S. cerevisiae

Mol Cell Biol, 1991 Jun, 11(6), 3009 - 19
SPT5, an essential gene important for normal transcription in Saccharomyces cerevisiae, encodes an acidic nuclear protein with a carboxy-terminal repeat; Swanson MS et al.; Mutations in the SPT5 gene of Saccharomyces cerevisiae were isolated previously as suppressors of delta insertion mutations at HIS4 and LYS2 . In this study we have shown that spt5 mutations suppress the his4-912 delta and lys2-128 delta alleles by altering transcription . We cloned the SPT5 gene and found that either an increase or a decrease in the copy number of the wild-type SPT5 gene caused an Spt- phenotype . Construction and analysis of an spt5 null mutation demonstrated that SPT5 is essential for growth, suggesting that SPT5 may be required for normal transcription of a large number of genes . The SPT5 DNA sequence was determined; it predicted a 116-kDa protein with an extremely acidic amino terminus and a novel six-amino-acid repeat at the carboxy terminus (consensus = S-T/A-W-G-G-A/Q) . By indirect immunofluorescence microscopy we showed that a bifunctional SPT5-beta-galactosidase protein was located in the yeast nucleus . This molecular analysis of the SPT5 gene revealed a number of interesting similarities to the previously characterized SPT6 gene of S . cerevisiae . These results suggest that SPT5 and SPT6 act in a related fashion to influence essential transcriptional processes in S . cerevisiae.

Cell, 1991 May 31, 65(5), 875 - 83
A potential positive feedback loop controlling CLN1 and CLN2 gene expression at the start of the yeast cell cycle; Cross FR et al.; The CLN1, CLN2, and CLN3 genes of S . cerevisiae form a redundant family essential for the G1-to-S phase transition . CLN1 and CLN2 mRNAs were previously shown to be negatively regulated by mating pheromone and by cell cycle progression out of G1, whereas CLN3 mRNA is not . The CLN3-2 (DAF1-1) allele prevents both cell cycle arrest and the turnoff of CLN1 and CLN2 mRNAs in response to mating pheromone, but only in the presence of an active CDC28 gene . An internally deleted nonfunctional cln2 gene was used as a reporter gene to demonstrate that in the absence of mating pheromone, efficient expression of cln2 mRNA requires both an active CDC28 gene and at least one functional CLN gene . mRNA from a nonfunctional cln1 gene was regulated similarly . Thus, CLN function and CDC28 activity jointly stimulate CLN1 and CLN2 mRNA levels, potentially forming a positive feedback loop for CLN1 and CLN2 expression.

Cell, 1991 May 31, 65(5), 785 - 95
SGV1 encodes a CDC28/cdc2-related kinase required for a G alpha subunit-mediated adaptive response to pheromone in S . cerevisiae; Irie K et al.; The GPA1 gene of S . cerevisiae encodes a G alpha subunit that plays a positive role in the transduction of signals stimulating recovery from pheromone-induced cell cycle arrest . The GPA1Val50 mutation, in which Gly-50 is replaced by valine, causes hyperadaptation to pheromone . However, GPA1Val50 cells do not recover from division arrest in the absence of both CLN1 and CLN3, which encode G1 cyclins, indicating that the recovery-promoting activity of GPA1Val50 requires the function of G1 cyclins . An sgv1 mutation suppresses the hyperadaptive response caused by GPA1Val50 and also confers cold- and temperature-sensitive growth . The SGV1 gene encodes an apparent protein kinase homologous to CDC28/cdc2 kinase: SGV1 is 42% identical to CDC28 . The activated mutation, CLN3-2, partially suppresses the growth defect of sgv1, suggesting that the SGV1 and CLN3 proteins may act in the same growth control pathway.

Gene, 1991 May 15, 101(1), 105 - 11
Mutations of the alpha-galactosidase signal peptide which greatly enhance secretion of heterologous proteins by yeast; Hofmann KJ et al.; The Saccharomyces carlsbergensis MEL1 gene encodes alpha-galactosidase (melibiase; MEL1) which is readily secreted by yeast cells into the culture medium . To evaluate the utility of the MEL1 signal peptide (sp) for the secretion of heterologous proteins by Saccharomyces cerevisiae, an expression vector was constructed which contains the MEL1 promoter and MEL1 sp coding sequence (MEL1sp) . The coding sequences for echistatin (Echis) and human plasminogen activator inhibitor type 1 (PAI-1) were inserted in-frame with the MEL1sp . S . cerevisiae transformants containing the resulting expression vectors secreted negligible amounts of either Echis or PAI-1 . Using site-directed mutagenesis, several mutations were introduced into the MEL1sp . Two mutations were identified which dramatically increased the secretion of both Echis and PAI-1 to levels similar to those achieved when using the yeast MF alpha 1 pre-pro secretory leader . In particular, increasing the hydrophobicity of the core region plus the addition of a positive charge to the N-terminal domain of the MEL1 sp resulted in the greatest increase in the secretion levels of those two proteins.

Cell, 1991 May 3, 65(3), 507 - 15
A yeast origin of replication is activated late in S phase; Ferguson BM et al.; The mechanism that causes large regions of eukaryotic chromosomes to remain unreplicated until late in S phase is not understood . We have found that 67 kb of telomere-adjacent DNA at the right end of chromosome V in S . cerevisiae is replicated late in S phase . An ARS element in this region, ARS501, was shown by two-dimensional gel analysis to be an active origin of replication . Kinetic analyses indicate that the rate of replication fork movement within this late region is similar to that in early replicating regions . Therefore, the delayed replication of the region is a consequence of late origin activation . The results also support the idea that the pattern of interspersed early and late replication along the chromosomes of higher eukaryotes is a consequence of the temporal regulation of origin activation.

Mol Gen Genet, 1991 May, 227(1), 72 - 80
The organization and expression of the Saccharomyces cerevisiae L4 ribosomal protein genes and their identification as the homologues of the mammalian ribosomal protein gene L7a; Yon J et al.; A cDNA for the mouse ribosomal protein (rp) L7a, formerly called Surf-3, was used as a probe to isolate two homologous genes from Saccharomyces cerevisiae . The two yeast genes (L4-1 and L4-2) were identified as encoding S . cerevisiae L4 by 2D gel analysis of the product of the in vitro translation of hybrid-selected mRNA and additionally by direct amino acid sequencing . The DNA sequences of the two yeast genes were highly homologous (95%) over the 771 bp that encode the 256 amino acids of the coding regions but showed little homology outside the coding region . L4-1 differed from L4-2 by 7 out of the 256 amino acids in the coding region, which is the greatest divergence between the products of any two duplicated yeast ribosomal protein genes so far reported . There is strong homology between the mouse rpL7a/Surf-3 and the yeast L4 genes -57% at the nucleic acid level and also 57% at the amino acid level (though some regions reach as much as 80-90% homology) . While most yeast ribosomal protein genes contain an intron in their 5' region both L4-1 and L4-2 are intronless . The mRNAs derived from each yeast gene contained heterogenous 5' and 3' ends but in each case the untranslated leaders were short . The L4-1 mRNA was found to be much more abundant than the L4-2 mRNA as assessed by cDNA and transcription analyses . Yeast cells containing a disruption of the L4-1 gene formed much smaller colonies than either wild-type or disrupted L4-2 strains . Disruption of both L4 genes is a lethal event, probably due to an inability to produce functional ribosomes.

EMBO J, 1991 May, 10(5), 1265 - 73
Mutation of the hamster cell cycle gene RCC1 is complemented by the homologous genes of Drosophila and S.cerevisiae; Ohtsubo M et al.; The RCC1 gene has been isolated from several vertebrates, including human, hamster and Xenopus . Genes similar to RCC1, namely BJ1 and SRM1/PRP20, have been isolated from the insect Drosophila and from the budding yeast Saccharomyces cerevisiae . A mutation of the RCC1 gene in the hamster BHK21 cell line, tsBN2, confers pleiotropic phenotypes, including G1 arrest and premature induction of mitosis in cells synchronized at the G1/S boundary . Similarly, mutations of the SRM1/PRP20 gene are pleiotropic; the srm1 mutant shows G1 arrest and suppression of the mating defect of mutants lacking pheromone receptors, and the prp20 mutant shows an alteration in mRNA metabolism . Here we show that both BJ1 and SRM1/PRP20 complement the temperature sensitive phenotype of the tsBN2 cells . Like RCC1 proteins of vertebrates, the protein products of the Drosophila and yeast RCC1 homologues were located in the nuclei of the mammalian cells . These results suggest that the BJ1 and SRM1/PRP20 genes are functionally equivalent to the vertebrate RCC1 genes, and that the RCC1 gene plays an important role in the regulation of gene expression in the eukaryotic cell cycle.

Mol Cell Biol, 1991 May, 11(5), 2744 - 51
A single base pair dominates over the novel identity of an Escherichia coli tyrosine tRNA in Saccharomyces cerevisiae; Trezeguet V et al.; The Escherichia coli su+3 tyrosine tRNA was shown recently to be a leucine-specific tRNA in Saccharomyces cerevisiae . This finding raises the possibility that some determinants for tRNA identity in E . coli may be different in S . cerevisiae . To investigate whether the fungal system is sensitive to the major determinant for alanine acceptance in E . coli, a single G3 . U70 base pair was introduced into the acceptor helix of the su+3 tyrosine tRNA . This substitution converts the identity of the E . coli suppressor in S . cerevisiae from leucine to alanine . Thus, as in E . coli, G3 . U70 is a strong determinant for alanine acceptance that can dominate over other features in a tRNA that might be recognized by alternative charging enzymes.

Mol Cell Biol, 1991 May, 11(5), 2723 - 35
Association of RAP1 binding sites with stringent control of ribosomal protein gene transcription in Saccharomyces cerevisiae; Moehle CM et al.; An amino acid limitation in bacteria elicits a global response, called stringent control, that leads to reduced synthesis of rRNA and ribosomal proteins and increased expression of amino acid biosynthetic operons . We have used the antimetabolite 3-amino-1,2,4-triazole to cause histidine limitation as a means to elicit the stringent response in the yeast Saccharomyces cerevisiae . Fusions of the yeast ribosomal protein genes RPL16A, CRY1, RPS16A, and RPL25 with the Escherichia coli lacZ gene were used to show that the expression of these genes is reduced by a factor of 2 to 5 during histidine-limited exponential growth and that this regulation occurs at the level of transcription . Stringent regulation of the four yeast ribosomal protein genes was shown to be associated with a nucleotide sequence, known as the UASrpg (upstream activating sequence for ribosomal protein genes), that binds the transcriptional regulatory protein RAP1 . The RAP1 binding sites also appeared to mediate the greater ribosomal protein gene expression observed in cells growing exponentially than in cells in stationary phase . Although expression of the ribosomal protein genes was reduced in response to histidine limitation, the level of RAP1 DNA-binding activity in cell extracts was unaffected . Yeast strains bearing a mutation in any one of the genes GCN1 to GCN4 are defective in derepression of amino acid biosynthetic genes in 10 different pathways under conditions of histidine limitation . These Gcn- mutants showed wild-type regulation of ribosomal protein gene expression, which suggests that separate regulatory pathways exist in S . cerevisiae for the derepression of amino acid biosynthetic genes and the repression of ribosomal protein genes in response to amino acid starvation.

Curr Genet, 1991 May, 19(5), 353 - 8
Cycloheximide resistance as a yeast cloning marker; del Pozo L et al.; In CYH2/cyh2 heterozygous diploids of the yeast Saccharomyces cerevisiae resistance is dominant over sensitivity at low (0.5-5 micrograms/ml) cycloheximide (cyh) concentrations . The cyh-resistant haploid strain MMY1 confers relatively high (10 micrograms/ml) cyh-resistance to heterozygous diploids constructed by mating this strain with cyh-sensitive haploid strains . We present here a genetic and biochemical study of strain MMY1 . Analysis of tetrads obtained from a MMY1 heterozygous diploid showed that two unlinked nuclear mutations, determining high- and low-cycloheximide resistance, were present in MMY1 . From a genomic library of this strain, constructed in vector YCp50, two plasmids (pRC1 and pRC13) have been isolated which, respectively, confer high- and low-resistance phenotypes to cyh-sensitive S . cerevisiae strains . The restriction maps of pRC1 and pRC13 are totally unrelated . This finding suggests that the genes harboring the two mutations encoding cyh-resistance from MMY1 were cloned in plasmids pRC1 and pRC13, respectively . Pulse field gel electrophoresis showed that the DNA insert of pRC1 maps at either chromosome VII or XV, whereas that from pRC13 maps at chromosome XI . This latter gene appears to define a previously unreported locus and has been named cyh5 . By restriction and nucleotide sequencing analysis, the cyh gene present in pRC1 has been shown to correspond to cyh2, which maps at chromosome VII . These results suggest that the dominant cyh-resistance phenotype conferred by MMY1 in heterozygous diploids is promoted by the presence of both cyh2 and cyh5.(ABSTRACT TRUNCATED AT 250 WORDS)

Curr Genet, 1991 May, 19(5), 339 - 42
The gene DIS2S1 is essential in Saccharomyces cerevisiae and is involved in glycogen phosphorylase activation; Clotet J et al.; S . cerevisiae gene DIS2S1, which codes for a protein very similar to the catalytic subunit of mammalian protein phosphatase 1, was disrupted "in vitro" . Diploid yeast cells were transformed and sporulated . Tetrad analysis demonstrated that disruption of DIS2S1 is lethal for the cell . Glycogen phosphorylase alpha and glycogen synthase activity ratio were measured in diploids carrying a disrupted allele of the gene . Phosphorylase was dramatically activated in mutant cells but, under the same conditions, glycogen synthase activity was essentially identical in both mutant and wild-type cells.

Curr Genet, 1991 May, 19(5), 333 - 7
Regulation of the pyrimidine salvage pathway by the FUR1 gene product of Saccharomyces cerevisiae; Kern L et al.; In Saccharomyces cerevisiae, the protein encoded by the FUR1 gene is absolutely required for the expression of uracil phosphoribosyl transferase activity . The occurrence of semi-dominant mutations for 5-fluorouracil-(5FU)-resistance at this locus led us to clone and sequence the semi-dominant fur1-5 allele . A single point mutation, resulting in the substitution of arginine 134 for serine, is responsible for this mutant phenotype . The fur1-5 allele is transcribed and expressed at the same level as the wild-type allele . But, in contrast with the wild-type, the UPRTase activity of the fur1-5 mutant strain is stimulated in vitro by UTP and does not, therefore, correspond to a loss of feedback of UPRTase activity . We found that uracil, as a free base, induces a significative increase in transcription and UPRTase activity in a wild-type strain as well as in uracil-overproducing mutants which principally explains the high efficiency of the pyrimidine salvage pathway in S . cerevisiae.

Antonie Van Leeuwenhoek, 1991 May, 59(4), 269 - 83
Physiological aspects of growth and recombinant DNA stability in Saccharomyces cerevisiae; Mason CA; Despite the fact that plasmid stability in the yeast Saccharomyces cerevisiae is influenced by both genetical and physiological parameters most attention has been focused on the former . Physiological factors affecting the stability of plasmids have been poorly characterized despite the need for such information in order to optimize the use of S . cerevisiae as a host for recombinant protein production processes . The physiology of wild type S . cerevisiae differs considerably when grown using different cultivation techniques . A limited amount of phenomenological data has been reported concerning plasmid instability effects under these different conditions and in this article these have been collected together with the intention of providing an overview to instability effects and to try and propose reasons as to how the physiological response to different growth conditions can be manifested as stability/instability effects.

Mol Cell Biol, 1991 May, 11(5), 2576 - 82
Isolation, DNA sequence, and regulation of a Saccharomyces cerevisiae gene that encodes DNA strand transfer protein alpha; Clark AB et al.; DNA strand transfer protein alpha (STP alpha) from meiotic Saccharomyces cerevisiae cells promotes homologous pairing of DNA without any nucleotide cofactor in the presence of yeast single-stranded DNA binding protein . This gene (DNA strand transferase 1, DST1) encodes a 309-amino-acid protein with a predicted molecular mass of 34,800 Da . The STP alpha protein level is constant in both mitotic and meiotic cells, but during meiosis the polypeptide is activated by an unknown mechanism, resulting in a large increase in its specific activity . A dst1::URA3/dst1::URA3 mutant grows normally in mitotic media; however, meiotic cells exhibit a greatly reduced induction of both DNA strand transfer activity and intragenic recombination between his1 heteroalleles . Spore viability is normal . These results suggest that DST1 is required for much of the observed induction of homologous recombination in S . cerevisiae during meiosis but not for normal sporulation.

J Bacteriol, 1991 May, 173(10), 3101 - 8
The phosphoinositol sphingolipids of Saccharomyces cerevisiae are highly localized in the plasma membrane; Patton JL et al.; To investigate the vital function(s) of the phosphoinositol-containing sphingolipids of Saccharomyces cerevisiae, we measured their intracellular distribution and found these lipids to be highly localized in the plasma membrane . Sphingolipids were assayed in organelles which had been uniformly labeled with {3H}inositol or 32P and by chemical measurements of alkali-stable lipid P, of long chain bases, and of very long chain fatty acids . We have developed an improved method for the preparation of plasma membranes which is based on the procedure of Duran et al . (Proc . Natl . Acad . Sci . USA 72:3952-3955, 1975) . On the basis of marker enzyme and DNA assays carried out with a number of preparations, the plasma membranes contained less than 10% vacuolar membranes (alpha-mannosidase) and nuclei (DNA); the contamination by the endoplasmic reticulum (NADPH-cytochrome c reductase) varied from 0 to 20% . The plasma membrane preparations showed a 13-fold increase in the specific activity of vanadate-sensitive ATPase, compared with that in the homogenate, with a yield ranging from 50 to 80% . A comparison of the distribution of the ATPase with that of sphingolipids assayed by a variety of methods showed that 80 to 100% of the sphingolipids are localized in the plasma membrane; the sphingolipids constitute about 30% of the total phospholipid content of the plasma membrane . Minor amounts of sphingolipids that were found in isolated mitochondria and nuclei can be attributed to the presence of small amounts of plasma membrane in these fractions . These results suggest that one or more essential functions of these lipids is in the plasma membrane . Furthermore, sphingolipids may be useful chemical markers of the plasma membrane of S . cerevisiae.

Mikrobiologiia, 1991 May-Jun, 60(3), 537 - 40
{Detection of a wild population of yeast of the biological species Saccharomyces cerevisiae in Siberia}; Naumov GI et al.; A wild yeast population of Saccharomyces cerevisiae was found for the first time in Siberia, as was confirmed by hybridological analysis . The European cultivated S . cerevisiae strains were demonstrated to take their origin from Asia.

J Mol Evol, 1991 May, 32(5), 396 - 404
Polymorphisms in tandemly repeated sequences of Saccharomyces cerevisiae mitochondrial DNA; Skelly PJ et al.; A spontaneously arising mitochondrial DNA (mtDNA) variant of Saccharomyces cerevisiae has been formed by two extra copies of a 14-bp sequence (TTAATTAAATTATC) being added to a tandem repeat of this unit . Similar polymorphisms in tandemly repeated sequences have been found in a comparison between mtDNAs from our strain and others . In 5850 bp of intergenic mtDNA sequence, polymorphisms in tandemly repeated sequences of three or more base pairs occur approximately every 400-500 bp whereas differences in 1-2 bp occur approximately every 60 bp . Some polymorphisms are associated with optional G + C-rich sequences (GC clusters) . Two such optional GC clusters and one A + T repeat polymorphism have been discovered in the tRNA synthesis locus . In addition, the variable presence of large open reading frames are documented and mechanisms for generating intergenic sequence diversity in S . cerevisiae mtDNA are discussed.

Clin Chem, 1991 May, 37(5), 662 - 6
Different constructs for the expression of mammalian gamma-glutamyltransferase cDNAs in Escherichia coli and in Saccharomyces cerevisiae; Angele C et al.; To prepare a reference material for gamma-glutamyltransferase (GGT; EC 2.3.2.2) measurements in clinical chemistry, we constructed different vectors containing either the rat kidney or the human hepatoma Hep G2 GGT cDNA downstream from an inducible promoter for expression in Escherichia coli and Saccharomyces cerevisiae . Transformed bacterial and yeast cells were tested for GGT production by use of Western blot analysis and enzymatic activity measurements . Both rat renal and Hep G2 GGT cDNAs were expressed in E . coli, producing active and nonglycosylated enzymes localized in the periplasmic space . Recombinant Hep G2 GGT was synthesized as a single-chain protein, unlike rat renal GGT, which presented two polypeptides of 62 and 30 kDa, identified as the precursor and a GGT heavy-subunit-like peptide, respectively . Rat renal GGT was produced in S . cerevisiae as two polypeptides, 55 and 30 kDa, detected by antisera against rat renal GGT . These results suggest maturation mechanisms such as glycosylation and cleavage steps, enhancing the interest of S . cerevisiae as a useful expression system for producing active mammalian proteins as reference materials.

J Biol Chem, 1991 Apr 25, 266(12), 7517 - 23
Characterization of ATP12, a yeast nuclear gene required for the assembly of the mitochondrial F1-ATPase; Bowman S et al.; Mitochondrial F1-ATPase is an oligomeric enzyme composed of five distinct subunit polypeptides . The alpha and beta subunits make up the bulk of protein mass of F1 . In Saccharomyces cerevisiae both subunits are synthesized as precursors with amino-terminal targeting signals that are removed upon translocation of the proteins to the matrix compartment . Recently, two different complementation groups (G13, G57), consisting of yeast nuclear mutants with defective F1, have been described . Biochemical analyses indicate that the mutational block in both groups of mutants affects a critical step needed for the assembly of the alpha and beta subunits into the F1 oligomer after their transport into mitochondria . In this study the ATP12 gene representative of the nuclear respiratory-deficient mutant of S . cerevisiae (pet) complementation group G57 has been cloned and the encoded product partially characterized . The ATP12 reading frame is 975 base pairs long and codes for a protein of Mr = 36,587 . The ATP12 protein is not homologous to the subunits of F1 whose sequences are known, nor does it exhibit significant primary structure similarity to any known protein . In vitro import assays indicate that ATP12 protein is synthesized as a precursor approximately 3 kDa larger than the mature protein . The mitochondrial localization of the protein has been confirmed by Western blot analysis of mitochondrial proteins with an antibody against a hybrid protein expressed from a trpE-ATP12 fusion . Fractionation of mitochondria indicates further that the ATP12 protein is either a minor component of the matrix compartment or is weakly bound to the matrix side of the inner membrane . The molecular weight of the native protein, estimated from its sedimentation properties in sucrose gradients, is at least two times larger than the monomer . This suggests that the ATP12 protein is probably part of a larger complex.

Eur J Biochem, 1991 Apr 23, 197(2), 399 - 405
Carboxypeptidase yscS: gene structure and function of the vacuolar enzyme; Spormann DO et al.; The gene encoding carboxypeptidase yscS in Saccharomyces cerevisiae, CPS1, was cloned by complementation of the cps1-3 mutation . The cloned CPS1 gene, which again enabled a leucine auxotrophic cps1-3 mutant to grow on the modified dipeptide Cbz-Gly-Leu (Cbz, benzyloxycarbonyl) as sole leucine source, was sequenced and found to consist of an open reading frame of 1728 bp encoding a protein of 576 amino acids . The putative protein contains a hydrophobic stretch of 20 amino acids and a putative signal sequence cleavage site . Five putative N-glycosylation sites are also in the protein sequence . This data is consistent with the previous finding of carboxypeptidase yscS being a vacuolar peptidase . Chromosomal disruption of the CPS1 gene completely abolishes carboxypeptidase yscS activity . This protein is yet another member of the peptidases in S . cerevisiae involved in nitrogen metabolism.

J Mol Biol, 1991 Apr 20, 218(4), 735 - 46
Incipient mitochondrial evolution in yeasts . I . The physical map and gene order of Saccharomyces douglasii mitochondrial DNA discloses a translocation of a segment of 15,000 base-pairs and the presence of new introns in comparison with Saccharomyces cerevisiae; Tian GL et al.; We have determined the physical and genetic map of the 73,000 base-pair mitochondrial genome of a novel yeast species Saccharomyces douglasii . Most of the protein and RNA-coding genes known to be present in the mitochondrial DNA of Saccharomyces cerevisiae have been identified and located on the S . douglasii mitochondrial genome . The nuclear genomes of the two species are thought to have diverged some 50 to 80 million years ago and their nucleo-mitochondrial hybrids are viable but respiratorily deficient . The mitochondrial genome of S . douglasii displays many interesting features in comparison with that of S . cerevisiae . The three mosaic genes present in both genomes are quite different with regard to their structure . The S . douglasii COXI gene has two new introns and is missing the five introns of the S . cerevisiae gene . The S . douglasii cytochrome b gene has one new intron and lacks two introns of the S . cerevisiae gene . Finally, the L-rRNA gene of S . douglasii, like that of S . cerevisiae, has one intron of which the structure is different . Another salient feature of the S . douglasii mitochondrial genome reported here is that the gene order is different in comparison with S . cerevisiae mitochondrial DNA . In particular, a segment of approximately 15,000 base-pairs including the genes coding for COXIII and S-rRNA has been translocated to a position between the genes coding for varl and L-rRNA.

J Mol Biol, 1991 Apr 20, 218(4), 747 - 60
Incipient mitochondrial evolution in yeasts . II . The complete sequence of the gene coding for cytochrome b in Saccharomyces douglasii reveals the presence of both new and conserved introns and discloses major differences in the fixation of mutations in evolution; Tian GL et al.; We have determined the complete sequence of the mitochondrial gene coding for cytochrome b in Saccharomyces douglasii . The gene is 6310 base-pairs long and is interrupted by four introns . The first one (1311 base-pairs) belongs to the group ID of secondary structure, contains a fragment open reading frame with a characteristic GIY .. . YIG motif, is absent from Saccharomyces cerevisiae and is inserted in the same site in which introns 1 and 2 are inserted in Neurospora crassa and Podospora anserina, respectively . The next three S . douglasii introns are homologous to the first three introns of S . cerevisiae, are inserted at the same positions and display various degrees of similarity ranging from an almost complete identity (intron 2 and 4) to a moderate one (intron 3) . We have compared secondary structures of intron RNAs, and nucleotide and amino acid sequences of cytochrome b exons and intron open reading frames in the two Saccharomyces species . The rules that govern fixation of mutations in exon and intron open reading frames are different: the relative proportion of mutations occurring in synonymous codons is low in some introns and high in exons . The overall frequency of mutations in cytochrome b exons is much smaller than in nuclear genes of yeasts, contrary to what has been found in vertebrates, where mitochondrial mutations are more frequent . The divergence of the cytochrome b gene is modular: various parts of the gene have changed with a different mode and tempo of evolution.

Cell, 1991 Apr 19, 65(2), 333 - 40
A highly conserved domain of TFIID displays species specificity in vivo; Gill G et al.; Recombinant TFIID proteins from yeast, Drosophila, and human function interchangeably in vitro to restore basal level transcription to a human HeLa extract depleted for TFIID . Here we report that the recently cloned human and Drosophila TFIID genes fail to substitute in vivo for the S . cerevisiae TFIID gene, SPT15, which is essential for viability . Analysis of yeast-human hybrid TFIID proteins reveals that the failure of human TFIID to functionally replace yeast TFIID maps to the highly conserved C-terminal domain . Thus, the C-terminal conserved domain of TFIID, as well as the N-terminal divergent domain, appears to be involved in species-specific interactions.

Nucleic Acids Res, 1991 Apr 11, 19(7), 1657 - 60
Alpha-sarcin cleavage of ribosomal RNA is inhibited by the binding of elongation factor G or thiostrepton to the ribosome; Miller SP et al.; The translocation reaction catalyzed by elongation factor G (EF-G) is inhibited either by alpha-sarcin cleavage of 23S rRNA or by the binding of thiostrepton to the E . coli ribosome . Here we show that the transitory binding of EF-G and GDP to the ribosome inhibited the rate of alpha-sarcin cleavage and that stabilization of this binding with fusidic acid completely prevented alpha-sarcin cleavage . A similar pattern of inhibition was seen upon the binding of elongation factor 2 to the S . cerevisiae ribosome . The irreversible binding of the antibiotic thiostrepton to the E . coli ribosome, on the other hand, decreased the rate of cleavage by alpha-sarcin approximately 2-fold . These results suggest that the alpha-sarcin site is located within the ribosomal domain for EF-G binding and that the conformation of this site is affected by the binding of thiostrepton.

