially proposed by Futcher . This capability may ensure stable maintenance of the plasmid by enabling it to correct downward deviations in copy number that result from imprecision of the plasmid-encoded partitioning system.

Biochem Biophys Res Commun, 1986 Aug 14, 138(3), 1110 - 5
Cloning of Lentinus edodes mitochondrial DNA fragment capable of autonomous replication in Saccharomyces cerevisiae; Katayose Y et al.; Mitochondrial (mt) DNA of the higher basidiomycetes Lentinus edodes with a molecular weight of about 69 kb was partially digested with Sau3AI, cloned with plasmid YIp32 (a hybrid of pBR322 and the yeast leu2 gene) and analyzed for sequences capable of autonomous replication (ARSs) in the eukaryote Saccharomyces cerevisiae . One recombinant plasmid was isolated which contained 3.2 kb fragment of the mtDNA with ARS activity . This plasmid (named pSK52) exhibited a high-frequency yeast transformation and was found to be maintained within the cell as an extrachromosomal element . The stability and copy number properties of pSK52 were similar to those of the recombinant plasmid of YIp32 and S . cerevisiae mt-ARS constructed as a reference . Subcloning experiments were carried out to assess the localization of ARS on the above 3.2 kb fragment, revealing that the fragment contains at least two ARSs.

FEBS Lett, 1986 Aug 11, 204(1), 129 - 33
Archaebacterial and eukaryotic ribosomal subunits can form active hybrid ribosomes; Altamura S et al.; Purified ribosomal subunits from the extremely thermoacidophilic archaebacterium Sulfolobus solfataricus are able to recognize ribosomal subunits from the yeast Saccharomyces cerevisiae forming hybrid monosomes that can be revealed by sucrose gradient analysis and are active in peptide bond formation . Both reciprocal combinations (archaebacterial 30 S + eukaryotic 60 S and archaebacterial 50 S + eukaryotic 40 S) are functional . In contrast, no hybrid couples are formed between subunits of yeast and Escherichia coli ribosomes . These results indicate that ribosomes of at least one archaebacterial species share specific structural features with those of the lower eukaryote S . cerevisiae.

Mol Cell Biol, 1986 Aug, 6(8), 2828 - 38
Genetic manipulation of Saccharomyces cerevisiae by use of the LYS2 gene; Barnes DA et al.; The structural gene for alpha-aminoadipate reductase (LYS2) was isolated from a Saccharomyces cerevisiae genomic DNA library by complementation of a lys2 mutant . Both genetic and biochemical criteria confirmed that the DNA obtained corresponds to the LYS2 locus on chromosome II . Subcloning and deletion analysis showed that a functional LYS2 gene is contained within a 4.6-kilobase (kb) EcoRI-HindIII fragment of the original insert, and the slightly larger EcoRI-ClaI segment (4.8 kb) was used to construct a series of cloning vehicles, including integrating, episomal, replicative, and centromeric vectors . The cloned DNA was also used to generate a genomic deletion that lacks all LYS2 coding sequences on chromosome II . The level of the LYS2 transcript (4.2 kb) was 10-fold higher in cells grown on minimal medium than in cells grown on complete medium and was not repressed by the presence of lysine alone . Gene disruption, gene replacement, and promoter analysis of the major alpha-factor structural gene (MF alpha 1) were performed to illustrate the utility of the LYS2 gene for the genetic manipulation of yeasts . Because all fungi synthesize lysine via the alpha-aminoadipate pathway, the techniques developed here for using the S . cerevisiae LYS2 gene should be directly applicable to other fungal systems.

Biochem Cell Biol, 1986 Aug, 64(8), 717 - 21
The RPO31 gene of Saccharomyces cerevisiae encodes the largest subunit of RNA polymerase III; Moyle M et al.; The 5' end of the RNA transcript of the Saccharomyces cerevisiae RNA polymerase related gene, RPO31, was localized on the RPO31 locus using Northern analyses and primer extension experiments . The amino terminal protein sequence was determined for the largest (relative mass approximately 160,000) subunit polypeptide of S . cerevisiae RNA polymerase III . The N-terminal amino acid sequence of this polypeptide was shown to be identical to that predicted for a putative RPO31 polypeptide initiated at the first ATG codon located 208-216 base pairs 3' to the major RPO31 mRNA start sites . The RPO31 locus thus encodes the largest subunit of S . cerevisiae RNA polymerase III.

J Bacteriol, 1986 Aug, 167(2), 713 - 5
In vivo phosphorylation of Saccharomyces cerevisiae ribosomal protein S10 by cyclic-AMP-dependent protein kinase; Otaka E et al.; Using wild-type Saccharomyces cerevisiae strains and mutants which are defective in the regulatory subunit of cyclic-AMP-dependent protein kinase (bcy1) and phosphoprotein phosphatase activity (ppd1), we demonstrated that a cyclic-AMP-dependent protein kinase phosphorylated the S . cerevisiae ribosomal protein S10 in vivo . S10 was not dephosphorylated in bcy1 or ppd1 mutants after heat shock . The phosphorylated forms of S10 were diminished during the stationary phase in bcy1 and ppd1 mutants as well as in wild-type cells.

J Bacteriol, 1986 Aug, 167(2), 551 - 5
Construction of a host-vector system in Candida maltosa by using an ARS site isolated from its genome; Takagi M et al.; To construct a host-vector system in an n-alkane-assimilating yeast, Candida maltosa, the isolation of an ARS site from its genome which replicates autonomously in C . maltosa was attempted . Leu- mutants of C . maltosa were transformed with a gene library prepared by using YEp13 (LEU2+) as a vector, and Leu+ transformants were obtained at a high frequency . A plasmid named pCS1 was isolated from the recipient cells . pCS1 contained a 6.3-kilobase (kb) fragment of the C . maltosa genome, and a 3.8-kb fragment with ARS activity was subcloned and designated the TRA (transformation ability) region . Vectors (pTRA1 and pTRA11) for C . maltosa J288 were constructed that contained this 3.8-kb fragment, pBR322, and the LEU2 gene of Saccharomyces cerevisiae . Transformation of C . maltosa J288 with these plasmids was successful by both spheroplast and lithium acetate methods . Southern blot analysis suggested that the copy number of pTRA1 in C . maltosa was between 10 and 20, and it was stably maintained during growth without selective pressure in the medium . It was also found that these vectors could transform S . cerevisiae leu2- to LEU2+, suggesting that the TRA region contained an ARS site(s) that was specific not only for C . maltosa but also for S . cerevisiae.

Cell, 1986 Aug 1, 46(3), 345 - 53
Down regulation of the alpha-factor pheromone receptor in S . cerevisiae; Jenness DD et al.; The peptide pheromone, alpha-factor, was found to elicit down regulation of receptor sites on yeast a cell targets . Cellular uptake of alpha-factor accompanied the loss of receptor sites . Receptor-deficient a cells bearing a deletion of the STE2 gene were unable to internalize alpha-factor . Cultures were found to reaccumulate receptor sites following the initial period of down regulation; reaccumulation was dependent upon protein synthesis . Pheromone-resistant mutants, ste4-3 and ste5-3, retained the ability to down regulate receptors but failed to show reaccumulation . Our results suggest that alpha-factor-receptor complexes enter the cell by receptor-mediated endocytosis and that receptors are continuously lost and resynthesized in the presence of alpha-factor . We found no reduction of alpha-factor binding capacity in a cell cultures that had adapted to alpha-factor.

Cell, 1986 Jul 18, 46(2), 235 - 43
The HTS1 gene encodes both the cytoplasmic and mitochondrial histidine tRNA synthetases of S . cerevisiae; Natsoulis G et al.; The gene encoding the histidine-tRNA synthetase (HTS1) has two in-frame translation start sites located 60 bp apart . One set of HTS1 transcripts (long) initiates upstream of both ATG codons, and the other set (short) initiates between the two ATG codons and therefore contains only the downstream ATG . A mutation that destroys the first AUG on the long message results in the Pet- (respiratory deficient) phenotype, but does not affect either the level of the cytoplasmic histidine-tRNA synthetase or viability . Mutations distal to the second ATG lead to loss of cytoplasmic synthetase function, lethality and respiratory deficiency . These phenotypes can be explained if the longer message were to encode the mitochondrial synthetase and the shorter message were to encode the cytoplasmic histidine-tRNA synthetase.

Cell, 1986 Jul 18, 46(2), 213 - 25
The S . cerevisiae structural gene for chitin synthase is not required for chitin synthesis in vivo; Bulawa CE et al.; The chitin synthase of Saccharomyces is a plasma membrane-bound zymogen . Following proteolytic activation, the enzyme synthesizes insoluble chitin that has chain length and other physical properties similar to chitin found in bud scars . We isolated mutants lacking chitin synthase activity (chs1) and used these to clone CHS1 . The gene has an open reading frame of 3400 bases and encodes a protein of 130 kd . The fission yeast S . pombe lacks chitin synthase and chitin . When a plasmid encoding a CHS1-lacZ fusion protein is introduced into S . pombe, both enzymatic activities are expressed in the same ratio as in S . cerevisiae, demonstrating that CHS1 encodes the structural gene of chitin synthase . Three CHS1 gene disruption experiments were performed . In all cases, strains with the disrupted gene have a recognizable phenotype, lack measurable chitin synthase activity in vitro but are viable, contain normal levels of chitin in vivo, and mate and sporulate efficiently.

Nucleic Acids Res, 1986 Jul 11, 14(13), 5187 - 97
Yeast omnipotent supressor SUP1 (SUP45): nucleotide sequence of the wildtype and a mutant gene; Breining P et al.; The primary structures of the yeast recessive omnipotent suppressor gene SUP1 (SUP45) and one of its mutant alleles (sup1-ts36) was determined . The gene codes for a protein of 49 kD . The mutant protein differs from the wildtype form in one amino acid residue (Ser instead of Leu) in the N-terminal part . The codon usage differs significantly from that of yeast ribosomal protein genes . However, an upstream element resembling a conserved oligonucleotide in the region 5' to ribosomal protein genes in S . cerevisiae has been found . A DNA probe internal to the SUP1 gene does not exhibit detectable homology to genomic DNA neither from higher eucaryotes nor from eu- or archaebacteria . The hypothetical function of this protein in control of translational fidelity is discussed.

J Gen Microbiol, 1986 Jul, 132 ( Pt 7), 2053 - 6
Water stress plating hypersensitivity of yeasts; Mackenzie KF et al.; Saccharomyces cerevisiae, when growing exponentially in batch culture, passed through a phase in which, on average, one cell in 10(4) survived plating onto a low water activity (aw) agar medium . Stationary phase cultures were resistant as were all other species tested, with the exception of Candida krusei . In continuous culture, S . cerevisiae was more resistant at low than at high dilution rates . Plating at low aw was lethal to those cells that were not protected by an adequate content of compatible solute . In naturally resistant yeasts and in S . cerevisiae that had been exposed to an adaptation process, the compatible solute was one or more types of polyhydric alcohol . Resistance in stationary phase was attributable to a different cause.

J Gen Microbiol, 1986 Jul, 132 ( Pt 7), 2023 - 34
Effect of osmotic stress on the ultrastructure and viability of the yeast Saccharomyces cerevisiae; Morris GJ et al.; Exposure of the yeast Saccharomyces cerevisiae to hypertonic solutions of non-permeating compounds resulted in cell shrinkage, without plasmolysis . The relationship between cell volume and osmolality was non-linear; between 1 and 4 osM there was a plateau in cell volume, with apparently a resistance to further shrinkage; beyond 4 osM cell volume was reduced further . The loss of viability of S . cerevisiae after hypertonic stress was directly related to the reduction in cell volume in the shrunken state . The plasma membrane is often considered to be the primary site of osmotic injury, but on resuspension from a hypertonic stress, which would have resulted in a major loss of viability, all cells were osmotically responsive . The effects of osmotic stress on mitochondrial activity and structure were investigated using the fluorescent probe rhodamine 123 . The patterns of rhodamine staining were altered only after extreme stress and are assumed to be a pathological feature rather than a primary cause of injury . Changes in the ultrastructure of the cell envelope were examined by freeze-fracture and scanning electron microscopy . In shrunken cells the wall increased in thickness, the outer surface remained unaltered, whilst the cytoplasmic side buckled with irregular projections into the cytoplasm . On return to isotonic solutions these structural alterations were reversible, suggesting a considerable degree of plasticity of the wall . However, the rate of enzyme digestion of the wall may have been modified, indicating that changes in wall structure persist.

Mol Cell Biol, 1986 Jul, 6(7), 2674 - 83
Intron mutations affect splicing of Saccharomyces cerevisiae SUP53 precursor tRNA; Strobel MC et al.; The Saccharomyces cerevisiae amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the role of intron structure and sequence on precursor tRNA splicing in vivo and in vitro . This gene encodes a pre-tRNA which contains a 32-base intervening sequence . Two types of SUP53 intron mutants were constructed: ones with an internal deletion of the natural SUP53 intron and ones with a novel intron . These mutant genes were transcribed in vitro, and the end-processed transcripts were analyzed for their ability to serve as substrates for the partially purified S . cerevisiae tRNA endonuclease and ligase . The in vitro phenotype of these mutant RNAs was correlated with the in vivo suppressor tRNA function of these SUP53 alleles after integration of the genes into the yeast genome . Analysis of these mutant pre-tRNAs, which exhibited no perturbation of the mature domain, clearly showed that intron structure and sequence can have profound effects on pre-tRNA splicing . All of the mutant RNAs, which were inefficiently spliced or unspliced, evidenced cleavage only at the 5' splice junction . Base changes in the intron proximal to the 3' splice junction could partially rescue the splicing defect . The implications of these data for tRNA endonuclease-substrate interactions are discussed.

Mol Cell Biol, 1986 Jul, 6(7), 2663 - 73
Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo; Strobel MC et al.; The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function . This gene encodes a pre-tRNA which contains a 32-base intron . The mature tRNASUP53 contains a 5-methylcytosine modification of the anticodon wobble base . Mutations were made in the SUP53 intron . These mutant genes were transcribed in an S . cerevisiae nuclear extract preparation . In this extract, primary tRNA gene transcripts are end-processed and base modified after addition of cofactors . The base modifications made in vitro were examined, and the mutant pre-tRNAs were analyzed for their ability to serve as substrates for partially purified S . cerevisiae tRNA endonuclease and ligase . Finally, the suppressor function of these mutant tRNA genes was assayed after their integration into the S . cerevisiae genome . Mutant analysis showed that the totally intact precursor tRNA, rather than any specific sequence or structure of the intron, was necessary for efficient nonsense suppression by tRNASUP53 . Less efficient suppressor activity correlated with the absence of the 5-methylcytosine modification . Most of the intron-altered precursor tRNAs were successfully spliced in vitro, indicating that modifications are not critical for recognition by the tRNA endonuclease and ligase.

Mol Cell Biol, 1986 Jul, 6(7), 2638 - 45
Proline utilization in Saccharomyces cerevisiae: analysis of the cloned PUT1 gene; Wang SS et al.; The PUT1 gene was isolated by functional complementation of a put1 (proline oxidase-deficient) mutation in Saccharomyces cerevisiae . Three independent clones with overlapping inserts of 6.8, 10.5, and 11 kilobases (kb) were isolated from S . cerevisiae genomic libraries in YEp24 (2 micron) and YCp50 (CEN) plasmids . The identity of the PUT1 gene was determined by a gene disruption technique, and Southern hybridization and genetic analyses confirmed that the bona fide gene had been cloned . Plasmids containing the PUT1 gene restored regulated levels of proline oxidase activity to put1 recipient strains . The PUT1 DNA was present in a single copy in the yeast genome and encoded a transcript of ca . 1.5 kb . S1 nuclease protection experiments were used to determine the direction of transcription of the PUT1 message and to localize its 5' and 3' termini within a subcloned 3-kb DNA fragment . Approximately 50-fold more PUT1-specific mRNA was detected in induced (proline-grown) cells than in uninduced (ammonia-grown) cells . A yeast strain carrying the previously identified put3 regulatory mutation that caused constitutive levels of proline oxidase activity was found to have sevenfold elevated PUT1 mRNA levels under noninducing conditions . The absence of a functional electron transport system in vegetative petite (rho-) strains interfered with their ability to use proline as a nitrogen source . Although these strains were Put- and made no detectable proline oxidase activity, PUT1 message was detected under inducing conditions . The PUT1 gene was mapped distal to the GAL2 gene on chromosome XII by tetrad analysis.

Mol Cell Biol, 1986 Jul, 6(7), 2429 - 35
Transcriptional regulation of ribosomal proteins during a nutritional upshift in Saccharomyces cerevisiae; Donovan DM et al.; The relative rates of synthesis of Saccharomyces cerevisiae ribosomal proteins increase coordinately during a nutritional upshift . We constructed a gene fusion which contained 528 base pairs of sequence upstream from and including the TATA box of ribosomal protein gene rp55-1 (S16A-1) fused to a CYC1-lacZ fusion . This fusion was integrated in single copy at the rp55-1 locus in the yeast genome . During a nutritional upshift, in which glucose was added to cells growing in an ethanol-based medium, we found that the increase in the relative rate of synthesis of the beta-galactosidase protein product followed the same kinetics as the change in relative rates of synthesis of several ribosomal proteins measured in the same experiment . This demonstrates that the nontranscribed sequences upstream from the rp55-1 gene, which are present in the fusion, are sufficient to mediate the change in rates of synthesis characteristic of ribosomal proteins under these conditions . The results also suggest that a change in transcription rates is mainly responsible for the increase in relative rates of synthesis of ribosomal proteins during a nutritional upshift in S . cerevisiae.

Exp Cell Res, 1986 Jul, 165(1), 199 - 206
Cytological characterization of NOR in the bivalent of Saccharomyces cerevisiae; Kuroiwa T et al.; We cytologically characterized the nucleolar organizer region (NOR) on the bivalent in the yeast Saccharomyces cerevisiae . We used staining with 4'-6-diamidino-2-phenylindole (DAPI), chromomycin A3, and silver nitrate and in situ hybridization technique and utilized a video-intensified microscope system with an ultra-high-sensitive video camera . The results showed that of 16 bivalents of S . cerevisiae, the longest was a recognizable nucleolar chromosome which has an annular and synaptonemal complexless NOR in its submedian portion . The NOR was comprised of 2.65 X 10(9) D DNA which corresponded to 118 copies per haploid of rDNA repeating units . This evidence is discussed in terms of the possible participation of the annular NOR in suppressing the meiotic recombination of the rDNA gene clusters.

Biochim Biophys Acta, 1986 Jun 26, 858(2), 309 - 11
Effect of tunicamycin on thiamine transport in Saccharomyces cerevisiae; Nosaka K et al.; The activity of thiamine transport in Saccharomyces cerevisiae was decreased by the treatment with tunicamycin without affecting the growth of yeast cells . Although the total activity of a soluble thiamine-binding protein in yeast periplasm, which is known to be a glycoprotein, was decreased by tunicamycin treatment, the activity of thiamine uptake by yeast protoplasts was inhibited as much as by whole cells . Furthermore, tunicamycin decreased the activity of the membrane-bound thiamine-binding protein in a dose dependent way and in parallel with the thiamine transport activity . These findings suggested that the membrane-bound thiamine-binding protein is a glycoprotein which plays a functional role in thiamine transport in S . cerevisiae.

Mol Cell Biol, 1986 Jun, 6(6), 2089 - 97
An RNA polymerase I enhancer in Saccharomyces cerevisiae; Elion EA et al.; By the use of an artificial gene coding for rRNA (rDNA gene), we found that transcription of the major precursor rRNA in Saccharomyces cerevisiae cells is stimulated 15-fold by a positive control element located 2 kilobases upstream of the transcription initiation site . Analysis of in vitro runon transcripts suggests that this promoter element increases the frequency of initiation by RNA polymerase I molecules . A 190-base-pair fragment encompassing the promoter element can stimulate transcription on a centromere plasmid in either orientation, upstream or downstream of the transcription initiation site, suggesting that it is an enhancer element . Integration of artificial rDNA genes into a nonribosomal locus in the genome demonstrates that the rDNA enhancer functions either 5' or 3' to an rRNA transcription unit, suggesting it may operate in both directions within the rDNA tandem array . This is the first observation in S . cerevisiae of the stimulation of transcription by an element placed downstream . Finally, enhancer activity is dependent upon sequences that lie at both boundaries of the 190-base-pair fragment . In particular, a 5-base-pair deletion at the extreme 3' boundary of the 190-base-pair fragment greatly reduces the activation of transcription and implicates a set of inverted repeats.

EMBO J, 1986 Jun, 5(6), 1193 - 7
Expression of murine and human granulocyte-macrophage colony-stimulating factors in S . cerevisiae: mutagenesis of the potential glycosylation sites; Miyajima A et al.; Murine (m) and human (h) granulocyte--macrophage colony-stimulating factors (GM-CSF) have been expressed in large quantities in Saccharomyces cerevisiae using a secretion vector containing the promoter and leader sequences of the mating pheromone alpha-factor . Functionally active mGM-CSF was identified by a proliferation assay with a factor-dependent cell line and by a granulocyte--macrophage colony formation assay using bone marrow cells . The activity of hGM-CSF was confirmed by stimulation of granulocyte--macrophage colony formation using human cord blood cells . Murine GM-CSF with various apparent mol . wts (13, 18, 24, 34 and 40 kd, as well as a smear of higher mol . wts) was detected in yeast culture medium by protein blotting using a rat monoclonal antibody specific for the mGM-CSF N-terminal region peptide . Protein blotting using a rat monoclonal antibody specific for the hGM-CSF N-terminal region demonstrated that a 15.6-kd and higher mol . wt heterogeneous species were secreted . Mutations introduced at each of the two potential N-linked glycosylation sites in mGM-CSF showed that the 13-kd protein is not glycosylated and the major 18-kd protein is mainly glycosylated at the more C-terminal site, whereas the heterogeneous higher mol . wt species were not affected by the mutations . The N-terminal amino acid of the 13-kd protein was shown to be Ser which was four amino acids in the C-terminal direction from the fusion point.