Eur J Biochem, 1991 Apr 10, 197(1), 1 - 7
The proteinase yscB inhibitor (PB12) gene of yeast and studies on the function of its protein product; Schu P et al.; The gene for proteinase yscB inhibitor I2B (PBI2) from Saccharomyces cerevisiae was isolated by oligonucleotide screening of a genomic DNA library, and was sequenced . The gene codes for a single protein of 75 amino acids . In contrast to the published amino acid sequence {Maier, K., Muller, H., Tesch, R., Trolp, T., Witt, I . & Holzer, H . (1979) J . Biol . Chem . 254, 12,555-12,561} the DNA sequence revealed a valine instead of a leucine at position 33 (32 of the mature protein) . Therefore the primary sequences of the isoinhibitors I2B of S . cerevisiae and I1B of Saccharomyces carlsbergensis differ only at position 34 (glutamic acid/lysine) . The open reading frame of PBI2 was replaced in vitro by the URA3 gene and a I2B null mutant of S . cerevisiae was constructed by gene replacement . The mutation resulted in an elevation of the protein degradation rate by 50% when grown under nutritional stress compared to the isogenic wild type . Growth and viability of the cells was not significantly affected by the absence of I2B.

Eur J Biochem, 1991 Apr 10, 197(1), 105 - 11
Isolation of the ATP synthase subunit 6 and sequence of the mitochondrial ATP6 gene of the yeast Candida parapsilosis; Guelin E et al.; The mitochondrially translated product called subunit 6 was extracted from the yeast Candida parapsilosis mitochondria using an organic solvent mixture and purified by reverse-phase HPLC . The partial N-terminal sequence of subunit 6 reveals a post-translational cleavage site as in Saccharomyces cerevisiae . The structural mitochondrial gene ATP6 was isolated form a mitochondrial DNA library using the oligonucleotide probe procedure . The gene and the surrounding regions were cloned into M13tg130 and M13tg131 phage vectors . The insert contained an open reading frame 738-bp encoding a 246-amino-acid polypeptide . Mature subunit 6 contains 243 amino acid residues and the predicted molecular mass is 26,511 Da . The subunit shows 52% similarity with ATP synthase subunit 6 of the yeast S . cerevisiae . Comparison between protein and DNA sequences shows that the CUN codon family codes for a leucine in C . parapsilosis mitochondria.

J Mol Biol, 1991 Apr 5, 218(3), 543 - 56
A general approach to the isolation of cell cycle-regulated genes in the budding yeast, Saccharomyces cerevisiae; Price C et al.; We describe a general approach to the isolation of cell cycle-dependently regulated transcripts in Saccharomyces cerevisiae . This approach is based on the physical identification of cell cycle-regulated transcripts by Northern hybridization using as probes yeast DNA isolated from an ordered S . cerevisiae genomic library . The purpose of this is twofold; first, to assess the importance of transcriptional regulation in cell cycle control; and second, to identify novel genes that may have important roles in the eukaryotic cell cycle . We report the isolation of two previously uncharacterized genes that are transcribed at points in the cell cycle to which specific transcriptional activation has not been assigned: namely, mitosis and early G1 phase . It is argued that these transcripts serve as important landmarks for cell cycle events that are not readily distinguished by either morphological or cytological criteria . The cell cycle-dependent transcription of the RNR1 and CLN1 genes is also described and the implications for cell cycle control, in G1, are discussed with reference to these two genes.

Cell, 1991 Apr 5, 65(1), 163 - 74
A cyclin B homolog in S . cerevisiae: chronic activation of the Cdc28 protein kinase by cyclin prevents exit from mitosis; Ghiara JB et al.; A cyclin B homolog was identified in Saccharomyces cerevisiae using degenerate oligonucleotides and the polymerase chain reaction . The protein, designated Scb1, has a high degree of similarity with B-type cyclins from organisms ranging from fission yeast to human . Levels of SCB1 mRNA and protein were found to be periodic through the cell cycle, with maximum accumulation late, most likely in the G2 interval . Deletion of the gene was found not to be lethal, and subsequently other B-type cyclins have been found in yeast functionally redundant with Scb1 . A mutant allele of SCB1 that removes an amino-terminal fragment of the encoded protein thought to be required for efficient degradation during mitosis confers a mitotic arrest phenotype . This arrest can be reversed by inactivation of the Cdc28 protein kinase, suggesting that cyclin-mediated arrest results from persistent protein kinase activation.

Cell, 1991 Apr 5, 65(1), 145 - 61
The role of CDC28 and cyclins during mitosis in the budding yeast S . cerevisiae; Surana U et al.; cdc28-1N is a conditional allele that has normal G1 (Start) function but confers a mitotic defect . We have isolated seven genes that in high dosage suppress the growth defect of cdc28-1N cells but not of Start-defective cdc28-4 cells . Three of these (CLB1, CLB2, and CLB4) encode proteins strongly homologous to G2-specific B-type cyclins . Another gene, CLB3, was cloned using PCR, CLB1 and CLB2 encode a pair of closely related proteins; CLB3 and CLB4 encode a second pair . Neither CLB1 nor CLB2 is essential; however, disruption of both is lethal and causes a mitotic defect . Furthermore, the double mutant cdc28-1N clb2::LEU2 is nonviable, whereas cdc28-4 clb2::LEU2 is viable, suggesting that the cdc28-1N protein may be defective in its interaction with B-type cyclins . Our results are consistent with CDC28 function being required in both G1 and mitosis . Its mitotic role, we believe, involves interaction with a family of at least four G2-specific cyclins.

Mol Gen Genet, 1991 Apr, 226(1-2), 310 - 4
The pentafunctional FAS1 genes of Saccharomyces cerevisiae and Yarrowia lipolytica are co-linear and considerably longer than previously estimated; Kottig H et al.; The fatty acid synthetase (FAS) gene FAS1 of the alkane-utilizing yeast Yarrowia lipolytica was cloned and sequenced . The gene is represented by an intron-free reading frame of 6228 bp encoding a protein of 2076 amino acids and 229,980 Da molecular weight . This protein exhibits a 58% sequence similarity to the corresponding Saccharomyces cerevisiae FAS beta-subunit . The sequential order of the five FAS1-encoded enzyme The sequential order of the five FAS1-encoded enzyme domains, acetyl transferase, enoyl reductase, dehydratase and malonyl/palmityl-transferase, is co-linear in both organisms . This finding agrees with available evidence that the functional organization of FAS genes is similar in related organisms but differs considerably between unrelated species . In addition, previously reported conflicting data concerning the 3' end of S . cerevisiae FAS1 were re-examined by genomic and cDNA sequencing of the relevant portion of the gene . Thereby, the translational stop codon was shown to lie considerably downstream of both published termination sites . The S . cerevisiae FAS1 gene thus has a corrected length of 6153 bp and encodes a protein of 2051 amino acids and 228,667 Da molecular weight.

EMBO J, 1991 Apr, 10(4), 981 - 5
A single-stranded DNA binding protein from S . cerevisiae specifically recognizes the T-rich strand of the core sequence of ARS elements and discriminates against mutant sequences; Schmidt AM et al.; A protein named ssARS-T binding protein has been purified from yeast that specifically binds to the T-rich strand of the consensus core sequence of yeast autonomously replicating sequence (ARS) elements . As assayed from gel mobility shift experiments the ssARS-T protein shows characteristics of a sequence specific single-stranded DNA binding protein . The complementary A-rich strand of the ARS core sequence is bound much more weakly and no binding can be detected for the double-stranded form of the core sequence . Three single base substitutions in the core sequence that are known to abolish ARS function in vivo also lead to weaker binding of the core sequence to the ssARS-T protein in vitro . The strong correlation between the binding of mutated sequences in vitro and the ARS properties of these sequences in vivo points to an essential function of the ssARS-T protein during replication initiation in yeast ARS elements.

Nucleic Acids Res, 1991 Mar 25, 19(6), 1189 - 95
Construction of an ordered clone bank and systematic analysis of the whole transcripts of chromosome VI of Saccharomyces cerevisiae; Yoshikawa A et al.; By comparing sequences of restriction enzyme cleavage sites and their distance data, we sorted 384 lambda phage clones containing segments of chromosome VI of S . cerevisiae and constructed an ordered clone bank for this chromosome . The physical length of this bank is 269.7 kb . The bank contains the entire chromosome including the left telomere, but it is not certain whether it contains the right telomere as well . To estimate the number of genes present on this chromosome, we performed a series of Northern hybridization experiments using 157 restriction enzyme fragments prepared from the bank as hybridization probes and total poly(A)+ RNA from vegetatively growing cells . Thus, 97 distinct transcripts were identified . The relative abundance levels of individual transcripts were measured by comparing their band intensity with that of the RPO41 transcript . It was found that the transcripts from the genes located in the telomeric and centromeric regions are less abundant as compared to those from the genes in the central regions of both arms.

Proc Natl Acad Sci U S A, 1991 Mar 15, 88(6), 2055 - 9
Incorporation of 12-methoxydodecanoate into the human immunodeficiency virus 1 gag polyprotein precursor inhibits its proteolytic processing and virus production in a chronically infected human lymphoid cell line; Bryant ML et al.; Covalent linkage of myristate (tetradecanoate; 14:0) to the NH2-terminal glycine residue of the human immunodeficiency virus 1 (HIV-1) 55-kDa gag polyprotein precursor (Pr55gag) is necessary for its proteolytic processing and viral assembly . We have shown recently that several analogs of myristate in which a methylene group is replaced by a single oxygen or sulfur atom are substrates for Saccharomyces cerevisiae and mammalian myristoyl-CoA:protein N-myristoyltransferase (EC 2.3.1.97; NMT) despite their reduced hydrophobicity . Some inhibit HIV-1 replication in acutely infected CD4+H9 cells without accompanying cellular toxicity . To examine the mechanism of their antiviral effects, we performed labeling studies with two analogs, 12-methoxydodecanoate (13-oxamyristate; 13-OxaMyr) and 5-octyloxypentanoate (6-oxamyristate; 6-OxaMyr), the former being much more effective than the latter in blocking virus production . {3H}Myristate and {3H}13-OxaMyr were incorporated into Pr55gag with comparable efficiency when it was coexpressed with S . cerevisiae NMT in Escherichia coli . {3H}6-OxaMyr was not incorporated, even though its substrate properties in vitro were similar to those of 13-OxaMyr and myristate . {3H}13-OxaMyr, but not {3H}6-OxaMyr, was also efficiently incorporated into HIV-1 Pr55gag and nef (negative factor) in chronically infected H9 cells . Analog incorporation produced a redistribution of Pr55gag from membrane to cytosolic fractions and markedly decreased its proteolytic processing by viral protease . 13-OxaMyr and 3'-azido-3'-deoxythymidine (AZT) act synergistically to reduce virus production in acutely infected H9 cells . Unlike AZT, the analog is able to inhibit virus production (up to 70%) in chronically infected H9 cells . Moreover, the inhibitory effect lasts 6-8 days . These results suggest that (i) its mechanism of action is distinct from that of AZT and involves a late step in virus assembly; (ii) the analog may allow reduction in the dose of AZT required to affect viral replication; and (iii) combinations of analog and HIV-1 protease inhibitors may have synergistic effects on the processing of Pr55gag.

Eur J Biochem, 1991 Mar 14, 196(2), 431 - 8
Purification and characterisation of plasminogen activator inhibitor 2 produced in Saccharomyces cerevisiae; Steven J et al.; Expression of plasminogen activator inhibitor 2 (PAI-2) under the control of the protease B gene promoter in a mutant strain of Saccharomyces cerevisiae, DS569, resulted in its accumulation intracellularly at up to 20% of the soluble cell protein . Provision of an N-terminal signal sequence resulted in the secretion of a hyperglycosylated molecule . The intracellularly produced PAI-2 was purified by copper-chelate and anion-exchange chromatography to greater than 95% pure and was fully active . The recombinant PAI-2 formed SDS-stable complexes with urokinase and tissue-type plasminogen activator and inhibited the proteases with similar reaction kinetics to placental PAI-2 (second-order rate constant for uPA, 2.4 x 10(6) M-1 s-1, and for two-chain tPA, 0.7 x 10(5) M-1 s-1) . As is the case for placental PAI-2, the N-terminus of the yeast-derived recombinant PAI-2 was blocked . The high productivity and consequent ease of purification mean that S . cerevisiae provides an excellent source of recombinant PAI-2 for investigation of its therapeutic potential in the treatment of neoplastic and inflammatory diseases.

Gene, 1991 Mar 1, 99(1), 39 - 46
Structural and functional conservation between the high-affinity K+ transporters of Saccharomyces uvarum and Saccharomyces cerevisiae; Anderson JA et al.; In Saccharomyces cerevisiae, high-affinity K+ uptake is dependent upon a 180-kDa plasma membrane protein encoded by TRK1 (c-TRK1) {Gaber et al., Mol . Cell . Biol . 8 (1988) 2848-2859)} . Although hybridization with a c-TRK1 probe revealed highly homologous sequences in the genomes of most Saccharomyces species, the TRK1 sequence in S . uvarum (u-TRK1) was detected only under low-stringency conditions . We cloned u-TRK1 and found it to confer high-affinity K+ uptake in S . cerevisiae . A comparison of the inferred amino acid sequences reveals 78% identity and 86% similarity between the two high-affinity transporters . The most highly conserved regions are the putative membrane-spanning domains (95% identical), suggesting that the structure of the transmembrane domains is important for high-affinity K+ transport.

Genetics, 1991 Mar, 127(3), 475 - 88
Distributive disjunction of authentic chromosomes in Saccharomyces cerevisiae; Guacci V et al.; Distributive disjunction is defined as the first division meiotic segregation of either nonhomologous chromosomes that lack homologs or homologous chromosomes that have not recombined . To determine if chromosomes from the yeast Saccharomyces cerevisiae were capable of distributive disjunction, we constructed a strain that was monosomic for both chromosome I and chromosome III and analyzed the meiotic segregation of the two monosomic chromosomes . In addition, we bisected chromosome I into two functional chromosome fragments, constructed strains that were monosomic for both chromosome fragments and examined meiotic segregation of the chromosome fragments in the monosomic strains . The two nonhomologous chromosomes or chromosome fragments appeared to segregate from each other in approximately 90% of the asci analyzed, indicating that yeast chromosomes were capable of distributive disjunction . We also examined the ability of a small nonhomologous centromere containing plasmid to participate in distributive disjunction with the two nonhomologous monosomic chromosomes . The plasmid appeared to efficiently participate with the two full length chromosomes suggesting that distributive disjunction in yeast is not dependent on chromosome size . Thus, distributive disjunction in S . cerevisiae appears to be different from Drosophila melanogaster where a different sized chromosome is excluded from distributive disjunction when two similar size nonhomologous chromosomes are present.

Oncogene, 1991 Mar, 6(3), 357 - 60
Ets1, when fused to the GAL4 DNA binding domain, efficiently enhances galactose promotor dependent gene expression in yeast; Seneca S et al.; Ets1, the translation product of the c-ets1 proto-oncogene and the related Ets2 protein, act as sequence-specific transcriptional factors in transient transfection experiments in animal cells . We report here that in S . cerevisiae, expression of a lacZ test gene placed under the control of the GAL1 promoter is stimulated efficiently by a fusion protein in which the chicken Ets1 sequence starting from amino acid 37, is linked to the DNA binding domain of the yeast GAL4 transcriptional activator . This suggests that Ets1 contains one or more intrinsic transcription activation domain(s) . However, the GAL4 integral of Ets1 fusion protein was unable to restore growth of a gal4 deletion mutant on galactose, implying that the fusion product cannot substitute for GAL4 enhancement on all GAL genes.

Curr Genet, 1991 Mar, 19(3), 169 - 74
Mitochondrial genome of Saccharomyces douglasii: genes coding for components of the protein synthetic apparatus; Ragnini A et al.; Mitochondrial genes coding for some components of the protein synthetic apparatus in S . douglasii have been studies in detail . A region containing stretches of high homology to the S . cerevisiae tRNA synthesis locus (TSL) and the tRNA(fmet) gene has been identified and sequenced . The organization of this region was very similar to that present in S . cerevisiae, including the presence of a possible transcription starting signal . The S . douglasii TSL gene is shorter due to several deletions which, however, do not involve the regions coding for RNA domains know to be required for the catalytic activity of mitochondrial RNAse P . The S . douglasii LSU rRNA gene has been shown to contain a typical group I intron highly homologous to its S . cerevisiae counterpart, except for the absence of the open reading frame which in S . cerevisiae codes for I-SceI endonuclease.

Proc Natl Acad Sci U S A, 1991 Mar 1, 88(5), 1731 - 5
Lambda YES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations; Elledge SJ et al.; This work describes a multifunctional phage lambda expression vector system, lambda YES, designed to facilitate gene isolation from eukaryotes by complementation of Escherichia coli and Saccharomyces cerevisiae mutations . lambda YES vectors have a selection for cDNA inserts using an oligo adaptor strategy and are capable of expressing genes in both E . coli and S . cerevisiae . They also allow conversion from phage lambda to plasmid clones by using the cre-lox site-specific recombination system, referred to here as automatic subcloning . A simple method has been developed for the conversion of any plasmid into a phage lambda cDNA cloning vector with automatic subcloning capability . cDNA libraries constructed in these vectors were used to isolate genes from humans and Arabidopsis thaliana by complementation of yeast and bacterial mutations, respectively.

Biochem Int, 1991 Mar, 23(5), 861 - 73
Activation of the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae by addition of hydrogen peroxide; Sigler K et al.; Addition of hydrogen peroxide (greater than 10 mM) to aerated derepressed cells of S . cerevisiae in the absence of substrate caused a boost of endogenous respiration and both intra- and extracellular acidification, without any significant change in cellular ATP level . Furthermore, a hyperpolarization of the plasma membrane was indicated by an enhanced accumulation of tetraphenylphosphonium in the cells . The extracellular pH attained was as low as 3.5 . The acidification could be suspended by the H(+)-ATPase inhibitors diethylstilbestrol and dicyclohexylcarbodiimide and was, in general, associated with an opposite flux of K+ . K+ also stimulated the H(+)-ATPase activity in the purified plasma membrane fraction . These results are consistent with the plasma membrane H(+)-ATPase being involved in the H+ extrusion induced by H2O2 in the absence of substrate . Extended exposure of cells to H2O2 led eventually to an arrest of both respiration and ion fluxes that could be again lifted by depolarizing the plasma membrane . Along with differences in the cellular NADH/NAD+ ratio and in the participation of organic acids, this makes the H2O2-induced acidification distinct from that induced by glucose.

FEBS Lett, 1991 Feb 25, 279(2), 341 - 5
Saccharomyces cerevisiae gene SIT4 is involved in the control of glycogen metabolism; Posas F et al.; The gene SIT4 of S . cerevisiae, which codes for a protein structurally related to the catalytic subunit of mammalian protein phosphatase 2A, was disrupted in vitro . Analysis of glycogen synthase activity ratio in mutant haploid cells indicated that the enzyme was less active than in wild-type cells . On the contrary, glycogen phosphorylase alpha activity was much higher . The activation of glycogen synthase observed in wild-type cells after incubation with lithium ions was not detected in mutant cells . These results suggest that the product of gene SIT4, a putative protein phosphatase, could be involved in the control of glycogen metabolism in yeast cells.

Cell, 1991 Feb 22, 64(4), 717 - 25
A 240 kd multisubunit protein complex, CBF3, is a major component of the budding yeast centromere; Lechner J et al.; A key protein component (CBF3) of the budding yeast (S . cerevisiae) centromere/kinetochore has been purified and characterized . CBF3 is a 240 kd multisubunit protein complex that binds specifically to the yeast wild-type centromere DNA (CEN), but not to nonfunctional CEN DNA containing a single base substitution in the critical CDEIII consensus sequence . When purified by affinity chromatography, CBF3 contains three protein components: CBF3A (110 kd), CBF3B (64 kd), and CBF3C (58 kd) . Highly purified CBF3 requires the presence of a separate assembly factor or chaperone activity to bind to CEN DNA . Treatment with phosphatase inactivates CBF3, indicating that at least one of the CBF3 subunits must be phosphorylated for DNA binding to occur . A 56 bp region including the 26 bp CDEIII consensus is protected from DNAase I cleavage in the CBF3-CEN DNA complex.

Cell, 1991 Feb 22, 64(4), 703 - 16
Pheromone-induced phosphorylation of a G protein beta subunit in S . cerevisiae is associated with an adaptive response to mating pheromone; Cole GM et al.; The mating pheromone response in S . cerevisiae is activated by a G protein-mediated signaling pathway in which G beta gamma is the active transducer of the signal . When exogenous pheromone is added to vegetatively growing cells, G beta is rapidly phosphorylated at several sites; phosphorylation does not require de novo protein synthesis . A mutation in G beta was constructed that eliminates signal-induced phosphorylation . This mutation leads to enhanced sensitivity to and impaired ability to recover from pheromone, but does not affect the ability of G beta gamma to transmit the mating signal . These phenotypes suggest that G protein phosphorylation mediates an adaptive response to pheromone-induced signaling . G beta phosphorylation does not require either the pheromone receptor C-terminus or the product of the SST2 gene, both of which mediate separate adaptive responses to pheromone . However, G beta phosphorylation is greatly facilitated by the presence of the G alpha subunit, which has also been shown to participate in an adaptation to pheromone.

Nature, 1991 Feb 21, 349(6311), 715 - 7
A suppressor of a yeast splicing mutation (prp8-1) encodes a putative ATP-dependent RNA helicase; Jamieson DJ et al.; Five small nuclear RNAs (snRNAs) are required for nuclear pre-messenger RNA splicing: U1, U2, U4, U5 and U6 . The yeast U1 and U2 snRNAs base-pair to the 5' splice site and branch-point sequences of introns respectively . The role of the U5 and U4/U6 small nuclear ribonucleoprotein particles (snRNPs) in splicing is not clear, though a catalytic role for the U6 snRNA has been proposed . Less is known about yeast splicing factors, but the availability of genetic techniques in Saccharomyces cerevisiae has led to the identification of mutants deficient in nuclear pre-mRNA splicing (prp2-prp27) . Several PRP genes have now been cloned and their protein products characterized . The PRP8 protein is a component of the U5 snRNP and associates with the U4/U6 snRNAs/snRNP to form a multi-snRNP particle believed to be important for spliceosome assembly . We have isolated extragenic suppressors of the prp8-1 mutation of S . cerevisiae and present here the preliminary characterization of one of these suppressors, spp81 . The predicted amino-acid sequence of the SPP81 protein shows extensive similarity to a recently identified family of proteins thought to possess ATP-dependent RNA helicase activity . The possible role of this putative helicase in nuclear pre-mRNA splicing is discussed.

Biochemistry, 1991 Feb 19, 30(7), 1986 - 96
Compound I radical in site-directed mutants of cytochrome c peroxidase as probed by electron paramagnetic resonance and electron-nuclear double resonance; Fishel LA et al.; The reaction of ferric cytochrome c peroxidase (CcP) from Saccharomyces cerevisiae with peroxide produces compound I, characterized by both an oxyferryl iron center and a protein-based free radical . The electron paramagnetic resonance (EPR) signal of the CcP compound I radical can be resolved into a broad majority component which accounts for approximately 90% of the spin intensity and a narrow minority component which accounts for approximately 10% of the integrated spin intensity {Hori, H., & Yonetani, T . (1985) J . Biol . Chem . 260, 3549-3555} . It was shown previously that the broad component of the compound I radical signal is eliminated by mutation of Trp-191 to Phe {Scholes, C . P., Liu, Y., Fishel, L . F., Farnum, M . F., Mauro, J . M., & Kraut, J . (1989) Isr . J . Chem . 29, 85-92} . The present work probed the effect of mutations in the vicinity of this residue by EPR and electron-nuclear double resonance (ENDOR) . These mutations were obtained from a plasmid-encoded form of S . cerevisiae expressed in Escherichia coli {Fishel, L . A., Villafranca, J . E., Mauro, J . M., & Kraut, J . (1987) Biochemistry 26, 351-360} . The EPR line shape and ENDOR signals of the compound I radical were perturbed only by mutations that alter Trp-191 or residues in its immediate vicinity: namely, Met-230 and Met-231, which have sulfur atoms within 4 A of the indole ring, and Asp-235, which forms a hydrogen bond with the indole nitrogen of Trp-191 . Mutations of other potential oxidizable sites (tryptophan, tyrosine, methionine, and cysteine) did not alter the EPR line shapes of the compound I radical, although the integrated spin intensities were weaker in some of these mutants . Mutations at Met-230 and/or -231 perturbed the EPR line shapes of the compound I radical signal but did not eliminate it . ENDOR of these two methionine mutants showed alteration to the hyperfine couplings of several strongly coupled protons, which are characteristic of the majority compound I radical electronic structure, and a change in weaker hyperfine couplings, which suggests a different orientation of the radical with respect to its surroundings in the presence of these methionine mutations . Besides the Trp-191----Phe mutation, only the Asp-235----Asn mutation eliminated the broad component of the compound I signal . Loss of the broad compound I EPR signal coincides with both the loss of the Asp----Trp-191 hydrogen-bonding interaction and alteration of the position of the indole ring of Trp-191.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1991 Feb 15, 266(5), 3005 - 15
Purification and characterization of the 180- and 86-kilodalton subunits of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex . The 180-kilodalton subunit has both DNA polymerase and 3'----5'-exonuclease activities; Brooke RG et al.; The yeast Saccharomyces cerevisiae catalytic DNA polymerase I 180-kDa subunit and the tightly associated 86-kDa polypeptide have been purified using immunoaffinity chromatography, permitting further characterization of the DNA polymerase activity of the DNA primase-DNA polymerase protein complex . The subunits were purified to apparent homogeneity from separate overproducing yeast strains using monoclonal antibodies specifically recognizing each subunit . When the individual subunits were recombined in vitro a p86p180 physical complex formed spontaneously, as judged by immunoprecipitation of 180-kDa polypeptide and DNA polymerase activity with the anti-86-kDa monoclonal antibody . The 86-kDa subunit stabilized the DNA polymerase activity of the 180-kDa catalytic subunit at 30 degrees C, the physiological temperature . The apparent DNA polymerase processivity of 50-60 nucleotides on poly(dA).oligo(dT)12 or poly(dT).oligo(A)8-12 template-primer was not affected by the presence of the 86-kDa subunit but was reduced by increased Mg2+ concentration . The Km of the catalytic 180-kDa subunit for dATP or DNA primer terminus was unaffected by the presence of the 86-kDa subunit . The isolated 180-kDa polypeptide was sufficient to catalyze all the DNA synthesis that had been observed previously in the DNA primase-DNA polymerase protein complex . The 180-kDa subunit possessed a 3'----5'-exonuclease activity that catalyzed degradation of polynucleotides, but degradation of oligonucleotide substrates of chain lengths up to 50 was not detected . This exonuclease activity was unaffected by the presence of the 86-kDa subunit . Despite the striking physical similarity of the DNA primase-DNA polymerase protein complex in all eukaryotes examined, the data presented here indicate differences in the enzymatic properties detected in preparations of the DNA polymerase subunits isolated from S . cerevisiae as compared with the properties of preparations from Drosophila cells . In particular, the 3'----5'-exonuclease activity associated with the yeast catalytic DNA polymerase subunit was not masked by the 86-kDa subunit.

FEBS Lett, 1991 Feb 11, 279(1), 41 - 4
Isolation of a new gene (SW A2) encoding an alpha-amylase from Schwanniomyces occidentalis and its expression in Saccharomyces cerevisiae; Abarca D et al.; A new gene (SW A2) encoding a secretory alpha-amylase activity from Schwanniomyces occidentalis has been cloned from this yeast and then expressed in Saccharomyces cerevisiae . Both Sw, occidentalis and a transformant of S . cerevisiae incorporating SW A2 contain a transcript of 2.1 kb which hybridizes to DNa carrying the SW A2 gene . This indicates that the transcript is a product of the SW A2 gene . Transcription of the SW A2 gene seems to be regulated in both Sw . occidentalis and S . cerevisiae . Furthermore, the secretion of alpha-amylase activity is drastically repressed by glucose in both Sw . occidentalis and a transformant of S . cerevisiae containing SW A2.

Cell, 1991 Feb 8, 64(3), 499 - 510
PAS1, a yeast gene required for peroxisome biogenesis, encodes a member of a novel family of putative ATPases; Erdmann R et al.; PAS genes are required for peroxisome biogenesis in the yeast S . cerevisiae . Here we describe the cloning, sequencing, and characterization of the PAS1 gene . Its gene product, Pas1p, has been identified as a rather hydrophilic 117 kd polypeptide . The predicted Pas1p sequence contains two putative ATP-binding sites and reveals a structural relationship to three other groups of proteins associated with different biological processes such as vesicle-mediated protein transport (NSF and Sec18p), control of cell cycle (Cdc48p, VCP, and p97-ATPase), and modulation of gene expression of the human immunodeficiency virus (TBP-1) . The proteins share a highly conserved domain of about 185 amino acids including a consensus sequence for ATP binding . We suggest that these proteins are members of a novel family of putative ATPases and may be descendants of one common ancestor.