J Bacteriol, 1986 Jun, 166(3), 763 - 8
Adenosine accumulation in Saccharomyces cerevisiae cultured in medium containing low levels of adenine; Laten HM et al.; By monitoring the in vivo incorporation of low concentrations of radiolabeled adenine into acid-soluble compounds, we observed the unusual accumulation of two nucleosides in Saccharomyces cerevisiae that were previously considered products of nucleotide degradation . Under the culture conditions used in the present study, radiolabeled adenosine was the major acid-soluble intracellular derivative, and radiolabeled inosine was initially detected as the second most prevalent derivative in a mutant lacking adenine aminohydrolase . The use of yeast mutants defective in the conversion of adenine to hypoxanthine or to AMP renders very unlikely the possibility that the presence of adenosine and inosine is attributable to nucleotide degradation . These data can be explained by postulating the existence of two enzyme activities not previously reported in S . cerevisiae . The first of these activities transfers ribose to the purine ring and may be attributable to purine nucleoside phosphorylase (EC 2.4.2.1) or adenosine phosphorylase (EC 2.4.2.-) . The second enzyme converts adenosine to inosine and in all likelihood is adenosine aminohydrolase (EC 3.5.4.4).

Mol Cell Biol, 1986 Jun, 6(6), 1936 - 42
Saccharomyces cerevisiae contains two functional citrate synthase genes; Kim KS et al.; The tricarboxylic acid cycle occurs within the mitochondria of the yeast Saccharomyces cerevisiae . A nuclear gene encoding the tricarboxylic acid cycle enzyme citrate synthase has previously been isolated (M . Suissa, K . Suda, and G . Schatz, EMBO J . 3:1773-1781, 1984) and is referred to here as CIT1 . We report here the isolation, by an immunological method, of a second nuclear gene encoding citrate synthase (CIT2) . Disruption of both genes in the yeast genome was necessary to produce classical citrate synthase-deficient phenotypes: glutamate auxotrophy and poor growth on rich medium containing lactate, a nonfermentable carbon source . Therefore, the citrate synthase produced from either gene was sufficient for these metabolic roles . Transcription of both genes was maximally repressed in medium containing both glucose and glutamate . However, transcription of CIT1 but not of CIT2 was derepressed in medium containing a nonfermentable carbon source . The significance of the presence of two genes encoding citrate synthase in S . cerevisiae is discussed.

Arch Microbiol, 1986 Jun, 145(1), 32 - 8
Properties of the purified APS-kinase from Escherichia coli and Saccharomyces cerevisiae; Schriek U et al.; Adenylylsulphate kinase (EC 2.7.1.25, ATP:adenylylsulphate 3'-phosphotransferase) has been isolated from Escherichia coli and from Saccharomyces cerevisiae . As major steps of purification, affinity chromatography on Sepharose CL 6B ("blue" or "red") and chromatofocusing on polybuffer PBE 94tm were employed . The proteins were obtained in nearly homogeneous state after five chromatographic steps . The isolated enzymes from both sources appeared predominantly to exist as dimers . Upon reduction of the protein with dithiothreitol, it disintegrated into assumingly identical smaller subunits (E . coli rom Mr 90-85,000 to 45-40,000 and S . cerevisiae from 52-49,500 to 28-29,500) . Both forms, dimer and monomer were found catalytically active . Preincubation of the isolated enzyme from either source in the presence of thioredoxin plus DTT, reduced glutathione or DTT increased the activity significantly . Treatment of the enzyme with SH-blocking reagents inactivated the enzyme irreversibly as compared to the inactivation caused by oxidants (2,6-dichlorophenol-indophenol, ferricyanide or oxydized glutathione) . This oxidant induced inactivation was less pronounced for the fungal enzyme than for the bacterial protein . The enzyme from E . coli required thioredoxin in order to alleviate the GSSG-induced inactivation.

Yeast, 1986 Jun, 2(2), 77 - 85
Vanadium metabolism in wild type and respiratory-deficient strains of S . cerevisiae; Willsky GR et al.; Vanadium metabolism was studied in a wild type and respiratory-deficient strain of S . cerevisiae . Inhibition of growth by vanadate {V(+5)}, vanadate accumulation, and conversion of medium vanadate {V(+5)} to both cell-associated and medium vanadyl {V(+4)} and vanadate {V(+5)} were compared . The growth of both the parental and respiratory-deficient strains was inhibited by vanadate at concentrations greater than or equal to 1 mM . Both parental and respiratory-deficient strains accumulated vanadate and converted medium vanadate to cellular vanadyl as detected using electron spin resonance (ESR) . The accumulation of cell-associated vanadyl was correlated with the loss of medium vanadate in both strains using a chemical assay . In contrast, the respiratory-deficient strain showed a greater amount of a cell-associated vanadate compound, as detected with vanadium-51 nuclear magnetic resonance (51V-NMR), than the wild type strain or a representative respiratory-competent vanadate-resistant mutant . These data imply that mitochondrial function may be directly involved in vanadium metabolism.

Mol Cell Biol, 1986 May, 6(5), 1535 - 44
Effects of progressive depletion of TCM1 or CYH2 mRNA on Saccharomyces cerevisiae ribosomal protein accumulation; Nam HG et al.; When present in excess, the mRNAs for Saccharomyces cerevisiae ribosomal proteins L3 and L29 are translated less efficiently, so that synthesis of these proteins remains commensurate with that of other ribosomal proteins (N.J . Pearson, H.M . Fried, and J.R . Warner, Cell 29:347-355, 1982; J.R . Warner, G . Mitra, W.F . Schwindinger, M . Studeny, and H.M . Fried, Mol . Cell . Biol . 5:1512-1521, 1985) . We used a yeast strain with a conditionally transcribed derivative of the L3 gene to deplete cells progressively of L3 mRNA . In this case translation of L3 mRNA did not become more efficient so that L3 was not maintained at a normal level . Even when there was an initial excess of L3 mRNA, interruption of its further transcription produced an immediate drop in L3 synthesis, suggesting that the translational efficiency of preexisting mRNA cannot be altered . Lack of L3 synthesis afforded an opportunity to examine coordinate accumulation of other ribosomal proteins . Without L3, apparent synthesis of several 60S subunit proteins diminished, and 60S subunits did not assemble . A similar phenomenon occurred when, in a second strain, synthesis of ribosomal protein L29 was prevented . Loss of 60S subunit assembly was accompanied by a destabilization of some 60S ribosomal protein mRNAs . These data suggest that synthesis of some S . cerevisiae ribosomal proteins may be regulated posttranscriptionally as a function of the extent to which they are assembled.

Antibiot Med Biotekhnol, 1986 May, 31(5), 374 - 8
{Toxic properties of the double-stranded RNA of virus-like particles from Saccharomyces cerevisiae killer yeasts}; Masychneva VI et al.; The toxic properties of dsRNA isolated from S . cerevisiae were studied on noninbred albino mice . The preparation was administered intraperitoneally . The lots of the yeast dsRNA consisted of highly and moderately toxic compounds . The toxicity level was directly associated with the content of the dsRNA in the preparation . Administration of the preparation in doses equal to the maximum tolerance dose and 1/5 of LD50 had no significant effect on the motor activity, temperature and weight of the animals . However, it induced marked morphological impairments in the gastrointestinal tract, liver and kidneys and pronounced shifts in the hematological indices . 1/50 of LD50 had no significant effect on the physiological, hematological and morphological indices of the organs . No capacity for the yeast dsRNA cumulation in the host was observed.

Nucleic Acids Res, 1986 Apr 25, 14(8), 3587 - 601
Expression of the major heat shock gene of Drosophila melanogaster in Saccharomyces cerevisiae; de Banzie JS et al.; A copy of the gene which encodes the major heat shock protein (hsp70) of D . melanogaster was integrated in both orientations into the genome of S . cerevisiae at the leu2 locus . The level of transcript from the D . melanogaster gene was measured under both normal conditions and conditions which are known to give rise to the heat shock response in S . cerevisiae . In both orientations the D . melanogaster gene gave rise to an abundant transcript in uninduced cells . The level of this transcript was increased transiently on heat shock, peaking after about 30 min at the elevated temperature . The average induction observed was around 5-fold . Although the D . melanogaster gene is heat inducible in S . cerevisiae, the transcripts are initiated at several sites which lie between 10 and 40 base pairs downstream of the initiation site in D . melanogaster . Thus, the transcriptional apparatus of S . cerevisiae appears to recognize the promoter and regulatory elements of the D . melanogaster major heat shock gene, although the manner in which transcription is initiated differs between the two species.

Cell, 1986 Apr 11, 45(1), 3 - 13
Uncoating ATPase is a member of the 70 kilodalton family of stress proteins; Chappell TG et al.; The synthetic peptide, VGIDLGTTYSC, derived from the heat shock-induced genes human hsp70, Drosophila hsp70, S . cerevisiae YG100, and E . coli dnaK, elicited antibodies that recognized two constitutive proteins in bovine extracts . One of these proteins, 71 kd, has previously been identified as uncoating ATPase, an enzyme that releases clathrin from coated vesicles . This immunological data complemented the result that uncoating ATPase was indistinguishable from the constitutive mammalian 71 kd stress protein by either partial proteolytic mapping or two-dimensional gel analysis . In addition, affinity-purified uncoating ATPase antibodies recognize proteins in yeast identified as the gene products of the heat shock or heat shock cognate genes YG100 and YG102 . The results show that uncoating ATPase is a member of the 70 kd heat shock protein family.

Mol Gen Genet, 1986 Apr, 203(1), 163 - 71
Partial complementation of the UV sensitivity of E . coli and yeast excision repair mutants by the cloned denV gene of bacteriophage T4; Chenevert JM et al.; The denV gene of bacteriophage T4 was reconstituted from two overlapping DNA fragments cloned in M13 vectors . The coding region of the intact gene was tailored into a series of plasmid vectors containing different promoters suitable for expression of the gene in E . coli and in yeast . Induction of the TAC promoter with IPTG resulted in overexpression of the gene, which was lethal to E . coli . Expression of the TACdenV gene in the absence of IPTG, or the use of the yeast GAL1 or ADH promoters resulted in partial complementation of the UV sensitivity of uvrA, uvrB, uvrC and recA mutants of E . coli and rad1, rad2, rad3, rad4 and rad10 mutants of S . cerevisiae . The extent of denV-mediated reactivation of excision-defective mutants was approximately equal to that of photoreactivation of such strains . Excision proficient E . coli cells transformed with a plasmid containing the denV gene were slightly more resistant to ultraviolet (UV) radiation than control cells without the denV gene . On the other hand, excision proficient yeast cells were slightly more sensitive to killing by UV radiation following transformation with a plasmid containing the denV gene . This effect was more pronounced in yeast mutants of the RAD52 epistasis group.

Genetika, 1986 Apr, 22(4), 549 - 56
{Cloning and physical mapping of the ADE1 gene of the yeast Saccharomyces cerevisiae}; Sasnauskas KV et al.; ADE1 gene of Saccharomyces cerevisiae codes for the primary structure of SAICAR-synthetase . Mutational changes of ADE1 gene result in the accumulation of red pigment in cells . Colour differences, thus, serve as a basis for the selection of mutants or transformants . ADE1 gene was cloned as a 4.0 kb HindIII fragment of yeast DNA in a shuttle vector by complementing the ade1 mutation in yeast . The study of ADE1 gene expression in Escherichia coli showed that the 4.0 kb fragment containing the ADE1 gene does not complement purC mutations in E . coli . However, prototrophic colonies appeared at a frequency of 10(-7)-10(-8) after incubating clones bearing the recombinant plasmid with ADE1 gene on selective media . The plasmid DNA isolated from such clones complements the purC mutation in E . coli and the ade1 mutation in S . cerevisiae . Structural analysis of the plasmid demonstrated that the cloned DNA fragment contained an additional insertion of the bacterial origin . Further restriction enzyme analysis proved the insertion to be the bacterial element IS1 . Expression of the cloned ADE1 gene in S . cerevisiae is controlled by its own promoter, whereas in E . coli it is controlled by the IS1 bacterial element.

Mol Gen Genet, 1986 Apr, 203(1), 89 - 94
Cloning of Candida pelliculosa beta-glucosidase gene and its expression in Saccharomyces cerevisiae; Kohchi C et al.; Candida pelliculosa var . acetaetherius is a strain of yeast which can utilize cellobiose as the carbon source . From a gene library prepared from this yeast, the beta-glucosidase gene has been cloned in a S . cerevisiae host using a chromogenic substrate, 5-bromo-4-chloro-3-indolyl-beta-glucoside as an indicator . It was proved by Southern analysis that the DNA fragment carrying the beta-glucosidase gene originated from C . pelliculosa . beta-Glucosidase produced by S . cerevisiae transformants was secreted into the periplasmic space . In Candida, beta-glucosidase was not induced by cellobiose but was derepressed by lowering the concentration of glucose . The regulation of beta-glucosidase synthesis in S . cerevisiae carrying the cloned beta-glucosidase was not clear compared with that in Candida, however, the enzyme activity in low glucose medium (0.05%) was reproducibly higher than in high glucose medium (2%) . We have found the sequence that controls the expression of the beta-glucosidase gene negatively in S . cerevisiae.

EMBO J, 1986 Apr, 5(4), 763 - 71
Structure of RNA polymerase II promoters . Conformational alterations and template properties of circularized Saccharomyces cerevisiae GAL1-GAL10 divergent promoters; Camilloni G et al.; A DNA fragment encompassing the Saccharomyces cerevisiae GAL1--GAL10 divergent promoters (914 bp) has been circularized in vitro with T4 DNA ligase . We have defined a set of conditions that allows the production of a series of nine topoisomers covering a range from relaxed to highly negatively supercoiled DNA . Topoisomers were recovered in pure form from agarose gels and were analysed singly for the presence of sites sensitive to the single strand-specific endonuclease Pl . In this way, the occurrence of conformational alterations as a function of the linking deficiency of the closed DNA domain has been determined . Interestingly, sites of Pl hypersensitivity localize on the three sequences identified as relevant for the in vitro transcription of the GAL1 moiety of the divergent promoter: the upstream activator sequence (UAS), the TATA sequence, and the RNA initiation site (RIS) . In vitro transcription with purified S . cerevisiae RNA polymerase II shows that activation of transcription parallels the appearance of conformational alterations on the UAS, the TATA and the RIS sequences.

Mol Gen Genet, 1986 Apr, 203(1), 73 - 8
Heme control region of the catalase T gene of the yeast Saccharomyces cerevisiae; Spevak W et al.; The 5'-flanking region of the Saccharomyces cerevisiae catalase T gene (CTT1) and the part of the gene coding for the N-terminus of catalase T were sequenced . 5'-Ends of transcripts of the region were located by S1 nuclease mapping and primer extension . To analyse control elements in the upstream region, a CTT1-lacZ gene fusion was constructed . Deletion analysis was carried out within a part of the 5'-flanking region showing homology to the upstream region of the yeast CYC1 gene . Like the CTT1 gene, this gene is controlled by heme, oxygen and glucose . The results obtained show that the CTT1 gene is positively controlled by heme . Tentative evidence has been obtained for the involvement of upstream sequences homologous to UAS1 and UAS2 of the CYC1 gene in heme control . Further, a negative site has been located between the upstream activator sites and the transcription start . Within this negative region a ten base-pair sequence was detected that shows high homology to a sequence located within a negative control region of the CYC1 gene and some homology to the negative control elements of the S . cerevisiae CAR1 and CAR2 genes.

J Biol Chem, 1986 Mar 15, 261(8), 3479 - 82
Identification of a glycoprotein involved in cell cycle progression in yeast; Popolo L et al.; The molecular events of start, the regulatory step that commits yeast cells to DNA replication, have recently begun to be investigated . One of the gene products required for completion of start has been found to have a significant structural homology with oncogenes endowed with protein kinase activity . Our experiments provide data on the biosynthetic pathway of a previously identified labile protein (p100, molecular weight 100,000, isoelectric point of approximately 4.8-5) involved in cell cycle progression at start, which appears to be specifically made during the release from cell cycle arrest of a temperature-sensitive mutant (cdc25) of Saccharomyces cerevisiae . On two-dimensional gel, p100 migrates very close to another 100-kDa labile protein (p100*) which behaves as a cell cycle modulated protein with reduced synthesis in G1 . Pulse and chase labeling of protein with {35S}methionine suggests that both p100 and p100* are processed to a protein (p115) of slightly higher molecular weight (Mr = 115,000) . Peptide mapping analysis indicates that p100 and p100 yield identical maps and that both p100 and p100* are very much similar to p115 . p115 is a glycosylated protein as shown by a labeling experiment with {3H}glucosamine and by the fact that the synthesis of both p100 and p115 is inhibited if cells are cultured in the presence of tunicamycin . A protein having the same heterogeneous aspect of migration on sodium dodecyl sulfate-polyacrylamide gel and the same apparent molecular weight and isoelectric point of p115 is abundantly present in a preparation of membranes from S . cerevisiae and the isolated radioactive p115 comigrates with it . Taken together these results favor the idea that terminal glycosylation of both p100 and p100* gives rise to the fully glycosylated p115 protein which appears to be a membrane-associated protein.

FEBS Lett, 1986 Mar 3, 197(1-2), 50 - 4
Killer toxin from Hansenula mrakii selectively inhibits cell wall synthesis in a sensitive yeast; Yamamoto T et al.; Hansenula mrakii secretes extracellularly a killer toxin which kills sensitive Saccharomyces cerevisiae . In protoplasts of this yeast, the killer toxin selectively inhibited the synthesis of alkali-insoluble acid-insoluble polysaccharides consisting mainly of beta-glucan, but did not inhibit either the synthesis of other cell wall polysaccharides, such as mannan, chitin and alkali-insoluble acid-soluble polysaccharides, or the synthesis of protein . Consistent with these results, the toxin was inhibitory to the beta-(1,3)-glucan synthetase activity of a cell-free extract from sensitive S . cerevisiae.

J Biochem (Tokyo), 1986 Mar, 99(3), 741 - 9
Monooxygenase activity of Saccharomyces cerevisiae cells transformed with expression plasmids carrying rat cytochrome P-450MC cDNA; Sakaki T et al.; The recombinant plasmids pAMC1 and pJMC1 were constructed; the former contained the cytochrome P-450MC (P-450MC) cDNA expression unit consisting of yeast alcohol dehydrogenase I (ADH) promoter, rat P-450MC cDNA and ADH terminator, and the Leu 2 marker gene, and the latter contained the same expression unit and the leu 2-d gene . Saccharomyces cerevisiae AH22 cells transformed with each of the recombinant plasmids were examined for plasmid copy number, P-450MC mRNA level, P-450MC content, and monooxygenase activity . The S . cerevisiae AH22/pJMC1 cells contained about 2-fold higher levels of the plasmid, P-450MC mRNA, and P-450MC than the AH22/pAMC1 cells . Monooxygenase activity towards 7-ethoxycoumarin and acetanilide of the AH22/pJMC1 cells was 1.7-fold and 1.5-fold higher than that of the AH22/pAMC1 cells, respectively, whereas the activity of the AH22/pAMC1 cells towards 7-ethoxycoumarin and acetanilide was more than 1,000-fold 10-fold higher than that of the control AH22/pAAH5 cells which contain no P-450MC cDNA, respectively . Therefore, it is likely that monooxygenase activity of the AH22 cells carrying rat P-450MC cDNA was approximately proportional to the expression level of P-450MC cDNA.

Yeast, 1986 Mar, 2(1), 59 - 67
Identification of the RNA2 protein of Saccharomyces cerevisiae; Lee MG et al.; The rna2-1 mutant of Saccharomyces cerevisiae has a conditional lethal phenotype, accumulating high molecular weight RNAs of intron-containing nuclear genes at 36 degrees C . The cloned RNA2 gene suppresses this phenotype and the RNA2 gene product has been implicated in RNA splicing . Rabbit antisera have been raised against an N-terminal synthetic peptide taken from the RNA2 gene DNA sequence data, and against a beta-galactosidase/RNA2 gene fusion protein . Both antisera identify the same 97-105 kd protein from S . cerevisiae cell extracts which is consistent with the predicted size of the RNA2 protein (from the 2800 nucleotide transcript size and DNA sequence data).

Mol Cell Biol, 1986 Mar, 6(3), 925 - 32
Size threshold for Saccharomyces cerevisiae chromosomes: generation of telocentric chromosomes from an unstable minichromosome; Zakian VA et al.; A 9-kilobase pair CEN4 linear minichromosome constructed in vitro transformed Saccharomyces cerevisiae with high frequency but duplicated or segregated inefficiently in most cells . Stable transformants were only produced by events which fundamentally altered the structure of the minichromosome: elimination of telomeres, alteration of the centromere, or an increase of fivefold or greater in its size . Half of the stable transformants arose via homologous recombination between an intact chromosome IV and the CEN4 minichromosome . This event generated a new chromosome from each arm of chromosome IV . The other "arm" of each new chromosome was identical to one "arm" of the unstable minichromosome . Unlike natural yeast chromosomes, these new chromosomes were telocentric: their centromeres were either 3.9 or 5.4 kilobases from one end of the chromosome . The mitotic stability of the telocentric chromosome derived from the right arm of chromosome IV was determined by a visual assay and found to be comparable to that of natural yeast chromosomes . Both new chromosomes duplicated, paired, and segregated properly in meiosis . Moreover, their structure, as deduced from mobilities in orthogonal field gels, did not change with continued mitotic growth or after passage through meiosis, indicating that they did not give rise to isochromosomes or suffer large deletions or additions . Thus, in S . cerevisiae the close spacing of centromeres and telomeres on a DNA molecule of chromosomal size does not markedly alter the efficiency with which it is maintained . Taken together these data suggest that there is a size threshold below which stable propagation of linear chromosomes is no longer possible.

Can J Genet Cytol, 1986 Feb, 28(1), 154 - 60
The effect of 2-micron DNA on survival and mutagenesis in Saccharomyces cerevisiae; Nestmann ER et al.; Strains of Saccharomyces cerevisiae, with and without endogenous 2-microns DNA, were studied in experiments designed to determine the effect of this plasmid on survival and mutagenesis in yeast . Comparison of the two strains exposed to ultraviolet light, 4-nitroquinoline oxide, or methyl methanesulfonate (MMS), revealed that the presence of 2-microns DNA slightly enhanced survival after exposure to each agent . Spontaneous frequencies of mutations (histidine reversion, canavanine resistance, and mitochondrial petites, but not adenine auxotrophy) were reduced by the presence of 2-microns DNA . MMS-induced His+ reversion was weak, and both strains responded similarly . No difference was found between the two strains when induced forward mutation to canavanine resistance was examined . The extent of induction of mitochondrial petites was about the same in both strains . Therefore, it appears that under these experimental conditions with these mutagens, 2-microns DNA has an effect on spontaneous mutation and survival after DNA damage but not on induced mutagenesis in S . cerevisiae.

DNA, 1986 Feb, 5(1), 1 - 10
Expression of rat NADPH-cytochrome P-450 reductase cDNA in Saccharomyces cerevisiae; Murakami H et al.; A full-length cDNA for rat NADPH-cytochrome P-450 reductase was cloned by the procedure of Okayama and Berg (1982) from hepatic poly(A)RNA prepared from phenobarbital-induced rats . Both cDNA and amino acid sequences agreed with the sequences reported by Porter and Kasper (1985) except for four single base differences . Three expression plasmids were constructed by insertion of the reductase cDNA between yeast alcohol dehydrogenase I (ADH) promoter and terminator regions . Plasmids pARF1 and pTRF2 were constructed with slightly different lengths between the ADH promoter and the initiation codon; on introduction into Saccharomyces cerevisiae AH22 cells, they synthesized about 1 X 10(3) and 5 X 10(3) reductase protein molecules per cell, respectively . A third plasmid, pARM1, containing a cytochrome P-450MC cDNA expression unit located between two reductase cDNA expression units synthesized 4 X 10(5) cytochrome P-450MC hemoprotein and 1 X 10(4) reductase protein molecules per cell . The cellular extracts of the AH22/pARM1 strain, which synthesized both rat enzymes, showed higher cytochrome c reductase and cytochrome P-450MC-dependent 7-ethoxycoumarin O-deethylation activities as compared to extracts of the AH22/pAMC1 strain, which synthesized only rat cytochrome P-450MC . 7-Ethoxycoumarin O-deethylation activity in the cellular extract of AH22/pARM1 strain was partly inhibited by the addition of anti-rat reductase IgG . In addition, whole AH22/pARM1 cells exhibited higher monooxygenase activity toward acetanilide and 7-ethoxycoumarin than control AH22/pAMC1 cells . These results indicated that a functional electron-transport chain consisting of rat NADPH-cytochrome P-450 reductase and rat cytochrome P-450MC was constructed in S . cerevisiae cells.

Mol Cell Biol, 1986 Feb, 6(2), 593 - 600
Three genes for the elongation factor EF-1 alpha in Mucor racemosus; Linz JE et al.; We cloned three genes from Mucor racemosus coding for protein synthesis elongation factor 1 alpha (EF-1 alpha) . A 110-base-pair (bp) EF-1 alpha-specific cDNA clone was identified by hybrid-selected translation . The nucleotide sequence of the cDNA showed significant homology to a region of the Saccharomyces cerevisiae genes for EF-1 alpha (TEF1 and TEF2) . The cDNA was used to isolate an 850-bp EcoRI genomic DNA fragment containing a portion of the EF-1 alpha gene . Screening of a lambda/M . racemosus genomic DNA bank with the 850-bp EcoRI probe resulted in the identification of three DNA fragments containing a common 850-bp EcoRI fragment within a short overlapping region . S1 nuclease analysis of the three EF-1 alpha DNA fragments showed that the EF-1 alpha transcript covered the short overlapping region in the clones . Restriction fragments purified from flanking regions in each clone were used to probe a HindIII digest of M . racemosus genomic DNA . Each flanking probe hybridized to one of three DNA fragments which hybridized to the 850-bp EF-1 alpha-specific probe . Nucleotide sequence data from two random "shotgun clones" of one of the three genes show good homology to two regions of S . cerevisiae TEF1 . The data indicate the presence of three genes for EF-1 alpha in M . racemosus located at unique sites in the genome.

J Cell Biol, 1986 Feb, 102(2), 523 - 33
The amino terminus of the yeast F1-ATPase beta-subunit precursor functions as a mitochondrial import signal; Emr SD et al.; The ATP2 gene of Saccharomyces cerevisiae codes for the cytoplasmically synthesized beta-subunit protein of the mitochondrial F1-ATPase . To define the amino acid sequence determinants necessary for the in vivo targeting and import of this protein into mitochondria, we have constructed gene fusions between the ATP2 gene and either the Escherichia coli lacZ gene or the S . cerevisiae SUC2 gene (which codes for invertase) . The ATP2-lacZ and ATP2-SUC2 gene fusions code for hybrid proteins that are efficiently targeted to yeast mitochondria in vivo . The mitochondrially associated hybrid proteins fractionate with the inner mitochondrial membrane and are resistant to proteinase digestion in the isolated organelle . Results obtained with the gene fusions and with targeting-defective ATP2 deletion mutants provide evidence that the amino-terminal 27 amino acids of the beta-subunit protein precursor are sufficient to direct both specific sorting of this protein to yeast mitochondria and its import into the organelle . Also, we have observed that certain of the mitochondrially associated Atp2-LacZ and Atp2-Suc2 hybrid proteins confer a novel respiration-defective phenotype to yeast cells.

Cell, 1986 Jan 17, 44(1), 53 - 63
Isolation of two genes that affect mitotic chromosome transmission in S . cerevisiae; Meeks-Wagner D et al.; Two DNA sequences that reduce mitotic fidelity of chromosome transmission have been identified: MIF1 and MIF2 . MIF1 is a unique sequence located on the right arm of chromosome XII that stimulates loss and recombination for both chromosomes V and VII when present in a high copy number plasmid . MIF1 is not essential for cell division but is necessary for the normal fidelity of chromosome transmission . MIF2 is a unique sequence located 15 cM distal to HIS6 on chromosome IX that induces a high frequency of chromosome VII loss and a lower frequency of chromosome V loss when present in high copy number; it has no effect on mitotic recombination . Disruption of the genomic MIF2 locus was lethal and cells lacking this function arrested division with a terminal phenotype characteristic of a block in DNA replication or nuclear division.

Biochem Biophys Res Commun, 1986 Jan 14, 134(1), 285 - 91
A method for determining the intracellular distribution of enzymes in yeast provides no evidence for the association of hexokinase with mitochondria; Kovac L et al.; A simple procedure based upon the principle discovered by Durr et al . (Arch . Microbiol . (1975) 105, 319-327) was used to measure the intracellular distribution of enzymes in S . cerevisiae grown under both glucose repression and derepression . No substantial hexokinase activity was found to be associated with cellular organelles . The result does not support the hypotheses that reversible binding of hexokinase to mitochondria is important in regulation of glycolysis and cell growth.

Gene, 1986, 46(2-3), 237 - 45
Construction of LYS2 cartridges for use in genetic manipulations of Saccharomyces cerevisiae; Fleig UN et al.; Linker arrays were added to the 5' and 3' boundaries of the Saccharomyces cerevisiae LYS2 gene, which allow the generation of 18 LYS2 cartridges with different sticky ends . As it was necessary to define the beginning and the end of the approx . 4.5-kb LYS2 gene, we sequenced 1 kb of its 5' and 1.5 kb of its 3' region and mapped the mRNA start point . The open reading frame (ORF) found by this analysis was proven to be the LYS2 ORF by exchanging the sequences upstream from the presumptive ATG with the S . cerevisiae CYC1 promoter and subsequent demonstration of LYS2 expression in vivo . The proper functioning of the LYS2 cartridges was demonstrated by the transformation of lys2 mutant strains to Lys+ prototrophy using plasmids furnished with a LYS2 cartridge.

Gene, 1986, 46(1), 71 - 8
Molecular cloning and regulation of the expression of the MET2 gene of Saccharomyces cerevisiae; Baroni M et al.; The MET2 gene of Saccharomyces cerevisiae, which codes for homoserine-O-acetyltransferase, a key enzyme in methionine biosynthesis, was isolated by complementation of a met2 mutant strain of S . cerevisiae with a yeast gene bank . A 3.9-kb genomic fragment contains the entire gene, as demonstrated by genetic and molecular analysis of the integrative transformants . A polyadenylated mRNA of 1700 nt is detected by Northern blot hybridization with a MET2 probe . The level of this mRNA decreases by addition of exogenous methionine or of S-adenosylmethionine, suggesting a transcriptional regulation . The level of specific mRNA and the enzyme activity found in transformants that bear the MET2 gene on a multicopy plasmid suggest that also a post-transcriptional regulatory mechanism may be operative in budding yeast.

Gene, 1986, 46(1), 135 - 41
Unusually high-level expression of a foreign gene (hepatitis B virus core antigen) in Saccharomyces cerevisiae; Kniskern PJ et al.; As a model system for the study of factors affecting gene expression, hepatitis B virus core antigen (HBcAg) has been expressed in the yeast Saccharomyces cerevisiae . The singularly high levels of expression achieved are approx . 40% of the soluble yeast protein . The HBcAg polypeptides are present as 28-nm particles which are morphologically indistinguishable from HBcAg particles in human plasma and are highly immunogenic in mice . The plasmid construction employed to achieve these very high levels of expression utilizes the constitutively active yeast promoter from the GAP491 gene which is fused in a way that all non-translated sequences flanking the HBcAg coding region are yeast-derived . Hybrid constructions containing 3'-nontranslated viral DNA (yeast 5') or 5'-nontranslated viral DNA (yeast 3') as well as a construction with both 5'- and 3'-nontranslated viral DNA also have been made . A comparison of these constructions for levels of HBcAg expression indicates that the strongest contributor to the high levels of protein is the presence of 5'-flanking sequences which are yeast-derived; secondarily, a significant improvement can be achieved if the 3'-flanking sequences also are yeast-derived . The high abundance of HBcAg in the highest producer is explicable in part on the basis of the very high stability in yeast cells of HBcAg polypeptides . Analysis of the HBcAg coding sequence reveals a very low index of codon bias for S . cerevisiae, largely discounting codon usage as a contributor to the high level of protein obtained.

Gene, 1986, 45(3), 237 - 45
Isolation of a cytochrome P-450 structural gene from Saccharomyces cerevisiae; Kalb VF et al.; We have transformed a Saccharomyces cerevisiae host with an S . cerevisiae genomic library contained in the shuttle vector YEp24 and screened the resultant transformants for resistance to ketoconazole (Kc), an inhibitor of the cytochrome P-450 (P-450) enzyme lanosterol 14 alpha-demethylase . Two plasmids were isolated which transformed yeast to both increased resistance to Kc and increased levels of total P-450 . Hybrid-selection and immunoprecipitation experiments showed that these plasmids, pVK1 and pVK2, contained the structural gene for an S . cerevisiae P-450 . This conclusion was confirmed by the nucleotide sequence of a portion of pVK2, which revealed an open reading frame encoding a characteristic P-450 heme-binding region.

CRC Crit Rev Biochem, 1986, 21(3), 225 - 48
Structure-activity relationships of the yeast alpha-factor; Naider F et al.; The yeast Saccharomyces cerevisiae produces a peptide pheromone, termed the alpha-factor, as a prelude to sexual conjugation . Haploid MAT alpha-cells, but not haploid MAT a-cells or MAT a/alpha-diploids, produce this tridecapeptide of the structure: Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr . Structural analogues of the alpha-factor have been prepared with alterations in many of the residues, derivatized peptides have been synthesized, and truncated and elongated peptides have been studied . These peptides have been analyzed for their biological activities by various assays . Mutants of S . cerevisiae have been isolated that do not respond to alpha-factor or are supersensitive to the pheromone and its analogues . The mating system of S . cerevisiae provides a powerful model in which genetics, biochemistry, and molecular biology can be used to unravel the mysteries of peptide hormone structure and function.

Microbios, 1986, 46(188-189), 179 - 85
Partial purification of polypeptides activating catalase in Saccharomyces cerevisiae; Maqueda M et al.; In a previous paper the existence of an activating factor for catalase biosynthesis in Saccharomyces cerevisiae was reported . The partial purification of this factor by ammonium sulphate precipitation and chromatography is described . Several fractions with different molecular weights manifesting catalase activity in repressed cultures of S . cerevisiae were obtained, and are discussed.

Microbios, 1986, 45(184-185), 169 - 79
Cd2+ accommodation by Saccharomyces cerevisiae; Joho M et al.; The Cd2+ accommodation mechanism of S . cerevisiae has been examined . In a synthetic medium containing 1 microM CdSO4, cell proliferation was observed following a time lag period of approximately 18 h . During the time lag period, the cell number and viable cell count did not change and the cells did not excrete organic acid as a Cd-chelator, neither did they produce inorganic sulphide in the form of CdS . Whereas approximately 30% of Cd2+ taken up by Cd-accommodated cells was distributed in the cytosol, only a small percentage of Cd2+ was found in the cytosol of unaccommodated cells following 1 h incubation in 1 microM Cd-medium . Furthermore, some Cd2+ in the soluble fraction from Cd-accommodated cells was bound to low molecular weight (less than 30,000) protein(s), while unaccommodated cells only contained small amounts of these proteins . Therefore, it is proposed that Cd2+ binds to the low molecular weight protein(s) present in the cytosol, resulting in a decrease of Cd-toxicity, although the exact mechanism of accommodation is not clear.

Antonie Van Leeuwenhoek, 1986, 52(1), 15 - 24
Molecular events associated with glucose repression of invertase in Saccharomyces cerevisiae; Mormeneo S et al.; When S . cerevisiae growing in the presence of glucose (repressive condition) was shifted to higher temperatures, invertase was secreted . This secretion required protein synthesis, but was independent of RNA formation (Mormeneo & Sentandreu 1982) . In addition accumulation of invertasespecific messenger RNA occurred in the absence of protein synthesis but was expressed only after synthesis of protein . Invertase mRNA was continuously synthesized under repressive conditions and the levels of this mRNA were regulated by the presence of glucose . The hexose regulated the concentration of this mRNA at the level of transcription and/or by sensitization of this messenger RNA . The expression of the invertase mRNA present in the cells under repressive conditions was also regulated by glucose at the level of translation and/or secretion . As a result of these processes, under repressive conditions invertase is eliminated before secretion takes place.

J Bacteriol, 1986 Jan, 165(1), 101 - 6
GAL3 gene product is required for maintenance of the induced state of the GAL cluster genes in Saccharomyces cerevisiae; Nogi Y; The activities of the first three enzymes for galactose catabolism normally become detectable within 15 min after the addition of galactose into a culture of the yeast Saccharomyces cerevisiae . In S . cerevisiae with a recessive mutation termed gal3, a longer-than-normal lag is observed before the appearance of the enzyme activities (O . Winge and C . Roberts, C . R . Trav . Lab . Carlsberg Ser . Physiol . 24:263-315, 1948) . I isolated two S . cerevisiae mutants with temperature-sensitive defects in the GAL3 gene . Temperature shift experiments with one of those mutants led to the conclusion that the GAL3 function is required not only for the initiation of enzyme induction but also for the maintenance of the induced state in galactose-nonfermenting S . cerevisiae because of a defect in any of the genes for the galactose-catabolizing enzymes, such as gal1 or gal10 . In contrast, the GAL3 function is phenotypically dispensable in galactose-metabolizing S . cerevisiae . Thus, the normal catabolism of galactose can substitute for the GAL3 function.

Curr Genet, 1986, 10(12), 887 - 92
Induction of heterothallic strains and their genetic and physiological characterization in a homothallic strain of the yeast Saccharomyces exiguus; Hisatomi T et al.; We isolated heterothallic strains from a homothallic strain of S . exiguus by mutagenization with UV or ethylmethanesulfonate (EMS) . A gene, not linked to the mating-type locus, was found to control homothallism in the yeast, as in S . cerevisiae . alpha Pheromone of S . exiguus (alpha se pheromone) induced formation of large pear-shaped cells (shmooing) in alpha strains of S . exiguus, S . cerevisiae, and S . kluyveri, and sexual agglutinability of an inducible alpha strain of S . cerevisiae . alpha se Pheromone is a peptidyl substance a little different from alpha pheromone of S . cerevisiae . alpha Pheromone of S . exiguus acts only on alpha cells of S . exiguus . Contrary to the above results, neither sexual agglutination nor zygote formation occurred among these three Saccharomyces yeasts.

Curr Genet, 1986, 10(10), 741 - 7
Evidence for autonomous replication and stabilization of recombinant plasmids in the transformants of yeast Hansenula polymorpha; Tikhomirova LP et al.; For the transformation of the yeast Hansenula polymorpha we have constructed a set of hybrid plasmids carrying the LEU2 gene of Saccharomyces cerevisiae as a selective marker and fragments of mitochondrial DNA of Candida utilis and H . polymorpha or chromosomal DNA fragments of H . polymorpha as replicator sequences . The replication properties of chimeric plasmids in the yeast H . polymorpha were investigated . We showed that for plasmids propagated autonomously in this yeast the plasmid monomers could be detected in the transformants only during the immediate time after the transformation event . Further growth under selective conditions led to the selection of polymeric forms of plasmid DNA as it was clearly shown for transformants carrying cosmid pL2 with mtDNA fragment of C . utilis . Such transformants carrying polymerized plasmids showed a remarkably increased stability of the transformed phenotype . Cosmid pL2 was able to shuttle between Escherichia coli, S . cerevisiae and H . polymorpha, whereas plasmids with DNA fragments from H . polymorpha did not transform S . cerevisiae effectively.

Curr Genet, 1986, 10(5), 371 - 9
An approach to yeast classification by mapping mitochondrial DNA from Dekkera/Brettanomyces and Eeniella genera; Hoeben P et al.; Sequences hybridizing to mitochondrial DNA probes from Saccharomyces cerevisiae have been mapped in six mitochondrial genomes from the Dekkera/Brettanomyces yeasts and in mtDNA from the closely related Eeniella nana . Sequence order for the 34.5 kbp mtDNA of E . nana is identical to that for mtDNAs from B . custersianus (28.5 kbp) and B . naardenensis (41.7 kbp) thereby suggesting that the former yeast is affiliated with the latter two species . A closer relationship is suggested for D . intermedia and D . bruxellensis as mtDNAs from these yeasts, 73.2 and 85.0 kbp respectively, have the same sequence order and mostly common restriction endonuclease sites . Differences between the two molecules are reminiscent of those found in mtDNA polymorphisms of S . cerevisiae strains thereby suggesting that the two Dekkera yeasts are variants of a single species . An unusual feature of the Dekkera species mtDNA is an inversion of the cytochrome b hybridizable region relative to the LrRNA sequence . Likewise mtDNA from B . anomalus (57.7 kbp) has an inversion of the cytochrome oxidase subunit 1 sequence with respect to the LrRNA sequence . By contrast the largest mtDNA (101.1 kbp) from B . custersii has the cytochrome b and LrRNA sequences in the same orientation . In addition hybridizable regions in this mtDNA are found in three clusters that are separated by several thousand base pairs of sequence deficient in restriction endonuclease sites . This observation together with the low guanine and cytosine content of the mtDNA suggests that the regions separating the sequence clusters are mostly adenine and thymine residues.

Curr Genet, 1986, 10(9), 671 - 6
Effects of T-2 toxin on induction of petite mutants and mitochondrial function in Saccharomyces cerevisiae; Schappert KT et al.; The influence of the trichothecene mycotoxin T-2 on the mitochondria of Saccharomyces cerevisiae was studied . T-2 is a cytotoxic molecule inhibiting growth and macromolecular synthesis in S . cerevisiae . At low concentrations, T-2 toxin arrested yeast growth on glycerol medium and at higher concentrations, it arrested growth on glucose medium . The toxin was not capable itself of inducing petite mutations . Its inhibitory effect on the growth of petite strains, of both chromosomally isogenic and non-isogenic strains was less than that of grande strains . One exception to this was equally low susceptibility of psi+ SUP4-3 strain in both rho+ and rho- state . T-2 toxin was also capable of retarding the petite inducing activity of the mutagen, ethidium bromide . T-2 toxin inhibited the polymerization of P-ribo-sylaminoimidazole in an ade2 strain of S . cerevisiae . These results show that T-2 toxin is capable of interfering with the activity of the mitochondria in addition to its well studied effects on cytoplasmic protein synthesis.

Curr Genet, 1986, 10(9), 647 - 55
Alkylation mutagenesis in Saccharomyces cerevisiae: lack of evidence for an adaptive response; Polakowska R et al.; We have found no evidence for an adaptive response for either lethality or mutagenesis following treatment of Saccharomyces cerevisiae with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . The rad6 and rad52 mutants of S . cerevisiae are highly defective in MNNG and ethyl methanesulfonate induced mutagenesis of both stationary and exponential phase cells . These and other observations indicate that the mechanisms of repair of alkylation damage and mutagenesis differ markedly between S . cerevisiae and Escherichia coli.

Curr Genet, 1986, 10(8), 639 - 42
Defective karyogamy in meiotic segregants of a Candida utilis-Saccharomyces cerevisiae hybrid; Perez C et al.; Meiotic segregants derived from a Candida utilis--Saccharomyces cerevisiae hybrid obtained by protoplast fusion were crossed to several standard S . cerevisiae laboratory strains . Random spore and tetrad analysis suggested that in these segregants, karyogamy is impaired and internuclear chromosome transfer occurs.