Curr Genet, 1991 Feb, 19(2), 89 - 94
Distribution of mitochondrial intron sequences among 21 yeast species; Skelly PJ et al.; The mitochondrial and nuclear genomes of 21 yeast species belonging to 12 genera have been tested for the presence of sequences similar to seven S . cerevisiae mitochondrial introns (Se cox1.1,2,3,4,5c, Sc cob.4 and Sc LSU.1) and one K . lactis mitochondrial intron (K1 cox1.2) . Some introns, (Sc cox1.4, Sc cob.4, Sc LSU.1 and Kl cox1.2-all group I type), are widely distributed and are found in species with either basidiomycete or ascomycete affinities . This distribution is suggestive of recent sequence transfer between species . The remaining S . cerevisiae introns cross react with an additional species but with no set pattern . Pulsed field gel electrophoretic studies confirm that none of the tested mitochondrial introns cross react with nuclear DNA . These introns are, therefore, mitochondria-specific . Seven strains of K . lactis exhibit striking variability in intron content . In contrast to all mitochondrial introns tested, two introns of nuclear genes (the K . lactis actin gene and the S . cerevisiae RP29B gene) are not detected beyond their source species.

Mol Cell Biol, 1991 Feb, 11(2), 754 - 64
Suppressor analysis of temperature-sensitive mutations of the largest subunit of RNA polymerase I in Saccharomyces cerevisiae: a suppressor gene encodes the second-largest subunit of RNA polymerase I; Yano R et al.; The SRP3-1 mutation is an allele-specific suppressor of temperature-sensitive mutations in the largest subunit (A190) of RNA polymerase I from Saccharomyces cerevisiae . Two mutations known to be suppressed by SRP3-1 are in the putative zinc-binding domain of A190 . We have cloned the SRP3 gene by using its suppressor activity and determined its complete nucleotide sequence . We conclude from the following evidence that the SRP3 gene encodes the second-largest subunit (A135) of RNA polymerase I . First, the deduced amino acid sequence of the gene product contains several regions with high homology to the corresponding regions of the second-largest subunits of RNA polymerases of various origins, including those of RNA polymerase II and III from S . cerevisiae . Second, the deduced amino acid sequence contains known amino acid sequences of two tryptic peptides from the A135 subunit of RNA polymerase I purified from S . cerevisiae . Finally, a strain was constructed in which transcription of the SRP3 gene was controlled by the inducible GAL7 promoter . When this strain, which can grow on galactose but not on glucose, was shifted from galactose medium to glucose medium, a large decrease in the cellular concentration of A135 was observed by Western blot analysis . We have also identified the specific amino acid alteration responsible for suppression by SRP3-1 and found that it is located within the putative zinc-binding domain conserved among the second-largest subunits of eucaryotic RNA polymerases . From these results, it is suggested that this putative zinc-binding domain is in physical proximity to and interacts with the putative zinc-binding domain of the A190 subunit.

Yeast, 1991 Feb, 7(2), 173 - 83
Quantitation of readthrough of termination codons in yeast using a novel gene fusion assay; Firoozan M et al.; A simple quantitative in vivo assay has been developed for measuring the efficiency of translation of one or other of the three termination codons . UAA, UAG and UGA in Saccharomyces cerevisiae . The assay employs a 3-phosphoglycerate kinase-beta-galactosidase gene fusion, carried on a multicopy plasmid, in which the otherwise retained reading frame is disrupted by one or other of the three termination codons . Termination readthrough is thus quantitated by measuring beta-galactosidase in transformed strains . Using these plasmids to quantitate the endogenous levels of termination readthrough we show that readthrough of all three codons can be detected in a non-suppressor (sup+) strain of S . cerevisiae . The efficiency of this endogenous readthrough is much higher in a {psi+} strain than in a {psi-} strain with the UGA codon being the leakiest in the nucleotide context used . The utility of the assay plasmids for studying genetic modifiers of nonsense suppressors is also shown by their use to demonstrate that the cytoplasmic genetic determinant {psi+} broadens the decoding properties of a serine-inserting UAA suppressor tRNA (SUQ5) to allow it to translate the other two termination codons in the order of efficiency UAA greater than UAG greater than UGA.

EMBO J, 1991 Feb, 10(2), 361 - 8
A functional role for nucleosomes in the repression of a yeast promoter; Straka C et al.; Induction of the PHO5 gene in S . cerevisiae was previously shown to be accompanied by the removal of four positioned nucleosomes from the promoter . In order to assess the role of nucleosomes in the cascade of gene activation, DNA corresponding to one of these nucleosomes was excised . In its place two foreign DNA segments of the same length were inserted: a fragment from the African green monkey alpha-satellite DNA which is known to associate with histones in a highly specific fashion to give a uniquely positioned nucleosome or, alternatively, a fragment derived from pBR322 DNA . The promoter constructs were fused to the lacZ gene on centromere plasmids and transformed into yeast cells . The satellite fragment formed a nucleosome which persisted under inducing conditions . At the same time the inducibility of the PHO5 promoter was virtually abolished . When various subfragments containing between 35 and 100 bp of the satellite segment were tested, they were all found to decrease the inducibility of the promoter, full repression required the full length molecule, however . In contrast, the pBR fragment made the promoter weakly constitutive, and induction proceeded to levels even higher than with a promoter lacking an insert . Analysis of the chromatin structure reveals a nucleosome on the pBR segment at noninducing conditions which is removed upon induction . It is concluded that the quality of the histone-DNA interactions at the promoter makes an intrinsic contribution to the regulation of the gene.

Mol Pharmacol, 1991 Feb, 39(2), 109 - 13
Expression of human recombinant cAMP phosphodiesterase isozyme IV reverses growth arrest phenotypes in phosphodiesterase-deficient yeast; McHale MM et al.; The low-Km cAMP-specific phosphodiesterases (PDEases) are of great pharmacological significance because of their involvement in regulating cAMP concentrations, which, in turn, are responsible for mediating the cellular response to extracellular signals such as hormones and neurotransmitters . We recently reported the isolation of a cDNA clone that encodes a human monocyte low-Km, rolipram-sensitive, cAMP PDEase (isozyme IV) . We have engineered the inducible expression of this human PDEase in yeast . Cells of Saccharomyces cerevisiae contain two genes that encode cAMP PDEases . PDEase-deficient mutants are viable but exhibit specific growth arrest phenotypes associated with elevated intracellular cAMP content; these phenotypes include heat shock sensitivity and the inability to grow on acetate as a carbon source . We show that functional expression of our human cAMP PDEase in a genetically engineered PDEase-deficient strain of S . cerevisiae reverses these aberrant phenotypes . Furthermore, under conditions for growth arrest, rolipram is cytotoxic to PDEase-deficient mutants expressing the human cAMP PDEase, indicating that it is capable of inhibiting the human recombinant enzyme in vivo . This system can be used in the development of a yeast cell-based assay for isozyme-selective inhibitors of the human recombinant cAMP PDEase.

Mol Cell Biol, 1991 Feb, 11(2), 620 - 31
Cloning and characterization of ERG8, an essential gene of Saccharomyces cerevisiae that encodes phosphomevalonate kinase; Tsay YH et al.; Saccharomyces cerevisiae strains that contain the ery8-1 mutation are temperature sensitive for growth due to a defect in phosphomevalonate kinase, an enzyme of isoprene and ergosterol biosynthesis . A plasmid bearing the yeast ERG8 gene was isolated from a YCp50 genomic library by functional complementation of the erg8-1 mutant strain . Genetic analysis demonstrated that integrated copies of an ERG8 plasmid mapped to the erg8 locus, confirming the identity of this clone . Southern analysis showed that ERG8 was a single-copy gene . Subcloning and DNA sequencing defined the functional ERG8 regulon as an 850-bp upstream region and an adjacent 1,272-bp open reading frame . The deduced 424-amino-acid ERG8 protein showed no homology to known proteins except within a putative ATP-binding domain present in many kinases . Disruption of the chromosomal ERG8 coding region by integration of URA3 or HIS3 marker fragments was lethal in haploid cells, indicating that this gene is essential . Expression of the ERG8 gene in S . cerevisiae from the galactose-inducible galactokinase (GAL1) promoter resulted in 1,000-fold-elevated levels of phosphomevalonate kinase enzyme activity . Overproduction of a soluble protein with the predicted 48-kDa size for phosphomevalonate kinase was also observed in the yeast cells.

Free Radic Res Commun, 1991, 12-13 Pt 1, 279 - 85
The positive charge at position 189 is essential for the catalytic activity of iron- and manganese-containing superoxide dismutases; Borders CL Jr et al.; We have previously shown (C.L . Borders, Jr . et al., (1989) Archives of Biochemistry and Biophysics, 268, 74-80) that the iron-containing (FeSOD) and manganese-containing (MnSOD) superoxide dismutases from Escherichia coli are extensively (greater than 98%) inactivated by treatment with phenylglyoxal, an arginine-specific reagent . Examination of the published primary sequences of these two enzymes shows that Arg-189 is the only conserved arginine . This arginine is also conserved in the three additional FeSODs and seven of the eight additional MnSODs sequenced to date, with the only exception being the MnSOD from Saccharomyces cerevisiae, in which it is conservatively replaced by lysine . Treatment of S . cerevisiae MnSOD with phenylglyoxal under the same conditions used for the E . coli enzymes gives very little inactivation . However, treatment with low levels of 2,4,6-trinitrobenzenesulfonate (TNBS) and acetic anhydride, two lysine-selective reagents that cause a maximum of 65-80% inactivation of the E . coli SODs, gives complete inactivation of the yeast enzyme . Total inactivation of yeast MnSOD with TNBS correlates with the modification of approximately 5 lysines per subunit, whereas 6-7 lysines per subunit are acylated with acetic anhydride on complete inactivation . It appears that the positive charge contributed by residue 189, lysine in yeast MnSOD and arginine in all other SODs, may be critical for the catalytic activity of MnSODs and FeSODs.

Antonie Van Leeuwenhoek, 1991 Jan, 59(1), 49 - 63
A theoretical evaluation of growth yields of yeasts; Verduyn C et al.; Growth yields of Saccharomyces cerevisiae and Candida utilis in carbon-limited chemostat cultures were evaluated . The yields on ethanol and acetate were much lower in S . cerevisiae, in line with earlier reports that site I phosphorylation is absent in this yeast . However, during aerobic growth on glucose both organisms had the same cell yield . This can be attributed to two factors: --S . cerevisiae had a lower protein content than C . utilis; --uptake of glucose by C . utilis requires energy whereas in S . cerevisiae it occurs via facilitated diffusion . Theoretical calculations showed that, as a result of these two factors, the ATP requirement for biomass formation in C . utilis is 35% higher than in S . cerevisiae (theoretical YATP values of 20.8 and 28.1, respectively) . The experimental YATP for anaerobic growth of S . cerevisiae on glucose was 16 g biomass.mol ATP-1 . In vivo P/O-ratios can be calculated for aerobic growth on ethanol and acetate, provided that the gap between the theoretical and experimental ATP requirements as observed for growth on glucose is taken into account . This was done in two ways: --via the assumption that the gap is independent of the growth substrate (i.e . a fixed amount of ATP bridges the difference between the theoretical and experimental values) . --alternatively, on the assumption that the difference is a fraction of the total ATP expenditure, that is dependent on the substrate . Calculations of P/O-ratios for growth of both yeasts on glucose, ethanol, and acetate made clear that only by assuming a fixed difference between theoretical and experimental ATP requirements, the P/O-ratios are more or less independent of the growth substrate . These P/O-ratios are approximately 30% lower than the calculated mechanistic values.

Curr Genet, 1991 Jan, 19(1), 1 - 8
A gene tightly linked to CEN6 is important for growth of Saccharomyces cerevisiae; Carbone ML et al.; Transcriptional analysis of the region flanking the left boundary of the centromere of chromosome VI revealed the presence of a gene immediately adjacent to CEN6 . The transcription of the gene is directed toward the centromere, and nucleotide sequence analysis showed that the coding region terminates only 50 bp away from CEN6 . Our results extend to chromosome VI the observation that centromere-flanking regions of S . cerevisiae are transcriptionally active . Disruption of the coding region of the gene showed that its product, whilst not essential for cell viability, is important for normal cell growth . The gene has been termed DEG1 (DEpressed Growth rate) . Comparison of the deduced amino acid sequence of DEG1 with a protein sequence databank revealed homology with the enzyme tRNA pseudouridine synthase I of E . coli.

Yi Chuan Xue Bao, 1991, 18(1), 90 - 6
{Inhibitory effects of Chinese medicines on SOS responses in E . coli and their mechanism}; Wang LH et al.; Sixty kinds of commonly used Chinese medicines have been examined for their ability to depress the release of Lambda phage from lysogenic strain in the inductest . 11 Chinese medicines showed an inhibitory effects . Among them, Codonopsis radix, Polygonatum radix and fractus Lycium were strong depressors . They also showed an inhibitory effect on SOS response in SOS chromotest with a dose-effect response . These medicines were also found to decrease the frequency of gene conversion in S . cerevisiae in the presence of hydroxyurea . The effective compound (s) of Polygonatum radix partially purified from the extract with Sephadex G-25 chromatography was a reductive carbohydrate with molecular weight less than 3,000 . The compound was shown to exert an inhibitory effect on SOS response occurred at 42 degrees C in E . coli GW1060 (recA441), but has no effect on SOS network gene expression in E . coli GW 1107 (lexA51), suggesting that Polygonatum radix may contain an inhibitor of RecA protease.

Oncogene, 1991 Jan, 6(1), 11 - 9
Two c-myb proteins differing by their aminotermini exhibit different transcriptional transactivation activities (yeast/reporter-effector system); Punyammalee B et al.; We assayed in the yeast S . cerevisiae the transcriptional transactivation activity of the c-myb products encoded by a normal thymus cDNA and of an aminoterminally truncated version of it (minus 58 amino acids) corresponding to the cDNAs isolated from lymphoma and leukemia cells from different origins . Both proto-oncogene products were expressed under the control of the galactose inducible GAL10 promoter . The reporter system used to monitor the transactivation potential of the myb products consisted of a CYCl-lacZ gene fusion in which the UASCYC signals were replaced by one or multiple copies of the myb recognition element (mRE) . As shown by Northern blot analyses and by primer extension experiments both c-myb products increase the level of beta-galactosidase transcription . Interestingly, the c-myb product corresponding to lymphoma cDNAs stimulates transcription four to five times more efficiently than does the normal thymic c-myb product.

Mol Cell Biol, 1991 Jan, 11(1), 510 - 22
Peroxisomes in Saccharomyces cerevisiae: immunofluorescence analysis and import of catalase A into isolated peroxisomes; Thieringer R et al.; To isolate peroxisomes from Saccharomyces cerevisiae of a quality sufficient for in vitro import studies, we optimized the conditions for cell growth and for cell fractionation . Stability of the isolated peroxisomes was monitored by catalase latency and sedimentability of marker enzymes . It was improved by (i) using cells that were shifted to oleic acid medium after growth to stationary phase in glucose precultures, (ii) shifting the pH from 7.2 to 6.0 during cell fractionation, and (iii) carrying out equilibrium density centrifugation with Nycodenz containing 0.25 M sucrose throughout the gradient . A concentrated peroxisomal fraction was used for in vitro import of catalase A . After 2 h of incubation, 62% of the catalase was associated with, and 16% was imported into, the organelle in a protease-resistant fashion . We introduced immunofluorescence microscopy for S . cerevisiae peroxisomes, using antibodies against thiolase, which allowed us to identify even the extremely small organelles in glucose-grown cells . Peroxisomes from media containing oleic acid were larger in size, were greater in number, and had a more intense fluorescence signal . The peroxisomes were located, sometimes in clusters, in the cell periphery, often immediately adjacent to the plasma membrane . Systematic immunofluorescence observations of glucose-grown S . cerevisiae demonstrated that all such cells contained at least one and usually several very small peroxisomes despite the glucose repression . This finding fits a central prediction of our model of peroxisome biogenesis: peroxisomes form by division of preexisting peroxisomes; therefore, every cell must have at least one peroxisome if additional organelles are to be induced in that cell.

Mol Cell Biol, 1991 Jan, 11(1), 476 - 85
ACE2, an activator of yeast metallothionein expression which is homologous to SWI5; Butler G et al.; Transcription of the Saccharomyces cerevisiae metallothionein gene CUP1 is induced in response to high environmental levels of copper . Induction requires the ACE1 gene product, which binds to specific sites in the promoter region of the CUP1 gene . In this study, we found that deleting the entire coding sequence of the ACE1 gene resulted in a decrease in basal-level transcription of CUP1 to low but detectable levels and conferred a copper-sensitive phenotype to the cells . We have isolated a gene, designated ACE2, which when present on a high-copy-number plasmid suppresses the copper-sensitive phenotype of an ace1-deletion strain . The presence of multiple copies of the ACE2 gene enhanced expression of an unlinked CUP1-lacZ fusion integrated in the yeast genome and resulted in an increase in the steady-state levels of CUP1 mRNA in an ace1-deletion background . A large deletion of the coding region of the genomic copy of ACE2 resulted in a decrease in steady-state levels of CUP1 mRNA, indicating that ACE2 plays a role in regulating basal-level expression of CUP1 . The ACE2 open reading frame encodes a polypeptide of 770 amino acids, with putative zinc finger structures near the carboxyl terminus . This protein is 37% identical to the SWI5 gene product, an activator of HO gene transcription in S . cerevisiae, suggesting that ACE2 and SWI5 may have functional similarities.

Mol Cell Biol, 1991 Jan, 11(1), 329 - 37
Characterization of a short, cis-acting DNA sequence which conveys cell cycle stage-dependent transcription in Saccharomyces cerevisiae; McIntosh EM et al.; Comparison of the 5'-flanking regions of several cell cycle-regulated DNA replication genes of Saccharomyces cerevisiae has revealed the presence of a common sequence, 5'-ACGCGT-3', which is upstream and proximal to mapped transcription initiation sites . This sequence, which is the cleavage site for the restriction endonuclease MluI, is present twice in the upstream region of the yeast thymidylate synthase gene TMP1 . Previous studies have implicated these MluI sites as critical components in the cell cycle-dependent transcription of TMP1 . In this study, we examined more closely the importance of the ACGCGT sequences for the transcription of this gene . Using site-directed mutagenesis in combination with deletion analysis and subcloning experiments, we found that (i) while both of the TMP1 MluI sites contribute to the total transcription of this gene, the distal site is predominant and (ii) the 9-bp sequence ACGCGTTAA encompassing the distal MluI site exhibits properties of a cell cycle-stage dependent upstream activation sequence element . The results of this study support the notion that the ACGCGT sequence is an integral component of a transcription system which coordinates the cell cycle-dependent expression of DNA replication genes in S . cerevisiae.

Mol Cell Biol, 1991 Jan, 11(1), 218 - 25
The yeast rad18 mutator specifically increases G.C----T.A transversions without reducing correction of G-A or C-T mismatches to G.C pairs; Kunz BA et al.; Inactivation of the Saccharomyces cerevisiae RAD18 gene confers a mutator phenotype . To determine the specificity of this effect, a collection of 212 spontaneous SUP4-o mutants arising in a rad18 strain was characterized by DNA sequencing . Comparison of the resulting mutational spectrum with that for an isogenic wild-type (RAD18) strain revealed that the rad18 mutator specifically enhanced the frequency of single base pair substitutions . Further analysis indicated that an increase in the frequency of G.C----T.A transversions accounted for the elevated SUP4-o mutation frequency . Thus, rad18 is the first eucaryotic mutator found to generate only a particular base pair substitution . The majority of G.C pairs that were not mutated in the rad18 background were at sites where G.C----T.A events can be detected in SUP4-o, suggesting that DNA sequence context influences the rad18 mutator effect . Transformation of heteroduplex plasmid DNAs into the two strains demonstrated that the rad18 mutator did not reduce the efficiency of correcting G-A or C-T mismatches to G.C pairs or preferentially correct the mismatches to A.T pairs . We propose that the RAD18 gene product might contribute to the fidelity of DNA replication in S . cerevisiae by involvement in a process that serves to limit the formation of G-A and C-T mismatches at template guanine and cytosine sites during DNA synthesis.

Dev Genet, 1991, 12(4), 281 - 92
Cellular morphogenesis in the Saccharomyces cerevisiae cell cycle: localization of the CDC11 gene product and the timing of events at the budding site; Ford SK et al.; The Saccharomyces cerevisiae CDC3, CDC10, CDC11, and CDC12 genes encode a family of homologous proteins that are not closely related to other known proteins {Haarer BK, Ketcham SR, Ford SK, Ashcroft DJ, and Pringle JR (submitted)} . Temperature-sensitive mutants defective in any of these four genes display essentially identical pleiotropic phenotypes that include abnormal cell-wall deposition and bud growth, an inability to complete cytokinesis, and a failure to form the ring of 10 nm filaments that normally lies directly subjacent to the plasma membrane in the neck region of budding cells . We showed previously that the CDC3 and CDC12 gene products localize to the region of the mother-bud neck and are probably constituents of the ring of 10 nm filaments . We now report the generation of polyclonal antibodies specific for the CDC11 product (Cdc11p) and the use of these antibodies in immunofluorescence experiments with wild-type and mutant cells . The results suggest that Cdc11p is also a constituent of the filament ring, and thus support the hypothesis that the S . cerevisiae 10 nm filaments represent a novel type of eukaryotic cytoskeletal element . Cdc11p and actin both localize to the budding site well in advance of bud emergence and at approximately the same time, and both proteins also remain localized at the old budding site for some time after cytokinesis . Cdc11p also localizes to regions of cell-wall reorganization in mating cells and in cells responding to purified mating pheromone . Surprisingly, most preparations of affinity purified Cdc11p-specific antibodies also stained the nuclear and cytoplasmic microtubules . Although this staining probably reflects the existence of an epitope shared by Cdc11p and some microtubule-associated protein, the possibility that a fraction of the Cdc11p is associated with the microtubules could not be eliminated.

Electron Microsc Rev, 1991, 4(2), 377 - 400
Ultracytochemistry of the secretory pathway in Saccharomyces cerevisiae defies the established pathway model; Vorisek J; The molecular and cell biologic data supporting the established model of the intracellular secretory (transport) pathway for glycoproteins in the yeast Saccharomyces cerevisiae have been reviewed and confronted with our electron-cytochemical findings . These in situ findings show a new class of constitutive intracellular conveyors--the coated globules--and also suggest substantial alternatives in the cellular mechanism of the vacuole biogenesis . The controversial question of the Golgi compartment identity in S . cerevisiae is revived.

Crit Rev Biochem Mol Biol, 1991, 26(1), 53 - 76
The glucoamylase multigene family in Saccharomyces cerevisiae var . diastaticus: an overview; Pretorius IS et al.; Saccharomyces cerevisiae has been used widely both as a model system for unraveling the biochemical, genetic, and molecular details of gene expression and the secretion process, and as a host for the production of heterologous proteins of biotechnological interest . The potential of starch as a renewable biological resource has stimulated research into amylolytic enzymes and the broadening of the substrate range of S . cerevisiae . The enzymatic hydrolysis of starch, consisting of linear (amylose) and branched glucose polymers (amylopectin), is catalyzed by alpha- and beta-amylases, glucoamylases, and debranching enzymes, e.g., pullulanases . Starch utilization in the yeast S . cerevisiae var . diastaticus depends on the expression of the three unlinked genes, STA1 (chr . IV), STA2 (chr . II), and STA3 (chr . XIV), each encoding one of the extracellular glycosylated glucoamylases isozymes GAI, GAII, or GAIII, respectively . The restriction endonuclease maps of STA1, STA2, and STA3 are identical . These genes are absent in S . cerevisiae, but a related gene, SGA1, encoding an intracellular, sporulation-specific glucoamylase (SGA), is present . SGA1 is homologous to the middle and 3' regions of the STA genes, but lacks a 5' sequence that encodes the domain for secretion of the extracellular glucoamylases . The STA genes are positively regulated by the presence of three GAM genes . In addition to positive regulation, the STA genes are regulated negatively at three levels . Whereas strains of S . diastaticus are capable of expressing the STA genes, most strains of S . cerevisiae contain STA10, whose presence represses the expression of the STA genes in an undefined manner . The STA genes are also repressed in diploid cells, presumably by the MATa/MAT alpha-encoded repressor . STA gene expression is reduced in liquid synthetic media, it is carbon catabolite repressed by glucose, and is inhibited in petite mutants.

Cold Spring Harb Symp Quant Biol, 1991, 56, 577 - 84
cdc25 M-phase inducer; Millar J et al.; In this paper, we have described the critical experiments leading to the discovery and analysis of the cdc25 M-phase inducer . We have shown that timing of mitosis is sensitive to the level of cdc25+ expression and that the cellular concentration of p80cdc25 increases as cells approach mitosis . From these observations we conclude that, in S . pombe, rate of accumulation of p80cdc25 plays an important role in determining the timing of mitosis . We postulate that under a given set of conditions, a critical level of p80cdc25 activity is required to undergo mitosis . The actual level that is required can vary depending on ploidy, growth rate, nutritional status of the cell, and perhaps other parameters . These signals may be monitored through the weel pathway leading to tyrosyl phosphorylation of p34cdc2 . We have shown that p80cdc25 encodes a phosphate that acts by directly dephosphorylating the Tyr-15 residue of p34cdc2 . Our studies strongly indicate that this aspect of the mitotic control network is generally conserved among eukaryotes . It is conceivable, however, that the mode of regulation of cdc25 activity may vary from species to species . Clearly, in S . cerevisiae the cdc25+ homolog, MIH1, in contrast to cdc25+, is not rate-limiting for M-phase onset . It will be important to determine whether the level of cdc25+ homologs in other organisms also oscillates during the cell cycle, or whether their activity is controlled by localization or posttranslational mechanisms, such as phosphorylation . Furthermore, our finding of more than one cdc25+ homolog in a single species suggests an additional level of complexity to the control of M-phase onset by cdc25 in higher eukaryotes that will require further investigation.

Acta Histochem Suppl, 1991, 41, 193 - 200
Evidence that the osmotically fragile yeast S . cerevisiae VY1160 is an actin mutant; Schade B et al.; The phenotype of the osmotically dependent S . cerevisiae mutant VY1160 is caused by a single chromosomal mutation, termed srb, with pleiotropic effect . Compared with cells of the parental strain S288C, it was shown that the size and surface structure of the mutant cells are changed . The latter are sensitive to elevated cultivation temperatures as well as to hypotonic pressure and mechanical stress . In these cases, specific plasma membrane alteration were revealed by freeze-fracture electron microscopy . The total actin content is only 88% (21.4 micrograms actin/mg protein) of that of S288C cells . Remarkably, the mutant cells contain only 2.2 micrograms F-actin/mg protein, whereas the S288C cells have 10.3 micrograms F-actin/mg protein . Moreover, the level of reduced glutathione is found to be higher in the mutant cells (23.4 nmole/10(10) cells) than in the parental cells (15.2 nmole/10(10) cells) . These results implicate that the srb mutation is localized in the actin gene.

DNA Seq, 1991, 2(1), 19 - 32
The urea amidolyase (DUR1,2) gene of Saccharomyces cerevisiae; Genbauffe FS et al.; The DNA sequence of the urea amidolyase (DUR1,2) gene from S . cerevisiae has been determined . The polypeptide structure deduced from the DNA sequence contains 1,835 amino acid residues and possesses a calculated weight of 201,665 daltons which favorably correlates with that predicted from compositional analysis of purified protein (1,881 amino acid residues and a molecular weight of 203,900) . The C-terminal 57 residues of the polypeptide exhibit significant homology with similarly situated sequences found in five other biotin carboxylases whose primary structures have been determined or deduced from protein and DNA sequence data, respectively . Major S1 nuclease protection fragments derived from DUR1,2 RNA-DNA hybrids exhibit apparent termini at positions -140 and -141 upstream of the coding region . The termini of minor protection fragments also occur at eleven other positions as well.

Nahrung, 1991, 35(6), 641 - 5
Factors affecting the production of Saccharomyces cerevisiae from sugar beet pulp; el-Makhzangy A et al.; Factors affecting the production of S . cerevisiae from sugar beet pulp after acid hydrolysis were investigated . Maximum yield and economic coefficient were obtained at sugar concentration of 7.15% after an incubation period of 72 h at 30 degrees C and pH 6.0 using a mixture (1:1) of ammonium sulphate and ammonium nitrate (0.2 g/g sugar) as nitrogen source . HPLC analysis of beet pulp hydrolyzate showed that hexoses especially glucose serve as the most suitable sugars with regard to S . cerevisiae activity.