Curr Genet, 1986, 10(8), 579 - 85
Inhibition of DNA replication in Saccharomyces cerevisiae by araCMP; McIntosh EM et al.; Cytosine arabinoside (araC), a potent inhibitor of DNA replication in mammalian cells, was found to be completely ineffective in Saccharomyces cerevisiae . The 5' monophosphate derivative, araCMP, is toxic and effectively inhibits both nuclear and mitochondrial DNA synthesis in this organism . Although wild-type strains can be inhibited by araCMP, dTMP permeable (tup-) strains were found to be much more sensitive to the analogue . In vivo labelling experiments indicate that araC enters yeast cells; however, it is extensively catabolized by deamination and breakage of the glycosidic bond . In addition, the analogue is not efficiently phosphorylated in S . cerevisiae owing to an apparent lack of deoxynucleoside kinase activity . These results provide further evidence that deoxyribonucleotides can be synthesized only through de novo pathways in this organism . Finally, araCMP was found to be recombinagenic in S . cerevisiae which suggests, together with other previous studies, that, in general, inhibition of DNA synthesis in yeast promotes mitotic recombination events.

Basic Life Sci, 1986, 40, 479 - 92
Structural studies on centromeres in the yeast Saccharomyces cerevisiae; Ng R et al.; In the yeast Saccharomyces cerevisiae, circular or linear plasmids containing a functional centromere (CEN) and a chromosomal replicator (ARS) are mitotically stable and segregate as ordinary yeast chromosomes in the first and second meiotic divisions . A centromere in S . cerevisiae consists of a region of DNA, approximately 150 bp in length, containing three important sequence elements, which are folded with proteins into a specific conformation in the chromatin (the yeast kinetochore) . Each of the functional CEN sequences contains a high (91% to 95%) AT region (element II), 78 to 86 bp in length, flanked on one side by the common sequence PuTCACPuTG (element I), and on the other by the sequence TGTTT.TG.TTTCCGAAA....AAA (element III) . Deletions in the element II region partially inactivate mitotic function and cause precocious separation of the sister chromatids in meiosis I . Element III appears to be a protein binding site, as evidenced by the following observations . Various point mutations in element III inactivate centromere function, especially in the central CCG (17) . One or more protein binding sites in the element III region can be demonstrated by an exonuclease III blocking assay . Wild-type CEN sequences compete strongly in this binding assay, whereas certain functionally inactive mutant CEN sequences do not . In addition, various DNA segments containing either CEN3 or the element III region strongly repress expression of the yeast GAL1 gene when inserted immediately upstream from the transcriptional start site . Helical DNA segments containing CEN3 or CEN14 are shown to be bent or distorted in shape in the high-AT element II region.

Gene, 1986, 45(3), 265 - 73
Structure of the two genes coding for polypeptide chain elongation factor 1 alpha (EF-1 alpha) from Saccharomyces cerevisiae; Nagashima K et al.; Polypeptide chain elongation factor 1 alpha (EF-1 alpha) of Saccharomyces cerevisiae is encoded by two distinct genes designated EF1 alpha A and EF1 alpha B {Nagata et al., EMBO J . 3 (1984) 1825-1830} . Both genes were cloned, and their nucleotide (nt) sequences were determined {see also Schirmaier and Philippsen, EMBO J . 3 (1984) 3311-3315, and Cottrelle et al., J . Biol . Chem . 260 (1985) 3090-3096} . They contain an open reading frame of 1374 nt coding for an identical protein of 458 amino acid residues, although their nt sequences differed at two positions . In this paper, we determined their 5'- and 3'-flanking sequences which were considerably different each other . From the S1 nuclease mapping of mRNA, both genes are found to be expressed almost to the same extent in exponentially growing cells . The transcription start points for EF1 alpha A and EF1 alpha B mRNAs were precisely located by primer extension procedure at 32 and 23 nt upstream of the start codons, respectively . The sequence which commonly exists in the 5'-flanking regions of ribosomal protein genes of S . cerevisiae was also present in the two EF1 alpha genes.

Mol Cell Biol, 1986 Jan, 6(1), 90 - 6
DNA damage and heat shock dually regulate genes in Saccharomyces cerevisiae; McClanahan T et al.; Two Saccharomyces cerevisiae genes isolated in a differential hybridization screening for DNA damage regulation (DDR genes) were also transcriptionally regulated by heat shock treatment . A 0.45-kilobase transcript homologous to the DDRA2 gene and a 1.25-kilobase transcript homologous to the DDR48 gene accumulated after exposure of cells to 4-nitroquinoline-1-oxide (NQO; 1 to 1.5 microgram/ml) or brief heat shock (20 min at 37 degrees C) . The DDRA2 transcript, which was undetectable in untreated cells, was induced to high levels by these treatments, and the DDR48 transcript increased more than 10-fold as demonstrated by Northern hybridization analysis . Two findings argue that dual regulation of stress-responsive genes is not common in S . cerevisiae . First, two members of the heat shock-inducible hsp70 family of S . cerevisiae, YG100 and YG102, were not induced by exposure to NQO . Second, at least one other DNA-damage-inducible gene, DIN1, was not regulated by heat shock treatment . We examined the structure of the induced RNA homologous to DDRA2 after heat shock and NQO treatments by S1 nuclease protection experiments . Our results demonstrated that the DDRA2 transcript initiates equally frequently at two sites separated by 5 base pairs . Both transcriptional start sites were utilized when cells were exposed to either NQO or heat shock treatment . These results indicate that DDRA2 and DDR48 are members of a unique dually regulated stress-responsive family of genes in S . cerevisiae.

Mol Cell Biol, 1986 Jan, 6(1), 70 - 9
Ty insertions at two loci account for most of the spontaneous antimycin A resistance mutations during growth at 15 degrees C of Saccharomyces cerevisiae strains lacking ADH1; Paquin CE et al.; The mutation rate to antimycin A resistance was determined for strains of Sacchromyces cerevisiae lacking a functional copy of the structural gene for alcohol dehydrogenase I (ADH1) . One type of mutation that can cause antimycin A resistance in these strains is insertion of the transposable element Ty 5' to ADH2, the structural gene for the glucose-repressed isozyme of alcohol dehydrogenase, resulting in expression of this gene during growth on glucose . Here we show that after growth at 15 or 20 degrees C on glucose, 30% of the antimycin A resistance mutations are Ty insertions at ADH2 and another 65% of the mutations are Ty insertions at ADH4, a new locus identified and cloned as described in this paper . At 30 degrees C only 6% of the mutations are Ty insertions at either of these two loci . In addition, we show that the transposition rate is lower in mating-incompetent (a/alpha) cells than in either haploid or diploid mating-competent cells . Our results suggest that under certain conditions Ty transposition may be a major cause of spontaneous mutations in S . cerevisiae.

Gene, 1986, 44(1), 171 - 5
Isolation of Trypanosoma cruzi DNA fragments which function as ARS elements in Saccharomyces cerevisiae; Teixeira SM et al.; Genomic banks from Trypanosoma cruzi total cell DNA and kinetoplast DNA were constructed in a bacterial plasmid carrying the yeast URA3 gene marker . This vector is by itself unable to be maintained in Saccharomyces cerevisiae since it can neither replicate as an extrachromosomal element, nor readily integrate into the chromosome of ura3-52 yeast hosts . Using this system, we isolated sequences from nuclear and kinetoplast DNA of T . cruzi which enabled the vector to replicate autonomously in S . cerevisiae . The cloned DNA fragments fit the description of ARS sequences previously isolated from other organisms.

Gene, 1986, 42(2), 133 - 9
Oligodeoxynucleotide-directed mutagenesis using the yeast transformation system; Walder RY et al.; In this report we describe a highly efficient method for site-specific mutagenesis using the yeast transformation system . The method is based on the observation that Saccharomyces cerevisiae can be transformed at high frequency with single-stranded circular DNA vectors {Singh et al., Gene 20 (1982) 441-449} . The model system studied was the TRP1 gene of S . cerevisiae cloned into a derivative of the phage M13mp9 vector containing the yeast URA3 gene . ARS1, located adjacent to the TRP1 gene, allows the plasmid to replicate autonomously in yeast . Synthetic 5'P-oligodeoxynucleotides, 19 and 35 nucleotides (nt) in length, designed to produce an A----T transversion mutation within the TRP1 gene, were annealed to ss DNA of the M13 vector at a molar ratio of 30:1 and directly transformed into yeast . The intended single nt mutation was obtained at frequencies of 24 and 43%, respectively . The latter approaches the theoretical limit of 50% . In the absence of the 5'-terminal phosphate, both the transformation frequency and the efficiency of mutagenesis by the synthetic oligodeoxynucleotide (oligo) were decreased by 2-4 fold . This procedure completely obviates the need for any enzymatic manipulations in vitro after forming the heteroduplex with the oligo primer containing the desired mutation . For yeast genes, direct phenotypic selection is possible in the recipient strain.

Gene, 1986, 41(2-3), 271 - 80
Isolation of the positive-acting regulatory gene PHO4 from Saccharomyces cerevisiae; Koren R et al.; We have isolated a 10.2-kb fragment of yeast DNA from a genomic library of recombinant centromeric YCp50 plasmids, which complements a mutation in the PHO4 gene of Saccharomyces cerevisiae . The identity of the PHO4 gene on this plasmid was established by integration of a subfragment into the PHO4 region of the yeast chromosome . Analysis of a series of plasmid subclones covering different regions of the original yeast DNA insert localized the PHO4 gene within a 2.25-kb sequence . Southern hybridization of total genomic DNA prepared from wild-type strains and from integrative transformants show that the PHO4 gene consists of unique yeast DNA sequences and is present at a single copy in the S . cerevisiae genome . RNA blot hybridization mapping of transcripts within this genomic region identify the PHO4 transcript as a 1.7-kb, low-abundancy, constitutively expressed and polyadenylated RNA.

Curr Genet, 1986, 11(3), 185 - 91
Cloning of a nuclear gene MRS1 involved in the excision of a single group I intron (bI3) from the mitochondrial COB transcript in S . cerevisiae; Kreike J et al.; The respiratory deficient yeast nuclear mutant MK3 is defective in the synthesis of the mature transcripts of the mitochondrial COB and OX13 genes, which code for apocytochrome b and subunit I of cytochrome c oxidase, resp . Introns 3 and 4 of the COB transcript (bI3 and bI4) and intron 4 (aI4) of the OXI3 transcript can not be excised (Pillar et al . 1983a, b) . When combined with mitochondrial genomes lacking introns bI1, bI2 and bI3, or lacking intron bI3 alone the mutant is respiratory competent . Thus, the non-excision of bI4 and aI4 turns out to be an indirect effect of the mutation . From a wild type yeast genebank a plasmid has been isolated with a 3.3 kb DNA insert, which complements the mutant . Subcloning experiments assigned the functional gene to a 1.6 kb HaeIII-Sau3A fragment . Hybridization experiments showed, that it is (i) a single copy gene, (ii) also present in strain D273-10B, containing the "short form" mitochondrial genome (lacking the COB introns bI1-bI3), and (iii) located on chromosome IX . The nuclear gene defective in mutant MK3, was named MRS1 (Mitochondrial RNA Splicing) . The involvement of this nuclear gene in the excision of a single group I mitochondrial intron (bI3) of the COB transcript is discussed.

Curr Genet, 1986, 11(1), 55 - 63
Expression of the mitochondrial split gene coding for cytochrome oxidase subunit I in S . cerevisiae: RNA splicing pathway; Carignani G et al.; We have studied the splicing pathway leading to the synthesis of cytochrome oxidase subunit I (COX I) mRNA, by analysing the transcription pattern of several oxi3- splicing deficient mutants located in the first four introns of the gene . The four introns contain long open reading frames (ORFs) in phase with the upstream exons . All the mutations block the excision of the mutated intervening sequence (IVS) from the pre-mRNA, and accumulate characteristic novel polypeptides of sizes which could correspond to the translation products of the intron's ORF . Most of the mutations do not affect the splicing of the following intervening sequences; only in the case of mutations in the aI1 intron is a polar effect observed on the splicing of the second intron, aI2 . Our results indicate that the splicing of these two intervening sequences which both belong to the class II of introns described by Michel et al . (1982), is controlled by the activity of the maturases encoded by their respective ORFs and that the translation of the aI2 maturase depends on the previous excision of aI1 IVS . (Moreover, the aI1 maturase, which accumulates in some mutants, can efficiently splice aI2 IVS when the translation of the latter's proper maturase cannot occur).

Curr Genet, 1986, 10(8), 573 - 8
Cloning of a Candida utilis gene which complements leu2 mutation in Saccharomyces cerevisiae; Zhang YZ et al.; DNA fragments containing the LEU2 gene of Candida utilis have been isolated, utilizing the genome library (constructed in YRp12) of this organism . Two recombinant plasmids pZR84 and pZR32, containing the cloned LEU2 gene, were 4.24 kb and 10.4 kb, respectively, and were shown to complement leu2 mutation in Saccharomyces cerevisiae and leuB mutation in Escherichia coli . The cloned fragment in pZR84 contained one restriction site each for EcoRI and PvuII, and two for HindIII, but none for SalI, BamHI or PstI . This cloned fragment hybridized with the total DNA from C . utilis and from Leu+ transformants of S . cerevisiae, but not with that from untransformed S . cerevisiae . Subcloning analyses showed that a 2.34 kb BamHI-HindIII fragment of the cloned C . utilis sequence contains the region essential for the expression of the LEU2 gene.

Acta Microbiol Pol, 1986, 35(3-4), 221 - 5
5S rRNA genes from Aspergillus nidulans are not transcribed in Saccharomyces cerevisiae; Bartnik E et al.; Several different 5S rRNA genes from Aspergillus nidulans cloned in an Escherichia coli--Saccharomyces cerevisiae shuttle vector were introduced into S . cerevisiae cells by transformation . The A . nidulans 5S rRNA genes were not transcribed in S . cerevisiae.

Biochemistry, 1985 Dec 3, 24(25), 7070 - 6
Biological activity and conformational isomerism in position 9 analogues of the des-1-tryptophan,3-beta-cyclohexylalanine-alpha-factor from Saccharomyces cerevisiae; Shenbagamurthi P et al.; Analogues of the des-1-tryptophan,3-beta-cyclohexylalanine-alpha-factor of Saccharomyces cerevisiae, where the glycyl residue of position 9 was replaced by D-Ala, L-Ala, D-Leu, and L-Leu, were synthesized and evaluated by morphogenesis assays and circular dichroism spectroscopy . Synthesis was accomplished in solution phase with mixed anhydrides and p-nitrophenyl active esters as the coupling agents . All crude dodecapeptides were purified to greater than 98% homogeneity by preparative high-performance liquid chromatography on a reversed-phase column . The Gly9, D-Ala9, and D-Leu9 analogues elicited morphogenic alterations in MATa strains of S . cerevisiae at concentrations of 1-2 micrograms/mL and exhibited similar CD patterns in both trifluoroethanol and tris(hydroxymethyl)aminomethane buffer, pH 7.4 . In contrast, the L-Ala9 and L-Leu9 analogues were more than 200 times less active in the morphogenesis assay and had markedly different CD spectra . These results demonstrate that the position 9 residue plays an important role in determining the biological activity and solution conformation of alpha-factor . We suggest the presence of a type II beta-turn in the Lys7-Gln10 region when the alpha-factor assumes its biologically active conformation.

Mol Cell Biol, 1985 Dec, 5(12), 3357 - 60
Diphtheria toxin-resistant mutants of Saccharomyces cerevisiae; Chen JY et al.; We developed a selection procedure based on the observation that diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae (Murakami et al., Mol . Cell . Biol . 2:588-592, 1982); this procedure yielded mutants resistant to the in vitro action of the toxin . Spheroplasts of mutagenized S . cerevisiae were transformed in the presence of diphtheria toxin, and the transformed survivors were screened in vitro for toxin-resistant elongation factor 2 . Thirty-one haploid ADP ribosylation-negative mutants comprising five complementation groups were obtained by this procedure . The mutants grew normally and were stable to prolonged storage . Heterozygous diploids produced by mating wild-type sensitive cells with the mutants revealed that in each case the resistant phenotype was recessive to the sensitive phenotype . Sporulation of these diploids yielded tetrads in which the resistant phenotype segregated as a single Mendelian character . From these observations, we concluded that these mutants are defective in the enzymatic steps responsible for the posttranslational modification of elongation factor 2 which is necessary for recognition by diphtheria toxin.

Arch Biochem Biophys, 1985 Dec, 243(2), 492 - 503
Pyridine nucleotide transhydrogenations in yeast; Evans TC et al.; Though previously described as very low or absent in yeast, we find significant pyridine nucleotide transhydrogenation (NADPH + acetyl pyridine-NAD+----NADP+ + acetyl pyridine-NADH) activity in yeast extracts when assayed at pH 8-9, and describe here the subcellular distribution and separation of the various molecular forms contributing to the total activity in two yeast species . Gentle subcellular fractionation reveals transhydrogenase activity only in the cytosolic fraction of both Saccharomyces cerevisiae and Candida utilis while intact mitochondria and microsomes are without activity . On sucrose gradient centrifugation, this soluble cytosolic activity proves to be primarily in a high-molecular-weight (greater than 10(6)) band which has salmon-colored fluorescence on uv illumination . Sonication of the particulate subcellular fractions solubilizes substantial transhydrogenase activity from mitochondria of C . utilis (but not from S . cerevisiae) which on sucrose gradients consists of both high (greater than 10(6))- and low-molecular-weight active fractions, each with yellow-green fluorescence . Ammonium sulfate fractionation and sucrose gradient centrifugation of protein solubilized from whole yeast of both species by vigorous homogenization with glass beads confirms the presence and fluorescence of these various molecular weight forms . The relationship of these activities to other enzymatic activities (especially the mitochondrial external NADH dehydrogenase) is discussed.

Genetics, 1985 Dec, 111(4), 715 - 34
Isolation and characterization of mutations in the beta-tubulin gene of Saccharomyces cerevisiae; Thomas JH et al.; Of 173 mutants of Saccharomyces cerevisiae resistant to the antimitotic drug benomyl (BenR), six also conferred cold-sensitivity for growth and three others conferred temperature-sensitivity for growth in the absence of benomyl . All of the benR mutations tested, including the nine conditional-lethal mutations, were shown to be in the same gene . This gene, TUB2, has previously been molecularly cloned and identified as the yeast structural gene encoding beta-tubulin . Four of the conditional-lethal alleles of TUB2 were mapped to particular restriction fragments within the gene . One of these mutations was cloned and sequenced, revealing a single amino acid change, from arginine to histidine at amino acid position 241, which is responsible for both the BenR and the cold-sensitive lethal phenotypes . The terminal arrest morphology of conditional-lethal alleles of TUB2 at their restrictive temperature showed a characteristic cell-division-cycle defect, suggesting a requirement for tubulin function primarily in mitosis during the vegetative growth cycle . The TUB2 gene was genetically mapped to the distal left arm of chromosome VI, very near the actin gene, ACT1; no CDC (cell-division-cycle) loci have been mapped previously to this location . TUB2 is thus the first cell-division-cycle gene known to encode a cytoskeletal protein that has been identified in S . cerevisiae.

Cell, 1985 Dec, 43(2 Pt 1), 493 - 505
DNA sequence and characterization of the S . cerevisiae gene encoding adenylate cyclase; Kataoka T et al.; We have cloned CYR1, the S . cerevisiae gene encoding adenylate cyclase . The DNA sequence of CYR1 can encode a protein of 2026 amino acids . This protein would contain a central region comprised of over twenty copies of a 23 amino acid repeating unit with remarkable homology to a 24 amino acid tandem repeating unit of a trace human serum glycoprotein . Gene disruption and biochemical experiments indicate that the catalytic domain of adenylate cyclase resides in the carboxyl terminal 400 amino acids . Elevated expression of adenylate cyclase suppresses the lethality that otherwise results from loss of RAS gene function in yeast . Yeast adenylate cyclase, made in E . coli, cannot be activated by added RAS protein.

Appl Environ Microbiol, 1985 Nov, 50(5), 1200 - 7
Phenotype character of the methylglyoxal resistance gene in Saccharomyces cerevisiae: expression in Escherichia coli and application to breeding wild-type yeast strains; Murata K et al.; The gene responsible for the methylglyoxal resistance of Saccharomyces cerevisiae was cloned, and its phenotypic characteristics were investigated . S . cerevisiae cells with the gene could accumulate large amounts of glutathione in the medium and should remarkably high resistance to various toxic compounds such as methylglyoxal, tetramethylthiuram disulfide, iodoacetamide, and heavy-metal ions . The gene was also expressed in Escherichia coli cells, and the resistance of E . coli cells to toxic compounds also increased as observed for S . cerevisiae cells . The phenotypic characteristics of the gene were applicable to the selection of the transformants of wild-type yeast strains having no genetic markers.

Mutat Res, 1985 Nov, 144(3), 165 - 9
Conditions that influence the genetic activity of potassium dichromate and chromium chloride in Saccharomyces cerevisiae; Galli A et al.; Potassium dichromate and chromium chloride were analyzed for their ability to induce mitotic gene conversion and point reverse mutation in D7 diploid strain of S . cerevisiae . We used cells from the stationary phase of growth with and without metabolic activation (S9 hepatic fraction) and cells from the logarithmic phase, that contain a high level of cytochrome P-450 and have a greater permeability . In the present work we confirmed the genetic activity of K2Cr2O7 in cells from the stationary phase, with and without S9 fraction and in cells from the logarithmic growth phase . A slight increase in genetic activity was observed in experiments with CrCl3 using phosphate buffer, but no genetic effects were noted in Tris-HCl buffer . Our studies suggest that phosphate ion may be the carrier responsible of the entrance of trivalent chromium in the cells . The higher cellular permeability may account for the different results obtained with both compounds in cells from the stationary and logarithmic phases of growth.