Biomed Sci, 1991, 2(3), 298 - 301
Region of hepatitis B virus DNA with a homology to the yeast ARS replication enhancer; Legchilina SP et al.; A computer analysis of the primary sequence of hepatitis B virus (HBV) subtype ayw DNA, cloned within the pVG2 recombinant plasmid, which raises its stability in Saccharomyces cerevisiae transformants, was performed . This revealed that the structure of the HBV DNA has: two bends in the termination regions of the HBs and HBc genes, and multiple sequences with a high degree of homology to the ARS (autonomously replicating sequence) core consensus in this region of the HBs gene . DNA fragments from the HBs region (330 bp) and from the HBc region (378 bp) have an abnormal electrophoretic mobility in 8% polyacrylamide gels . The similarity of the structural motifs in the stop-region of HBs gene with the B-domain of the S . cerevisiae ARS element is discussed.

Curr Genet, 1991 Jan, 19(1), 15 - 9
A cyclin protein modulates mitosis in the budding yeast Saccharomyces cerevisiae; Veinot-Drebot LM et al.; For the budding yeast Saccharomyces cerevisiae the mitotic cell cycle is coordinated with cell mass at the regulatory step "start" . The threshold amount of cell mass (reflected as a "critical size") necessary for "start" is proportional to nutrient quality . This relationship leads to a transient accumulation of cells at "start", termed nutrient modulation, upon enrichment of nutrient conditions . Nutrient enrichment abruptly increases the critical size needed for "start", causing the smaller cells, produced in the previous cell cycle, to be delayed at "start" while growing larger . Here we show that, in S . cerevisiae, a second cell-cycle step, at mitosis, also exhibits nutrient modulation, and is, therefore, another point of cell-cycle regulation . At both mitosis and "start", nutrient modulation was found through mutation to be regulated by the activity of the cyclin-related WHI1 (CLN3) gene product.

Cell Mol Biol, 1991, 37(7), 739 - 44
Inhibition of topoisomerase I by NAD and enhancement of cytotoxicity of MMS by inhibitors of poly(ADP-ribose) polymerase in Saccharomyces cerevisiae; Park JK et al.; The activity of DNA topoisomerase I present in the nuclear extract of yeast, Saccharomyces cerevisiae, was inhibited by additions of NAD, the substrate of poly (ADP-ribose) polymerase . This NAD-inhibited topoisomerase activity was restored to the normal level in a dose-dependent manner by adding 3-aminobenzamide (3-AB), an inhibitor of the polymerase . The 3-AB sensitive polymerase enzyme activity, as determined by the rate of incorporation of the radiolabelled NAD in permeabilized cells, increased by treatment of cells with methyl methanesulfonate (MMS) in a dose-dependent manner . While the additions of MMS increased the polymerase activity, it has caused a decrease in cell survival . However, this cell killing activity of MMS was markedly potentiated by adding benzamide, another inhibitor of polymerase . Thus, these results suggest that the mode of modification of nuclear proteins by altering the poly(ADP-ribosylation) in S . cerevisiae resembles with those observed in mammalian cells.

Agric Biol Chem, 1991 Jan, 55(1), 59 - 65
Isolation and sequencing of a gene, C-ADE1, and its use for a host-vector system in Candida maltosa with two genetic markers; Kawai S et al.; The host-vector systems of an n-alkane-assimilating-yeast, Candida maltosa, that we previously constructed consisted of a vector replicating with an ARS region of this yeast, and C . maltosa strains J288 (leu2) or CH1 (his5) as hosts . Since each of these hosts has a single genetic marker, we have developed a new host-vector system using two genetic markers . By UV irradiation of strain CH1, an adenine auxotrophic mutant, CHA1, forming red colonies was isolated . A DNA fragment complementing this deficiency was isolated from the C . maltosa genome . Since the DNA fragment also complemented the ade1 mutation of S . cerevisiae, we termed a gene contained in this DNA fragment C-ADE1 . The nucleotides of C-ADE1 were sequenced . The deduced amino acid sequence (291 residues) had 65.6% homology with that of ADE1 of S . cerevisiae (306 residues) . Having the cloned C-ADE1 DNA, we improved the host-vector system of C . maltosa.

Biotechnology (N Y), 1991 Jan, 9(1), 57 - 61
Synthesis of wild type and mutant human hemoglobins in Saccharomyces cerevisiae; Wagenbach M et al.; We have expressed human alpha and beta-globin cDNA clones from separate, synthetic galactose-regulated hybrid promoters contained on a single plasmid in Saccharomyces cerevisiae . Co-expression of the alpha and beta-globin chains in S . cerevisiae results in the assembly of these proteins into soluble tetrameric hemoglobin that accumulates to 3-5 percent of the total cell protein . Endogenously produced heme is incorporated into the tetramer and the protein produced is functionally and structurally indistinguishable from human Ao hemoglobin . This expression system has been used to produce both wild type hemoglobin and a low O2-affinity hemoglobin mutant that has oxygen binding and dissociation characteristics similar to human whole blood . The yeast expression system we describe may be suitable for the production of a recombinant hemoglobin based blood substitute as well as for detailed structure-activity studies of human hemoglobin.

Cell, 1990 Dec 21, 63(6), 1277 - 86
The viral erbA oncogene protein, a constitutive repressor in animal cells, is a hormone-regulated activator in yeast; Privalsky ML et al.; The v-erbA oncogene is a retrovirus-transduced and altered copy of a cellular gene for a thyroid hormone receptor . In animal cells, the v-erbA protein fails to respond to hormone and acts as a dominant negative allele, inhibiting gene activation normally conferred by the wild-type thyroid hormone receptor . We report here that, unexpectedly, the v-erbA protein acts as a hormone-regulated transcriptional activator in S . cerevisiae . We suggest that the ability of v-erbA protein to function as a transcriptional repressor or an activator is determined by interaction with, or modification by, other cellular factors, and that this phenomenon may be relevant to understanding ligand regulation of the normal thyroid and steroid hormone receptors.

Gene, 1990 Dec 15, 96(2), 197 - 203
Characterisation of a repetitive DNA family from Entamoeba histolytica containing Saccharomyces cerevisiae ARS consensus sequences; Lohia A et al.; Several repetitive DNA families were identified in Entamoeba histolytica DNA digested with Sau3AI . Characterisation of one of these repetitive DNA families showed the presence of multiple copies of Saccharomyces cerevisiae autonomously replicating sequence (ARS) core consensus sequences . The E . histolytica ARS consensus sequences allowed a yeast-integrating plasmid, YIP5, to replicate autonomously in S . cerevisiae . A 'bent DNA' fragment was located in one member of this E . histolytica repetitive DNA family.

Gene, 1990 Dec 15, 96(2), 171 - 6
Cloning, sequencing and chromosomal assignment of a gene from Saccharomyces cerevisiae which is negatively regulated by glucose and positively by lipids; Stone RL et al.; We report the molecular cloning, nucleotide (nt) sequence and chromosomal assignment of the Saccharomyces cerevisiae gene GLP1 . This gene encoded a 15-kDa protein that was synthesized at a low level during growth on glucose and was induced ninefold upon glucose deprivation . When glucose withdrawal was accompanied by the addition of fatty acids the induction was enhanced an additional two- to threefold . The GLP1 gene product was identified as a soluble protein and purified using a combination of gel permeation and ion exchange chromatography . Using oligodeoxyribonucleotides as hybridization probes we have isolated the GLP1 gene and sequenced the single, long open reading frame which is 351 nt in length and is not interrupted by introns . The GLP1 gene directed the transcription of a 700-nt mRNA in response to glucose deprivation . The accumulation of the mRNA was further enhanced twofold by the addition of oleate . We have localized the GLP1 gene to S . cerevisiae chromosome VI.

J Biol Chem, 1990 Dec 5, 265(34), 20879 - 86
Isolation of the GSY1 gene encoding yeast glycogen synthase and evidence for the existence of a second gene; Farkas I et al.; Glycogen synthase preparations from Saccharomyces cerevisiae contained two polypeptides of molecular weights 85,000 and 77,000 . Oligonucleotides based on protein sequence were utilized to clone a S . cerevisiae glycogen synthase gene, GSY1 . The gene would encode a protein of 707 residues, molecular mass 80,501 daltons, with 50% overall identity to mammalian muscle glycogen synthases . The amino-terminal sequence obtained from the 85,000-dalton species matched the NH2 terminus predicted by the GSY1 sequence . Disruption of the GSY1 gene resulted in a viable haploid with glycogen synthase activity, and purification of glycogen synthase from this mutant strain resulted in an enzyme that contained the 77,000-dalton polypeptide . Southern hybridization of genomic DNA using the GSY1 coding sequence as a probe revealed a second weakly hybridizing fragment, present also in the strain with the GSY1 gene disrupted . However, the sequences of several tryptic peptides derived from the 77,000-dalton polypeptide were identical or similar to the sequence predicted by the GSY1 gene . The data are explained if S . cerevisiae has two glycogen synthase genes encoding proteins with significant sequence similarity The protein sequence predicted by the GSY1 gene lacks the extreme NH2-terminal phosphorylation sites of the mammalian enzymes . The COOH-terminal phosphorylated region of the mammalian enzyme over-all displayed low identity to the yeast COOH terminus, but there was homology in the region of the mammalian phosphorylation sites 3 and 4 . Three potential cyclic AMP-dependent protein kinase sites are located in this region of the yeast enzyme . The region of glycogen synthase likely to be involved in covalent regulation are thus more variable than the catalytic center of the molecule.

J Bacteriol, 1990 Dec, 172(12), 7275 - 7
Lysis protein S of phage lambda functions in Saccharomyces cerevisiae; Garrett J et al.; The lambda S lysis gene was cloned into a Saccharomyces cerevisiae expression vector under GAL1 control . Induction with galactose in S . cerevisiae terminated cell growth and prevented colony formation . Several membrane proteins immunoreactive with anti-S antibody accumulated in the membranes, indicating that sodium dodecyl sulfate-resistant oligomers of S are formed, similar to those observed in the membranes of Escherichia coli cells killed by expression of the S gene . These observations suggest that the S gene product functions as a cytotoxic protein in the yeast cytoplasmic membrane as it does in the bacterial membrane.

Proc Natl Acad Sci U S A, 1990 Dec, 87(24), 9853 - 7
Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42; Shinjo K et al.; We have isolated cDNA clones from a human placental library that code for a low molecular weight GTP-binding protein originally designated Gp (also called G25K) . This identification is based on comparisons with the available peptide sequences for the purified human Gp protein and the use of two highly specific anti-peptide antibodies . The predicted amino acid sequence of the protein is very similar to those of various members of the ras superfamily of low molecular weight GTP-binding proteins, including the N-, Ki-, and Ha-ras proteins (30-35% identical), the rho proteins (approximately 50% identical), and the rac proteins (approximately 70% identical) . The highest degree of sequence identity (80%) is found with the Saccharomyces cerevisiae cell-division-cycle protein CDC42 . The human placental gene, which we designate CDC42Hs, complements the cdc42-1 mutation in S . cerevisiae, which suggests that this GTP-binding protein is the human homolog of the yeast protein.

Mol Cell Biol, 1990 Dec, 10(12), 6690 - 9
ADP ribosylation factor is an essential protein in Saccharomyces cerevisiae and is encoded by two genes; Stearns T et al.; ADP ribosylation factor (ARF) is a ubiquitous 21-kDa GTP-binding protein in eucaryotes . ARF was first identified in animal cells as the protein factor required for the efficient ADP-ribosylation of the mammalian G protein Gs by cholera toxin in vitro . A gene (ARF1) encoding a protein homologous to mammalian ARF was recently cloned from Saccharomyces cerevisiae (Sewell and Kahn, Proc . Natl . Acad . Sci . USA, 85:4620-4624, 1988) . We have found a second gene encoding ARF in S . cerevisiae, ARF2 . The two ARF genes are within 28 centimorgans of each other on chromosome IV, and the proteins encoded by them are 96% identical . Disruption of ARF1 causes slow growth, cold sensitivity, and sensitivity to normally sublethal concentrations of fluoride ion in the medium . Disruption of ARF2 causes no detectable phenotype . Disruption of both genes is lethal; thus, ARF is essential for mitotic growth . The ARF1 and ARF2 proteins are functionally homologous, and the phenotypic differences between mutations in the two genes can be accounted for by the level of expression; ARF1 produces approximately 90% of total ARF . Among revertants of the fluoride sensitivity of an arf1 null mutation were ARF1-ARF2 fusion genes created by a gene conversion event in which the deleted ARF1 sequences were repaired by recombination with ARF2.

Genetika, 1990 Dec, 26(12), 2135 - 46
{Chromosome polymorphism in the yeast Saccharomyces}; Davydenko SG et al.; The variability of chromosomal band patterns was determined by pulse electrophoresis . The natural strains differed by the quantity and electrophoretic mobility of chromosomal DNA bands . The strains of independent genetic stocks originated from the XII race of Saccharomyces cerevisiae showed less significant difference in band patterns than the strains of different species of the Saccharomyces genus . The progeny of among strains with different karyotypes hybrid showed non-regular segregation of parental bands, the occurrence of new bands and the bands with altered mobility . Reverse crosses of hybrid progeny with strains of Peterhoff genetic stocks of S . cerevisiae led to decrease in chromosomal polymorphism . Homozygotization for ski5 allele and selection for increasing the copy number of killer plasmids was accompanied with repeated splash of polymorphism in 1-2 generations of intratetrad and intrafamily crossed hybrid progeny . Subsequent stabilization of electrophoretic karyotype took place, excluding the mendelian dimorphism of chromosome III, with was a stable trait of the last 6 generations of that progeny.

Curr Genet, 1990 Dec, 18(5), 413 - 9
Two substitutions at the same position in the mitochondrial cytochrome b gene of S . cerevisiae induce a mitochondrial myxothiazol resistance and impair the respiratory growth of the mutated strains abbeit maintaining a good electron transfer activity; Tron T et al.; Two cytochrome b respiratory-deficient mutants were sequenced and their DNA base change identified, leading to the replacement of glycine (G137 by valine or glutamic acid . No variation in their cytochrome b content with regard to cytochrome oxidase and cytochrome (c + c1) was found to have occurred . Their cellular respiratory activity with various substrates was partly conserved and was totally inhibited by antimycin A . Their ubiquinol (QH2)-cytochrome c reductase/mole cytochrome b activity decreased by about 50% . Paradoxically their growth on respiratory substrate was abolished . Both mutants retained a high-affinity binding site for antimycin A, and exhibited a myxothiazol-resistance at the mitochondrial level . It seems likely that the mutated position (137), which belongs to the ubiquinol oxidizing domain of the bc1 complex, interferes, directly or indirectly, with the respiratory growth capacity of the cell.

Curr Genet, 1990 Dec, 18(5), 395 - 400
Molecular cloning of the DAC2/FUS3 gene essential for pheromone-induced G1-arrest of the cell cycle in Saccharomyces cerevisiae; Fujimura H; Mating pheromones, known as a and alpha-factors, arrest the division of cells of opposite mating types, alpha and a respectively, in Saccharomyces cerevisiae . I have cloned the DAC2 gene, which is required for both pheromone-induced division-arrest and cell-fusion during conjugation . The constructed dac2::LEU2 null mutation leads to defects in both pheromone-induced division-arrest and cell-fusion during conjugation; it also suppresses the growth defect caused by the gpa1 mutation (a mutation in the alpha subunit of the S . cerevisiae G protein) . These results indicate that DAC2 may be the same gene as FUS3, which was recently isolated by Elion et al . (1990) as a gene essential for cell-fusion during conjugation . The dac2::LEU2 null mutant also showed morphological alterations in response to mating pheromones . I show here that the DAC2 product plays an essential role in both the division-arrest signalling pathway of the yeast pheromone response and in cell-fusion during conjugation.

Curr Genet, 1990 Dec, 18(6), 485 - 91
Ty virus-like particles in the Saccharomyces cerevisiae strain NCYC74; Capsey LJ et al.; Electron microscopic analysis of thin sections of Saccharomyces cerevisiae NCYC74 has revealed the presence of many clumped cytoplasmic particles that morphologically resemble Ty element virus-like particles (VLPs) . Accumulation of Ty VLPs has only previously been observed in S . cerevisiae strains that over-express a cloned Ty element . The particles in NCYC74 co-purify with Ty RNA, Ty-specific antigens and a reverse transcriptase activity . Furthermore, they appear to be recognized by antibodies to Ty VLPs during indirect immunofluorescence experiments . These observations provide compelling evidence that the cytoplasmic particle in NCYC74 are indeed Ty VLPs.

Cell, 1990 Nov 30, 63(5), 1039 - 51
Courtship in S . cerevisiae: both cell types choose mating partners by responding to the strongest pheromone signal; Jackson CL et al.; We demonstrate that during the courtship stage of conjugation, S . cerevisiae a cells choose the alpha cell producing the highest level of pheromone from among potential mating partners . From this result and that for alpha cells we conclude that both a and alpha cells act as signaling cells during courtship, that both cell types respond by discriminating different levels of signal, and that the signals are the mating pheromones . Responding cells that are supersensitive to signal fail to discriminate pheromone-producing from nonproducing cells to an extent that depends on their degree of supersensitivity . We propose that partner selection in S . cerevisiae results from polarized morphogenesis of a responding cell in the direction of highest pheromone concentration and that cells defective in discriminating this gradient execute a default pathway in which an adjacent cell is selected at random.

Cell, 1990 Nov 30, 63(5), 999 - 1011
Identification of a gene necessary for cell cycle arrest by a negative growth factor of yeast: FAR1 is an inhibitor of a G1 cyclin, CLN2; Chang F et al.; alpha factor is a negative growth factor and differentiation factor that induces G1 arrest and increases transcription of mating genes in S . cerevisiae a cells . We have identified a gene, FAR1 (for "factor arrest"), which is necessary for cell cycle arrest but not for other responses to alpha factor: far1- mutants are insensitive to arrest despite having an intact signal transduction pathway . FAR1 is a nonessential gene whose expression is induced 4- to 5-fold in a cells by alpha factor . The sequence of FAR1 indicates no significant similarities to known proteins . A null mutation in the CLN2 gene, which codes for a G1 cyclin, reverses the effect of a far1 null mutation: far1- cln2- strains arrest in response to alpha factor . We thus propose that FAR1 contributes to cell cycle arrest by inhibiting CLN2 . The behavior of far1- cln2- strains indicates that products other than FAR1 are responsible for inhibiting the other G1 cyclins, CLN1 and CLN3.

J Biol Chem, 1990 Nov 25, 265(33), 20098 - 105
Sorting of peroxisomal membrane protein PMP47 from Candida boidinii into peroxisomal membranes of Saccharomyces cerevisiae; McCammon MT et al.; A gene encoding PMP47, a peroxisomal integral membrane protein of the methylotrophic yeast Candida boidinii, was isolated from a genomic library . DNA sequencing of PMP47 revealed an open reading frame of 1269 base pairs capable of encoding a protein of 46,873 Da . At least two membrane-spanning regions in the protein are predicted from the sequence . Since the 3 amino acids at the carboxyl terminus are -AKE, PMP47 lacks a typical peroxisomal sorting signal . No significant similarities in primary structure between PMP47 and known proteins were observed, including PMP70, a rat peroxisomal membrane protein whose sequence has recently been reported (Kamijo, K., Taketani, S., Yokota, S., Osumi, T., and Hashimoto, T . (1990) . J . Biol . Chem . 265, 4534-4540) . In order to study the import and assembly of PMP47 into peroxisomes by genetic approaches, the gene was expressed in the yeast Saccharomyces cerevisiae . When PMP47 was expressed in cells grown on oleic acid to induce peroxisomes, the protein was observed exclusively in peroxisomes as determined by marker enzyme analysis of organelle fractions . Most of the PMP47 co-purified with the endogenous peroxisomal membrane proteins on isopycnic sucrose gradients . Either in the native host or when expressed in S . cerevisiae, PMP47 was not extractable from peroxisomal membranes by sodium carbonate at pH 11, indicating an integral membrane association . These results indicate that PMP47 is competent for sorting to and assembling into peroxisomal membranes in S . cerevisiae.

Cell, 1990 Nov 16, 63(4), 751 - 62
Position effect at S . cerevisiae telomeres: reversible repression of Pol II transcription; Gottschling DE et al.; S . cerevisiae chromosomes end with the telomeric repeat (TG1-3)n . When any of four Pol II genes was placed immediately adjacent to the telomeric repeats, expression of the gene was reversibly repressed as demonstrated by phenotype and mRNA analyses . For example, cells bearing a telomere-linked copy of ADE2 produced predominantly red colonies (a phenotype characteristic of ade2- cells) containing white sectors (characteristic of ADE2+ cells) . Repression was due to proximity to the telomere itself since an 81 bp tract of (TG1-3)n positioned downstream of URA3 when URA3 was approximately 20 kb from the end of chromosome VII did not alter expression of the gene . However, this internal tract of (TG1-3)n could spontaneously become telomeric, in which case expression of the URA3 gene was repressed . These data demonstrate that yeast telomeres exert a position effect on the transcription of nearby genes, an effect that is under epigenetic control.

Cell, 1990 Nov 16, 63(4), 739 - 50
RAP1 protein interacts with yeast telomeres in vivo: overproduction alters telomere structure and decreases chromosome stability; Conrad MN et al.; The protein encoded by the RAP1 gene of S . cerevisiae binds in vitro to a consensus sequence occurring at a number of sites in the yeast genome, including the repeated sequence C2-3A(CA)1-6 found at yeast telomeres . We present two lines of evidence for the in vivo binding of RAP1 protein at telomeres: first, RAP1 is present in telomeric chromatin and second, alterations in the level of RAP1 protein affect telomere length . The length changes seen with under- and overexpression of RAP1 are consistent with the interpretation that RAP1 binding to telomeres protects them from degradation . Unexpectedly, overproduction of the RAP1 protein was also shown to decrease greatly chromosome stability, suggesting that RAP1 mediates interactions that have a more global effect on chromosome behavior than simply protecting telomeres from degradation . Such interactions may involve telomere associations both with other telomeres and/or with structural elements of the nucleus.

Cell, 1990 Nov 16, 63(4), 851 - 9
The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins; Ballester R et al.; The von Recklinghausen neurofibromatosis locus, NF1, encodes a protein with homology restricted to the catalytic region of the RAS GTPase-activating protein, GAP, and with extensive homology to the IRA1 and IRA2 gene products of the yeast S . cerevisiae . A segment of the NF1 cDNA gene, expressed in yeast, can complement loss of IRA function and can inhibit both wild-type and mutant activated human H-ras genes that are coexpressed in yeast . Yeast expressing the NF1 segment have increased H-ras GTPase-stimulating activity . These studies indicate that the NF1 gene product can interact with RAS proteins and demonstrate structural and functional similarities and differences among the GAP, IRA1, IRA2, and NF1 proteins.

Cell, 1990 Nov 16, 63(4), 843 - 9
The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21; Martin GA et al.; The neurofibromatosis type 1 (NF1) protein contains a region of significant sequence similarity to ras p21 GTPase-activating protein (GAP) and the yeast IRA1 gene product . A fragment of NF1 cDNA encoding the GAP-related domain (NF1 GRD) was expressed, immunoaffinity purified, and assayed for effects on N-ras p21 GTPase activity . The GTPase of wild-type ras p21 was stimulated by NF1 GRD, but oncogenic mutants of ras p21 (Asp-12 and Val-12) were unaffected, and the GTPase of an effector mutant (Ala-38) was only weakly stimulated . NF1 GRD also down-regulated RAS function in S . cerevisiae . The affinity of NF1 GRD for ras p21 was estimated to be 250 nM: this is more than 20-fold higher than the affinity of GAP for ras p21 . However, its specific activity was about 30 times lower . These kinetic measurements suggest that NF1 may be a significant regulator of ras p21 activity, particularly at low ras p21 concentrations.

Cell, 1990 Nov 16, 63(4), 835 - 41
The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S . cerevisiae; Xu GF et al.; Sequencing of the neurofibromatosis gene (NF1) revealed a striking similarity among NF1, yeast IRA proteins, and mammalian GAP (GTPase-activating protein) . Using both genetic and biochemical assays, we demonstrate that this homology domain of the NF1 protein interacts with ras proteins . First, expression of this NF1 domain suppressed the heat shock-sensitive phenotype of yeast ira1 and ira2 mutants . Second, this NF1 domain, after purification as a glutathione S-transferase (GST) fusion protein, strongly stimulated the GTPase activity of yeast RAS2 and human H-ras proteins . The GST-NF1 protein, however, did not stimulate the GTPase activity of oncogenic mutant ras proteins, H-rasVal-12 and yeast RAS2Val-19 mutants, or a yeast RAS2 effector mutant . These results establish that this NF1 domain has ras GAP activity similar to that found with IRA2 protein and mammalian GAP, and therefore may also regulate ras function in vivo.

J Biol Chem, 1990 Nov 15, 265(32), 19736 - 41
Mitochondrial phosphate transport . N-ethylmaleimide insensitivity correlates with absence of beef heart-like Cys42 from the Saccharomyces cerevisiae phosphate transport protein; Guerin B et al.; The mitochondrial phosphate transport protein (PTP) has been purified in a reconstitutively active form from Saccharomyces cerevisiae and Candida parapsilosis . ADP/ATP carriers that copurify have been identified . The PTP from S . cerevisiae migrates as a single band (35 kDa) in sodium dodecyl sulfate gels with the same mobility as the N-ethylmaleimide-alkylated beef heart PTP . It does not cross-react with anti-sera against beef heart PTP . The CNBr peptide maps of the yeast and beef proteins are very different . The rate of unidirectional phosphate uptake into reconstituted proteoliposomes is stimulated about 2.5-fold to a Vmax of 170 mumol of phosphate min-1 (mg PTP)-1 (22 degrees C) by increasing the pHi of the proteoliposomes from 6.8 (same as pHe) to 8.0 . The Km for Pi of this reconstituted activity is 2.2 mM . The transport is sensitive to mersalyl (50% inhibition at 60 microM) and insensitive to N-ethylmaleimide . We have purified peptides matching the highly conserved motif Pro-X-(Asp/glu)-X-X-(Lys/Arg)-X-(Arg/lys) (X is an unspecified amino acid) of the triplicate gene structure sequence of the beef heart PTP . The N-ethylmaleimide-reactive Cys42 of the beef heart protein, located between the two basic amino acids of this motif (Lys41-Cys42-Arg43), is replaced with a Thr in the yeast protein . This substitution most likely is responsible for the lack of N-ethylmaleimide sensitivity of the yeast protein and mersalyl thus reacts with another cysteine to inhibit the transport . Finally it is concluded that Cys42 has no essential role in the catalysis of inorganic phosphate transport by the mitochondrial phosphate transport protein.

J Biol Chem, 1990 Nov 5, 265(31), 18912 - 21
Structure and regulation of the SSA4 HSP70 gene of Saccharomyces cerevisiae; Boorstein WR et al.; SSA4 is the only one of five heat-inducible HSP70 genes in Saccharomyces cerevisiae whose expression is restricted to conditions of stress . Comparison of the nucleotide sequences of the SSA4 gene with other HSP70 genes indicates that it diverged from its most closely related yeast homologues hundreds of millions of years ago . However, a high degree of identity has been maintained between Ssa4p and other yeast 70-kDa heat-shock proteins at the amino acid level suggesting, in light of its distinct pattern of regulation, that it performs an important function . A 44-base pair region of the SSA4 promoter containing an extended match to the conserved eukaryotic heat-shock element (HSE) is necessary and sufficient to mediate heat-inducible regulation . HSESSA4 is capable of promoting only a low level of transcription under nonstress conditions . We present evidence in support of a revised definition of the functional HSE in S . cerevisiae, similar to the recently proposed modular Drosophila HSE . Elevated expression of several heat-shock proteins in an ssa1ssa2 double-mutant strain has previously been reported . The SSA4 promoter is activated in this strain . The increase in expression of SSA4 caused by deletion of these closely related genes is mediated via the same upstream activating sequences that activate transcription in response to heat shock . Activation of HSE-mediated transcription by disruption of constitutively expressed HSP70 genes supports an autoregulatory model of control of the heat-shock response.

Curr Genet, 1990 Nov, 18(4), 385 - 6
Electrophoretic karyotyping without the need for generating protoplasts; McCluskey K et al.; Chromosome samples for pulsed-field electrophoresis have been prepared without first generating protoplasts . The technique involves treatment of intact, agarose-solidified cell material with protease in the presence of EDTA and SDS . Saccharomyces cerevisiae, Ustilago hordei, Tilletia caries, and T . controversa karyotypes are clearly resolved with this technique . Colonies of U . hordei and S . cerevisiae removed from the surface of agar-solidified media and prepared for PFGE by this abbreviated method also yield well resolved karyotypes.

Genetics, 1990 Nov, 126(3), 549 - 62
The translational activator GCN3 functions downstream from GCN1 and GCN2 in the regulatory pathway that couples GCN4 expression to amino acid availability in Saccharomyces cerevisiae; Hannig EM et al.; The GCN4 protein of S . cerevisiae is a transcriptional activator of amino acid biosynthetic genes which are subject to general amino acid control . GCN3, a positive regulator required for increased GCN4 expression in amino acid-starved cells, is thought to function by antagonism of one or more negative regulators encoded by GCD genes . We isolated gcn3c alleles that lead to constitutively derepressed expression of GCN4 and amino acid biosynthetic genes under its control . These mutations map in the protein-coding sequences and, with only one exception, do not increase the steady-state level of GCN3 protein . All of the gcn3c alleles lead to derepression of genes under the general control in the absence of GCN1 and GCN2, two other positive regulators of GCN4 expression . This finding suggests that GCN3 functions downstream from GCN1 and GCN2 in the general control pathway . In accord with this idea, constitutively derepressing alleles of GCN2 are greatly dependent on GCN3 for their derepressed phenotype . The gcn3c alleles that are least dependent on GCN1 and GCN2 for derepression cause slow-growth under nonstarvation conditions . In addition, all of the gcn3c alleles are less effective than wild-type GCN3 in overcoming the temperature-sensitive lethality associated with certain mutations in the negative regulator GCD2 . These results suggest that activation of GCN3 positive regulatory function by the gcn3c mutations involves constitutive antagonism of GCD2 function, leading to reduced growth rates and derepression of GCN4 expression in the absence of amino acid starvation.

Mol Cell Biol, 1990 Nov, 10(11), 5903 - 13
The HXT2 gene of Saccharomyces cerevisiae is required for high-affinity glucose transport; Kruckeberg AL et al.; The HXT2 gene of the yeast Saccharomyces cerevisiae was identified on the basis of its ability to complement the defect in glucose transport of a snf3 mutant when present on the multicopy plasmid pSC2 . Analysis of the DNA sequence of HXT2 revealed an open reading frame of 541 codons, capable of encoding a protein of Mr 59,840 . The predicted protein displayed high sequence and structural homology to a large family of procaryotic and eucaryotic sugar transporters . These proteins have 12 highly hydrophobic regions that could form transmembrane domains; the spacing of these putative transmembrane domains is also highly conserved . Several amino acid motifs characteristic of this sugar transporter family are also present in the HXT2 protein . An hxt2 null mutant strain lacked a significant component of high-affinity glucose transport when under derepressing (low-glucose) conditions . However, the hxt2 null mutation did not incur a major growth defect on glucose-containing media . Genetic and biochemical analyses suggest that wild-type levels of high-affinity glucose transport require the products of both the HXT2 and SNF3 genes; these genes are not linked . Low-stringency Southern blot analysis revealed a number of other sequences that cross-hybridize with HXT2, suggesting that S . cerevisiae possesses a large family of sugar transporter genes.

Mol Cell Biol, 1990 Nov, 10(11), 5977 - 82
Molecular cloning and expression of a G25K cDNA, the human homolog of the yeast cell cycle gene CDC42; Munemitsu S et al.; G25K is a low-molecular-mass GTP-binding protein with a broad distribution in mammalian tissues . A cDNA clone was isolated by using oligonucleotides corresponding to the partial amino acid sequence of purified human G25K . The cDNA encodes an 191-amino-acid polypeptide containing GTP-binding consensus sequences and a putative farnesylation site at the C terminus . The sequence exhibits 50 and 70% identities to the mammalian rho and rac proteins, respectively, and an 80% identity to the Saccharomyces cerevisiae CDC42 gene product . Insect Sf9 cells infected with recombinant baculovirus vectors expressing the G25K cDNA produced a 25-kDa protein that bound GTP and was recognized by antibodies specifically reactive to G25K . G25K appears to be the human homolog of the CDC42 gene product, since expression of the G25K cDNA in S . cerevisiae suppressed both cdc42-1 and cdc24-4 temperature-sensitive lethal mutations.

Mol Biol (Mosk), 1990 Nov-Dec, 24(6), 1624 - 30
{Factor-free translation of poly(dT) by 80S ribosomes from Saccharomyces cerevisiae}; Ovcharenko GV et al.; The conditions for preparation of 80S ribosomes from S . cerevisiae are suggested . The ribosomes can bind Phe-tRNAPhe in poly(U)-or poly(dT)-directed manner and are shown to be able to translate poly(dT) in the absence of elongation factor and GTP . Effects of different antibiotics on the factor-free translation have been studied.

Gene, 1990 Oct 30, 95(1), 73 - 7
Single-strand-binding factor(s) which interact with ARS1 of Saccharomyces cerevisiae; Kuno K et al.; Since plasmids containing autonomously replicating sequence(s) (ARS) can transform Saccharomyces cerevisiae cells at high frequency, ARS are considered to be the replication origins of chromosomes . To study the mechanism of initiation of eukaryotic chromosomal replication, we examined protein factors which interact with the ARS1 region located near the centromere of chromosome IV in S . cerevisiae . Using the gel-shift assay, we found protein factors which bound to a single-stranded, 97-bp fragment of the ARS1 region containing the core consensus . Competition experiments with various oligodeoxyribonucleotides (oligos) suggest that a site recognized by the factor(s) was within the element containing the core consensus and adjacent close matches to the core consensus of the minus strand . Indeed, when the oligo containing the minus strand of this element was used as a probe, two oligo-protein complexes were detected . Mutations in the core consensus reduced these binding activities . When the plus-strand oligo of the same region was used as a probe, a retarded band was also detected, but with less specific binding . Considering the fact that the core consensus and close matches to the core consensus are important for ARS function, these results imply that the protein factors detected in this experiment may participate in DNA replication.

Gene, 1990 Oct 30, 95(1), 111 - 21
Cloning of the Schwanniomyces occidentalis glucoamylase gene (GAM1) and its expression in Saccharomyces cerevisiae; Dohmen RJ et al.; The Schwanniomyces occidentalis glucoamylase (GAM)-encoding gene (GAM1) was isolated from a lambda Charon4A genomic library using synthetic oligodeoxynucleotides as probes . GAM1 contains an ORF of 2874 nucleotides (nt) coding for 958 amino acids . S1 mapping revealed that the transcript has only a very short 5'-untranslated leader of 8-12 nt . Disruption and displacement of the GAM1 gene in Sc . occidentalis resulted in loss of the ability to grow on starch efficiently . The gam1 strains still exhibit low GAM activity suggesting that at least a second weakly expressed GAM-encoding gene (GAM2) is present in Sc . occidentalis . Expression of the Sc . occidentalis GAM1 gene in Saccharomyces cerevisiae was achieved after promoter exchange . S . cerevisiae cells transformed with centromere plasmids carrying the GAM1 gene fused to promoters of different S . cerevisiae genes, namely GAL1, PDC1 and ADH1, efficiently secrete GAM and are able to grow with soluble starch as a sole carbon source . The essential enzymatic properties of the GAMs secreted from S . cerevisiae and Sc . occidentalis are identical, although the modifications of the proteins are different.

Nucleic Acids Res, 1990 Oct 25, 18(20), 6083 - 8
P alpha-chiral phosphorothioate analogues of bis(5'-adenosyl)tetraphosphate (Ap4A); their enzymatic synthesis and degradation; Lazewska D et al.; Synthesis of Sp and Rp diastereomers of Ap4A alpha S has been characterized in two enzymatic systems, the lysyl-tRNA synthetase from Escherichia coli and the Ap4A alpha, beta-phosphorylase from Saccharomyces cerevisiae . The synthetase was able to use both (Sp)ATP alpha S and (Rp)ATP alpha S as acceptors of adenylate thus yielding corresponding monothioanalogues of Ap4A,(Sp) Ap4A alpha S and (Rp)Ap4A alpha S . No dithiophosphate analogue was formed . Relative synthetase velocities of the formation of Ap4A,(Sp) Ap4A alpha S and (Rp)Ap4A alpha S were 1:0.38:0.15, and the computed Km values for (Sp)ATP alpha S and (Rp)ATP alpha S were 0.48 and 1.34 mM, respectively . The yeast Ap4A phosphorylase synthesized (Sp)Ap4A alpha S and (Rp)Ap4A alpha S using adenosine 5'-phosphosulfate (APS) as source of adenylate . The adenylate was accepted by corresponding thioanalogues of ATP . In that system, relative velocities of Ap4A, (Sp)Ap4A alpha S and (Rp)Ap4A alpha S formation were 1:0.15:0.60 . The two isomeric phosphorothioate analogues of Ap4A were tested as substrates for the following specific Ap4A-degrading enzymes: (asymmetrical) Ap4A hydrolase (EC 3.6.1.17) from yellow lupin (Lupinus luteus) seeds hydrolyzed each of the analogues to AMP and the corresponding isomer of ATP alpha S; (symmetrical) Ap4A hydrolase (EC 3.6.1.41) from E . coli produced ADP and the corresponding diastereomer of ADP alpha S; and Ap4A phosphorylase (EC 2.7.7.53) from S . cerevisiae cleaved the Rp isomer only at the unmodified end yielding ADP and (Rp)ATP alpha S whereas the Sp isomer was degraded non-specifically yielding a mixture of ADP, (Sp)ADP alpha S, ATP and (Sp)ATP alpha S . For all the Ap4A-degrading enzymes, the Rp isomer of Ap4A alpha S appeared to be a better substrate than its Sp counterpart; stereoselectivity of the three enzymes for the Ap4A alpha S diastereomers is 51, 6 and 2.5, respectively . Basic kinetic parameters of the degradation reactions are presented and structural requirements of the Ap4A-metabolizing enzymes with respect to the potential substrates modified at the Ap4A-P alpha are discussed.

J Biol Chem, 1990 Oct 15, 265(29), 17746 - 52
Characterization of recombinant tick anticoagulant peptide . A highly selective inhibitor of blood coagulation factor Xa; Neeper MP et al.; Tick anticoagulant peptide (TAP) is a potent, highly selective inhibitor of blood coagulation factor Xa (Waxman, L., Smith, D . E., Arcuri, K . E., and Vlasuk, G . P . (1990) Science, 248, 593-596) . Further detailed studies pertaining to the in vitro and in vivo evaluation of TAP require quantities of the inhibitor which cannot be isolated from ticks . To overcome this limitation we describe here the characterization of recombinant TAP (rTAP) secreted by Saccharomyces cerevisiae . Expression of rTAP was obtained using a chimeric gene containing a fusion between sequences encoding the secretory preproleader of the yeast mating pheromone alpha-factor and a synthetic sequence encoding the 60-amino acid inhibitor under the transcriptional control of a galactose-inducible promoter . Recombinant S . cerevisiae were found to secrete biologically active rTAP into the extracellular medium at levels of 0.1-0.15 g/liter . The secreted inhibitor was purified to homogeneity and found to be indistinguishable from the native inhibitor with respect to several criteria, including primary structure, amino acid composition, and electrophoretic mobility . In addition, purified rTAP and native TAP exhibited similar stoichiometric inhibition of factor Xa in vitro . The in vivo efficacy of rTAP was demonstrated using a model of low grade disseminated intravascular coagulation where the purified inhibitor was shown to significantly inhibit thromboplastin-induced fibrinopeptide A generation following an infusion into conscious rhesus monkeys . The availability of rTAP will allow a detailed evaluation of the in vitro and in vivo properties of this highly specific and potent factor Xa inhibitor.

Eur J Biochem, 1990 Oct 5, 193(1), 97 - 103
The three cysteine residues of cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae are not essential for its activity; Kern D et al.; Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is a dimer made up of identical subunits (Mr 63,000) each of these containing three cysteines (residues 255, 512 and 519 in the amino acid sequence) . Thiol-specific probes were used to label these cysteines and study the resulting effect of the modification on the kinetic parameters of both the ATP/PPi exchange and tRNA aminoacylation reactions . Using the classical techniques of protein chemistry it was shown that none of the three cysteines was labelled with iodoacetic acid, whilst N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoate) reacted with Cys512 and Cys255, respectively . Only the latter modification was accompanied by a decrease in the rates of both enzyme activities whilst the Km values for the various substrates remained unaffected . Site-directed mutagenesis was also used to replace each of the three cysteines by other residues, either individually or simultaneously . For these experiments the enzyme was expressed in Escherichia coli using an expression vector bearing the structural gene in which the first 13 codons were replaced by the first 14 of the CII lambda gene . The resulting substitution in the amino-terminal part of the expressed enzyme had no effect on the kinetic parameters, compared to those of the enzyme purified from S . cerevisiae . Taking into account the consequences of such substitutions, as well as those of chemical modifications on the two reactions catalysed by the enzyme . ATP/PPi exchange and tRNA aminoacylation, it could be concluded that none of these three cysteines plays any essential role in either substrate binding or catalysis.

Biochim Biophys Acta, 1990 Oct 5, 1028(2), 161 - 4
Transport overshoot during 2-methyl-4-amino-5-hydroxymethylpyrimidine uptake by Saccharomyces cerevisiae; Iwashima A et al.; The transport overshoot during 2-methyl-4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine) uptake by the thiamin transport system in Saccharomyces cerevisiae was investigated . The overshoot was found to be temperature- and energy-dependent and affected by the growth phase of the yeast . The efflux system for hydroxymethylpyrimidine appeared to be more sensitive to 2,4-dinitrophenol than the influx system, resulting in the loss of the overshoot of the pyrimidine in the presence of the uncoupler . Furthermore, the overshoot did not occur after the preincubation of yeast cells with inhibitors of protein synthesis such as cycloheximide and anisomycin . These results suggest that an active efflux system for hydroxymethylpyrimidine, which is rapidly synthesized, is involved in the overshoot of this pyrimidine during its transport in S . cerevisiae.

J Biol Chem, 1990 Oct 5, 265(28), 17274 - 80
Secretion of killer toxin encoded on the linear DNA plasmid pGKL1 from Saccharomyces cerevisiae; Tokunaga M et al.; By the kar1-mediated cytoduction, linear double-stranded DNA plasmids pGKL1 and pGKL2, encoding killer toxin complex, have been successfully transferred to the recipient strains with about 30% frequency . The killer toxin was found to be secreted through the normal yeast secretory pathway by introducing pGKL plasmids into the several Saccharomyces cerevisiae sec mutants and examining the secretion of killer toxin . S . cerevisiae cells, harboring newly isolated deletion plasmid pGKL1D, expressed only the 28K protein among three killer subunits, and secreted the 28K subunit at a level of zero to 20% efficiency of the cells containing intact pGKL1 plasmid . These data indicated that subunit interaction (cosecretion) of killer proteins is required for the efficient secretion of 28K subunit . The 28K precursor protein was found to translocate across the canine pancreatic endoplasmic reticulum membrane under the direction of its own signal peptide in vitro without any other subunits . From kex2 mutant cells harboring pGKL1 plasmid, the 97K subunit, and its precursor 128K protein were not secreted, however, the 28K subunit was secreted in the same amount as that secreted from KEX2 cells . These lines of evidence suggest that the final assembly of killer toxin complex after KEX2 site of Golgi apparatus is not essential for the secretion of 28K subunit, and therefore, that putative interaction between 128K protein and 28K subunit for the transport between endoplasmic reticulum and Golgi apparatus may be required for the efficient secretion of 28K subunit.

FEMS Microbiol Immunol, 1990 Oct, 2(3), 155 - 67
Use of live Saccharomyces cerevisiae cells as a biological response modifier in experimental infections; Bizzini B et al.; A short-term oral administration of live Saccharomyces cerevisiae cells, strain Sillix Hansen DSM 1883, resulted in enhanced resistance of mice toward infections with K . pneumoniae . S . pneumoniae and S . pyogenes A produced by intranasal inoculation . Yeast pre-treatment also increased the efficacy of antibiotic therapy in bacterial infections and of antiviral drugs in viral infections . Yeast treatment of animals stimulated phagocytosis, activated the complement system and induced interferon which are likely to represent the main mechanisms of action whereby pretreatment of mice with live S . cerevisiae cells increases resistance to infection . It is concluded that preventive administration of live Saccharomyces cerevisiae cells should be used for increasing resistance to bacterial infections, in particular of the respiratory tract, or to viral infections, as well as an adjunct to antibiotic and antiviral drug therapy.

Jpn J Genet, 1990 Oct, 65(5), 323 - 34
Structural and functional stability of IncP plasmids during stepwise transmission by trans-kingdom mating: promiscuous conjugation of Escherichia coli and Saccharomyces cerevisiae; Nishikawa M et al.; In order to establish a gene transfer system for yeast by promiscuous conjugation, we constructed plasmid pAY101 which contained an oriT sequence derived from RK2 (IncP) and the yeast TRP1 and ARS1 genes . A conjugation mixture consisted of yeast Saccharomyces cerevisiae, E . coli harboring pAY101, and E . coli carrying a helper plasmid with mob and tra . In the conjugation mixture a tryptophan-requiring yeast mutant (trp1) was converted to be prototrophic for tryptophan at a frequency of about 10(-5) to 10(-3) per recipient cell . This E . coli-yeast conjugation system required the mob, tra, oriT, TRP1 and ARS1 genes . The mob and tra genes were trans-acting elements as in an E . coli conjugation system . The mobilization was inhibited by nalidixic acid as in a typical bacterial conjugation . DNA analysis indicated that the plasmid pAY101 was transferred from E . coli to S . cerevisiae, and retained its original structure and function in yeast host cells.

Mutat Res, 1990 Oct, 232(2), 327 - 36
Analysis of interactions between mutagens, II . Ethyl methanesulfonate and ultraviolet light in Saccharomyces cerevisiae; Ager DD et al.; The results of this study indicate the existence of a strong interaction between ethyl methanesulfonate (EMS) and ultraviolet light (UV) for cell killing in the yeast Saccharomyces cerevisiae . Conversely, mutation and gene conversion frequencies observed for the combined treatment of EMS and UV do not deviate significantly from that expected on the basis of simple additivity . Studies involving repair-deficient mutants (rad mutants) reveal that the synergistic interaction for cell killing depends on RAD52 function (recombinational repair), but not on RAD3 function (excision repair) . On the basis of this analysis, the interaction between EMS and UV in S . cerevisiae might arise from the inhibition of double-strand break repair by one, or both agents.

J Bacteriol, 1990 Oct, 172(10), 6145 - 7
Regulation of thiamine biosynthesis in Saccharomyces cerevisiae; Kawasaki Y et al.; A pho6 mutant of Saccharomyces cerevisiae, lacking a regulatory gene for the synthesis of periplasmic thiamine-repressible acid phosphatase activity, was found to be auxotrophic for thiamine . The activities of four enzymes involved in the synthesis of thiamine monophosphate were hardly detectable in the crude extract from the pho6 mutant . On the other hand, the activities of these enzymes and thiamine-repressible acid phosphatase in a wild-type strain of S . cerevisiae, H42, decreased with the increase in the concentration of thiamine in yeast cells . These results suggest that thiamine synthesis in S . cerevisiae is subject to a positive regulatory gene, PHO6, whereas it is controlled negatively by the intracellular thiamine level.

Arch Biol Med Exp (Santiago), 1990 Oct, 23(2), 165 - 72
{Genetic engineering in filamentous fungi: cloning of the invertase gene from Neurospora crassa}; Caru M; The invertase wild type gene of N . crassa was cloned into the YRp7 yeast vector . This recombinant plasmid was selected by functional complementation of an invertaseless mutant strain of S . cerevisiae . The isolated recombinant plasmid (named pNC2) carries a 7.6 Kb BamHI DNA fragment from N . crassa . The cloned DNA hybridized with the N . crassa genomic DNA and transformed an invertase mutant of N . crassa Inv- to Inv+ . Transformation of N . crassa Inv- to Inv+ seems to take at least two different integration events . One of them involves an integration closely linked to inv locus, and the other one apparently involves an integration of cloned DNA at a genomic site different that the inv locus.

Microbiologica, 1990 Oct, 13(4), 283 - 95
Isolation and characterization of carbon catabolite repression mutants in Saccharomyces cerevisiae; Donnini C et al.; Two carbon catabolite repression mutants of S . cerevisiae were isolated and characterized . In spite of the selection procedure (red colonies after tetrazolium overlay at high glucose concentration) the mutants exhibited a respiration which was as repressed as that of the parental strain or even more repressed . When grown at high glucose concentration the mutants display hyper-repression of cytochrome aa3 and of certain mitochondrial enzymes (L- and D-lactate dehydrogenases) but not of others (malate dehydrogenase, succinate dehydrogenase), indicating the existence of separate control sites for the different genes involved in the mitochondrial biogenesis . The data obtained pointed out that the same mutation affects both repression and derepression . In addition, the mutation(s) give rise to the complete derepression of the cytoplasmic enzyme NAD-glutamate dehydrogenase at 10% glucose whereas the enzyme is normally repressed at 3% glucose . The results of the genetic analysis indicate the mitochondrial nature of the mutation(s).

Mol Microbiol, 1990 Oct, 4(10), 1765 - 9
Effects of N-methyl-D-aspartate receptor ligands on yeast sporulation; Pinon R; Earlier studies have suggested that glutamate may play an important role in the transition between the mitotic (vegetative) and meiotic (sporulative) stages of the life cycle in the yeast Saccharomyces cerevisiae . Glutamate is also a major excitatory neurotransmitter in the vertebrate brain, and its actions are mediated by the excitatory amino acid (EAA) family of receptors, the three best-characterized of which are the N-methyl-D-aspartate (NMDA), quisqualate (Q), and kainate (K) receptors . As an initial test of the possibility that glutamate action in S . cerevisiae might be mediated by an EAA-like receptor mechanism, the effects of ligands that define the functional domains of the vertebrate NMDA receptor have been examined . The responses of S . cerevisiae cells to ligands that act at four distinct sites on the NMDA receptor provide the first evidence for an NMDA-like receptor-mediated system involved in the control of yeast sporulation.

Nucleic Acids Res, 1990 Sep 25, 18(18), 5413 - 7
Identification of a putative RNA helicase in E.coli; Iggo R et al.; The human p68 protein, an SV40 large T related antigen, is an RNA dependent ATPase and RNA helicase . It belongs to a new large and highly conserved gene family, the DEAD box proteins, whose members are involved in a variety of processes requiring manipulation of RNA secondary structure such as translation and splicing . Multiple DEAD box genes are present in S.cerevisiae, but only one has previously been described in E.coli . Low stringency screening of an E.coli genomic library with a p68 cDNA probe led to the identification of dbpA, a new E.coli DEAD box gene located at 29.6 minutes on the W3110 chromosome . We report here the nucleotide and deduced amino acid sequences of the gene . We have overexpressed dbpA from its own promoter on a high copy number plasmid and identified the gene product as a approximately 50 kD protein by immunoblotting with an anti-DEAD antibody.

Eur J Biochem, 1990 Sep 24, 192(3), 791 - 5
The role of fructose 2,6-bisphosphate in glycolytic oscillations in extracts and cells of Saccharomyces cerevisiae; Yuan Z et al.; Fructose 2,6-bisphosphate is physiologically one of the most potent activators of yeast 6-phosphofructo-1-kinase . The glycolytic oscillation observed in cell-free cytoplasmic extracts of the yeast Saccharomyces cerevisiae responds to the addition of fructose 2,6-bisphosphate in micromolar concentrations by showing a pronounced decrease of both the amplitude and the period . The oscillations can be suppressed completely by 10 microM and above of this activator but recovers almost fully (95%) to the unperturbed state after 3 h . Fructose 2,6-bisphosphate shifts the phases of the oscillations by a maximal +/- 60 degrees . Oscillations in concentration of endogenous fructose 2,6-bisphosphate in the extract were also observed . Fructose 2,6-bisphosphate alters the dynamic properties of 6-phosphofructo-1-kinase which are vital for its role as the 'oscillophore' . However, the minute amount (approximately 0.3 microM) of endogenous fructose 2,6-bisphosphate and the phase relationship of its oscillations compared with other metabolites indicate that this activator is not an essential component of the oscillatory mechanism . Further support for this conclusion is the observation of sustained oscillations in both the extracts and a population of intact cells of a mutant strain (YFA) of S . cerevisiae with no detectable fructose 2,6-bisphosphate (less than 5 nM).

Cell, 1990 Sep 21, 62(6), 1143 - 51
A third essential DNA polymerase in S . cerevisiae; Morrison A et al.; DNA polymerases I and III are essential for viability of S . cerevisiae . We have cloned and analyzed POL2, the gene encoding the catalytic subunit of the third nuclear DNA polymerase, DNA polymerase II . POL2 expressed a transcript of approximately 7.5 kb and contained a reading frame that encoded a protein of calculated Mr 255,649 . The N-terminal half of the predicted protein displayed relatively weak similarity of sequence to eukaryotic DNA polymerases . Disruption of the coding sequence at midpoint led to viable, slowly growing cells, which yielded a truncated polypeptide with DNA polymerase II activity, free from subunits B or C . Deletion of the reading frame resulted in inviability and the dumbbell terminal morphology that typically follows arrest of DNA replication . We conclude that three DNA polymerases are essential in yeast and argue that all three are replicases, a possibility that challenges existing models of eukaryotic DNA replication.

Biochemistry, 1990 Sep 18, 29(37), 8614 - 9
Overexpression and mutagenesis of the catalytic domain of dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae; Niu XD et al.; The inner core domain (residues approximately 221-454) of the dihydrolipoamide acetyltransferase component (E2P) of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae has been overexpressed in Escherichia coli strain JM105 via the expression vector pKK233-2 . The truncated E2p was purified to apparent homogeneity . It exhibited catalytic activity (acetyl transfer from {1-14C}acetyl-CoA to dihydrolipoamide) very similar to that of wild-type E2p . The appearance of the truncated and wild-type E2p was also very similar, as observed by negative-stain electron microscopy, namely, a pentagonal dodecahedron . These findings demonstrate that the active site of E2p from S . cerevisiae resides in the inner core domain, i.e., catalytic domain, and that this domain alone can undergo self-assembly . The purified truncated E2p showed a tendency to aggregate . Aggregation was prevented by genetically engineered attachment of the interdomain linker segment (residues approximately 181-220) to the catalytic domain . All dihydrolipoamide acyltransferases contain the sequence His-Xaa-Xaa-Xaa-Asp-Gly near their carboxyl termini . By analogy with chloramphenicol acetyltransferase, the highly conserved His and Asp residues were postulated to be involved in the catalytic mechanism {Guest, J . R . (1987) FEMS Microbiol . Lett . 44, 417-422} . Substitution of the sole His residue in the S . cerevisiae truncated E2p, His-427, by Asn or Ala by site-directed mutagenesis did not have a significant effect on the kcat or Km values of the truncated E2p . However, the Asp-431----Asn, Ala, or Glu substitutions resulted in a 16-, 24-, and 3.7-fold reduction, respectively, in kcat, with little change in Km values.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1990 Sep 15, 265(26), 15518 - 24
Gene-enzyme relationship in the sulfate assimilation pathway of Saccharomyces cerevisiae . Study of the 3'-phosphoadenylylsulfate reductase structural gene; Thomas D et al.; In yeast, mutations in six different loci (MET1, MET4, MET8, MET16, MET22, and MET25) have been reported to result in the absence of 3'-phosphoadenylylsulfate (PAPS) reductase activity . In the present study, we show that MET16 is the structural gene for PAPS reductase and that the yeast and the Escherichia coli enzymes display significant similarities . Thioredoxin has been implicated in the reduction of PAPS in Saccharomyces cerevisiae as well as in E . coli . One of the generally accepted mechanisms for the action of thioredoxin as a hydrogen donor involves a redox-active sulfhydryl group in the catalytic site of PAPS reductase . However, the present study shows that the site-directed mutagenesis of the unique cysteine from PAPS reductase leads to an enzyme which remains active in vivo . This result would rather support the hypothesis of thioredoxin playing the role of a thiol carrier in the reduction of PAPS into sulfite . Strains separately mutated in the six different loci cited above were examined for the expression of different genes . A mutation in the MET4 gene abolishes transcription of both genes MET16 and MET25 . In contrast, mutations in MET1, MET8, and MET25 do not impair MET16 transcription, yet strains bearing these mutations are devoid of PAPS reductase activity . To account for the latter result, we postulate that the enzymes involved in sulfate assimilation may occur as a multienzyme complex in S . cerevisiae.