Mol Cell Biol, 1985 Oct, 5(10), 2746 - 52
Expression of normal and activated human Ha-ras cDNAs in Saccharomyces cerevisiae; Clark SG et al.; We expressed normal and activated human cellular Ha-ras cDNAs which encode 21,000-dalton polypeptides (p21s) in Saccharomyces cerevisiae by their insertion into a 2 micron-based replicating plasmid vector under 3-phosphoglycerate kinase promoter control . We found that newly synthesized p21 in S . cerevisiae was produced as a soluble precursor (pro-p21) which matured into a form electrophoretically indistinguishable from the processed form (p21) observed in mammalian cells . Coincident with the processing event was translocation to a membrane component, suggesting a coupling of the two events . Using vectors that direct the synthesis of p21 variants possessing the ability to autophosphorylate in vitro, we found that processing of p21 did not significantly affect this autophosphorylation reaction . In contrast to Escherichia coli, marked phenotypic changes were observed in S . cerevisiae as a consequence of the synthesis of p21, including reduction in growth rate and induction of flocculation . Accompanying these phenotypic alterations was a significant elevation of adenylate cyclase activity.

EMBO J, 1985 Oct, 4(10), 2649 - 56
In vivo modulation of yeast tRNA gene expression by 5'-flanking sequences; Raymond KC et al.; A pentadecanucleotide sequence, TTTCAACAAATAAGT, contiguous with the 5'-end of Saccharomyces cerevisiae tRNA-Leu3 coding sequence acts as a positive modulator of transcription in a homologous in vitro system . To determine whether modulation also takes place in vivo, the amber suppressor forms of tRNA-Leu3 genes with different 5'-flanking sequences were generated by site-specific mutagenesis and cloned into YCp19, a yeast vector maintained at 1-2 copies per cell . These plasmids were transformed into S . cerevisiae strains marked with amber mutations lys2-801, met8-1, and tyr7-1 . The ability of the tRNA-Leu3 amber suppressor genes (tDNA-Leu3A) to suppress functionally lys2-801 and tyr7-1 mutations in the yeast host strain correlated well with template activities measured in vitro . We conclude that the plasmid-borne tRNA gene acts as an effective suppressor from the plasmid and the conserved pentadecanucleotide sequence modulated the expression of yeast tRNA-Leu3 in vivo as well as in vitro . This regulatory sequence if found associated with genes coding for a number of tRNAs which are abundant in yeast . We postulate that this sequence represents a mechanism by which production of specific tRNAs can be enhanced to match demand created by codon use preferences.

J Mol Biol, 1985 Sep 20, 185(2), 451 - 5
Electron-microscopic examination of the binding of a large RNA polymerase III transcription factor to a tRNA gene; Stillman DJ et al.; A Saccharomyces cerevisiae RNA polymerase III transcription factor was previously shown to bind stably to tRNA genes . This transcription factor has been further purified on the basis of its large size and its binding to a S . cerevisiae tRNALeu3 gene has been examined by electron microscopy . Site-specific binding of the factor to the tRNALeu3 gene sharply bends the DNA.

Nucleic Acids Res, 1985 Sep 11, 13(17), 6273 - 82
Nucleotide sequence of Candida pelliculosa beta-glucosidase gene; Kohchi C et al.; The nucleotide sequence of the DNA fragment containing the beta-glucosidase gene of Candida pelliculosa was determined . Analysis of the sequence revealed three open reading frames which could encode 65,825, and 412 amino acid residues . The presence of the second frame was found to be sufficient for the expression of the beta-glucosidase gene in a heterologous host Saccharomyces cerevisiae . Putative protein encoded by this gene had hydrophobic amino acids, resembling a signal peptide, at its N-terminal region and 19 potential glycosylation sites . Codon usage of Candida genes had the similar pattern shown in S.cerevisiae . Codon bias of the beta-glucosidase gene of Candida was relatively low, compared with that of the highly expressed genes of S . cerevisiae.

Mol Cell Biol, 1985 Sep, 5(9), 2369 - 80
Identification of autonomously replicating circular subtelomeric Y' elements in Saccharomyces cerevisiae; Horowitz H et al.; We marked a large number of yeast telomeres within their Y' regions by transforming strains with a fragment of Y' DNA into which the URA3 gene had been inserted . A few of the Ura+ transformants obtained were very unstable and were found to contain autonomously replicating URA3-marked circular Y' elements in high copy number . These marked extrachromosomal circles were capable of reintegrating into the chromosome at other telomeric locations . In contrast, most of the Ura+ transformants obtained were quite stable mitotically and were marked at bona fide chromosomal ends . These stable transformants gave rise to mitotically unstable URA3-marked circular Y' elements at a low frequency (up to 2.5%) . The likelihood that such excisions and integrations represent a natural process in Saccharomyces cerevisiae is supported by our identification of putative Y' circles in untransformed strains . The transfer of Y' information among telomeres via a circular intermediate may be important for homogenizing the sequences at the ends of yeast chromosomes and for generating the frequent telomeric rearrangements that have been observed in S . cerevisiae.

Mol Cell Biol, 1985 Sep, 5(9), 2349 - 60
A hierarchy of trans-acting factors modulates translation of an activator of amino acid biosynthetic genes in Saccharomyces cerevisiae; Hinnebusch AG; The GCN4 gene encodes a positive effector of amino acid biosynthetic genes in Saccharomyces cerevisiae . Genetic analysis has suggested that GCN4 is regulated by a hierarchy of interacting positive and negative effectors in response to amino acid starvation . Results presented here for a GCN4-lacZ gene fusion support this regulatory model and suggest that the regulators of GCN4 exert their effects primarily at the level of translation of GCN4 mRNA . Both the GCN2 and GCN3 products appear to stimulate translation of GCN4 mRNA in response to amino acid starvation, because a recessive mutation in either gene blocked derepression of GCN4-lacZ fusion enzyme levels but did not reduce the fusion transcript level relative to that in wild-type cells grown in the same conditions . The GCD1 product appears to inhibit translation of GCN4 mRNA because under certain growth conditions, the gcd1-101 mutation led to derepression of the GCN4-lacZ fusion enzyme level in the absence of any increase in the fusion transcript level . In addition, the gcd1-101 mutation suppressed the low translational efficiency of GCN4-lacZ mRNA observed in gcn2- and gcn3- cells . A deletion of four small open reading frames in the 5' leader of GCN4-lacZ mRNA mimicked the effect of a gcd1 mutation and derepressed translation of the fusion transcript in the absence of either starvation conditions or the GCN2 and GCN3 products . By contrast, in a gcd1- strain, the deletion resulted in little additional increase in the translational efficiency of the fusion transcript . These results suggest that GCD1 mediates the translational repression normally exerted by the GCN4 leader sequences and that GCN2 and GCN3 antagonize these negative elements in response to amino acid starvation . The effects of the trans-acting mutations on the translation of GCN4-lacZ mRNA remained intact even when transcription of the fusion gene was placed under the control of the S . cerevisiae GAL1 transcriptional control element.

Appl Environ Microbiol, 1985 Sep, 50(3), 685 - 9
Recovery of Saccharomyces cerevisiae from ethanol-induced growth inhibition; Walker-Caprioglio HM et al.; Ethanol caused altered mobility of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene in plasma membrane preparations of Saccharomyces cerevisiae . Because lipids had been shown to protect yeast cells against ethanol toxicity, sterols, fatty acids, proteins, and combinations of these were tested; however, protection from growth inhibition was not seen . Ethanol-induced, prolonged lag periods and diminished growth rates in S . cerevisiae were reduced by an autoconditioning of the medium by the inoculum.

J Mol Biol, 1985 Aug 20, 184(4), 565 - 76
var1 Gene on the mitochondrial genome of Torulopsis glabrata; Ainley WM et al.; We have cloned and sequenced a region of the Torulopsis glabrata mitochondrial genome homologous to the Saccharomyces cerevisiae var1 gene (var1Sc) . An open reading frame that could encode a protein of 339 amino acids was found with 72.7% amino acid and 85.3% nucleotide sequence homology to the S . cerevisiae var1 gene . The T . glabrata gene (var1Tg) is transcribed yielding two stable RNAs, a more abundant 13.5 S RNA and a less abundant 18 S species . We have also identified a candidate for a T . glabrata var1 protein among mitochondrial translation products labeled in isolated mitochondria . The var1Tg gene is even more A + T-rich (93%) than var1Sc (89.6%) and has conserved the strong codon bias of var1Sc . Major differences between the two sequences were found . Significant among these are that no GC clusters are found in var1Tg and the sequences surrounding each of the sites where known polymorphisms exist in var1Sc have deletions at the corresponding sites in var1Tg . These data are discussed with respect to possible origins of these var1 genes and translocation of GC clusters in S . cerevisiae mitochondrial DNA.

Genetics, 1985 Aug, 110(4), 591 - 607
Mapping CDC mutations in the yeast S . cerevisiae by rad52-mediated chromosome loss; Hanic-Joyce PJ; Using the chromosome loss-mapping method of Schild and Mortimer, I have mapped several new temperature-sensitive mutations that define five CDC genes . Modified procedures were used to facilitate mapping temperature-sensitive mutations in general, and these modifications are discussed . The mutations were assigned to specific chromosomes by chromosome loss procedures, and linkage relationships were determined subsequently by standard tetrad analysis . Four of the mutations define new loci . The fifth mutation, cdc63-1, is shown to be allelic to previously known mutations in the PRT1 gene.

Proc Natl Acad Sci U S A, 1985 Aug, 82(15), 5060 - 3
Isolation of the gene encoding adenylate cyclase in Saccharomyces cerevisiae; Casperson GF et al.; By complementation of the cyr1-1 mutation in Saccharomyces cerevisiae, we have isolated yeast genomic DNA containing the structural gene that encodes the catalytic unit of adenylate cyclase (EC 4.6.1.1) . The isolated DNA restored adenylate cyclase activity to cyr1-1 mutants and directed integration at the CYR1 locus . Wild-type strains transformed with CYR1 DNA on the high copy number vector YEp24 contained 4- to 6-fold more adenylate cyclase activity than strains carrying the plasmid with no insert . This result suggests that expression of the CYR1 gene product, rather than that of other polypeptide components of the adenylate cyclase system, limits total adenylate cyclase activity in S . cerevisiae . CYR1-containing plasmids also complemented the temperature-sensitive growth defect of the cell division cycle mutation cdc35-1, which confers a phenotype under restrictive conditions similar to that of cyr1-1 and maps to the same locus . Further, cdc35-1 cam mutants, which contain mutations that enable them to take up cAMP from the medium, grew at the restrictive temperature in the presence of exogenous cAMP . These observations support the view that CDC35 and CYR1 are allelic and confirm the hypothesis that cAMP synthesis is required for cells to pass through the "start" position of the cell division cycle.

J Bacteriol, 1985 Aug, 163(2), 560 - 7
Regulation of phospholipid synthesis in phosphatidylserine synthase-deficient (chol) mutants of Saccharomyces cerevisiae; Letts VA et al.; chol mutants of Saccharomyces cerevisiae are deficient in the synthesis of the phospholipid phosphatidylserine owing to lowered activity of the membrane-associated enzyme phosphatidylserine synthase . chol mutants are auxotrophic for ethanolamine or choline and, in the absence of these supplements, cannot synthesize phosphatidylethanolamine or phosphatidylcholine (PC) . We exploited these characteristics of the chol mutants to examine the regulation of phospholipid metabolism in S . cerevisiae . Macromolecular synthesis and phospholipid metabolism were examined in chol cells starved for ethanolamine . As expected, when chol mutants were starved for ethanolamine, the rates of synthesis of the phospholipids phosphatidylethanolamine and PC declined rapidly . Surprisingly, however, coupled to the decline in PC biosynthesis was a simultaneous decrease in the overall rate of phospholipid synthesis . In particular, the rate of synthesis of phosphatidylinositol decreased in parallel with the decline in PC biosynthesis . The results obtained suggest that the slowing of PC biosynthesis in ethanolamine-starved chol cells leads to a coordinated decrease in the synthesis of all phospholipids . However, under conditions of ethanolamine deprivation in chol cells, the cytoplasmic enzyme inositol-1-phosphate synthase could not be repressed by exogenous inositol, and the endogenous synthesis of the phospholipid precursor inositol appeared to be elevated . The implications of these findings with respect to the coordinated regulation of phospholipid synthesis are discussed.

Eur J Biochem, 1985 Aug 1, 150(3), 435 - 9
Cytochrome b, the var 1 protein, and subunits I and III of cytochrome c oxidase are synthesized without transient presequences in Saccharomyces cerevisiae; Mannhaupt G et al.; The N-termini of four mitochondrial translation products, the var 1 protein, cytochrome b, and subunits I and III of cytochrome c oxidase have been characterized in Saccharomyces cerevisiae and compared with the known DNA sequences of the respective structural genes . The four mature proteins correspond to the predicted primary translation products and retain the formylated methionine residue . Thus, subunit II of cytochrome c oxidase studied previously {Pratje et al . (1983) EMBO J.2, 1049-1054} is so far the only mitochondrial translation product carrying a N-terminal-extended transient presequence in S . cerevisiae.

Biochem Int, 1985 Aug, 11(2), 255 - 63
High performance liquid chromatography purification and amino terminal sequence analysis of the subunits of bovine heart mitochondrial F1-ATPase; Crabb JW et al.; All five subunits of bovine heart mitochondrial F1-ATPase have been isolated by reverse-phase HPLC and NH2-terminal sequences determined by gas phase Edman degradations . Bovine gamma exhibits 16 identities in the first 30 residues compared with the NH2-terminus of gamma from E.coli F1 . Bovine delta exhibit about 27% identity with residues 28-59 of precursor delta from N.crassa and in the first six residues is identical with delta from S.cerevisiae . Approximately half of bovine epsilon has been sequenced . Possibly significant sequence similarities exist between bovine gamma and epsilon and kinase-related gene and oncogene products . The bovine alpha subunit has a blocked NH2-terminus.

Genetics, 1985 Jul, 110(3), 381 - 95
Altered fidelity of mitotic chromosome transmission in cell cycle mutants of S . cerevisiae; Hartwell LH et al.; Thirteen of 14 temperature-sensitive mutants deficient in successive steps of mitotic chromosome transmission (cdc2, 4, 5, 6, 7, 8, 9, 13, 14, 15, 16, 17 and 20) from spindle pole body separation to a late stage of nuclear division exhibited a dramatic increase in the frequency of chromosome loss and/or mitotic recombination when they were grown at their maximum permissive temperatures . The increase in chromosome loss and/or recombination is likely to be due to the deficiency of functional gene product rather than to an aberrant function of the mutant gene product since the mutant alleles are, with one exception, recessive to the wild-type allele for this phenotype . The generality of this result suggests that a delay in almost any stage of chromosome replication or segregation leads to a decrease in the fidelity of mitotic chromosome transmission . In contrast, temperature-sensitive mutants defective in the control step of the cell cycle (cdc28), in cytokinesis (cdc3) or in protein synthesis (ils1) did not exhibit increased recombination or chromosome loss.--Based upon previous results with mutants and DNA-damaging agents in a variety of organisms, we suggest that the induction of mitotic recombination in certain mutants is due to the action of a repair pathway upon nicks or gaps left in the DNA . This interpretation is supported by the fact that the induced recombination is dependent upon the RAD52 gene product, as essential component in the recombinogenic DNA repair pathway . Gene products whose deficiency leads to induced recombination are, therefore, strong candidates for proteins that function in DNA metabolism . Among the mutants that induce recombination are those known to be defective in some aspect of DNA replication (cdc2, 6, 8, 9) as well as some mutants defective in the G2 (cdc13 and 17) and M (cdc5 and 14) phases of the mitotic cycle . We suggest that special aspects of DNA metabolism may be occurring in G2 and M in order to prepare the chromosomes for proper segregation.

J Bacteriol, 1985 Jul, 163(1), 199 - 207
Involvement of heme biosynthesis in control of sterol uptake by Saccharomyces cerevisiae; Lewis TA et al.; Wild-type Saccharomyces cerevisiae do not accumulate exogenous sterols under aerobic conditions, and a mutant allele conferring sterol auxotrophy (erg7) could be isolated only in strains with a heme deficiency . delta-Aminolevulinic acid (ALA) fed to a hem1 (ALA synthetase-) erg7 (2,3-oxidosqualene cyclase-) sterol-auxotrophic strain of S . cerevisiae inhibited sterol uptake, and growth was negatively affected when intracellular sterol was depleted . The inhibition of sterol uptake (and growth of sterol auxotrophs) by ALA was dependent on the ability to synthesize heme from ALA . A procedure was developed which allowed selection of strains which would take up exogenous sterols but had no apparent defect in heme or ergosterol biosynthesis . One of these sterol uptake control mutants possessed an allele which allowed phenotypic expression of sterol auxotrophy in a heme-competent background.

Mutat Res, 1985 Jul, 154(1), 1 - 27
Spontaneous mutagenesis: the roles of DNA repair, replication, and recombination; Sargentini NJ et al.; There appears to be no dearth of mechanisms to explain spontaneous mutagenesis . In the case of base substitutions, data for bacteriophage T4 and especially for E . coli and S . cerevisiae suggest important roles in spontaneous mutagenesis for the error-prone repair of DNA damage (to produce mutations) and for error-free repair of DNA damage (to avoid mutagenesis) . Data from the very limited number of studies on the subject suggest that about 50% of the spontaneous base substitutions in E . coli, and perhaps 90% in S . cerevisiae are due to error-prone DNA repair . On the other hand, spontaneous frameshifts and deletions seem to result from mechanisms involving recombination and replication . Spontaneous insertions have been shown to be important in the strongly polar inactivation of certain loci, but it is less important at other loci . Perhaps with continued study, the term "spontaneous mutagenesis" will be replaced by more specific terms such as 5-methylcytosine deamination mutagenesis, fatty acid oxidation mutagenesis, phenylalanine mutagenesis, and imprecise-recombination mutagenesis . While most studies have concentrated on mutator mutations, the most conclusive data for the actual source of spontaneous mutations have come from the study of antimutator mutations . Further study in this area, perhaps along with an understanding of chemical antimutagens, should be invaluable in clarifying the bases of spontaneous mutagenesis.

Mol Gen Mikrobiol Virusol, 1985 Jul, (7), 26 - 31
{New yeast vectors containing the autonomously replicating sequences from Candida maltosa genome}; Polumienko AL et al.; Two different DNA sequences from the yeast Candida maltosa confer the ability to replicate autonomously to the yeast integrative vector pLD700 on which they are cloned . The recombinant plasmids pLD701 and pLD702 with autonomously replicating sequences (ARS) from Candida maltosa and LEU2 gene from Saccharomyces cerevisiae transform the auxotrophic strain S . cerevisiae DC5 with the efficiency 3-5 x 10(3) per microgram of DNA . Like other yeast vectors harbouring ARS, these plasmids are not stable in yeast cells . Restriction and hybridization analyses have revealed the pLD701 plasmid to contain ARS from chromosomal DNA of C . maltosa . Plasmid pLD701 appears to be a useful vector for yeast transformation.

EMBO J, 1985 Jul, 4(7), 1855 - 60
Endocytosis in Saccharomyces cerevisiae: internalization of enveloped viruses into spheroplasts; Makarow M; When vesicular stomatitis virus was incubated with Saccharomyces cerevisiae spheroplasts at 37 degrees C, part of the virus was internalized by the spheroplasts as shown by the following criteria . (i) The spheroplast-associated virus was protected from proteinase K digestion, which releases surface-bound virus by degrading the envelope glycoproteins . (ii) The spheroplast-associated virus was resistant to mild Triton X-100 treatment, which readily solubilizes the virus . The same results were obtained with Semliki Forest virus . Internalization of the two viruses followed linear kinetics up to 90 min at 37 degrees C . Internalization was concentration- and temperature-dependent . At 11 degrees C no uptake could be detected for at least 2 h . Homogenization and organelle fractionation protocols were designed for the S . cerevisiae spheroplasts to study the compartments into which the virions were internalized . Three compartments containing both marker viruses could be separated in density gradients . One coincided with vacuole markers, one banded at a slightly higher and one at a similar density to the plasma membrane markers . Thus, S . cerevisiae spheroplasts appear to have the capability of endocytosing particulate markers like viruses . The companion paper describes internalization of two soluble macromolecules, alpha-amylase and fluorescent dextran, into intact cells.

EMBO J, 1985 Jul, 4(7), 1861 - 6
Endocytosis in Saccharomyces cerevisiae: internalization of alpha-amylase and fluorescent dextran into cells; Makarow M; In the preceding paper I reported that Saccharomyces cerevisiae spheroplasts were able to internalize particulate markers, enveloped viruses, into intracellular organelles . Here the internalization of soluble macromolecules into cells having an intact cell wall is described . alpha-Amylase was taken up into cells in a temperature- and concentration-dependent way . The kinetics of accumulation were linear for the first 20-40 min at 37 degrees C and then started to level off . Internalization of alpha-amylase into spheroplasts displayed similar characteristics, but the accumulation rate was about four times higher than into cells . Fluorescent dextran was used to mark morphologically the compartment into which internalization occurred . This marker was accumulated into the vacuole of the cells in a time-, temperature- and concentration-dependent way . A temperature-sensitive mutant deficient in exocytosis was found to be defective in intracellular accumulation of alpha-amylase and dextran . At the restrictive temperature, very little alpha-amylase accumulated into the cells and only faint staining of intracellular organelles with fluorescent dextran could be detected . At the permissive temperatures, accumulation of alpha-amylase and dextran into the mutant cells was comparable with accumulation into wild-type cells . I conclude that alpha-amylase and fluorescent dextran were internalized into S . cerevisiae cells and directed into the vacuoles.

J Bacteriol, 1985 Jun, 162(3), 1135 - 41
Coordinate regulation of phospholipid biosynthesis in Saccharomyces cerevisiae: pleiotropically constitutive opi1 mutant; Klig LS et al.; Phospholipid metabolism in the Saccharomyces cerevisiae opi1 mutant, which excretes inositol and is constitutive for the biosynthetic enzyme inositol-1-phosphate synthase (M . Greenberg, P . Goldwasser, and S . Henry, Mol . Gen . Genet . 186:157-163, 1982), was examined and compared to that of a wild-type strain . In wild-type S . cerevisiae, the phospholipid composition and the relative rates of synthesis of individual phospholipids change in response to the availability of exogenous supplies of soluble phospholipid precursors, particularly inositol . The opi1 mutant, in contrast, displays a relatively invariant phospholipid composition, and its pattern of phospholipid synthesis does not change in response to exogenous phospholipid precursors . Phosphatidylinositol synthase was not found to be regulated in either wild-type or opi1 cells . In wild-type cells, phosphatidylserine synthase and the phospholipid N-methyltransferases are coordinately repressed in response to a combination of inositol and choline . However, in opi1 cells these activities are expressed constitutively . These results suggest that the gene product of the OPI1 locus participates in the coordinate regulation of phospholipid synthesis.

Proc Natl Acad Sci U S A, 1985 Jun, 82(12), 4167 - 71
Caenorhabditis elegans DNA that directs segregation in yeast cells; Stinchcomb DT et al.; We have isolated seven DNA fragments from Caenorhabditis elegans that enhance the mitotic segregation of autonomously replicating plasmids in the yeast Saccharomyces cerevisiae . These segregators, designated SEG1-SEG7, behave like isolated yeast chromosomes: they increase the stability and simultaneously lower the copy number of circular plasmids during mitotic growth in yeast . During meiosis, plasmids containing the C . elegans segregators show higher levels of precocious or aberrant disjunction than do plasmids bearing isolated yeast centromeres . Yet one of the segregators improved the meiotic segregation of the parental plasmid . We estimate that there may be as many as 30 segregator sequences in the C . elegans genome, a value that is consistent with the polycentric nature of C . elegans chromosomes . Five of the seven segregators are linked to sequences that are repeated in the worm genome, and four of these five segregators cross-hybridize . Other members of this family of repetitive DNA do not contain segregator function . Segregator sequences may prove useful for probing the structure of centromeres of both C . elegans and S . cerevisiae chromosomes.

Biochem Int, 1985 Jun, 10(6), 907 - 15
Chemical synthesis of a mitochondrial gene designed for expression in the yeast nucleus; Gearing DP et al.; A novel DNA sequence coding for subunit 8 of the mitochondrial ATPase of Saccharomyces cerevisiae has been constructed by chemical synthesis . The synthetic gene, termed NAP1, is designed for expression in the yeast nucleus and codes for a 48 amino acid polypeptide identical to that encoded by the mitochondrial aap1 gene of S . cerevisiae . The codons chosen for the NAP1 sequence correspond almost exclusively to those most frequently occurring in highly expressed yeast genes . The NAP1 coding region differs in 31 codons from that of aap1, and is flanked by sequences carrying restriction enzyme sites useful for cloning and for gene expression . A 170 bp double stranded DNA molecule was constructed by assembling 12 oligonucleotides (12 to 45 bases in length) in a single annealing/ligation mixture . This synthetic gene will provide a route for the systematic manipulation, through in vitro mutagenesis, of the structure of a protein normally encoded by mitochondrial DNA.

FEBS Lett, 1985 May 20, 184(2), 313 - 7
Apparent inhibition of glycoprotein synthesis by S.cerevisiae mating pheromones; Orlean P et al.; Saccharomyces cerevisiae mating pheromones a and alpha factor strongly inhibit the incorporation of radiolabelled glucosamine into N-glycosylated proteins of corresponding haploid cells . This observation was erroneously interpreted as an inhibition of glycoprotein synthesis . It has turned out that alpha factor causes a 4-5-fold dilution of incorporated {14C}glucosamine with non-radioactive endogenous precursor . In the case of the {14C}chitin synthesized, which does not show inhibition by alpha factor, the lowering of the specific activity of the precursor is exactly compensated for by an increased rate of chitin synthesis caused by alpha factor.

FEBS Lett, 1985 May 6, 184(1), 1 - 5
Introns, protein syntheses and aging; Picard-Bennoun M; In the fungus Podospora, a correlation has recently been established between the presence of circular DNA molecules arising from the mitochondrial genome (SEN-DNAs) and the senescence syndrome . Here, I propose a hypothesis which accounts for the initial event which leads to the first SEN-DNA . A molecule in the most frequent situation where the SEN-DNA is an intron which might code for a maturase . This hypothesis is based upon several observations made either in Podospora or in the yeast S . cerevisiae . It assumes that mitochondrially synthesized maturases are unspecific nucleases able to work at the level of RNA and DNA molecules . Their specificity for RNA splicing instead of DNA is given by cytoplasmic proteins . Therefore, if the balance between cytoplasmic and mitochondrial protein syntheses is disturbed in favour of the mitochondrial compartment, the maturase would be accumulated and allowed to splice introns from DNA instead of RNA molecules . This hypothesis can account for aging of higher eucaryotic cells by postulating analogous processes in their nuclear compartment.

Mol Gen Mikrobiol Virusol, 1985 May, (5), 28 - 31
{Transformation of the yeast Hansenula polymorpha by a hybrid plasmid containing the fragment of host DNA}; Makhina EN et al.; Spheroplasts of Hansenula polymorpha strain deficient in 2-isopropylmalate dehydrogenase have been shown to be transformed by the DNA of a hybrid plasmid pHRI, carrying the LEU2 gene from S . cerevisiae and 2.0 kilobase HindIII fragment of H . polymorpha genomic DNA . The frequency of transformants has reached 10(3) per 1 microgram of transforming DNA . Plasmid pHRI is maintained in transformants as an autonomous circular DNA molecule and is inherited by 1-2% fraction of cells from the population growing under the selective conditions . Transformation takes place under the same conditions that are required for spheroplast fusion . Thus, H . polymorpha becomes one more species of yeast susceptible to hybrid plasmid-mediated gene transfer in the process of DNA transformation.

Nucleic Acids Res, 1985 Apr 25, 13(8), 2803 - 14
Transcription of a Drosophila tRNAArg gene in yeast extract: 5'-flanking sequence dependence for transcription in a heterologous system; Schaack J et al.; The Drosophila tRNA gene encoded on pArg is efficiently transcribed in extracts of Saccharomyces cerevisiae, but the efficiency is 5'-flanking sequence dependent: deletion to between positions -21 and -17 (relative to position +1 of the mature coding sequence) reduces transcription to a very low level . This demonstrates that requirement for wild-type 5'-flanking sequence exists in the case of a heterologous combination of a tRNA gene and transcription extract . Expression of pArg in vivo in S . cerevisiae is also dependent on the wild-type 5'-flanking sequence, but only with deletion to between -17 and -11 is the steady-state level of pArg transcripts reduced to near zero . The 5'-flanking sequence requirement in S . cerevisiae extract is similar to that found in Drosophila Kc cell extract . However, transcription kinetics distinguish S . cerevisiae extract from that of Drosophila Kc cells . tRNA genes added to S . cerevisiae extract exhibit a lag phase before initiation of active transcription, but this lag is much shorter and much less temperature dependent than is the lag phase in Drosophila Kc cell extract.

Nucleic Acids Res, 1985 Apr 25, 13(8), 3005 - 14
A mitochondrial reading frame which may code for a maturase-like protein in Saccharomyces cerevisiae; Seraphin B et al.; In S . cerevisiae, the large oxi3/oli2 mitochondrial transcript contains the products of the oxi3, aap1 and oli2 genes and an unassigned reading frame, RF3 . In the work presented here, we have completed the nucleotide sequence of RF3 . We have shown that RF3 is composed of four fairly large ORFs which overlap within GC rich sequences . Furthermore, a shift of +1 base was found between each pair of consecutive reading frames . We discuss how these frameshifts could be removed to produce a 500 aminoacid long protein containing the two well conserved P1 and P2 oligopeptide sequences featuring several mitochondrial intron reading frames, suggesting, thereby, a RNA-maturase-like activity for the putative RF3 protein . In addition, we suggest that the insertion of GC clusters in a gene could provide a novel way of regulating its expression.

Can J Microbiol, 1985 Apr, 31(4), 322 - 6
Phospholipid enrichment of Saccharomyces cerevisiae and its effect on polyene sensitivity; Rao TV et al.; Sensitivity to polyene antibiotics, e.g., nystatin, amphotericin B, and filipin, was determined in phosphatidylcholine (PC) or phosphatidylethanolamine (PE) or phosphatidylserine (PS) enriched Saccharomyces cerevisiae cells, using glutamic acid, phenylalanine, glycine, and lysine transport as an index of polyene antibiotic action . As compared with normal cells, phospholipid-enriched cells acquired resistance towards different polyenes . However, the sensitivity of glutamic acid transport towards nystatin remained unaffected in PC-, PE-, or PS-enriched cells . In contrast to nystatin, the other two polyenes were more effective in checking the influx of amino acids . Results demonstrated that the specific enrichment of PC, PE, or PS could selectively protect S . cerevisiae cells from polyene antibiotic action.

Mol Cell Biol, 1985 Apr, 5(4), 751 - 61
Saccharomyces cerevisiae exhibits a sporulation-specific temporal pattern of transcript accumulation; Kaback DB et al.; Cultures of the yeast Saccharomyces cerevisiae that are heterozygous for the mating type (MATa/MAT alpha) undergo synchronous meiosis and spore formation when starved for nitrogen and supplied with a nonfermentable carbon source such as acetate . Haploid and homozygous MAT alpha/MAT alpha and MATa/MATa diploid cells incubated under the same conditions fail to undergo meiosis and are asporogenous . It has not yet been firmly established that gene expression during sporulation is controlled at the level of transcript accumulation . To examine this question, we used cloned genes that encode a variety of "housekeeping" functions to probe Northern blots to assay the appearance of specific transcripts in both sporulating and asporogenous S . cerevisiae . In sporulating cells, each transcript showed a characteristic pattern of accumulation, reaching a maximum relative abundance at one of several different periods . In contrast, in both asporogenous haploid MATa and diploid MAT alpha/MAT alpha cells, all transcripts accumulated with similar kinetics . These results suggest a sporulation-specific pattern for transcript appearance . During these studies, high levels of several different transcripts were observed at unexpected times in sporulating cells . Histone (H)2A and (H)2B1 transcripts, although most abundant during premeiotic DNA synthesis, remained at one-third to one-half maximal levels after its end and were found in mature ascospores . Their appearance at this time is in sharp contrast to vegetative cells in which these histone transcripts are only found just before and during the period of DNA synthesis . Furthermore, transcripts from GAL10 and CDC10 genes, which are believed to be dispensable for sporulation, were much more abundant in sporulating cells than in asporogenous cells and vegetative cells grown on glucose or acetate . The presence of these transcripts did not appear to be due to a general activation of transcription because each accumulated with different kinetics . In addition, the transcript for at least one gene, HO, that is also dispensable for sporulation was not detected . The increased abundance of transcripts from some genes not required for sporulation leads us to propose that genes preferentially expressed during sporulation need not be essential for this differentiation.

Exp Cell Res, 1985 Apr, 157(2), 387 - 96
Growth and the DNA-division sequence in the yeast Saccharomyces cerevisiae; Singer RA et al.; Cells of the yeast S . cerevisiae can be cultured under conditions in which the DNA-division sequence, and not cellular growth, is the rate-limiting feature for cell proliferation . Relief of these limiting conditions, which has been shown to allow accelerated cell division, did not result in increased rates of cell mass accumulation during the time of rapid cell division . Moreover, under conditions of constant DNA-division sequence constraint, populations of smaller cells produced by slowing growth with cycloheximide gave rise to large cells when cycloheximide was removed . These observations suggest that in proliferating cells of S . cerevisiae the DNA-division sequences does not affect cellular growth.

Nucleic Acids Res, 1985 Mar 25, 13(6), 1841 - 53
Structure of the galactokinase gene of Escherichia coli, the last (?) gene of the gal operon; Debouck C et al.; We present the nucleotide sequence of the galactokinase gene (galK) of Escherichia coli including its 5' and 3' flanking regions . This DNA sequence derives from the lambda gal8 transducing phage and is identical to the sequence present in the galK gene fusion vectors, pKO and pKG, commonly used to study transcriptional regulatory elements . We define the precise 3' junction between the bacterial and phage sequences in lambda gal8 and demonstrate that this junction probably results from a homologous recombination event between identical 9 bp sequences common to the gal operon and phage lambda . Moreover, we examine the 300 bp region located immediately beyond galK for transcription termination function and find no gal operon terminator . Lastly, we compare the galK genes of E . coli and the yeast S . cerevisiae and find several regions of strong homology among which is a potential ATP-binding site homology shared by a variety of ATP-binding proteins including protein kinases encoded by mammalian oncogenes.

FEBS Lett, 1985 Mar 25, 182(2), 413 - 4
'Red pigment' from ADE-2 mutants of S . cerevisiae prevents DNA cleavage by restriction endonucleases; Meskauskas A et al.; Protection of DNA from cleavage by restriction endonucleases EcoRI, HindIII, BamHI, and Bg/II with red pigment, produced by ADE-2 mutants of Saccharomyces cerevisiae is demonstrated . Purification of yeast DNA from pigment can be achieved by chromatography on hydroxyapatite columns.

Biochem Biophys Res Commun, 1985 Mar 15, 127(2), 623 - 8
Spectral properties of a novel cytochrome P-450 of a Saccharomyces cerevisiae mutant SG1 . A cytochrome P-450 species having a nitrogenous ligand trans to thiolate; Yoshida Y et al.; An altered cytochrome P-450 (SG1 P-450) was partially purified from Saccharomyces cerevisiae mutant SG1 which is defective in lanosterol 14 alpha-demethylation . Oxidized SG1 P-450 showed a Soret peak at 422 nm and the alpha peak was lower than the beta peak . This spectrum was considerably different from those of known low-spin P-450s, indicating a unique ligand structure of SG1 P-450 . The absorption spectrum of ferric SG1 P-450 was superimposable on that of the imidazole complex of ferric P-450, suggesting the presence of a nitrogenous ligand such as histidine of the apoprotein at the 6th coordination position . SG1 P-450 was immunochemically indistinguishable from cytochrome P-450 of S . cerevisiae catalyzing lanosterol 14 alpha-demethylation (P-45014DM) but had no lanosterol 14 alpha-demethylase activity.

Mutat Res, 1985 Mar, 155(3), 113 - 5
Mutagenic and recombinogenic activity of hydrazine sulphate in Saccharomyces cerevisiae; Vasudeva M et al.; Treatment of diploid cells of S . cerevisiae with 0.1 and 0.2 M hydrazine sulphate induced reverse mutation, mitotic crossing-over and gene conversion at exposures ranging over 4 orders of magnitude . The percentage of induced mitotic cross-overs and total aberrant colonies as well as the frequencies of revertants and convertants were dependent on the concentration used and the duration of treatment . These genetic events are induced without post-treatment cell division and at exposures which cause a minimal level of cell death.

Mol Cell Biol, 1985 Mar, 5(3), 563 - 8
Cloned mouse DNA fragments can replicate in a simian virus 40 T antigen-dependent system in vivo and in vitro; Ariga H et al.; Mouse liver DNA was cut out with BamHI and cloned into YIp5, which contained the URA3 gene of Saccharomyces cerevisiae in pBR322 . Of the several plasmids isolated, two plasmids, pMU65 and pMU111, could transform S . cerevisiae from the URA- to the URA+ phenotype and could replicate autonomously within the transformant, indicating that mouse DNA fragments present in pMU65 or pMU111 contain autonomously replicating sequences (ARS) for replication in S . cerevisiae . Furthermore, to determine the correlation between ARS function in yeast cells and that in much higher organisms, we tried to challenge these plasmids with the simian virus 40 (SV40) DNA replication system . Of the two plasmids tested, the EcoRI-BglII region of pMU65 could be hybridized with a chemically synthesized 13-nucleotide fragment corresponding to the origin region of SV40 DNA . Both pMU65 (the EcoRI-BglII region cloned in pBR322) and its subclone pMU65EB could replicate semiconservatively, and initiation of DNA replication started from the EcoRI-BglII region when the replicating activity of these plasmids was tested in the in vitro SV40 DNA replication system we have established before . Furthermore, pMU65 and pMU65EB could replicate autonomously within monkey Cos cells which produce SV40 T antigen constitutively . These results show that a 2.5-kilobase fragment of the EcoRI-BglII region in pMU65 contains the ARS needed for replication in the SV40 DNA replication system.

J Bacteriol, 1985 Feb, 161(2), 574 - 82
Polymorphic extracellular glucoamylase genes and their evolutionary origin in the yeast Saccharomyces diastaticus; Yamashita I et al.; DNA coding for extracellular glucoamylase genes STA1 and STA3 was isolated from DNA libraries of two Saccharomyces diastaticus strains, each carrying STA1 or STA3 . Cells transformed with a plasmid carrying either the STA1 or STA3 gene secreted glucoamylases having the same enzymatic and immunological properties and the same electrophoretic mobilities in acrylamide gel electrophoresis as those of authentic glucoamylases . Southern blot analysis of genomic DNA from S . diastaticus and a glucoamylase-non-secreting yeast, Saccharomyces cerevisiae, revealed that the STA1 and STA3 loci of S . diastaticus showed a high degree of homology, and that both yeast species (S . diastaticus and S . cerevisiae) contained DNA segments highly homologous to those of the extracellular glucoamylase genes . Restriction maps of the homologous DNA segments suggested that the extracellular glucoamylase genes of S . diastaticus may have arisen from recombination among the resident DNA segments in S . cerevisiae.

Cell, 1985 Feb, 40(2), 393 - 403
Genetic analysis of the mitotic transmission of minichromosomes; Koshland D et al.; The fidelity of the mitotic transmission of minichromosomes in S . cerevisiae is monitored by a novel visual assay that allows one to detect changes in plasmid copy number in individual mitotic divisions . This assay is used to investigate the mitotic transmission of a plasmid containing a putative yeast origin of replication (ARS 1) and a centromere (CEN3) . The rate of improper segregation for the minichromosome is 200-fold higher than observed for a normal chromosome . However, the replication of the minichromosome is stringently controlled; it overreplicates less than once per one thousand mitotic divisions . We also use this assay to isolate and characterize mutations in ARS 1 and CEN3 . The mutations in ARS 1 define a new domain required for its optimal activity, and the mutations in CEN3 suggest that the integrity of element II is not essential for centromere function . Finally, the phenotypes of the mutations in ARS 1 and CEN3 are consistent with their function in replication and segregation, respectively.

J Biol Chem, 1985 Jan 25, 260(2), 1090 - 5
Purification and properties of H+-translocating, Mg2+-adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae; Uchida E et al.; H+-translocating, Mg2+-ATPase was solubilized from vacuolar membranes of Saccharomyces cerevisiae with the zwitterionic detergent N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate and purified by glycerol density gradient centrifugation . Partially purified vacuolar membrane H+-ATPase, which had a specific activity of 18 units/mg of protein, was separated almost completely from acid phosphatase and alkaline phosphatase . The purified enzyme required phospholipids for maximal activity and hydrolyzed ATP, GTP, UTP, and CTP, with this order of preference . Its Km value for Mg2+-ATP was determined to be 0.21 mM and its optimal pH was 6.9 . ADP inhibited the enzyme activity competitively, with a Ki value of 0.31 mM . The activity of purified ATPase was strongly inhibited by N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, tributyltin, 7-chloro-4-nitrobenzoxazole, diethylstilbestrol, and quercetin, but was not affected by oligomycin, sodium azide, sodium vanadate, or miconazole . It was not inhibited at all by antiserum against mitochondrial F1-ATPase or mitochondrial F1-ATPase inhibitor protein . These results indicated that vacuolar membrane H+-ATPase is different from either yeast plasma membrane H+-ATPase or mitochondrial F1-ATPase . The vacuolar membrane H+-ATPase was found to be composed of two major polypeptides a and b of Mr = 89,000 and 64,000, respectively, and a N,N'-dicyclohexylcarbodiimide binding polypeptide c of Mr = 19,500, whose polypeptide composition was also different from those of either plasma membrane H+-ATPase or mitochondrial F1-ATPase of S . cerevisiae.

Mol Gen Genet, 1985, 200(2), 313 - 21
Response of S . cerevisiae to N-methyl-N'-nitro-N-nitrosoguanidine: mutagenesis, survival and DDR gene expression; Maga JA et al.; We have examined the effects of low concentrations of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on survival and mutagenesis of logarithmically growing Saccharomyces cerevisiae . Pretreatment of cells with nontoxic and submutagenic concentrations of MNNG for several generations does not reduce the cytotoxic and mutagenic effects of exposure to a high concentration of drug . This lack of 'adaptation' in S . cerevisiae was further investigated biochemically and cell extracts prepared from pretreated cells were shown to be deficient in the ability to remove O6-methyl guanine from alkylated DNA . Moreover, we could not detect transfer of the methyl group from the DNA to a protein acceptor in yeast cell extracts suggesting that the level of O6-methyl guanine transferase is below 200 molecules/cell following pretreatment . Exposure of S . cerevisiae to mutagenic concentrations of MNNG stimulates transcription of at least three DNA damage responsive genes that also respond to UV-irradiation and 4-nitroquinoline-1-oxide treatment . These results support the contention that in Saccharomyces alkylation damage is processed by repair pathways that operate on a variety of lesions, and that one or more of these pathways is inducible.

Gene, 1985, 34(2-3), 325 - 34
A DEX gene conferring production of extracellular amyloglucosidase on yeast; Meaden P et al.; A DEX gene from Saccharomyces diastaticus (strain BRG536 alpha DEX1) has been cloned in the hybrid vector pJDB207 . The gene is included within a 3.6-kb fragment and confers production of extracellular amylo-alpha-1,4-glucosidase (AMG) and, thereby the ability to hydrolyse starch or dextrins on Dex- strains of Saccharomyces cerevisiae . The cloned gene hybridizes to three fragments produced by ClaI digestion of DNA from BRG536; one of these (11 kb) cosegregates in crosses with DEX1, while another (10 kb) is present in all Dex+ and Dex- strains examined . Accumulation of extracellular AMG by Dex+ transformants is up to five-fold that of BRG536, and escapes regulation by the CDX1 gene under conditions of excess glucose . The enzyme produced by Dex+ transformants resembles that of BRG536 with respect to Mr (approx . 150 X 10(3)) and effects of temperature and pH . The cloned DEX gene can be used as a selectable marker for introducing recombinant plasmids into wild-type strains of S . cerevisiae.