Biochem Biophys Res Commun, 1990 Sep 14, 171(2), 705 - 10
Several mammalian ubiquitin carrier proteins, but not E2(20K), are related to the 20-kDa yeast E2, RAD6; Berleth ES et al.; Western blot analysis was used to probe the relationships between the multiple ubiquitin carrier proteins (E2 s) of rabbit reticulocytes and the 20-kDa E2 encoded by the RAD6 gene of the yeast S . cerevisiae . Reticulocyte E2-14K, E2-17K, and E2-25K each reacted with two or more polyclonal anti-RAD6 antibody preparations; E2-20K, E2-35K, and E2-230K did not cross-react . These results suggest that some, but not all, reticulocyte E2 s are members of a RAD6-like protein family which is conserved within and across species . RAD6 and E2-20K were also shown to multi-ubiquitinate histones by different mechanisms.

Cell, 1990 Sep 7, 62(5), 913 - 25
The fission yeast cut1+ gene regulates spindle pole body duplication and has homology to the budding yeast ESP1 gene; Uzawa S et al.; Mutations in the fission yeast cut1+, cut2+, and cut10+ genes uncouple normally coordinated mitotic events and deregulate, rather than arrest, mitosis . DNA synthesis continues, making polyploid nuclei with several spindles . Multiple, aberrant spindle pole bodies (SPBs) are produced in cut1 mutant cells . The cut1+ and cut2+ genes are cloned by transformation . High gene dosage of cut1+ also complements cut2 and cut10 mutants . The cut2+ gene, however, complements only cut2 . The 210 kd cut1+ gene product contains putative ATP binding and helical coil regions followed by a COOH-terminal domain homologous to the S . cerevisiae gene ESP1 . Mutations in the ESP1 gene also result in many SPBs . The cut1+ product is shown by anti-cut1 antibody to be a rare component of the insoluble nuclear fraction . It may play a key role in coupling chromosome disjunction with other cell cycle events and is potentially a component, regulator, or motor for the SPB and/or kinetochores.

Genetics, 1990 Sep, 126(1), 53 - 62
Mobility of two optional G + C-rich clusters of the var1 gene of yeast mitochondrial DNA; Wenzlau JM et al.; Yeast mtDNA contains two different kinds of mobile optional sequences, two group I introns and a short G + C-rich insertion to some var1 genes . Movement of each element in crosses has been called gene conversion though little is known about the mechanism of G + C cluster conversion . A new allele of the var1 gene found in mtDNA of Saccharomyces capensis is described that permitted a more detailed comparison between intron mobility and G + C cluster conversion . The S . capensis var1 gene lacks the cc+ element present in all S . cerevisiae var 1 genes and the previously described optional a+ element . In crosses with cc+ a- and cc+ a+ S . cerevisiae strains, both clusters were found to be mobile and, in the latter cross, appear to convert independently and only to homologous insertion sites . No evidence for flanking marker coconversion (a hallmark feature of intron conversion) was obtained despite the availability of nearby physical markers on both sides of cluster conversion sites . These data indicate that G + C cluster conversion has only a superficial resemblance to intron mobility; analogies to procaryotic transposition mechanisms are considered.

Microbiol Rev, 1990 Sep, 54(3), 211 - 25
PET genes of Saccharomyces cerevisiae; Tzagoloff A et al.; We describe a collection of nuclear respiratory-defective mutants (pet mutants) of Saccharomyces cerevisiae consisting of 215 complementation groups . This set of mutants probably represents a substantial fraction of the total genetic information of the nucleus required for the maintenance of functional mitochondria in S . cerevisiae . The biochemical lesions of mutants in approximately 50 complementation groups have been related to single enzymes or biosynthetic pathways, and the corresponding wild-type genes have been cloned and their structures have been determined . The genes defined by an additional 20 complementation groups were identified by allelism tests with mutants characterized in other laboratories . Mutants representative of the remaining complementation groups have been assigned to one of the following five phenotypic classes: (i) deficiency in cytochrome oxidase, (ii) deficiency in coenzyme QH2-cytochrome c reductase, (iii) deficiency in mitochondrial ATPase, (iv) absence of mitochondrial protein synthesis, and (v) normal composition of respiratory-chain complexes and of oligomycin-sensitive ATPase . In addition to the genes identified through biochemical and genetic analyses of the pet mutants, we have cataloged PET genes not matched to complementation groups in the mutant collection and other genes whose products function in the mitochondria but are not necessary for respiration . Together, this information provides an up-to-date list of the known genes coding for mitochondrial constituents and for proteins whose expression is vital for the respiratory competence of S . cerevisiae.

Mol Cell Biol, 1990 Sep, 10(9), 4905 - 11
A conserved sequence in histone H2A which is a ubiquitination site in higher eucaryotes is not required for growth in Saccharomyces cerevisiae; Swerdlow PS et al.; Histones H2A and H2B are modified by ubiquitination of specific lysine residues in higher and lower eucaryotes . To identify functions of ubiquitinated histone H2A, we studied an organism in which genetic analysis of histones is feasible, the yeast Saccharomyces cerevisiae . Surprisingly, immunoblotting experiments using both anti-ubiquitin and anti-H2A antibodies gave no evidence that S . cerevisiae contains ubiquitinated histone H2A . The immunoblot detected a variety of other ubiquitinated species . A sequence of five residues in S . cerevisiae histone H2A that is identical to the site of H2A ubiquitination in higher eucaryotes was mutated to substitute arginines for lysines . Any ubiquitination at this site would be prevented by these mutations . Yeast organisms carrying this mutation were indistinguishable from the wild type under a variety of conditions . Thus, despite the existence in S . cerevisiae of several gene products, such as RAD6 and CDC34, which are capable of ubiquitinating histone H2A in vitro, ubiquitinated histone H2A is either scarce in or absent from S . cerevisiae . Furthermore, the histone H2A sequence which serves as a ubiquitination site in higher eucaryotes is not essential for yeast growth, sporulation, or resistance to either heat stress or UV radiation.

Mol Cell Biol, 1990 Sep, 10(9), 4807 - 15
K28, a unique double-stranded RNA killer virus of Saccharomyces cerevisiae; Schmitt MJ et al.; The double-stranded RNA (dsRNA) viruses of Saccharomyces cerevisiae consist of 4.5-kilobase-pair (kb) L species and 1.7- to 2.1-kb M species, both found in cytoplasmic viruslike particles (VLPs) . The L species encode their own capsid protein, and one (LA) has been shown to encode a putative capsid-polymerase fusion protein (cap-pol) that presumably provides VLPs with their transcriptase and replicase functions . The M1 and M2 dsRNAs encode the K1 and K2 toxins and specific immunity mechanisms . Maintenance of M1 and M2 is dependent on the presence of LA, which provides capsid and cap-pol for M dsRNA maintenance . Although a number of different S . cerevisiae killers have been described, only K1 and K2 have been studied in any detail . Their secreted polypeptide toxins disrupt cytoplasmic membrane functions in sensitive yeast cells . K28, named for the wine S . cerevisiae strain 28, appears to be unique; its toxin is unusually stable and disrupts DNA synthesis in sensitive cells . We have now demonstrated that 4.5-kb L28 and 2.1-kb M28 dsRNAs can be isolated from strain 28 in typical VLPs, that these VLPs are sufficient to confer K28 toxin and immunity phenotypes on transfected spheroplasts, and that the immunity of the transfectants is distinct from that of either M1 or M2 . In vitro transcripts from the M28 VLPs show no cross-hybridization to denatured M1 or M2 dsRNAs, while L28 is an LA species competent for maintenance of M1 . K28, encoded by M28, is thus the third unique killer system in S . cerevisiae to be clearly defined . It is now amenable to genetic analysis in standard laboratory strains.

Mol Cell Biol, 1990 Sep, 10(9), 4612 - 22
The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line; Beck PJ et al.; The Saccharomyces cerevisiae DPM1 gene product, dolichol-phosphate-mannose (Dol-P-Man) synthase, is involved in the coupled processes of synthesis and membrane translocation of Dol-P-Man . Dol-P-Man is the lipid-linked sugar donor of the last four mannose residues that are added to the core oligosaccharide transferred to protein during N-linked glycosylation in the endoplasmic reticulum . We present evidence that the S . cerevisiae gene DPM1, when stably transfected into a mutant Chinese hamster ovary cell line, B4-2-1, is able to correct the glycosylation defect of the cells . Evidence for complementation includes (i) fluorescence-activated cell sorter analysis of differential lectin binding to cell surface glycoproteins, (ii) restoration of Dol-P-Man synthase enzymatic activity in crude cell lysates, (iii) isolation and high-performance liquid chromatography fractionation of the lipid-linked oligosaccharides synthesized in the transfected and control cell lines, and (iv) the restoration of endoglycosidase H sensitivity to the oligosaccharides transferred to a specific glycoprotein synthesized in the DPM1 CHO transfectants . Indirect immunofluorescence with a primary antibody directed against the DPM1 protein shows a reticular staining pattern of protein localization in transfected hamster and monkey cell lines.

Exp Cell Res, 1990 Sep, 190(1), 57 - 64
Acquired thermotolerance following heat shock protein synthesis prevents impairment of mitochondrial ATPase activity at elevated temperatures in Saccharomyces cerevisiae; Patriarca EJ et al.; The complex molecular response of cells to sudden temperature changes is a well-characterized phenomenon . Although it is clear that the induction of heat shock proteins provides protection from heat in all of the organisms so far tested, very little is known about the role that this set of proteins plays in cellular homeostasis . Recently, putative roles for hsp60 and hsp70-like proteins have been proposed in Saccharomyces cerevisiae . hsp70-like proteins have been shown to be necessary for translocation of precursor polypeptides into mitochondria and endoplasmic reticulum, while hsp60 is required for the assembly of precursor polypeptides into oligomeric complexes following incorporation into the mitochondrial matrix . In this paper, we report that a brief temperature shock (44 degrees C) impairs coupling of oxidative phosphorylation in S . cerevisiae as measured indirectly by the Cl-CCP/oligomycin assay . Furthermore, at high temperature oligomycin stimulates rather than inhibits oxygen uptake under nonthermotolerant conditions . Pretreatment of cells for a short period of time at 37 degrees C, prior to exposure to higher temperatures rescues the capacity to maintain coupling between oxidative phosphorylation and electron transport . Inhibition of cytoplasmic RNA or protein synthesis during heat shock prevents the protection of this mitochondrial activity . We propose that one of the roles of the induction of heat shock proteins (or related activities) is to protect mitochondrial ATPase activity under conditions of further increase in temperature.

Mol Biol (Mosk), 1990 Sep-Oct, 24(5), 1411 - 8
{Cloning, structure and features of a Saccharomyces cerevisiae DNA fragment causing the expression of reporter genes}; Fodor I et al.; A DNA fragment "PX" of 240 b.p . was isolated at random from the genomic sequences of S . cerevisiae by using a plasmid that contained a promoterless reporter gene lacZ of E . coli . "PX" was capable of activating synthesis of three reporter genes (pho5 from yeast and bacterial lacZ and neo) in yeast cells bidirectionally . Results of Southern-blot hybridization with yeast genomic DNA suggest that the cloned sequence is represented in at least ten copies per cell . Nucleotide sequence of "PX" shows that DNA contains putative TATA elements and four tandems of inverted repeats: {sequence: see text}.

Br J Dermatol, 1990 Sep, 123(3), 355 - 63
White piedra: evidence for a synergistic infection; Ellner KM et al.; To determine the relative roles of coryneform bacteria and Trichosporon beigelii in the pathogenesis of genital white piedra, scrotal hair from 10 subjects was studied . Hairs were examined by light microscopy to determine the relative proportions of each organism, and were also cultured for coryneforms and yeast . Histologically, hair nodules from five out of nine cases showed a mixture of yeasts and bacteria, four had bacteria alone, and none showed yeast alone . Five strains of T . beigelii were cultured, two strains of Saccharomyces cerevisiae and 22 strains of coryneforms . The isolates were tested for synergism by a plate-overlay method . Growth of coryneforms occurred over and around sections of the plate inoculated with T . beigelii but not around the control yeast, S . cerevisiae . There were strain differences in the stimulatory response of both T . beigelii and coryneform strains . In reverse experiments coryneforms did not enhance growth of T . beigelii . It was concluded that white piedra is a mixed infection caused by the synergistic action between T . beigelii and a specific coryneform bacteria resulting in invasion of the hair cuticle and cortex.

Mol Cell Biol, 1990 Sep, 10(9), 4630 - 7
Expression of the yeast PHR1 gene is induced by DNA-damaging agents; Sebastian J et al.; The PHR1 gene of Saccharomyces cerevisiae encodes a photolyase which repairs specifically and exclusively pyrimidine dimers, the most frequent lesions induced in DNA by far-UV radiation . We have asked whether expression of PHR1 is modulated in response to UV-induced DNA damage and to DNA-damaging agents that induce lesions structurally dissimilar to pyrimidine dimers . Using a PHR1-lacZ fusion gene in which expression of beta-galactosidase is regulated by PHR1 5' regulatory elements, we found that exposure of cells to 254-nm light, 4-nitroquinoline-N-oxide, methyl methanesulfonate, and N-methyl-N'-nitro-N-nitrosoguanidine induced synthesis of increased amounts of fusion protein . In contrast to these DNA-damaging agents, neither heat shock nor exposure to photoreactivating light elicited a response . Induction by far-UV radiation was evident both when the fusion gene was carried on a multicopy plasmid and when it replaced the endogenous chromosomal copy of PHR1, and it was accompanied by an increase in the steady-state concentration of PHR1-lacZ mRNA . Northern (RNA) blot analysis of PHR1 mRNA encoded by the chromosomal locus was consistent with either enhanced transcription of PHR1 after DNA damage or stabilization of the transcripts . Neither the intact PHR1 or RAD2 gene was required for induction . Comparison of the region of PHR1 implicated in regulation of its expression with other damage-inducible genes from yeast cells revealed a common conserved sequence that is present in the PHR1, RAD2, and RNR2 genes and is required for damage inducibility of the latter two genes . These sequences may constitute elements of a damage-responsive regulon in S . cerevisiae.

Mikrobiologiia, 1990 Sep-Oct, 59(5), 756 - 63
{Effect of an abrupt rise in 2,4-dinitrophenol concentration on the growth of a turbidostat Saccharomyces cerevisiae culture}; Kaliuzhin VA; The purpose of the work was to study the effect of an abrupt rise in 2,4-dinitrophenol concentration on the growth of a turbidostat Saccharomyces cerevisiae 14 culture . The action of DNP caused three types of response . The growth of S . cerevisiae 14 ceased when DNP concentration was raised once to 10 mg per litre at 30 degrees C and pH 4, but did not stop if that was done in a gradual manner . It took at least 0.25 to 0.5 h for the culture to develop a protection against the action of DNP at a concentration of 10 mg per litre, and a minimal DNP concentration inducing the protection was from 0.6 to 0.9 mg per litre . The pH factor could also induce DNP protection . The dynamics of changes in DNP concentration was followed up in the cells in the course of transient processes . Apparently, the ability of yeast cells to grow in media with DNP is determined by the inhibitor concentration within the first 1.5 h . Apparently, adaptation of the culture to DNP is realised due to changes in the properties of the cell membrane complex.

Biochemistry, 1990 Aug 28, 29(34), 7953 - 8
Fidelity in the aminoacylation of tRNA(Val) with hydroxy analogues of valine, leucine, and isoleucine by valyl-tRNA synthetases from Saccharomyces cerevisiae and Escherichia coli; Englisch-Peters S et al.; Several analogues of valine, leucine, and isoleucine carrying hydroxyl groups in the gamma- or delta-position have been tested in the aminoacylation of tRNA by valyl-tRNA synthetases from Saccharomyces cerevisiae and Escherichia coli . Results of the ATP/PPi exchange and of the aminoacylation reactions indicate that the amino acid analogues not only can form the aminoacyl adenylate intermediate but are also transferred to tRNA . However, the fact that the reaction consumes an excess of ATP indicates that the misactivated amino acid analogue is hydrolytically removed . Thus, valyl-tRNA synthetase from S . cerevisiae shows a high fidelity in forming valyl-tRNA . Although the much bulkier amino acid analogues allo- and iso-gamma-hydroxyvaline and allo- and iso-gamma-hydroxyisoleucine are initially charged to tRNA, the misaminoacylated tRNA(Val) is enzymatically deacylated . This cleavage reaction is mediated by the hydroxyl groups of the amino acid analogues which are converted into the corresponding lactones.

Cell, 1990 Aug 24, 62(4), 631 - 47
The identification of a second cell cycle control on the HO promoter in yeast: cell cycle regulation of SW15 nuclear entry; Nasmyth K et al.; HO encodes a site-specific endonuclease that initiates mating type switching in S . cerevisiae . It is expressed only transiently during the cell cycle of mother cells, as they undergo Start, but not in daughter cells . Since SWI5 appears to be the only HO transcription factor missing when daughter cells undergo Start, we were interested in the intracellular distribution of SWI5 at cell division . We discovered that SWI5 is found equally concentrated in the nuclei of both mother and daughter cells at the end of anaphase, suggesting that its subsequent fate must somehow differ . Prior to the end of anaphase, SWI5 accumulates in the cytoplasm and only moves into the nucleus when cells enter G1 . A version of the HO promoter that has lost its dependence on Start is nevertheless still strongly cell cycle regulated and is activated when SWI5 moves into the nucleus.

Cell, 1990 Aug 24, 62(4), 819 - 28
Portable encapsidation signal of the L-A double-stranded RNA virus of S . cerevisiae; Fujimura T et al.; The (+) single-stranded RNA (ssRNA) of the L-A virus is the species packaged to form new viral particles . Empty L-A viral particles specifically bind viral (+) ssRNA, and a sequence 400 bases from the 3' end is necessary for this activity . We show that its stem-loop structure, the A residue protruding from the stem, and the loop sequence are all important for the binding, and that this 34 base region is sufficient for the binding . M1, a satellite virus of L-A, has a similar structure on its (+) strand that is likewise sufficient for the binding . Heterologous RNA with the binding sequence from L-A or M1, when expressed in vivo, was packaged in L-A viral particles . Thus, the sites necessary to bind to empty particles are encapsidation signals for the L-A virus . Since the pol domain of the 180 kd minor coat protein appears to be responsible for the binding, this result suggests that the RNA polymerase molecule recognizes the viral genome for packaging.

J Gen Microbiol, 1990 Aug, 136 ( Pt 8), 1469 - 74
Temperature adaptation in yeasts: the role of fatty acids; Suutari M et al.; Studies on the yeasts Candida oleophila, Candida utilis, Lipomyces starkeyi, Rhodosporidium toruloides and Saccharomyces cerevisiae revealed the existence of three different temperature adaptation responses involving changes in fatty acid composition . These conclusions were drawn by determining the growth rates, total cellular fatty acid content, fatty acid composition, degree of unsaturation, and the mean chain length of fatty acids over a range of growth temperatures . Within temperatures permitting growth, there were no changes in the major fatty acids of any of the yeasts, but the absolute amounts and relative compositions of the fatty acids did alter . In S . cerevisiae there were temperature-induced changes in the mean fatty acid chain length, whereas in R . toruloides there were changes in the degree of unsaturation . C . oleophila, C . utilis and L . starkeyi showed both responses, depending on whether the growth temperature was above or below 20-26 degrees C . Below 20-26 degrees C temperature-dependent changes were observed in the mean chain length whereas above 20-26 degrees C there were changes in the degree of unsaturation.

Mol Gen Genet, 1990 Aug, 223(1), 24 - 32
Expression in Saccharomyces cerevisiae of a gene associated with cytoplasmic male sterility from maize: respiratory dysfunction and uncoupling of yeast mitochondria; Glab N et al.; We asked whether the mitochondrial T-urf13 gene, associated with the male sterility phenotype of T cytoplasm in maize, can be expressed in Saccharomyces cerevisiae and whether this expression can mimic the effects observed in maize . We introduced the universal code equivalent of the T-urf13 gene into the S . cerevisiae nucleus by transformation and directed its translation product into mitochondria by means of a fusion with the targeting presequence from Neurospora crassa AT-Pase subunit 9 . We show that expression of the universal code equivalent of the T-urf13 gene in the yeast nucleus does indeed mimic its effects in maize: respiratory growth of yeast is inhibited, respiration-deficient cytoplasmic mutants accumulate and NADH oxidation of isolated mitochondria is uncoupled . All these effects are observed only if the mitochondrial targeting peptide and methomyl or HmT toxin are present.

Mol Gen Genet, 1990 Aug, 223(1), 11 - 6
Correct splicing of modified introns of a Rhizopus proteinase gene in Saccharomyces cerevisiae; Ashikari T et al.; The intron of the Rhizopus aspartic proteinase gene (RNAP-I) was modified by in vitro mutagenesis and examined for its splicing efficiency in Saccharomyces cerevisiae . The wild-type intron of the RNAP-I gene was not spliced at all in spite of its structural similarity to introns of S . cerevisiae . The primary transcript of the RNAP-I gene was converted to correctly translatable mRNA only when the complete consensus sequence of S . cerevisiae introns (i.e . 5'-GTATGT-----TACTAAC-----TAG-3') was introduced into its intron, although the efficiency of splicing was low . It is also shown that transformants carrying the RNAP-I gene with the complete consensus sequence of S . cerevisiae introns produce active RNAP-I protein.

Biotechnol Appl Biochem, 1990 Aug, 12(4), 357 - 63
Mass spectrometric characterization of recombinant human interleukins 1 beta; Pucci P et al.; The cDNA coding for amino acid residues 121-269 of human interleukin 1 beta was cloned and expressed at Sclavo Research Center by C . Baldari et al . (1987, EMBO J . 6, 229-234) using Saccharomyces cerevisiae as the host cell . The purified protein showed a very low specific activity when compared to that of a mature recombinant interleukin expressed from Escherichia coli . Preliminary experiments indicated the occurrence of post-translational events in the yeast-derived protein . In an attempt to correlate the structural modifications and the biological activity of recombinant interleukins, the two proteins were characterized by fast atom bombardment mass spectrometry (FAB/MS) following the FAB-mapping procedure . The amino acid sequence of interleukin expressed in E . coli was identical to that expected whereas the mass spectrometric analysis of the recombinant S . cerevisiae protein confirmed the occurrence of covalent modifications . In particular, the asparagine residue at position 7 (numbering follows the mature active protein sequence) was shown to be glycosylated and the two cysteine residues at position 8 and 71 were involved in an S-S bridge . The results demonstrate the value of FAB/MS in the quality control of recombinant interleukins.

J Bacteriol, 1990 Aug, 172(8), 4280 - 7
Subunit structure, expression, and function of NAD(H)-specific isocitrate dehydrogenase in Saccharomyces cerevisiae; Keys DA et al.; Mitochondrial NAD(H)-specific isocitrate dehydrogenase was purified from Saccharomyces cerevisiae for analyses of subunit structure and expression . Two subunits of the enzyme with different molecular weights (39,000 and 40,000) and slightly different isoelectric points were resolved by denaturing electrophoretic techniques . Sequence analysis of the purified subunits showed that the polypeptides have different amino termini . By using an antiserum to the native enzyme prepared in rabbits, subunit-specific immunoglobulin G fractions were obtained by affinity purification, indicating that the subunits are also immunochemically distinct . The levels of NAD(H)-specific isocitrate dehydrogenase activity and immunoreactivity were found to correlate closely with those of a second tricarboxylic acid cycle enzyme, malate dehydrogenase, in yeast cells grown under a variety of conditions . S . cerevisiae mutants with defects in NAD(H)-specific isocitrate dehydrogenase were identified by screening a collection of yeast mutants with acetate-negative growth phenotypes . Immunochemical assays were used to demonstrate that one mutant strain lacks the 40,000-molecular-weight subunit (IDH1) and that a second strain lacks the 39,000-molecular-weight subunit (IDH2) . Mitochondria isolated from the IDH1 and IDH2 mutants exhibited a markedly reduced capacity for utilization of either isocitrate or citrate for respiratory O2 consumption . This confirms an essential role for NAD(H)-specific isocitrate dehydrogenase in oxidative functions in the tricarboxylic acid cycle.

EMBO J, 1990 Aug, 9(8), 2523 - 8
Role of trans-activating proteins in the generation of active chromatin at the PHO5 promoter in S . cerevisiae; Fascher KD et al.; Induction of the PHO5 gene in Saccharomyces cerevisiae by phosphate starvation was previously shown to be accompanied by the removal of four positioned nucleosomes from the promoter . We have now investigated the role of two trans-activating proteins, encoded by PHO2 and PHO4, which bind to the PHO5 promoter . Both proteins are absolutely required for the chromatin transition to occur as shown by analysis of null mutants of the two genes . Transformation of these mutant strains with plasmids containing the respective genes restores the wild type chromatin response . Increasing the gene dosage of PHO2 and of PHO4 makes it possible to differentiate functionally between the two proteins . From over-expressing PHO4 in a wild type and also in a pho2 null mutant strain and complementary experiments with PHO2, it is concluded that the PHO4 protein is the primary trigger for the chromatin transition, consistent with one of its two binding sites being located between positioned nucleosomes in repressed chromatin and thereby accessible . PHO2, the binding site of which is located within a nucleosome under conditions of PHO5 repression, contributes to the chromatin transition either by destabilizing histone-DNA interactions or by under-going interactions with PHO4.

Eur J Biochem, 1990 Jul 31, 191(2), 257 - 61
Translation and regulation of translation in the yeast Saccharomyces cerevisiae; Muller PP et al.; In recent years the yeast Saccharomyces cerevisiae has become a model system for studies of eukaryotic translation and translation regulation . Analysis of mRNA structure, translation initiation factor sequences and the translation initiation pathway indicate, that translation in S . cerevisiae is very similar to translation in higher eukaryotes . The availability of powerful genetic techniques lead to the dissection in yeast of individual steps in the translation pathway, the detection of biochemical interactions between components involved in translation and the unravelling of complex regulation phenomena.

Cell, 1990 Jul 27, 62(2), 213 - 24
A candidate protein kinase C gene, PKC1, is required for the S . cerevisiae cell cycle; Levin DE et al.; Probes derived from cDNAs encoding isozymes of rat protein kinase C (PKC) were used to screen the genome of the budding yeast S . cerevisiae . A single gene (PKC1) was isolated that encodes a putative protein kinase closely related to the alpha, beta, and gamma subspecies of mammalian PKC . Deletion of PKC1 resulted in recessive lethality . Cells depleted of the PKC1 gene product displayed a uniform phenotype, a characteristic of cell division cycle (cdc) mutants, and arrested cell division at a point subsequent to DNA replication, but prior to mitosis . Unlike most cdc mutants, which continue to grow in the absence of cell division, PKC1-depleted cells arrested growth with small buds . PKC1 may regulate a previously unrecognized checkpoint in the cell cycle.

Cell, 1990 Jul 27, 62(2), 225 - 37
G1-specific cyclins of S . cerevisiae: cell cycle periodicity, regulation by mating pheromone, and association with the p34CDC28 protein kinase; Wittenberg C et al.; The S . cerevisiae CLN genes encode cyclin homologs essential for progression from G1 to S phase . The CLN2 gene encodes a 62 kd polypeptide that accumulates periodically, peaking during G1 and decreasing rapidly thereafter, and is rapidly lost following exposure of cells to mating pheromone . Cln2 abundance can be explained by the G1-specific accumulation of the CLN2 transcript coupled with instability of the Cln2 protein . The abundance of the CLN1 and CLN2 transcripts increases greater than 5-fold during the G1 interval, decreasing dramatically as cells enter S phase . Both transcripts decrease in cells responding to mating pheromone . Finally, we demonstrate that the Cln2 polypeptide interacts with p34CDC28 to form an active protein kinase complex . This physical interaction is consistent with the genetic interaction between the CLN genes and CDC28 and suggests that Cln proteins are an essential component of the active protein kinase complex required for the G1 to S transition.

Nucleic Acids Res, 1990 Jul 25, 18(14), 4215 - 21
Yeast tRNATrp genes with anticodons corresponding to UAA and UGA nonsense codons; Kim D et al.; Naturally occurring suppressor mutants derived from tRNATrp genes have never been identified in S . cerevisiae . Oligonucleotide-directed mutagenesis was used to generate potential ochre and opal suppressors from a cloned tRNATrp gene . In vitro transcription analyses show the ochre suppressor form of the gene, TRPO, accumulates precursors and tRNA in amounts comparable to the parent . The opal suppressor, TRPOP, accumulates 4-5 fold less tRNA . Both forms of the gene are processed and spliced in vitro to produce tRNAs with the expected base sequences . The altered genes were subcloned into yeast vectors and introduced into yeast strains carrying a variety of amber, ochre, and opal mutations . When introduced on a CEN vector, neither ochre nor opal suppressor forms show suppressor activity . Deletion of the CEN region from the clones increases the copy number to 10-20/cell . The opal suppressor form shows moderate suppressor activity when the gene is introduced on this vector, however, the ochre suppressor form exhibits no detectable biological activity regardless of gene copy number . Northern blot analyses of the steady state levels of tRNATrp in cells containing the high copy-number clones reveal 20-100% increases in the abundance of tRNATrp.