Mol Cell Biol, 1985 Jan, 5(1), 75 - 84
Specific Saccharomyces cerevisiae genes are expressed in response to DNA-damaging agents; Ruby SW et al.; When exposed to DNA-damaging agents, the yeast Saccharomyces cerevisiae induces the expression of at least six specific genes . We have previously identified one damage inducible (DIN) gene as a gene fusion (din-lacZ fusion) whose expression increases in response to DNA-damaging treatments . We describe here the identification of five additional DIN genes as din-lacZ fusions and the responses of all six DIN genes to DNA-damaging agents . Northern blot analyses of the transcripts of two of the DIN genes show that their levels increase after exposure to DNA-damaging agents . Five of the din-lacZ fusions are induced in S . cerevisiae cells exposed to UV light, gamma rays, methotrexate, or alkylating agents . One of the din-lacZ fusions is induced by either UV or methotrexate but not by the other agents . This finding suggests that there are sets of DIN genes that are regulated differently.

Curr Genet, 1985, 10(4), 261 - 7
Differences in the cytochrome P-450 enzymes of sterol C-14 demethylase mutants of Saccharomyces cerevisiae; King DJ et al.; A number of nystatin-resistant strains of S . cerevisiae have been isolated which are defective in lanosterol C-14 demethylation, a reaction normally catalysed by cytochrome P-450 . In this paper two of these strains have been compared and found to have differences in their reduced-CO difference spectra indicating different distortions in the enzyme molecule . Nystatin resistance in the C-14 demethylation deficient SG1 in shown to be determined by a single gene, and a sterol 5,6-desaturase defect does not appear to be required for viability of SG1, was reported for the C-14 demethylase deficient isolate JR4 by Taylor et al . (1983) . There are at least two discernable mutant phenotypes for the yeast cytochrome P-450 structural gene which give a C-14 demethylase defect.

Curr Genet, 1985, 10(3), 171 - 7
Nuclear mutations affecting the stability of the mitochondrial genome in S . cerevisiae; Rayko E et al.; We have studied a pleiotropic mutation petD in S . cerevisiae which both confers the inability to grow on glycerol (Gly-) and greatly increases the frequency of cytoplasmic petites (Het) . The first phenotype, Gly-, is recessive, whereas the second, Het, is dominant . Genetic and biochemical analysis showed that the majority of the petites in petD strains are not of the rho degree type (completely lacking mit-DNA), but of the rho- type (containing partially deleted mit-DNA) . This finding and the fact that the phenotype Het is dominant argue in favour of the involvement of the petD product in the excision process of the mit-DNA . Another nuclear mutation, mod, was shown to exhibit a dominant epistasy with respect to the Het phenotype of the mutation petD . Two types of Gly+ revertants from petD mutants were isolated: rpa revertants, which restore completely the wild-type phenotype, and rpb revertants, which restore only the growth on glycerol, but still allow the production of high frequencies of cytoplasmic petites . Thus the mutations mod and rpb permit the genetic uncoupling of two phenotypes induced by the mutation petD.

Curr Genet, 1985, 9(5), 333 - 9
Regulation of ureaamidolyase synthesis in Saccharomyces cerevisiae, RNA analysis, and cloning of the positive regulatory gene DURM; Jacobs E et al.; In S . cerevisiae, the synthesis of ureaamidolyase is subject to at least two different forms of regulation: nitrogen catabolite repression and induction by allophanate . Two positive regulatory genes DURM and DURL are involved in the induction process . We have measured the levels of mRNA homologous to the DUR2,1 gene in conditions of ureaamidolyase induction and in regulatory mutants . The amounts of DUR2,1 enzyme and messengers are well coordinated; moreover, the half life of DUR2,1 messengers is identical in the presence or absence of inducer . These data suggest that the ureaamidolyase production is probably controlled at the level of transcription . From a pool of hybrid plasmids carrying Sau3A fragments representing the entire yeast genome, a 13 kb DNA fragment containing the regulatory gene DURM was cloned by complementation of a durM mutation which prevents the growth on allantoin as sole nitrogen source . Cells containing the cloned DNA recover the inducibility of ureaamidolyase by allophanate . Four RNA transcripts have homology to this 13 kb DNA fragment but the study of subcloned restriction endonuclease fragments allowed us to map the DURM regulatory gene within a 4 kilobase pair region . This fragment encodes a 1 kb transcript . The level of this RNA is the same in induced and non-induced cells.

Curr Genet, 1985, 9(2), 179 - 81
Characterization of blasticidin S-resistant mutants of Saccharomyces cerevisiae; Ishiguro J et al.; Blasticidin S-resistant mutants of S . cerevisiae were isolated and characterized . Resistant mutations were found to fall into two complementation groups . A single recessive nuclear gene was responsible for each group, donated as bls1 and bls2, respectively . A gene bls1 was linked to an ilv3 gene located on the right arm of chromosome X . The resistant phenotypes from both genes were not associated with ribosomes known to be target sites of Blasticidin S, when analyzed by poly(U)-directed polyphenylalanine synthesis . The resistant mechanisms of the mutations are discussed in this paper.

Gene, 1985, 37(1-3), 155 - 61
Secretion of mature mouse interleukin-2 by Saccharomyces cerevisiae: use of a general secretion vector containing promoter and leader sequences of the mating pheromone alpha-factor; Miyajima A et al.; We have constructed a general expression vector which allows the synthesis and secretion of processed gene products in Saccharomyces cerevisiae . This vector contains yeast DNA, including the promoter of the mating pheromone (alpha-factor), its downstream leader sequence, and the TRP5 terminator . A cDNA {encoding mature mouse interleukin-2 (IL-2); Yokota et al., Proc . Natl . Acad . Sci . USA 82 (1984) 68-72} was fused immediately downstream to the alpha-factor leader sequence . The resulting recombinant plasmid directed the synthesis of mature mouse IL-2 in S . cerevisiae, with most of the T-cell growth-factor (TCGF) activity secreted into the culture fluid and extracellular space . TCGF activities in the cell extract, as well as in the culture fluid, increased in parallel with cell growth . Production of mature mouse IL-2 was inhibited by tunicamycin (TM), with precursor molecules accumulating in the cell extract . The precursor was processed accurately at the junction between the alpha-factor peptide leader sequence and the coding sequence downstream, yielding mature IL-2 . The Mr of the secreted mouse IL-2 determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was 17 kDal, a value expected for the mature mouse IL-2 polypeptide based on the nucleotide (nt) sequence.

Gene, 1985, 35(1-2), 27 - 32
Plasmids pEMBLY: new single-stranded shuttle vectors for the recovery and analysis of yeast DNA sequences; Baldari C et al.; We describe the construction and properties of pEMBLY plasmids . They belong to a new family of yeast shuttle vectors which are derived from plasmid vector pEMBL9 and offer the following improvement: relatively small size; large number of cloning sites; screening for insert-containing plasmids on indicator plates; different combinations of genes which complement auxotrophic deficiencies and sequences that support DNA replication in Saccharomyces cerevisiae; and ability to isolate the plasmid DNA in single-stranded (ss) form . The yeast S . cerevisiae can be efficiently transformed by these plasmids in both the ss and double-stranded (ds) forms . Finally, the presence of the phage f1 intergenic region allows one to obtain the cloned sequences in the ss form upon infection with the wild-type ss phage {Dotto et al., Virology 114 (1981) 463-473}.

Gene, 1985, 34(2-3), 179 - 86
Cloning of the CDC7 gene of Saccharomyces cerevisiae in association with centromeric DNA; Meddle CC et al.; The cell-division-cycle gene, CDC7, of Saccharomyces cerevisiae has been cloned in a large plasmid (pCM6) containing an insert of about 45 kb of yeast chromosomal DNA in the yeast-Escherichia coli shuttle vector, YRp7 . A subclone (pCM39), having a 7-kb insert with a unique BamHI site, was capable of conferring the ts+ phenotype on a cdc7ts- strain of S . cerevisiae . When this insert was placed in the vector Y1p28, and then cut with BamHI and integrated into the ts- strain, genetic analysis showed the CDC7-complementing activity and an associated vector marker to be linked to the TRP1 locus, i.e., to chromosome IV . Analysis of subclones covering the pCM39 insert showed that pCM52, in which a 3.3-kb EcoRI-HpaI fragment with the unique BamHI site has been cloned, possessed the full complementing activity . Although the CDC7 gene lies across the BamHI site, the main part of the gene appears to be in the 2.2-kb BamHI-HpaI segment of the pCM52 insert . A Northern blot analysis showed this region to give rise to a 1.7-kb mRNA which we conclude is the transcript of CDC7 . The large insert of pCM6 also contains a 1.5-kb XhoI fragment which several observations suggest is that containing the centromere (CEN4) of chromosome IV to which the CDC7 gene is closely linked . The 45-kb DNA fragment in pCM6 is believed to be an uninterrupted piece of chromosome IV.

Mol Cell Biol, 1985 Jan, 5(1), 161 - 6
Expression of the gene for ornithine decarboxylase of Saccharomyces cerevisiae in Escherichia coli; Fonzi WA et al.; Diploid cells of Saccharomyces cerevisiae homozygous for the spe1A mutation, which eliminates ornithine decarboxylase activity, were found to sporulate at a greatly reduced frequency in the absence of polyamines . Plasmids which complement the spe1A mutation were isolated by their ability to restore sporulation competence to these cells . Three distinct plasmids were isolated . Each plasmid insert overlapped the same 8.0-kilobase region, and each plasmid restored ornithine decarboxylase activity to spe1A mutants . These plasmids also conferred ornithine decarboxylase activity to Escherichia coli EWH319 from which the ornithine decarboxylase gene is deleted . The plasmid-encoded activity expressed in E . coli resembled S . cerevisiae ornithine decarboxylase in its kinetic characteristics, indicating that the yeast ornithine decarboxylase gene was cloned . Southern blot analysis suggested that ornithine decarboxylase is a single-copy gene in S . cerevisiae . A single 2.1-kilobase transcript was demonstrated by Northern blot analysis.

Mol Gen Genet, 1985, 199(3), 495 - 9
Mitochondrial resistance to miconazole in Saccharomyces cerevisiae; Portillo F et al.; One mutant of mitochondrial origin resistant to miconazole has been isolated and characterized in S . cerevisiae . The mutation is linked to the locus oli1, the structural gene for subunit 9 of ATPase on mitochondrial DNA . Miconazole inhibited the mitochondrial ATPase of the wild type while the enzyme of the resistant mutant was insensitive to this effect . Levels of ATP decreased to one-third of the control in the wild type in the presence of miconazole, while they were unaffected in the mutant.

Gene, 1985, 40(2-3), 349 - 52
Isolation and characterization of the yeast aspartyl-tRNA synthetase gene; Sellami M et al.; A yeast genomic library in Escherichia coli, constructed by insertion of Sau3A restriction fragments into the hybrid Saccharomyces cerevisiae-E . coli plasmid pFL1, was screened by a radioimmunoassay (RIA) for colonies expressing yeast aspartyl-tRNA synthetase (AspRS) . Four clones were isolated by this technique . Data obtained by Southern and restriction analysis of the inserts showed a common 3.8-kb BamHI restriction fragment which, when inserted into the plasmid pFL1, gave a positive RIA . Several controls showed that this 3.8-kb insert codes for the entire AspRS: (i) S . cerevisiae transformed by the PFL1 plasmid carrying the 3.8-kb fragment overproduces AspRS activity by a factor of ten compared to the wild-type yeast strain; and (ii) a new protein with electrophoretic behaviour similar to AspRS and immuno-reactive toward anti-AspRS appears in crude extracts of transformed yeast and E . coli.

Gene, 1985, 36(3), 235 - 40
Transformation of Rhodosporidium toruloides; Tully M et al.; Rhodosporidium toruloides protoplasts could be transformed, in the presence of polyethylene glycol (PEG), at frequencies of approx . 1 X 10(3) transformants/micrograms of DNA . The plasmid used, pHG2, which contains the phenylalanine ammonia-lyase (PAL)-coding gene (PAL) of R . toruloides, could replicate as an unstable plasmid in the yeast, or could integrate at the PAL locus to give stable transformants . Plasmids that function in R . toruloides were constructed using either the PAL gene or LEU2 gene of Saccharomyces cerevisiae as dominant selectable markers . R . toruloides transformed with pHG8, which contains both genes, coinherited the two markers . It is also shown that the 2mu replicon of S . cerevisiae does not function in R . toruloides; neither is the PAL gene expressed in S . cerevisiae.

Mol Gen Genet, 1985, 199(3), 524 - 33
Regulation of the expression of iso 2-cytochrome c gene in S . cerevisiae: cloning of the positive regulatory gene CYP1 and identification of the region of its target sequence on the structural gene CYP3; Verdiere J et al.; CYP1 is a trans acting regulatory locus modulating both iso 1- and iso 2-cytochrome c synthesis . Genetical analysis of various mutated alleles has allowed us to identify the gene product as a positive regulatory element . The region of the target sequence of the CYP1 product on the iso 2-cytochrome c structural gene was located by molecular and genetic analysis of two cis acting mutations located at the CYP3 locus: CYP3-36 and CYP3-4, which have been shown to arise from the integration of TY1 elements near the promoter site . Determination of the amount of iso 2-cytochrome c synthesized by strains bearing various genetic constructions, in which the cis acting mutations were associated with different alleles of the CYP1 trans acting locus, showed that TY1 inserted into CYP3-36 extinguishes the activation function due to a mutated overproducer allele CYP1-18, while CYP3-4 amplifies this function . This result identifies at least a part of the target sequence of the CYP1 product within the region separating the two TY1 insertions . To clone the CYP1 gene, we took advantage of the iso 2-cytochrome c overproducer phenotype of the mutated allele CYP1-18, which confers a Lactate+ phenotype on an iso 1-cytochrome c-deficient strain . Such a phenotype allowed the isolation of a recombinant plasmid YEpJFM1 carrying the mutated allele, able to complement on lactate medium a lactate- recipient strain . The identity of the YEpJFM1 sequence with the chromosomal gene was confirmed by homologous recombination at the CYP1 locus.

Cell, 1985 Jan, 40(1), 27 - 36
In yeast, RAS proteins are controlling elements of adenylate cyclase; Toda T et al.; S . cerevisiae strains containing RAS2val19, a RAS2 gene with a missense mutation analogous to one that activates the transforming potential of mammalian ras genes, have growth and biochemical properties strikingly similar to yeast strains carrying IAC or bcy1 . Yeast strains carrying the IAC mutation have elevated levels of adenylate cyclase activity . bcy1 is a mutation that suppresses the lethality in adenylate cyclase deficient yeast . Yeast strains deficient in RAS function exhibit properties similar to adenylate cyclase deficient yeast . bcy1 suppresses lethality in ras1- ras2- yeast . Compared to wild-type yeast strains, intracellular cyclic AMP levels are significantly elevated in RAS2val19 strains, significantly depressed in ras2- strains, and virtually undetectable in ras1- ras2- bcy1 strains . Membranes from ras1- ras2- bcy1 yeast lack the GTP-stimulated adenylate cyclase activity present in membranes from wild-type cells, and membranes from RAS2val19 yeast strains have elevated levels of an apparently GTP-independent adenylate cyclase activity . Mixing membranes from ras1- ras2- yeast with membranes from adenylate cyclase deficient yeast reconstitutes a GTP-dependent adenylate cyclase.

Gene, 1985, 37(1-3), 247 - 53
Nucleotide sequence of the GDH gene coding for the NADP-specific glutamate dehydrogenase of Saccharomyces cerevisiae; Nagasu T et al.; The isolation of the Saccharomyces cerevisiae gene for NADP-dependent glutamate dehydrogenase (NADP-GDH) by cross hybridization to the Neurospora crassa am gene, known to encode for NADP-GDH is described . Two DNA fragments selected from a yeast genomic library in phage lambda gt11 were shown by restriction analysis to share 2.5 kb of common sequence . A yeast shuttle vector (CV13) carrying either to the cloned fragments complements the gdh- strain of S . cerevisiae and directs substantial overproduction of NADP-GDH . One of the cloned fragments was sequenced, and the deduced amino acid (aa) sequence of the yeast NADP-GDH is 64% homologous to N . crassa, 51% to Escherichia coli and 24% to bovine NADP-GDHs.

Gene, 1985, 36(1-2), 123 - 9
Cloning of a DNA sequence that complements glutamine auxotrophy in Saccharomyces cerevisiae; Gonzalez A et al.; Glutamine (gln) requiring mutants of Saccharomyces cerevisiae have been isolated . They synthesize small amounts of glutamine synthetase (GS), which is more thermolabile than the enzyme from the parental strain . The gln auxotrophy was complemented in transformation experiments using an S . cerevisiae gene library constructed in the plasmid vector YEp13 . The transformants were mitotically unstable and synthesized almost tenfold higher amounts of GS than wild-type cells . This activity was as thermoresistant as that from the wild-type strain . A recombinant plasmid was isolated from one of the transformants and partially mapped . Upon reintroduction into the auxotrophic strain, the transformation frequency to gln prototrophy was the same as that for the marker LEU2 gene . The evidence presented suggests that we have cloned the structural gene for GS from S . cerevisiae.

Biol Cell, 1985, 53(1), 75 - 9
Relationship between growth inhibition and mitochondrial function in petite-negative yeasts . II . Effects of central nervous system drugs upon pathogenic and non-pathogenic Candida species; Marmiroli N et al.; Six nervous system drugs which inhibited vegetative reproduction of S . cerevisiae arrested also mitotic division of C . utilis, C . albicans and C . tropicalis . Chlorpromazine and chlorpheniramine which proved to be the most effective, affected respiration and cytochrome biosynthesis . Electrophoretic bands with MW congruent to 100 K were faint in silver-stained electrophoregrams of proteins from cells grown in the presence of a sub-inhibitory concentration of chlorpromazine.

Curr Genet, 1985, 9(5), 351 - 60
Nucleotide sequence of the mitochondrial cytochrome oxidase subunit II gene in the yeast Hansenula saturnus; Lawson JE et al.; The gene for subunit II of cytochrome oxidase in the yeast Hansenula saturnus was previously shown to be located on a 1.7 kb HindIII-BamHI fragment of mitochondrial DNA (Lawson and Deters, accompanying paper) . In this paper, we report the nucleotide sequence of a large part of this fragment, covering the coding region of the subunit II gene, designated coxII, and its 5' and 3' flanking regions . The coding region of the coxII gene consists of a continuous open reading frame, 744 nucleotides long, containing 6 in frame TGA codons . Examination of the sequence and alignment with known homologous gene sequences of other organisms indicates that TGA codes for tryptophan in H . saturnus mitochondria as it does in several other mitochondria . Despite considerable homology to subunit II of Saccharomyces cerevisiae, there are 9 codons used in coxII that are not used in the corresponding S . cerevisiae gene . CTT, which is believed to code for threonine in S . cerevisiae mitochondria, appears 3 times in coxII and probably codes for leucine . While the CGN family is rarely, if ever, used in S . cerevisiae mitochondria, CGT appears 4 times in coxII and probably codes for arginine . The deduced amino acid sequence, excluding the first ten amino acids at the N-terminus, is 81% homologous to the amino acid sequence of the S . cerevisiae subunit II protein . The first ten amino acids at the N-terminus are not homologous to the N-terminus of the S . cerevisiae protein but are highly homologous to the first ten amino acids of the deduced amino acid sequence of subunit II of Neurospora crassa . Minor variations of a transcription initiation signal and an end of message or processing signal reported in S . cerevisiae are found in the regions flanking the H . saturnus coxII gene . The subunit II gene contains numerous symmetrical elements, i.e . palindromes, inverted repeats, and direct repeats . Some of these have conserved counterparts in the S . cerevisiae subunit II gene, suggesting that they may be functionally or structurally important.

Curr Genet, 1985, 9(5), 345 - 50
Identification and isolation of the cytochrome oxidase subunit II gene in mitochondria of the yeast Hansenula saturnus; Lawson JE et al.; Mitochondrial DNA from the petite negative yeast Hansenula saturnus has been isolated and sized by digestion with restriction enzymes . The size of the mitochondrial genome is approximately 47 kb . The gene for subunit II of cytochrome oxidase was localized in the genome by Southern blotting using a {32P}-labeled probe containing the subunit II gene of the yeast Saccharomyces cerevisiae . The probe hybridized to a 1.7 kb HindIII-BamHI fragment under stringent conditions (65 degrees C), indicating a high degree of homology between the S . cerevisiae and H . saturnus mitochondrial DNA fragments . The 1.7 kb fragment from H . saturnus was cloned into pBR322 and physically mapped . The map was used to obtain the nucleotide sequence of the subunit II gene (Lawson and Deters presented in the accompanying paper).

EMBO J, 1984 Dec 20, 3(13), 3311 - 5
Identification of two genes coding for the translation elongation factor EF-1 alpha of S . cerevisiae; Schirmaier F et al.; The translation elongation factor EF-1 alpha of the yeast Saccharomyces cerevisiae is coded for by two genes, called TEF1 and TEF2 . Both genes were cloned . TEF1 maps on chromosome II close to LYS2 . The location of TEF2 is unknown . TEF2 alone is sufficient to promote growth of the cells as shown with a strain deleted for TEF1 . TEF1 and TEF2 were originally identified as two strongly transcribed genes, which most likely code for an identical or nearly identical protein as judged from S1 nuclease protection experiments with mRNA-DNA hybrids . The DNA sequence analysis of TEF1 allowed the prediction of the protein sequence . This was shown, by a search in the Dayhoff protein data bank, to represent the translation elongation factor EF-1 alpha due to the striking similarity to EF-1 alpha from the shrimp Artemia . A search for TEF1 homologous sequences in several yeast species shows, in most cases, duplicated genes and a much higher sequence conservation than among genes encoding amino acid biosynthetic enzymes.