Biochim Biophys Acta, 1990 Jul 16, 1045(2), 156 - 63
Phosphatidylethanolamine methyltransferase and phospholipid methyltransferase activities from Saccharomyces cerevisiae . Enzymological and kinetic properties; Gaynor PM et al.; In the yeast Saccharomyces cerevisiae, two membrane-associated enzymes catalyze the three-step methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) . Phosphatidylethanolamine methyltransferase (PEMT) catalyzes the first methylation reactions (PE----phosphatidylmonomethylethanolamine (PMME} and phospholipid methyltransferase (PLMT) catalyzes the second two methylation reactions (PMME----phosphatidyldimethylethanolamine (PDME)----PC) . Using gene disruption mutants of the S . cerevisiae OP13 and CHO2 genes, we independently studied the enzymological properties of microsome-associated PEMT and PLMT, respectively . The enzymological properties of the enzymes differed with respect to their pH optima, cofactor requirements and thermal lability . For the PEMT reactions, the apparent Km values for PE and S-Adenosylmethionine (AdoMet) were 57 microM and 110 microM, respectively . For the PLMT reactions, the apparent Km values for PMME and PDME were 380 microM and 180 microM, respectively . The apparent Km values for AdoMet were 54 microM and 59 microM with PMME and PDME as substrates, respectively . S-Adenosylhomocysteine (AdoHcy) was a competitive inhibitor of PEMT (Ki = 12 microM) and PLMT (Ki = 57 microM and Ki = 54 microM for PMME and PDME, respectively) with respect to AdoMet . AdoHcy was a noncompetitive inhibitor of PEMT (Ki = 160 microM) and PLMT (Ki = 120 microM) with respect to PE and PMME and PDME, respectively.

Gene, 1990 Jul 16, 91(2), 255 - 9
Drosophila melanogaster tRNA(Ser) suppressor genes function with strict codon specificity when introduced into Saccharomyces cerevisiae; Pappu SS et al.; The anticodon of the wild-type tRNA(7Ser) gene of Drosophila melanogaster was mutated using oligodeoxyribonucleotide-directed, site-specific mutagenesis, and all three nonsense suppressor derivatives of the gene were constructed . These constructs were cloned into an Escherichia coli-yeast shuttle vector (YRp7), and used to transform a Saccharomyces cerevisiae strain {JG 369-3B(alpha)} containing an array of nonsense alleles . When tested on appropriate omission media, the D . melanogaster suppressor genes were found to function in the yeast with strict codon specificity . Subsequent Northern hybridization analyses revealed that the D . melanogaster suppressor genes were transcribed and processed well, when in S . cerevisiae.

Nucleic Acids Res, 1990 Jul 11, 18(13), 3745 - 52
Detection of latent sequence periodicities; Pizzi E et al.; A method is proposed for the automatic detection of serial periodicities in a linear sequence . Its application to DNA subtelomeric sequences from two lower eukaryotes, P.falciparum and S.cerevisiae, reveals ordered patterns organised in hierarchical periodicities, not easily recognizable by other methods . The possible implications concerning the evolution of tandemly repetitive arrays are discussed in light of a model which involves, as successive steps, random repeat modification, the fusion of differently modified repeat versions into longer units, and the amplification of (and/or homogenization to) the more recent repeat units.

J Biol Chem, 1990 Jul 5, 265(19), 10981 - 7
The Neurospora crassa arg-2 locus . Structure and expression of the gene encoding the small subunit of arginine-specific carbamoyl phosphate synthetase; Orbach MJ et al.; We have characterized genomic and cDNA clones for arg-2, the gene encoding the small subunit of the Neurospora crassa arginine-specific carbamoyl phosphate synthetase (CPS-A), and examined its transcriptional regulation . The polypeptide's predicted amino acid sequence (453 residues) is 56% and 36% identical with the sequences of the homologous polypeptides of Saccharomyces cerevisiae and Escherichia coli, respectively . The ARG2 polypeptide has an additional amino-terminal domain with the hallmark features of a mitochondrial signal sequence . The arg-2 mRNA also encodes a 24-residue peptide in the segment upstream of the coding region for the ARG2 polypeptide . This upstream open reading frame (uORF) strongly resembles the uORF in the homologous S . cerevisiae transcript . Northern analyses indicate that arg-2 mRNA levels are reduced by arginine supplementation and increased by amino acid limitation . The large increase in arg-2 mRNA levels that occurs in response to amino acid limitation is not observed in a strain containing the cpc-1 mutation, indicating that the cross-pathway control system participates in arg-2 regulation . Four copies of the sequence TGACTC, the binding site for the CPC1 regulatory protein, are found in the arg-2 genetic region . Two copies are located upstream of the mRNA start sites, and two are present within introns in the arg-2 uORF.

Appl Environ Microbiol, 1990 Jul, 56(7), 2114 - 9
Effects of cyclohexane, an industrial solvent, on the yeast Saccharomyces cerevisiae and on isolated yeast mitochondria; Uribe S et al.; Little information on the effects of cyclohexane at the cellular or subcellular level is available . In Saccharomyces cerevisiae, cyclohexane inhibited respiration and diverse energy-dependent processes . In mitochondria isolated from S . cerevisiae, oxygen uptake and ATP synthesis were inhibited, although ATPase activity was not affected . Cyclohexane effects were similar to those reported for beta-pinene and limonene, suggesting that the cyclohexane ring in these monoterpenes may be a determinant for their biological activities.

Mutat Res, 1990 Jul, 241(3), 225 - 42
Detection of induced mitotic chromosome loss in Saccharomyces cerevisiae--an interlaboratory assessment of 12 chemicals; Whittaker SG et al.; Induced mitotic chromosome loss was assayed using diploid yeast strain S . cerevisiae D61.M . The test relies upon the uncovering and expression of multiple recessive markers reflecting the presumptive loss of the chromosome VII homologue carrying the corresponding wild-type alleles . An interlaboratory study was performed in which 12 chemicals were tested under code in 2 laboratories . The results generated by the Berkeley and the Darmstadt laboratories were in close agreement . The solvents benzonitrile and methyl ethyl ketone induced significantly elevated chromosome loss levels . However, a treatment regime that included overnight storage at 0 degree C was required to optimize chromosome loss induction . Hence, these agents are postulated to induce chromosome loss via perturbation of microtubular assembly . Fumaronitrile yielded inconsistent results: induction of chromosome loss and respiratory deficiency was observed in both laboratories, but the response was much more pronounced in the Darmstadt trial than that observed in Berkeley . The mammalian carcinogens, benzene, acrylonitrile, trichloroethylene, 1,1,1-trichloroethane and 1,1,1,2-tetrachloroethane failed to induce chromosome loss but elicited high levels of respiratory deficiency, reflecting anti-mitochondrial activity . Trifluralin, cyclophosphamide monohydrate, diazepam and diethylstilbestrol dipropionate failed to induce any detectable genetic effects . These data suggest that the D61.M system is a reproducible method for detecting induced chromosome loss in yeast.

Mol Cell Biol, 1990 Jul, 10(7), 3397 - 404
Expression of a proteolipid gene from a high-copy-number plasmid confers trifluoperazine resistance to Saccharomyces cerevisiae; Shih CK et al.; A wild-type haploid yeast strain was transformed with a library of wild-type yeast DNA fragments ligated into a high-copy-number plasmid vector (YEp24) . The pooled URA+ transformants were plated on rich medium containing a lethal concentration of trifluoperazine (TFP) . Plasmids rescued into Escherichia coli from TFP-resistant yeast colonies contained overlapping DNA fragments from a unique region of yeast chromosome XVI . Deletion and disruption experiments, mini-Tn10 LUK hop analysis, and DNA sequencing defined a novel gene with significant amino acid identity to bovine and yeast vacuoletype proteolipid subunits . This is the second locus identified that can be altered to confer TFP resistance to Saccharomyces cerevisiae and that has significant amino acid identity to a vacuolar ATPase subunit . This suggests that a target for TFP in S . cerevisiae is the electrogenic membranes of the vacuolar network and that alteration of expression or activity of vacuolar proton ATPase subunits is a general mechanism for TFP resistance in this yeast.

Curr Genet, 1990 Jul, 18(1), 29 - 39
Analysis of interchromosomal mitotic recombination; McGill CB et al.; A novel synthetic locus is described that provides a simple assay system for characterizing mitotic recombinants . The locus consists of the TRP1 and HIS3 genes inserted into chromosome III of S . cerevisiae between the CRY1 and MAT loci . Defined trp1 and his3 alleles have been generated that allow the selection of interchromosomal recombinants in this interval . Trp+ or His+ recombinants can be divided into several classes based on coupling of the other alleles in the interval . The tight linkage of the CRY1 and MAT loci, combined with the drug resistance and cell type phenotypes that they respectively control, facilitates the classification of the recombinants without resorting to tetrad dissection . We present the distribution of spontaneous recombinants among the classes defined by this analysis . The data suggest that the recombination intermediate can have regions of symmetric strand exchange and that co-conversion tracts can extend over 1-3 kb . Continuous conversion tracts are favored over discontinuous tracts . The distribution among the classes defined by this analysis is altered in recombinants induced by UV irradiation.

Curr Genet, 1990 Jul, 18(1), 93 - 6
Allelism of SNQ1 and ATR1, genes of the yeast Saccharomyces cerevisiae required for controlling sensitivity to 4-nitroquinoline-N-oxide and aminotriazole; Gompel-Klein P et al.; SNQ1 gene function is required for the expression of resistance to 4NQO in wild-type yeast . The sequence of a 3.7 kb yeast DNA containing the gene SNQ1 was determined . The SNQ1 gene consists of an open reading frame of 1641 bp and encodes, according to the hydrophobicity analysis of the putative protein, a transmembrane protein of 547 amino acids . Homology searches in yeast genome databanks revealed a 100% sequence homology with gene ATR1 which controls resistance to aminotriazole in S . cerevisiae . Pre-treatment of wild-type yeast, but not of snq1-0::LEU2 disruption mutants, with sublethal doses of aminotriazole induced hyper-resistance to 4-nitroquinoline-N-oxide . Partial deletion of the nucleotide sequence coding for a putative ATP-binding site has no, or little, influence on resistance to 4NQO whereas total deletion of the region coding for this ATP-binding domain leads to 4NQO-sensitive null-mutants.

EMBO J, 1990 Jul, 9(7), 2197 - 205
The subunit structure of Saccharomyces cerevisiae transcription factor IIIC probed with a novel photocrosslinking reagent; Bartholomew B et al.; A photocrosslinking nucleotide, 5-{N-(p-azidobenzoyl)-3-aminoallyl}-deoxyuridine monophosphate (N3Rd-UMP), has been used to identify four polypeptides that are associated with the large Saccharomyces cerevisiae RNA polymerase III transcription factor TFIIIC, and to map the locations of these subunits along DNA when TFIIIC binds to the S.cerevisiae SUP4 tRNA(Tyr) gene . The 145 kd subunit of TFIIIC is primarily accessible to photocrosslinking from the vicinity of the box B + internal promoter element; 95 and 55 kd subunits are located on opposite sides of the DNA helix in the vicinity of the box A internal promoter element; a 135 kd subunit is less strongly crosslinked to the box A region and to a DNA segment between boxes B and A . DNA probes containing more than one N3RdUMP residue can form crosslinks between polypeptide chains . The specific circumstances of formation and the apparent mol . wts of two of these products lead to the tentative suggestion that a protomer of TFIIIC may contain two 95 kd subunits.

Agric Biol Chem, 1990 Jul, 54(7), 1771 - 9
High-level secretion of a Rhizopus niveus aspartic proteinase in Saccharomyces cerevisiae; Horiuchi H et al.; The gene encoding an extracellular Rhizopus niveus aspartic proteinase I (RNAP-I) was introduced into Saccharomyces cerevisiae . The yeast cell carrying a plasmid containing the intact RNAP-I gene under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene promoter of S . cerevisiae did not synthesize RNAP-I at all . On the other hand, when the intron of the RNAP-I gene had been removed from the gene in the plasmid, the cell secreted RNAP-I with high efficiency . Processing of the pro-sequence occurred at the same region of the pro-enzyme during cultivation as observed in the culture of R . niveus . Moreover, the promoter and the terminator of the original RNAP-I gene were found to be weakly functional in the yeast cell with respect to expression of the intronless RNAP-I gene, although the initiation and termination sites were heterogeneous . The effects of vector-types on the extracellular production of RNAP-I were also investigated.

Cell, 1990 Jun 29, 61(7), 1359 - 63
The ERD2 gene determines the specificity of the luminal ER protein retention system; Lewis MJ et al.; Luminal ER proteins carry a signal at their C terminus that prevents their secretion; in S . cerevisiae this signal is the tetrapeptide HDEL . Indirect evidence suggests that HDEL is recognized by a receptor that retrieves ER proteins from the secretory pathway and returns them to the ER, and a candidate for this receptor is the product of the ERD2 gene (see accompanying paper) . We show here that presumptive ER proteins from the budding yeast K . lactis can terminate either with HDEL or, in the case of BiP, with DDEL . S . cerevisiae does not efficiently recognize DDEL as a retention signal, but exchange of its ERD2 gene for the corresponding gene from K . lactis allows equal recognition of DDEL and HDEL . Thus the specificity of the retention system is determined by the ERD2 gene . We conclude that ERD2 encodes the receptor that sorts luminal ER proteins.

Cell, 1990 Jun 29, 61(7), 1171 - 8
Analysis of structure-function relationships of yeast TATA box binding factor TFIID; Horikoshi M et al.; A systematic series of N-terminal, C-terminal, and internal deletion mutants of S . cerevisiae TFIID were expressed in vitro and tested for TATA box binding and basal level transcription activities using, respectively, DNA mobility shift and in vitro transcription assays . The domains responsible for these activities were colocalized to a surprisingly large region containing C-terminal residues 63-240 . This region was noted previously to contain potentially interesting structural motifs (central basic core, direct repeats, and sigma factor homology) and, more recently, to be highly conserved among TFIID from different species . Deletion mutant cotranslation studies revealed that TFIID binds DNA as a monomer . The implications of these results for TFIID structure and function are discussed.

Nucleic Acids Res, 1990 Jun 25, 18(12), 3529 - 35
Transcriptional slippage occurs during elongation at runs of adenine or thymine in Escherichia coli; Wagner LA et al.; A run of 11 adenine or thymine residues at the 5' end of an out-of-frame lacZ gene causes a high level of beta-galactosidase expression in E . coli . This effect was not observed for a run of guanine residues . Reverse transcription of mRNA isolated from E . coli containing the run of 11 A's reveals heterogeneity of transcript length while reverse transcription of mRNA isolated from S . cerevisiae containing the same gene shows no heterogeneity . Protein sequencing of the beta-galactosidase molecules derived from the out-of-frame construct containing a run of adenines reveals the addition of a lysine at the run . A new method was developed where messages small enough to allow resolution of single nucleotide differences on an acrylamide gel are electrophoresed, electroblotted onto nylon and probed . This confirmed the reverse transcription results and showed that additional residues can be added to transcripts derived from DNA containing 10 or 11 thymine residues . A mechanism for slippage is discussed where the A-U rich RNA-DNA hybrid can denature during elongation and rehybridize in an offset position, causing the addition of extra residues to the transcript.

FEBS Lett, 1990 Jun 18, 266(1-2), 21 - 5
Expression and pharmacological characterization of the human M1 muscarinic receptor in Saccharomyces cerevisiae; Payette P et al.; The yeast S . cerevisiae has been examined as a heterologous host for the expression of mammalian neurotransmitter receptors which couple to guanine nucleotide regulatory (G) proteins . A cloned gene encoding the M1 subtype of human muscarinic receptor (HM1) was transformed into S . cerevisiae on a high copy plasmid under the control of the promoter for the yeast alcohol dehydrogenase (ADH) gene . Northern blotting demonstrated the presence of HM1 transcripts in transformants, and crude membranes prepared from these cells showed saturable binding of the muscarinic antagonist {3H}N-methyl scopolamine with a Kd of 179 pM and Bmax of 20 fmol/mg protein . Competition binding studies revealed pharmacological properties for these sites which were comparable to those reported for the M1 site in mammalian tissues . Yeast expressing HM1 did not exhibit high affinity agonist binding or cell-cycle arrest in the presence of muscarinic agonists, indicating that the mammalian receptor did not couple to the endogenous yeast G protein.

FEMS Microbiol Lett, 1990 Jun 15, 58(1), 67 - 72
Transmission of killer activity into laboratory and industrial strains of Saccharomyces cerevisiae by electroinjection; Salek A et al.; The killer character was electrically introduced into protoplasts of three yeast strains . These were the killer-negative variant of the K1 killer strain Saccharomyces cerevisiae T 158 C (his-); the killer-sensitive laboratory strain S . cerevisiae AH 215 (leu-, his-); and the killer-sensitive industrial strain S . cerevisiae AS 4/H2 (rho-) . The killer dsRNA used for electroinjection was isolated from the super-killer strain S . cerevisiae T 158 C . Optimum numbers of transformed cells were obtained after regeneration and selection in appropriate media if the protoplasts were exposed to three exponentially decaying field pulses of 18.2 kV/cm strength and 40 microseconds duration at 4 degrees C . In the case of the killer-negative variant of S . cerevisiae T 158 C the majority of the protoplasts were transformed, whereas in the case of the two other strains the yield of transformed clones was much less . This latter result is expected if the expression of the electroinjected dsRNA was diminished in these two strains . Gel electrophoresis of the dsRNA of the clones of the three strains supported the conclusion that the transformed clones exhibited killer activity . The transformed clones of all three species were stable.

Cell, 1990 Jun 15, 61(6), 965 - 78
The NUP1 gene encodes an essential component of the yeast nuclear pore complex; Davis LI et al.; Monoclonal antibodies generated against a family of related nuclear pore complex proteins (nucleoporins) from rat liver nuclei cross-react with several proteins in the yeast S . cerevisiae and show punctate nuclear envelope staining similar to the pattern seen in mammalian cells . We have cloned a gene encoding one of these proteins (NUP1) and have confirmed the localization of the NUP1 protein to the pore complex by immunofluorescence, using an epitope-tagged construct to differentiate it from other members of this family . The NUP1 protein is essential for cell viability, and overexpression from the yeast GAL10 promoter prevents further cell growth . The central domain of NUP1 consists of a series of degenerate repeats similar to those found in the nucleoskeletal protein NSP1, a protein that cross-reacts with monoclonal antibodies against NUP1 . We propose that the repetitive domain is a feature common to the nucleoporins.

Cell, 1990 Jun 15, 61(6), 1089 - 101
A pathway for generation and processing of double-strand breaks during meiotic recombination in S . cerevisiae; Cao L et al.; We have identified and analyzed a meiotic reciprocal recombination hot spot in S . cerevisiae . We find that double-strand breaks occur at two specific sites associated with the hot spot and that occurrence of these breaks depends upon meiotic recombination functions RAD50 and SPO11 . Furthermore, these breaks occur in a processed form in wild-type cells and in a discrete, unprocessed form in certain nonnull rad50 mutants, rad50S, which block meiotic prophase at an intermediate stage . The breaks observed in wild-type cells are similar to those identified independently at another recombination hot spot, ARG4 . We show here that the breaks at ARG4 also occur in discrete form in rad50S mutants . Occurrence of breaks in rad50S mutants is also dependent upon SPO11 function . These observations provide additional evidence that double-strand breaks are a prominent feature of meiotic recombination in yeast . More importantly, these observations begin to define a pathway for the physical changes in DNA that lead to recombination and to define the roles of meiotic recombination functions in that pathway.

Biochem Biophys Res Commun, 1990 Jun 15, 169(2), 369 - 76
Yeast acetyl-CoA carboxylase: in vitro phosphorylation by mammalian and yeast protein kinases; Witters LA et al.; Acetyl-CoA carboxylase (ACC) is regulated in mammalian tissues, in part, by multisite enzyme phosphorylation . Yeast ACC (Y-ACC) has been highly purified from S . cerevisiae by monomeric avidin-Sepharose chromatography, revealing an enzyme subunit species of molecular mass 265,000 Da . Unlike mammalian enzyme, Y-ACC is citrate-independent, and reacts weakly or not at all with a panel of anti-rat liver ACC antibodies . Like rat ACC, Y-ACC is rapidly phosphorylated and inactivated by two mammalian carboxylase kinases, the cAMP-dependent protein kinase and 5'-AMP-stimulated kinase . It is also phosphorylated by rat liver casein kinase II, but without any change in catalytic activity . Three major yeast protein kinases active on ACC have been fractionated; all co-elute with kinases active on casein, but each appears to be a distinct catalytic species . Like the mammalian casein kinases, however, phosphorylation of ACC by these yeast kinases does not alter yeast ACC activity . Taken together, these data indicate that Y-ACC possesses at least two classes of phosphorylation sites, one or more of which acutely regulates enzyme activity . Alterations in Y-ACC phosphorylation in yeast, as in mammalian tissues, could be an important modulator of the rates of fatty acid synthesis.

Curr Genet, 1990 Jun, 17(6), 499 - 506
Primary and secondary structure of the 25S rRNA from the dimorphic fungus Mucor racemosus; Ji GE et al.; A 9.76 Kb ribosomal DNA repeat unit from the nuclear genome of the dimorphic fungus Mucor racemosus (Zygomycetes) was identified using a hybridization probe from the yeast Saccharomyces cerevisiae (Ascomycetes) . This material was cloned in Escherichia coli plasmids as four overlapping pieces and mapped with respect to cleavage sites for 12 restriction endonucleases . The nucleotide sequence of the complete 25S rRNA gene and flanking regions was determined . The 5' and 3' ends of the structural gene were identified by comparison with the published sequence for the S . cerevisiae gene . The Mucor gene was found to possess 3469 bp and have a GC content of 42.8% . It was compared with the homologous gene from several other eukaryotes and found to be most similar to that from Saccharomyces . A potential secondary structure of the putative RNA transcript consistent with the structures proposed for the E . coli and Saccharomyces molecules was constructed by computer modelling.

Mol Gen Genet, 1990 Jun, 222(1), 87 - 96
Differential regulation of STA genes of Saccharomyces cerevisiae; Pugh TA et al.; The single glucoamylase gene (SGA1) of the yeast Saccharomyces cerevisiae is expressed exclusively during the sporulation phase of the life cycle . Enzymatic studies and nucleic acid sequence comparisons have shown that the SGA1 glucoamylase is closely related to the secreted enzymes of S . cerevisiae var . diastaticus . The latter are encoded by any of three unlinked STA genes, which have been proposed to derive from the ancestral SGA1 form by genomic rearrangement . We show that the regulation of SGA1 is distinct from that of the other members of the STA gene family . SGA1 expression did not respond to STA10, the primary determinant of glucoamylase expression from STA2 . Unlike STA2, SGA1 was not regulated directly by the mating type locus . Expression of SGA1 depended on the function of the MAT products in supporting sporulation and not on the formation of haploid progeny spores or on the composition of the mating type locus per se . We conclude that the STA genes acquired regulation by STA10 and MAT by the genomic rearrangements that led to their formation . This regulation is thus distinct from that of the ancestral SGA1 gene.

J Lipid Res, 1990 Jun, 31(6), 1121 - 9
Novel fatty acyl substrates for myristoyl-CoA:protein N-myristoyl-transferase; Heuckeroth RO et al.; Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristic acid to the NH2-terminal Gly residues of a number of viral and cellular proteins . The remarkable specificity of this enzyme for myristoyl CoA observed in vivo appears to arise in large part from a cooperativity between NMT's acylCoA and peptide binding sites: the length of the acylCoA bound to NMT influences the interactions of peptide substrates with NMT . We have previously synthesized analogs of myristic acid with single oxygen or sulfur for methylene substitutions . These heteroatom substitutions produce significant reductions in acyl chain hydrophobicity without accompanying alterations in chain length or stereochemical restrictions . In vitro studies have shown that the CoA thioesters of these analogs are substrates for S . cerevisiae NMT and that the efficiency of their transfer to octapeptide substrates is peptide sequence-dependent . In vivo studies with cultured mammalian cells have confirmed that these fatty acid analogs are selectively incorporated into a subset of cellular N-myristoylproteins, that only a subset of analog-substituted proteins undergo redistribution from membrane to cytosolic fractions, and that these analogs can inhibit the replication of human immunodeficiency virus I and Moloney murine leukemia viruses--two retroviruses that depend upon N-myristoylation of their gag polyprotein precursors for assembly . We have now extended our analysis of NMT-acylCoA interactions by synthesizing additional analogs of myristic acid and testing them in a coupled in vitro assay system . Myristic acid analogs with two oxygen or two sulfur substitutions have hydrophobicities comparable to that of hexanoic acid and decanoic acid, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biomol Struct Dyn, 1990 Jun, 7(6), 1279 - 89
Translation-initiation promoting site on transcripts of highly expressed genes from Saccharomyces cerevisiae and the role of hairpin stems to position the site near the initiation codon; Thanaraj TA et al.; It is reported that the AUG-upstream region on mRNAs of highly expressed genes from S . cerevisiae invariably possesses a translation-initiation promoting site, that can base pair with a well-conserved site on 18S rRNA during the formation of 40S initiation complex . Weak hairpin stem in the mRNA region between such a site and the initiation codon brings the site nearer to the initiation codon and also extends the length of base pairing . Such a base pairing can lead to a comparatively more stable 40S initiation complex, resulting in a higher rate of formation of the 80S initiation complex and consequently in an enhancement of the rate of initiation of translation . The site on 18S rRNA can interchange its base pairing between the site on mRNA and a well-conserved site on 25S rRNA in the formation of the 80S initiation complex.

Mol Cell Biol, 1990 Jun, 10(6), 2832 - 9
Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription; Ponticelli AS et al.; The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13 . On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms . To begin a study of his3 transcription in vitro, we used S . cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo . We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters . In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein . In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo . The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element . However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript . This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro . The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

J Virol, 1990 Jun, 64(6), 2594 - 8
The cauliflower mosaic virus open reading frame VII product can be expressed in Saccharomyces cerevisiae but is not detected in infected plants; Wurch T et al.; Antiserum was prepared against a synthetic peptide corresponding to the N-terminal 20 amino acids of the protein encoded by cauliflower mosaic virus (CaMV) open reading frame VII (ORF VII) . This antiserum was used to detect the expression of CaMV ORF VII either in Saccharomyces cerevisiae transformed by an expression vector containing CaMV ORF VII or in CaMV-infected plants . Only in S . cerevisiae has a 14-kilodalton protein been detected.

Biotechniques, 1990 Jun, 8(6), 628 - 32
Detection of picogram amounts of nucleic acid by dot blot hybridization; Pepin RA et al.; An increasing number of human proteins isolated from cell sources are being produced for pharmaceutical use . Consequently, federal agencies have required the quantitative determination of residual nucleic acids that copurify with the potential protein products . We have conducted these assays in connection with our application for licensure of Alferon Injection . We report a sensitive dot blot hybridization assay that was used to quantitate picogram (or less) amounts of nucleic acids which copurified with human proteins isolated from recombinant (S . cerevisiae) or natural (leukocytes) sources.

Mol Cell Biol, 1990 Jun, 10(6), 2599 - 605
RNA processing in vitro produces mature 3' ends of a variety of Saccharomyces cerevisiae mRNAs; Butler JS et al.; Ammonium sulfate fractionation of a Saccharomyces cerevisiae whole-cell extract yielded a preparation which carried out correct and efficient endonucleolytic cleavage and polyadenylation of yeast precursor mRNA substrates corresponding to a variety of yeast genes . These included CYC1 (iso-1-cytochrome c), HIS4 (histidine biosynthesis), GAL7 (galactose-1-phosphate uridyltransferase), H2B2 (histone H2B2), PRT2 (a protein of unknown function), and CBP1 (cytochrome b mRNA processing) . The reaction processed these pre-mRNAs with varying efficiencies, with cleavage and polyadenylation exceeding 70% in some cases . In each case, the poly(A) tail corresponded to the addition of approximately 60 adenosine residues, which agrees with the usual length of poly(A) tails formed in vivo . Addition of cordycepin triphosphate or substitution of CTP for ATP in these reactions inhibited polyadenylation but not endonucleolytic cleavage and resulted in accumulation of the cleaved RNA product . Although this system readily generated yeast mRNA 3' ends, no processing occurred on a human alpha-globin pre-mRNA containing the highly conserved AAUAAA polyadenylation signal of higher eucaryotes . This sequence and adjacent signals used in mammalian systems are thus not sufficient to direct mRNA 3' end formation in yeast . Despite the lack of a highly conserved nucleotide sequence signal, the same purified fraction processed the 3' ends of a variety of unrelated yeast pre-mRNAs, suggesting that endonuclease cleavage and polyadenylation may produce the mature 3' ends of all mRNAs in S . cerevisiae.