Nature, 1984 Dec 13-19, 312(5995), 663 - 6
The yeast ubiquitin gene: head-to-tail repeats encoding a polyubiquitin precursor protein; Ozkaynak E et al.; Ubiquitin, a 76-residue protein, occurs in cells either free or covalently joined to a variety of protein species, from chromosomal histones to cytoplasmic proteins . Conjugation of ubiquitin to proteolytic substrates is essential for the selective degradation of intracellular proteins in higher eukaryotes . We show here that a protein homologous to human ubiquitin exists in the yeast Saccharomyces cerevisiae, and that yeast extracts conjugate human ubiquitin to a variety of endogenous proteins in an ATP-dependent reaction . We have isolated the S . cerevisiae ubiquitin gene and found it to contain six consecutive ubiquitin-coding repeats in a found it to contain six consecutive ubiquitin-coding repeats in a head-to-tail arrangement . This apparently unique gene organization suggests that yeast ubiquitin is generated by processing of a precursor protein in which several exact repeats of the ubiquitin amino acid sequence are joined directly via Gly-Met peptide bonds between the last and first residues of mature ubiquitin, respectively . Ubiquitin-coding yeast DNA repeats are restricted to a single genomic locus; although the sequenced repeats differ in up to 27 of 228 bases per repeat, they encode identical amino acid sequences . As this predicted amino acid sequence differs in only 3 of 76 residues from that of ubiquitin in higher eukaryotes, ubiquitin is apparently the most conserved of known proteins.

Mol Cell Biol, 1984 Dec, 4(12), 2858 - 64
Cell cycle-dependent expression of thymidylate synthase in Saccharomyces cerevisiae; Storms RK et al.; Synchronous populations of Saccharomyces cerevisiae cells, generated by two independent methods, have been used to show that thymidylate synthase, in contrast to the vast majority of cellular proteins thus far examined, fluctuates periodically during the S . cerevisiae cell cycle . The enzyme, as assayed by two different methods, accumulated during S period and peaked in mid to late S phase, and then its level dropped . These observations suggest that both periodic synthesis and the instability of the enzyme contribute to the activity profile seen during the cell cycle . Accumulation of thymidylate synthase is determined at the level of its transcript, with synthase-specific mRNA levels increasing at least 10-fold to peak near the beginning of S period and then falling dramatically to basal levels after the onset of DNA synthesis . This mRNA peak coincided with the time during the cell cycle when thymidylate synthase levels were increasing maximally and immediately preceded the peak of DNA synthesis, for which the enzyme provides precursor dTMP.

Mol Cell Biol, 1984 Dec, 4(12), 2818 - 27
Virus-like particle capsid proteins encoded by different L double-stranded RNAs of Saccharomyces cerevisiae: their roles in maintenance of M double-stranded killer plasmids; El-Sherbeini M et al.; The plasmid determinants of killer phenotypes in type K1 and K2 killer yeast cells are the 1.9-kilobase (kb) M1 and 1.7-kb M2 double-stranded RNAs (dsRNAs), respectively . These are dependent for their maintenance and encapsidation, in Saccharomyces cerevisiae virus ScV-M1 or ScV-M2 virus-like particles, on the capsid provided by one of a group of moderately related 4.7-kb dsRNAs called LA . The L1A and L2A dsRNAs found in naturally isolated K1 and K2 killers encode 88-kilodalton VL1A-P1 and 86-kilodalton VL2A-P1 capsids, respectively . These are competent for encapsidating homologous LA dsRNAs as well as M dsRNAs . Most strains of S . cerevisiae, including killers, contain one of a second group of closely related 4.7-kb dsRNAs called LBC . These encode their own 82-kilodalton capsid protein, VLBC-P1, which, at least in strains containing only LBC, encapsidates homologous dsRNA in ScV-LBC virus-like particles . In a K1 killer strain containing both L1A and LBC, ScV-M1 particles contain only VL1A-P1 . In such strains it is probable that each virus-like particle contains a single capsid type and that each L dsRNA is encapsidated by a homologous capsid.

Cell, 1984 Dec, 39(3 Pt 2), 675 - 82
The SPT3 gene is required for normal transcription of Ty elements in S . cerevisiae; Winston F et al.; The transposable Ty elements consist of a central core, epsilon, flanked by direct repeats called deltas . In wild-type strains Ty transcripts initiate in one delta and terminate in the other . Insertion mutations caused by Ty elements have a wide variety of phenotypes, ranging from inhibition of gene expression to constitutive gene expression . Mutations in the SPT3 gene suppress these effects of Ty and delta insertion mutations on adjacent genes . In spt3 null mutants the Ty transcription pattern for the entire ensemble of Ty elements is changed . The delta-delta transcripts are absent and initiation begins at a position 800 bp into the epsilon region . In these spt3 strains, transcription that initiates in solo deltas and proceeds into adjacent structural genes is also abolished . The requirement of SPT3 for normal transcription from delta can explain the ability of spt3 mutations to suppress the mutations caused by Ty and delta insertions . In SPT3 strains transcription from the delta into adjacent sequences interferes with normal expression of those sequences, whereas in spt3 strains the aberrant transcript is not made . spt3 mutations also lead to defects in diploid formation and sporulation, suggesting that SPT3 is important for the expression of genes in addition to Ty elements.

Cell, 1984 Dec, 39(2 Pt 1), 377 - 86
Cis-acting, recombination-stimulating activity in a fragment of the ribosomal DNA of S . cerevisiae; Keil RL et al.; Special mechanisms for stimulating recombination among the nearly identical repeat units of certain multigene families may exist in order to maintain their sequence homogeneity . We have found evidence for such a recombination-stimulating activity in the tandemly repeated ribosomal RNA genes of yeast . A fragment of the yeast ribosomal DNA (rDNA), containing the 5S gene, nontranscribed spacer DNA, and part of the 25S gene, causes a localized stimulation of recombination when inserted at novel locations in the yeast genome . The rDNA fragment stimulates both interchromosomal and intrachromosomal mitotic recombination but not meiotic recombination . To stimulate mitotic recombination, the fragment must act on both copies of the recombining gene . Furthermore, the rDNA fragment stimulates exchange only when inserted with the 5S gene proximal to, and the 25S gene distal to, the recombining alleles.

Biochim Biophys Acta, 1984 Nov 21, 778(1), 1 - 16
Kinetics and pH-dependence of glycine-proton symport in Saccharomyces cerevisiae; Ballarin-Denti A et al.; Interactions between intracellular pH (pHi) and H+-coupled transmembrane transport of glycine have been studied by means of 31P-NMR, using both aerobic and 'energy starved' cells of the yeast Saccharomyces cerevisiae . The general features of glycine transport in the yeast strain used (NCYC 239) are similar to those already reported for Saccharomyces carlsbergensis and S . cerevisiae, there being two kinetically distinct glycine uptake systems, with pH-independent K1/2 values near 14 and 0.4mM, respectively, but pH-dependent maximal velocities . Glycine transport itself has no measurable effect on pHi in aerobic cells, and only a marginal effect in energy-starved cells, but changes of pHi, imposed by extracellular addition of butyric acid, strongly influence glycine transport . Indeed, the dependence of glycine influx (in energy-starved cells) upon cytoplasmic H+ concentration appears to be third order, showing Hill slopes of 2.7-3.0 . A crucial kinetic role for cytoplasmic pH in glycine transport is further indicated by a proportionality between the decline of flux and the decline of pHi produced by various metabolic inhibitors and uncouplers . Extracellular pH (pHo), by contrast, has only a weak effect on glycine influx, showing a Hill slope of 0.5 . The major observations can be accommodated by a simple cyclic carrier scheme, in which 2 or more protons are transported along with glycine, but only one extracellular proton binding site dissociates in the testing range, with a pK near 5.5 . The model requires a finite membrane potential, which must be somewhat sensitive to both pHi and pHo, and accommodates the discrepancy between measured net proton flux (one per glycine) and the kinetically required proton flux (two or more per glycine) by shunting through other proton-conducting pathways in the yeast membrane.

Eur J Biochem, 1984 Nov 15, 145(1), 187 - 93
The mechanism by which glucose increases fructose 2,6-bisphosphate concentration in Saccharomyces cerevisiae . A cyclic-AMP-dependent activation of phosphofructokinase 2; Francois J et al.; When glucose was added to a suspension of Saccharomyces cerevisiae in stationary phase, it caused a transient increase in the concentration of cyclic AMP and a more persistent increase in the concentration of hexose 6-phosphate and of fructose 2,6-bisphosphate . These effects of glucose on cyclic AMP and fructose 2,6-bisphosphate but not that on hexose 6-phosphate were greatly decreased in the presence of 0.15 mM acridine orange or when a temperature-sensitive mutant deficient in adenylate cyclase was used at the restrictive temperature . Incubation of the cells in the presence of dinitrophenol and in the absence of glucose increased the concentration of both cyclic AMP and fructose 2,6-bisphosphate, but with a minimal change in that of hexose 6-phosphate . Glucose induced also in less than 3 min a severalfold increase in the activity of 6-phosphofructo-2-kinase and this effect was counteracted by the presence of acridine orange . When a cell-free extract of yeast in the stationary phase was incubated with ATP-Mg and cyclic AMP, there was a 10-fold activation of 6-phosphofructo-2-kinase . Finally, the latter enzyme was purified 150-fold and its activity could then be increased about 10-fold upon incubation with ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase . This activation resulted from a 4.3-fold increase in V and a 2-fold decrease in Km . Both forms of the enzyme were inhibited by sn-glycerol 3-phosphate . From these results it is concluded that the effect of glucose in increasing the concentration of fructose 2,6-bisphosphate in S . cerevisiae is mediated by the successive activation of adenylate cyclase and of cyclic-AMP-dependent protein kinase and by the phosphorylation of 6-phosphofructo-2-kinase by the latter enzyme . In deep contrast with what is known of the liver enzyme, yeast 6-phosphofructo-2-kinase is activated by phosphorylation instead of being inactivated.

Mol Cell Biol, 1984 Nov, 4(11), 2467 - 78
Saccharomyces cerevisiae GAL1-GAL10 divergent promoter region: location and function of the upstream activating sequence UASG; West RW Jr et al.; The GAL1 and GAL10 genes, separated by 680 base pairs and divergently transcribed on chromosome 2 of Saccharomyces cerevisiae, were separately fused to the lacZ gene of Escherichia coli so that beta-galactosidase synthesis in S . cerevisiae reflected GAL1 and GAL10 promoter function . Analysis of two sets of deletions defined a 75-base-pair sequence, located ca . midway between the transcription initiation regions of GAL1 and GAL10, that mediates GAL4-dependent induction of both genes . Deletion of various parts of this sequence (called the GAL upstream activating sequence or UASG) reduced GAL1 and GAL10 induction about equally . Sequences in the GAL10-proximal half of UASG in some sequence contexts functioned independently of sequences in the GAL1-proximal half of UASG . A 33-base-pair deletion of the GAL10-proximal half of UASG drastically reduced induction . Deletions between UASG and the GAL1 TATA box caused beta-galactosidase to be synthesized at an unexpectedly high basal level, that is, in the absence of galactose and GAL4 product . Some of these mutations also reduced the repression caused by glucose.

Mol Cell Biol, 1984 Nov, 4(11), 2455 - 66
Deletion mutations affecting autonomously replicating sequence ARS1 of Saccharomyces cerevisiae; Celniker SE et al.; DNAs that contain specific yeast chromosomal sequences called ARSs transform Saccharomyces cerevisiae at high frequency and can replicate extrachromosomally as plasmids when introduced into S . cerevisiae by transformation . To determine the boundaries of the minimal sequences required for autonomous replication in S . cerevisiae, we have carried out in vitro mutagenesis of the first chromosomal ARS described, ARS1 . Rather than identifying a distinct and continuous segment that mediates the ARS+ phenotype, we find three different functional domains within ARS1 . We define domain A as the 11-base-pair (bp) sequence that is also found at most other ARS regions . It is necessary but not sufficient for high-frequency transformation . Domain B, which cannot mediate high-frequency transformation, or replicate by itself, is required for efficient, stable replication of plasmids containing domain A . Domain B, as we define it, is continuous with domain A in ARS1, but insertions of 4 bp between the two do not affect replication . The extent of domain B has an upper limit of 109 bp and a lower limit of 46 bp in size . There is no obvious sequence homology between domain B of ARS1 and any other ARS sequence . Finally, domain C is defined on the basis of our deletions as at least 200 bp flanking domain A on the opposite side from domain B and is also required for the stability of domain A in S . cerevisiae . The effect of deletions of domain C can be observed only in the absence of domain B, at least by the assays used in the current study, and the significance of this finding is discussed.

Mol Cell Biol, 1984 Nov, 4(11), 2298 - 305
Expression and characterization of ras mRNAs from Saccharomyces cerevisiae; Temeles GL et al.; The cellular homologs of the Harvey and Kirsten murine sarcoma virus oncogenes comprise a multigene family, ras, that displays striking evolutionary conservation . We recently reported {DeFeo-Jones et al., Nature (London) 306:707-709, 1983} the cloning of two ras homologs from the yeast Saccharomyces cerevisiae . The nucleotide sequences of these genes predict polypeptides that show remarkable homology to p21, the mammalian ras gene product . We have also found proteins in yeast lysates with serological cross-reactivity to p21 (Papageorge et al., Mol . Cell . Biol . 4:23-29, 1984) . In this work, we explored the relationship between the immunoprecipitated proteins and the yeast ras genes . We show that both ras genes are expressed in the wild-type cell . Furthermore, we demonstrate by in vitro translation of hybrid-selected RASsc1 mRNA and immunoprecipitation of the translation products that the cloned RASsc1 gene encodes the proteins immunoprecipitated from yeast lysates by anti-p21 monoclonal antibody . Finally, we used anti-p21 monoclonal antibodies to detect a guanine nucleotide binding activity in yeast lysates . The structural and biochemical homologies between ras gene products of S . cerevisiae and mammalian cells suggest that information obtained by genetic analysis of ras function in a lower eucaryote should be applicable to higher organisms as well.

Cell Biol Int Rep, 1984 Nov, 8(11), 987 - 92
Detection of an antigen to primary biliary cirrhosis in wild type and petite mutant Saccharomyces cerevisiae; Uzoegwu PN et al.; Antigens to primary biliary cirrhosis (PBC) appeared to be identical in wild type (rho+) and petite (rho o) mutant S.cerevisiae . As the latter mutants lack functional mitochondria, the PBC antigens, which are associated with mitochondrial ATPase in other cells, may be of nucleocytoplasmic origin.

Nucleic Acids Res, 1984 Oct 25, 12(20), 7721 - 39
Structural analysis of the two tandemly repeated acid phosphatase genes in yeast; Bajwa W et al.; We have sequenced the genetically linked genes for repressible (PHO5) and and constitutive (PHO3) acid phosphatase from S . cerevisiae . Both genes are located on a 3.91 Kb BamHI and HpaI fragment, in the order (5') PHO5, PHO3 (3') . The mRNA transcripts have been analysed by S1-nuclease mapping . They show heterogenous initiation sites . Each of the PHO5 and PHO3 genes codes for 467 amino acids as deduced from the DNA sequence . The coding regions of the two genes show homology both at the nucleotide (82%) and the amino acid (87%) level . In the coding sequences, long stretches of homologous regions are flanked by small non-homologous regions . The nucleotide homology (65%) extends to some length into the 5' and 3' non-coding flanking sequences . Further upstream sequences are unrelated . The comparison of the NH2-terminal amino acid sequence deduced from the nucleotide sequence, with that of purified repressible acid phosphatase revealed the presence of a putative signal peptide.

Biochim Biophys Acta, 1984 Oct 24, 796(1), 110 - 7
Regulatory role of phosphatidate phosphatase in triacylglycerol synthesis of Saccharomyces cerevisiae; Hosaka K et al.; The regulatory mechanism of triacylglycerol synthesis in Saccharomyces cerevisiae was studied . The triacylglycerol content increased markedly during the entry of cells into the stationary growth phase, whereas the content of phospholipids remained unchanged . Pulse-labeling experiments to measure {14C}acetate incorporation into triacylglycerol revealed that the synthesis of triacylglycerol increased in the stationary growth phase . An increase in fatty acid synthesis was observed only in the later stage of the stationary growth phase and thus does not seem to be the principal causative factor for the triacylglycerol accumulation . Among various triacylglycerol-synthetic enzymes tested, the increase in the phosphatidate phosphatase (EC 3.1.3.4) activity was most closely correlated with the accumulation of triacylglycerol in the stationary phase . Our results show that phosphatidate phosphatase plays an important role in the regulation of triacylglycerol synthesis in S . cerevisiae.

Nucleic Acids Res, 1984 Oct 11, 12(19), 7317 - 26
Initiation of transcription of a mitochondrial tRNA gene cluster in S . cerevisiae; Palleschi C et al.; In Saccharomyces cerevisiae most mitochondrial tRNA genes are clustered in a 9 kbp region between the cap and oxil genes . Polygenic transcripts of this region have been previously identified . A transcriptional initiation site at a TTATAAGTA box, located upstream from the tRNAcys gene, has now been detected by S1 mapping experiments and by the capping of primary transcripts . Results are consistent with the hypothesis that this box represents the initiation site for transcription of a cluster of tRNA genes, while the adjacent tRNA2thr is cotranscribed with the 21S rRNA . Results obtained with various strains are compared, and the efficiency of this sequence as a transcriptional initiation site in different mitochondrial contexts is discussed.

Clin Exp Immunol, 1984 Oct, 58(1), 229 - 36
Familial defect of polymorph neutrophil phagocytosis associated with absence of a surface glycoprotein antigen (OKMI); Thompson RA et al.; Two siblings with delayed separation of the umbilical cord, recurrent skin ulceration and dental sepsis were shown to have defective neutrophil phagocytosis of opsonized yeast (S . cerevisiae) and respiratory burst to opsonized and unopsonized zymosan . Increased activity in the NBT reduction test, normal ingestion and killing of S . aureus, and normal spontaneous and directional motility were also demonstrated . These abnormalities of neutrophil phagocytosis were confined to the affected siblings; their healthy parents and brother showed normal neutrophil function . Both children had a polymorph neutrophil leucocytosis, and had normal humoral and cell-mediated immunity . SDS electrophoresis of neutrophil cell membrane preparations showed absence of a glycoprotein band of 175,000 daltons, which was present in the parents' neutrophils in reduced amounts . OKMI monoclonal antibody, which recognized the C3bi receptor (CR3) failed to bind to the affected siblings neutrophils . The findings in these children emphasize the importance of this receptor in phagocytosis, and possibly other neutrophil functions.

Cell, 1984 Oct, 38(3), 841 - 9
Mutations of the heat inducible 70 kilodalton genes of yeast confer temperature sensitive growth; Craig EA et al.; S . cerevisiae contains a family of genes related to the major heat shock induced gene of Drosophila (Hsp70) . Two members of this family, YG100 and YG102, are 97% identical to each other in their protein coding regions . RNA transcribed from YG100 increases markedly after a heat shock, while transcripts of YG102 increase minimally . Mutants of the two genes were constructed in vitro and substituted in the yeast genome in place of the wild-type alleles . No phenotypic effect of single mutations of either gene was detected . However, cells containing both the YG100 and YG102 mutations grew slowly at 30 degrees C and could not form colonies at 37 degrees C . The temperature sensitive phenotype can be overcome by inserting either an intact YG100 or YG102 gene into the genome . Pretreatment at 37 degrees C before shift to a normally lethal temperature of 51 degrees C protected the double mutant as well as the wild type, indicating that YG100 and YG102 gene products are not needed for resistance to high temperatures for short periods.

J Gen Microbiol, 1984 Oct, 130 ( Pt 10), 2527 - 34
Plasmids resembling 2-micrometers DNA in the osmotolerant yeasts Saccharomyces bailii and Saccharomyces bisporus; Toh-e A et al.; Five of eight strains of Saccharomyces bailii and one of 13 strains of S . bisporus were found to harbour DNA plasmids . pSB1 and pSB2 plasmids were isolated from S . bailii strains IFO 0488 and IFO 1047, respectively, and pSB3 and pSB4 from S . bisporus strain IFO 1730 . All four plasmids resemble 2-micrometers DNA of S . cerevisiae in that their molecular sizes are about 6 kb, each molecule possesses a pair of inverted repeats, they exist as a mixture of two isomers and their copy numbers in the native host are similar . None of them showed homology with 2-micrometers DNA or with each other by Southern hybridization under moderately stringent conditions, but pSB4 hybridized with the pSR1 DNA, which was found previously in a strain of S . rouxii . Each of the pSB plasmids has DNA sequence(s) effective for autonomous replication in S . cerevisiae . In S . cerevisiae, pSB3 and pSB4 showed intramolecular recombination but neither supported isomerization of 2-micrometers DNA.

Nucleic Acids Res, 1984 Sep 25, 12(18), 7199 - 213
Correlation between suppressed meiotic recombination and the lack of DNA strand-breaks in the rRNA genes of Saccharomyces cerevisiae; Hogset A et al.; We have examined whether the suppressed homologous meiotic recombination within the rDNA of S . cerevisiae is reflected by a lack of possibly recombination-initiating strand-breaks in this part of the genome . Our findings indicate that bulk DNA in the ds-break repair deficient mutant rad52/rad52 accumulates a limited number of both ss- and ds-breaks during meiosis as compared to a RAD+/rad52 heterozygote . The rDNA-containing chromosome is however protected against these breaks, and thus this may be an explanation for the suppression of recombination in the rDNA . The fact that ds-breaks seem to be involved gives indirect support to the ds-break-repair model for recombination.

Farmaco {Sci}, 1984 Sep, 39(9), 739 - 51
The metabolism of 8-methoxypsoralen by Saccharomyces cerevisiae . Evidence for an inducing effect of ethanol; Prognon P et al.; The metabolism of 8-methoxypsoralen (8-MOP) by the yeast Saccharomyces cerevisiae has been investigated in order to determine if this cell system could provide a simple model to study the metabolism of new photosensitizing drugs in vitro . 8-MOP was found to be rapidly metabolized by S . cerevisiae in non growing conditions . A metabolite was detected which exhibits a structure similar to that of a metabolite previously isolated in dog and man . Ethanol showed a strong inducing effect on 8-MOP metabolization.

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