Mol Cell Biol, 1990 Jun, 10(6), 3174 - 84
Structure of the DNA damage-inducible gene DDR48 and evidence for its role in mutagenesis in Saccharomyces cerevisiae; Treger JM et al.; The DDR48 gene of Saccharomyces cerevisiae is a member of a set of genes that displays increased transcription in response to treatments that produce DNA lesions or to heat-shock stress . Other members of this group include the DDRA2 and UBI4 genes . DNA sequence analysis of the DDR48 gene demonstrates the presence of two overlapping open reading frames, each of which has the capacity to encode a protein with a molecular mass of approximately 45 kilodaltons . Fusions of the DDR48 coding sequences to lacZ demonstrates that only one of these frames is expressed in yeast cells . The protein predicted from this sequence is extremely hydrophilic and contains multiple repeats of the peptide sequence Ser-Asn-Asn-X-Asp-Ser-Tyr-Gly where X is either Asn or Asp . Additionally, closely related sequences are found throughout the primary sequence . Primer extension data indicate that, after 4-nitroquinoline-1-oxide and heat-shock treatments, there are three major and two minor transcriptional start sites which are utilized . The function of the DDR48 gene was investigated by disrupting this gene in diploid cells . Viable haploid cells containing the DDR48 gene disruption were isolated after tetrad analysis . Although the ddr48 mutant showed a slightly altered sensitivity to killing by 4-nitroquinoline-1-oxide and to heat shock compared with the DDR48 haploid, the spontaneous mutation rate of reversion of a his4 mutation was reduced 6- to 14-fold in the ddr48 strain . These results implicate the DDR48 gene in the production or recovery of mutations in S . cerevisiae.

Enzyme Microb Technol, 1990 Jun, 12(6), 419 - 30
Hydrodynamic deposition: a novel method of cell immobilization; Salter GJ et al.; A novel method of cell immobilization is described . The cell support consists of ceramic microspheres of approximately 50-75 microns diameter . The spheres are hollow, having a wall thickness of 10-15 microns and one entrance (ca . 20 microns diameter) . The walls are porous with a mean pore size of approximately 90 nm . When a cell suspension (of S . cerevisiae) is passed through a column of such particles, cells are immobilized . Conditions are devised such that the overwhelming majority of cells are held in the central cavity of the support and not between the particles . Provided turbulence is avoided, the distribution of cells along the column length in the steady state is rather homogeneous . The facts that (a) essentially all particles, regardless of orientation, entrap cells, and (b) nonporous particles also entrap cells with high efficiency, indicate that filtration effects are irrelevant and that heretofore unrecognized hydrodynamic forces are alone responsible for the cell immobilization . Cells can be immobilized to high biomass densities, while the hydrodynamic properties of columns containing such immobilized cells are excellent . We describe an on-line electronic method for the real-time measurement of immobilized cellular biomass . Cell growth (so recorded) and metabolism continue to occur in such particles at high rates . Using the glycolytic production of ethanol by S . cerevisiae as a model reaction, volumetric productivities as great as any published are obtained . Thus the "lobster-pot effect" or "hydrodynamic deposition" represents a novel, promising, and generally applicable method of cell immobilization.

Gene, 1990 May 31, 90(1), 69 - 78
Escherichia coli and Saccharomyces cerevisiae acetylornithine aminotransferase: evolutionary relationship with ornithine aminotransferase; Heimberg H et al.; Genes argD and ARG8, encoding the acetylornithine aminotransferase (ACOAT) subunit in Escherichia coli and Saccharomyces cerevisiae, respectively, have been cloned and sequenced . The deduced amino acid sequences show substantial similarity . Moreover, they resemble ornithine aminotransferase (OAT) sequences (i.e., those from yeast, rat and man); the observed similarities are statistically significant, indicating that the enzymes are homologous . However, in contrast to OATs, which appear to be substrate (i.e., ornithine)-specific, S . cerevisiae ACOAT transaminates ornithine about as efficiently as E . coli does . The evolutionary relationship between ACOATs and OATs is discussed in terms of substrate ambiguity.

Gene, 1990 May 31, 90(1), 105 - 14
Transcriptional control by galactose of a yeast gene encoding a protein homologous to mammalian aldo/keto reductases; Magdolen V et al.; Expression of the S . cerevisiae gene, GCY, encoding a 35-kDa protein with striking homology to mammalian aldo/keto reductases, is under the control of galactose: the intracellular concentration of the respective mRNA (about 1300 nt in length) varies strongly with the carbon source . It is particularly high when galactose is the sole energy source but is low as soon as glucose is present . Lactate, glycerol and raffinose lead to intermediate expression . Both Northern blot analyses and lacZ fusion data indicate a 20- to 50-fold increase in the steady state concentrations of mRNA and beta Gal activity, respectively, when grown on galactose as compared to glucose . The gene is derepressed after cultivation on glycerol in the wt and in a gal80 mutant background but remains uninducible by galactose in strains carrying either a gal2 or a gal4 mutation, affecting galactose permease and the GAL gene trans-activator, respectively . Analysis of GCY expression in gal regulatory mutants reveals epistasis interactions of the gal4 and the gal80 mutations as expected if GCY is regulated by the Gal control system . Repression of GCY transcription by glucose is observed in all three above gal mutant strains . The results suggest that the gene is both positively controlled by galactose and negatively by glucose . Analysis of a set of upstream deletions identifies a single UAS matching the consensus for GAL gene upstream regulation sites . By contrast to other genes regulated by galactose, disruption mutants of GCY exhibit no obvious phenotype, and in particular do not lose the ability to grow on and adapt to galactose . Enzyme tests with AKR-specific substrates suggest that GCY encodes a carbonyl reductase.

J Biol Chem, 1990 May 25, 265(15), 8802 - 7
The ANB1 locus of Saccharomyces cerevisiae encodes the protein synthesis initiation factor eIF-4D; Mehta KD et al.; The Saccharomyces cerevisiae anaerobic gene (ANB1) is negatively regulated both by oxygen and heme . We have shown recently that an upstream repressor site located in the 5'-flanking region of this gene controls its expression (Mehta, K.D., and Smith, M . (1989) J . Biol . Chem . 265, 8670-8675) . In this paper, we present the complete genomic sequence of the ANB1 locus of S . cerevisiae . The ANB1 locus encodes a protein of 157 residues with an Mr of 17, 134 . The deduced amino acid sequence of the ANB1 gene product shows strikingly extensive sequence and structural homology (63.5% identical residues and an additional 15% conservative substitutions) to the 154-amino-acid-long human and rabbit eukaryotic translation initiator factor (eIF)-4D . Factor eIF-4D is the only known mammalian protein that undergoes a unique post-translational modification of Lys-50 to the amino acid hypusine, and interestingly the same lysine is also present in the ANB1 gene product . Results presented provide strong evidence that the ANB1 locus that encodes a transcript, tr-2, and a second locus encoding a transcript, tr-1, together encode two forms of yeast eIF-4D . Interestingly, heme regulates both the loci in an opposite manner; as a result it can dictate the isoform available under conditions of high and low oxygen tension . The ROX1 locus of S . cerevisiae is known to regulate CYC1, COXVb, and ANB1 genes at the transcriptional level; the ROX1 locus thus regulates all known anaerobically expressed genes that are involved in different cellular functions such as respiration and protein synthesis.

J Biol Chem, 1990 May 25, 265(15), 8782 - 7
Transfer RNA pseudouridine synthases in Saccharomyces cerevisiae; Samuelsson T et al.; A transfer RNA lacking modified nucleosides was produced by transcription in vitro of a cloned gene that encodes a Saccharomyces cerevisiae glycine tRNA . At least three different uridines (in nucleotide positions 13, 32, and 55) of this transcript tRNA are modified to pseudouridine by an extract of S . cerevisiae . Variants of the RNA substrate were also constructed that each had only one of these sites, thus allowing specific monitoring of pseudouridylation at different nucleotide positions . Using such RNAs to assay pseudouridine synthesis, enzymes producing this nucleoside were purified from an extract of S . cerevisiae . The activities corresponding to positions 13, 32, and 55 in the tRNA substrate could all be separated chromatographically, indicating that there is a separate enzyme for each of these sites . The enzyme specific for position 55 (denoted pseudouridine synthase 55) was purified approximately 4000-fold using a combination of DEAE-Sepharose, heparin-Sepharose, and hydroxylapatite.

Science, 1990 May 18, 248(4957), 866 - 8
Enhancement of the GDP-GTP exchange of RAS proteins by the carboxyl-terminal domain of SCD25; Crechet JB et al.; In Saccharomyces cerevisiae, the product of the CDC25 gene controls the RAS-mediated production of adenosine 3',5'-monophosphate (cAMP) . In vivo the carboxyl-terminal third of the CDC25 gene product is sufficient for the activation of adenylate cyclase . The 3'-terminal part of SCD25, a gene of S . cerevisiae structurally related to CDC25, can suppress the requirement for CDC25 . Partially purified preparations of the carboxy-terminal domain of the SCD25 gene product enhanced the exchange rate of guanosine diphosphate (GDP) to guanosine triphosphate (GTP) of pure RAS2 protein by stimulating the release of GDP . This protein fragment had a similar effect on the human c-H-ras-encoded p21 protein . Thus, the SCD25 carboxyl-terminal domain can enhance the regeneration of the active form of RAS proteins.

Cell, 1990 May 18, 61(4), 697 - 708
In vivo degradation of a transcriptional regulator: the yeast alpha 2 repressor; Hochstrasser M et al.; Metabolic instability is characteristic of regulatory proteins whose in vivo concentrations must vary as a function of time . The cell type-specific alpha 2 repressor of the yeast S . cerevisiae is shown here to have a half-life of only approximately 5 min . Each of the two structural domains of alpha 2 carries a sequence that can independently target a normally long-lived protein for rapid destruction . Moreover, these two degradation signals are shown to operate via distinct mechanisms . Mutants deficient in the degradation of alpha 2 have been isolated and found to have a number of additional defects, indicating that the pathways responsible for alpha 2 turnover include components with multiple functions . Finally, we demonstrate that a short-lived subunit of an oligomeric protein can be degraded in vivo without destabilizing other, long-lived subunits of the same protein . This subunit-specific degradation makes possible a novel type of posttranslational remodeling in which a heteromeric protein could be functionally modified by selective, degradation-mediated replacement of its subunits.

Gene, 1990 May 14, 89(2), 231 - 7
Expression of a cDNA encoding human 5-lipoxygenase under control of the STA1 promoter in Saccharomyces cerevisiae; Nakamura M et al.; A yeast-expression vector utilizing the STA1 promoter was constructed, and shown to be useful for the expression of a heterologous gene . A cDNA, encoding human 5-lipoxygenase (5LO), was inserted into the vector and expressed in Saccharomyces cerevisiae . The enzyme (yh5LO) produced in the transformant was purified to homogeneity from the cellular soluble fraction . The purified enzyme showed both 5LO and leukotriene A4 synthase activities, which were stimulated by Ca2+ and ATP . The N-terminal end of yh5LO contained five extra amino acids not present in 5LO purified from human leukocytes . A human 5LO-secretion vector containing the STA1 signal sequence was also constructed . When this hybrid gene was expressed in S . cerevisiae, its product was glycosylated and accumulated in the fractions related to the secretory pathway.

Nucleic Acids Res, 1990 May 11, 18(9), 2769 - 76
Yeast general transcription factor GFI: sequence requirements for binding to DNA and evolutionary conservation; Dorsman JC et al.; GFI is an abundant DNA binding protein in the yeast S . cerevisiae . The protein binds to specific sequences in both ARS elements and the upstream regions of a large number of genes and is likely to play an important role in yeast cell growth . To get insight into the relative strength of the various GFI-DNA binding sites within the yeast genome, we have determined dissociation rates for several GFI-DNA complexes and found them to vary over a 70-fold range . Strong binding sites for GFI are present in the upstream activating sequences of the gene encoding the 40 kDa subunit II of the QH2:cytochrome c reductase, the gene encoding ribosomal protein S33 and in the intron of the actin gene . The binding site in the ARS1-TRP1 region is of intermediate strength . All strong binding sites conform to the sequence 5' RTCRYYYNNNACG-3' . Modification interference experiments and studies with mutant binding sites indicate that critical bases for GFI recognition are within the two elements of the consensus DNA recognition sequence . Proteins with the DNA binding specificities of GFI and GFII can also be detected in the yeast K . lactis, suggesting evolutionary conservation of at least the respective DNA-binding domains in both yeasts.

Cell, 1990 May 4, 61(3), 437 - 46
Yeast centromere binding protein CBF1, of the helix-loop-helix protein family, is required for chromosome stability and methionine prototrophy; Cai M et al.; The centromere and its binding proteins constitute the kinetochore structure of metaphase chromosomes, which is crucial for the high accuracy of the chromosome segregation process . Isolation and analysis of the gene encoding a centromere binding protein from the yeast S . cerevisiae, CBF1, are described in this paper . DNA sequence analysis of the CBF1 gene reveals homology with the transforming protein myc and a family of regulatory proteins known as the helix-loop-helix (HLH) proteins . Disruption of the CBF1 gene caused a decrease in the growth rate, an increase in the rate of chromosome loss/nondisjunction, and hypersensitivity to the antimitotic drug thiabendazole . Unexpectedly, the cbf1 null mutation concomitantly resulted in a methionine auxotrophic phenotype, which suggests that CBF1, like other HLH proteins in higher eukaryotic cells, participates in the regulation of gene expression.

Cell, 1990 May 4, 61(3), 419 - 36
Analysis of wild-type and rad50 mutants of yeast suggests an intimate relationship between meiotic chromosome synapsis and recombination; Alani E et al.; The RAD50 gene of S . cerevisiae is required during meiosis for both recombination and chromosome synapsis and is also required for repair of double strand breaks during vegetative growth . We present below the isolation and analysis of several types of rad50 mutants . We show that null mutations block both meiotic recombination and formation of synaptonemal complex (SC) at early stages, while nonnull mutations block both processes at intermediate stages . These observations suggest that recombination and SC formation involve a series of intimately related events . Furthermore, all rad50 mutants block formation of tripartite SC structure but permit other aspects of SC development, i.e., formation of axial cores . In light of this and other observations, the meiotic and mitotic defects of rad50 mutants can be accounted for economically by the proposal that meiotic recombination, meiotic chromosome pairing, and vegetative DNA repair all use a common chromosomal homology search that involves RAD50 function.

J Gen Microbiol, 1990 May, 136 ( Pt 5), 861 - 6
Activation of Ca2+ influx by metabolic substrates in Saccharomyces cerevisiae: role of membrane potential and cellular ATP levels; Eilam Y et al.; Influx of Ca2+ into cells of Saccharomyces cerevisiae was measured under non-steady-state conditions, which enable measurements of the initial rate of transport across plasma membranes without interference by the vacuolar Ca2+ transport system . Removal of glucose from the incubation medium led to inactivation of Ca2+ influx within 5 min . Readdition of glucose led to a transient increase in the rate of Ca2+ transport, reaching a peak after 3-5 min . A second increase was observed 60-80 min later . To examine whether the first transient activation of Ca2+ influx by glucose was mediated by membrane hyperpolarization, influx of 45Ca2+ was measured in the presence and absence of metabolic substrates (glucose, glycerol, and glucose plus antimycin A) in cells hyperpolarized to different values of membrane potential (delta psi) . Logarithms of the rate of Ca2+ influx were plotted against values of delta psi . Two different slopes were obtained, depending upon whether the metabolic substrate was present or absent . Ca2+ influx in the presence of the metabolic substrates was always higher than expected by their effect on delta psi . Glycerol plus antimycin A did not affect Ca2+ influx . It was concluded that metabolized substrates activate Ca2+ influx not only by effects on delta psi but also by additional mechanism(s) . Since no simple correlation between Ca2+ influx and intracellular ATP levels was observed, it was concluded that ATP levels do not affect the initial rates of Ca2+ transport across the plasma membrane of S . cerevisiae.

Biol Chem Hoppe Seyler, 1990 May, 371 Suppl, 37 - 42
Aprotinin and aprotinin analogues expressed in yeast; Norris K et al.; Synthetic genes encoding aprotinin and aprotinin analogues were constructed and fused in frame to the S . cerevisiae mating factor alpha 1 signal-leader (1-85) sequence . Expression in yeast resulted in secretion into the culture medium of a moderate yield of correctly processed aprotinin (1-58) together with two N-terminally extended forms . Des-Arg1, Pro2-aprotinin was expressed in a higher yield . In this case only the correct N-terminal amino acid sequence was found . Substitution of Ser42 for Arg42 in the potential internal KEX2 processing site improved the secretion yield . The aprotinins are characterized by an inhibition profile similar to that of native aprotinin . Des-Arg1, Pro2-{Arg15, Ser42} aprotinin has a strongly increased plasma kallikrein inhibition profile.

Gene, 1990 Apr 30, 89(1), 139 - 44
Heterogeneity among the 2 microns plasmids in Saccharomyces cerevisiae: a new sequence for the REP1 gene; Neuville P et al.; Some species of yeasts contain naturally-occurring circular DNA plasmids . The most studied of these plasmids is the 2 microns circle of Saccharomyces cerevisiae . Three variants of this plasmid, Scp1, Scp2 and Scp3, have been described according to their restriction maps {Cameron et al., Nucleic Acids Res . 4 (1977) 1429-1448; Livingston, Genetics 86 (1977) 73-84} . The entire nucleotide (nt) sequence of the Scp1 variant from strain A364A has been published {Hartley and Donelson, Nature 286 (1980) 860-864} . We report here the nt sequence of the 2 microns plasmid REP1 gene from S . cerevisiae strain SKQ2n . According to the restriction analysis, this plasmid is the Scp3 variant previously described . The only observed differences between the Scp1 and Scp3 variants were the loss of one EcoRI restriction site and an apparent deletion in Scp3 . The nt sequence we report differs significantly from the previously published one for Scp1 . The differences correspond to 128 (about 8.5%) substituted, deleted or additional nt of 1510 nt compared . These differences affect the coding region (8%) as well as the noncoding regions (9.7%) . Regarding the putative encoded proteins, 38 (about 10%) amino acids (aa) are modified or deleted in our sequence and 11 are added . Most of these aa modifications are not randomly distributed but are concentrated in certain regions . These observations are indicative of important intraspecific evolution between the two 2 microns plasmid variants considered, as well as of conservative selection pressure on some domains of the REP1 protein.

Cell, 1990 Apr 20, 61(2), 319 - 27
Cloning and characterization of CAP, the S . cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein; Field J et al.; Adenylyl cyclase from S . cerevisiae contains at least two subunits, a 200 kd catalytic subunit and a subunit with an apparent molecular size of 70 kd, which we now call CAP (cyclase-associated protein) . We cloned a cDNA encoding CAP by screening a yeast cDNA expression library in E . coli with antisera raised against the purified protein . The cDNA contained an open reading frame capable of encoding a 526 amino acid protein that is not homologous to any sequences in the current data bases . Adenylyl cyclase activity in membranes from cells that lacked CAP was not stimulated by RAS2 proteins in vitro . These results suggest that CAP is required for at least some aspects of the RAS-responsive signaling system . Mutants lacking CAP had four additional phenotypes that appear to be unrelated to effects of the RAS/adenylyl cyclase pathway: the inability to grow on rich medium (YPD), temperature sensitivity on minimal medium, sensitivity to nitrogen starvation, and a swollen cell morphology.

Biochim Biophys Acta, 1990 Apr 6, 1048(2-3), 265 - 9
The complex between ribosomal proteins and aminoacyl-tRNA: the interactions and hydrolytic activities are not confined to the proteins L2 and L16 of Escherichia coli ribosomes; Sumpter VG et al.; The capacity of some Escherichia coli (E . coli) ribosomal proteins to bind to tRNA and to hydrolyse their aminoacylated derivatives has been analysed . The following results were obtained: (1) The basic proteins L2, L16 and L33 and S20 bound f{3H}Met-tRNA to a similar extent as the total proteins from 30 S (TP30) or 50 S (TP50) when tested by nitrocellulose filtration, in contrast to the more acidic proteins L7/L12 and S8 . (2) The proteins of the peptidyltransferase centre, L2 and L16, showed no distinct specificity, binding various charged tRNAs from E . coli and Saccharomyces cerevisiae (S . cerevisiae) . (3) A number of isolated ribosomal proteins hydrolysed aminoacyl-tRNA as assessed by trichloroacetic acid precipitation, in contrast to the TP30 and TP50 . (4) The loss of radiolabel from Ac{14C}Phe-tRNA and from {14C}tRNA in the presence of these proteins could not be prevented by RNasin, a ribonuclease inhibitor, whereas that mediated by a sample of non-RNase-free bovine serum albumin was inhibited . (5) When double-labelled, Ac{3H}Phe-{14C}tRNA was incubated with L2 both radiolabels were lost, indicating that this potential candidate for a peptidyltransferase enzyme does not specifically cleave the ester bond between the aminoacyl residue and the tRNA.

Mol Cell Biol, 1990 Apr, 10(4), 1439 - 51
HPR1, a novel yeast gene that prevents intrachromosomal excision recombination, shows carboxy-terminal homology to the Saccharomyces cerevisiae TOP1 gene; Aguilera A et al.; The HPR1 gene has been cloned by complementation of the hyperrecombination phenotype of hpr1-1 strains by using a color assay system . HPR1 is a gene that is in single copy on chromosome IV of Saccharomyces cerevisiae, closely linked to ARO1, and it codes for a putative protein of 752 amino acids (molecular mass, 88 kilodaltons) . Computer searches revealed homology (48.8% conserved homology; 24.8% identity) with the S . cerevisiae TOP1 gene in an alpha-helical stretch of 129 amino acids near the carboxy-terminal region of both proteins . The ethyl methanesulfonate-induced hpr1-1 mutation is a single-base change that produces a stop codon at amino acid 559 coding for a protein that lacks the carboxy-terminal TOP1 homologous region . Haploid strains carrying deletions of the HPR1 gene show a slightly reduced mitotic growth rate and extremely high rates of intrachromosomal excision recombination (frequency, 10 to 15%) but have a undetectable effect on rDNA recombination . Double-null mutants hpr1 top1 grow very poorly . We conclude that Hpr1 is a novel eucaryotic protein, mutation of which causes an increase in mitotic intrachromosomal excision recombination, and that it may be functionally related to an activity of the topoisomerase I protein.

Mol Cell Biol, 1990 Apr, 10(4), 1373 - 81
Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins; Tao J et al.; There are a number of yeasts that secrete killer toxins, i.e., proteins lethal to sensitive cells of the same or related species . Ustilago maydis, a fungal pathogen of maize, also secretes killer toxins . The best characterized of the U . maydis killer toxins is the KP6 toxin, which consists of two small polypeptides that are not covalently linked . In this work, we show that both are encoded by one segment of the genome of a double-stranded RNA virus . They are synthesized as a preprotoxin that is processed in a manner very similar to that of the Saccharomyces cerevisiae k1 killer toxin, also encoded by a double-strand RNA virus . Active U . maydis KP6 toxin was secreted from S . cerevisiae transformants expressing the KP6 preprotoxin . The two secreted polypeptides were not glycosylated in U . maydis, but one was glycosylated in S . cerevisiae . Comparison of known and predicted cleavage sites among the five killer toxins of known sequence established a three-amino-acid specificity for a KEX2-like enzyme and predicted a new, undescribed processing enzyme in the secretory pathway in the fungi . The mature KP6 toxin polypeptides had hydrophobicity profiles similar to those of other known cellular toxins.

Curr Genet, 1990 Apr, 17(4), 281 - 8
A genetic analysis of glucoamylase activity in the diastatic yeast Saccharomyces cerevisiae NCYC 625; Patel D et al.; The wild diastatic yeast Saccharomyces cerevisiae NCYC 625 has been shown to be homozygous for the glucoamylase-specifying gene STA2 . spoII-1-mapping has positioned STA2 on chromosome II . Expression of STA2 is suppressed in some but not all diploids capable of sporulation, and is also inhibited by unlinked nuclear suppressor genes (SGL) found in some S . cerevisiae tester strains . EMS-induced glucoamylase-negative mutants often contain STA2-suppressor mutations . Depending on the allelic status of GEP1, a nuclear gene which also appears able to antagonise SGL-mediated suppression, STA2 expression can be blocked in petite mutants.

Mol Cell Biol, 1990 Apr, 10(4), 1633 - 41
A bacterial amber suppressor in Saccharomyces cerevisiae is selectively recognized by a bacterial aminoacyl-tRNA synthetase; Edwards H et al.; Little is known about the conservation of determinants for the identities of tRNAs between organisms . We showed previously that Escherichia coli tyrosine tRNA synthetase can charge the Saccharomyces cerevisiae mitochondrial tyrosine tRNA in vivo, even though there are substantial sequence differences between the yeast mitochondrial and bacterial tRNAs . The S . cerevisiae cytoplasmic tyrosine tRNA differs in sequence from both its yeast mitochondrial and E . coli counterparts . To test whether the yeast cytoplasmic tyrosyl-tRNA synthetase recognizes the E . coli tRNA, we expressed various amounts of an E . coli tyrosine tRNA amber suppressor in S . cerevisiae . The bacterial tRNA did not suppress any of three yeast amber alleles, suggesting that the yeast enzymes retain high specificity in vivo for their homologous tRNAs . Moreover, the nucleotides in the sequence of the E . coli suppressor that are not shared with the yeast cytoplasmic tyrosine tRNA do not create determinants which are efficiently recognized by other yeast charging enzymes . Therefore, at least some of the determinants that influence in vivo recognition of the tyrosine tRNA are specific to the cell compartment and organism . In contrast, expression of the cognate bacterial tyrosyl-tRNA synthetase together with the bacterial suppressor tRNA led to suppression of all three amber alleles . The bacterial enzyme recognized its substrate in vivo, even when the amount of bacterial tRNA was less than about 0.05% of that of the total cytoplasmic tRNA.

Nucleic Acids Res, 1990 Mar 25, 18(6), 1447 - 9
Ribosomal protein L4 of Saccharomyces cerevisiae: the gene and its protein; Arevalo SG et al.; The sequence of a gene for ribosomal protein L4 of Saccharomyces cerevisiae has been determined . Unlike most ribosomal protein genes of S . cerevisiae this gene has no intron . The single open reading frame predicts that L4 is highly homologous to mammalian ribosomal protein L7a . There appear to be two genes for L4, both of which are active.

Nucleic Acids Res, 1990 Mar 25, 18(6), 1395 - 400
The basidiomycete Lentinus edodes linear mitochondrial DNA plasmid contains a segment exhibiting a high autonomously replicating sequence activity in Saccharomyces cerevisiae; Katayose Y et al.; A linear DNA plasmid, designated pLLE1, has been isolated from a mitochondrial fraction of a strain of Lentinus edodes . pLLE1(11.0 kbp) was sensitive to the 3'----5'-acting exonuclease III and resistant to the 5'----3'-acting lambda exonuclease . It showed no homology with mitochondrial and nuclear genomic DNAs of plasmidless strain as well as the pLLE1-harboring host strain of L . edodes . The 1434-bp fragment (sequences) capable of autonomous replication in the yeast Saccharomyces cerevisiae (ARSs) was cloned from pLLE1 DNA with YIp32 (pBR322 containing yeast LEU2 DNA), which displayed a high ARS activity . The cloned 1434-bp fragment was shown to lie near to the end of pLLE1 DNA (nucleotides about 800-2200) and contained three consecutive ARS consensus sequences (A/T)TTTAT(A/G)TTT(A/T) of S . cerevisiae and dispersive eight ARS consensus-like sequences . The subcloned 366-bp fragment containing the three ARSs retained original ARS activity of the 1434-bp fragment.

Cell, 1990 Mar 23, 60(6), 981 - 90
The product of fem-1, a nematode sex-determining gene, contains a motif found in cell cycle control proteins and receptors for cell-cell interactions; Spence AM et al.; We report the cloning and sequencing of fem-1, a gene required for sex determination in both germline and somatic tissues in the nematode C . elegans . Clones carrying a 5.5 kb fragment are able to rescue the progeny of a fem-1 mutant when injected into its oocytes . The major fem-1 transcript in both sexes is 2.4 kb and comprises 11 exons . It encodes a soluble, intracellular protein of 656 amino acids that includes near its N-terminus six contiguous copies of a motif found in the products of the cdc10 gene of S . pombe, the SWI6 gene of S . cerevisiae, the Notch gene of Drosophila, and the lin-12 and glp-1 genes of C . elegans.

FEMS Microbiol Lett, 1990 Mar 15, 56(3), 229 - 32
Transcriptional control of SSL1, a gene controlling alpha-specific inactivation of a-factor in Saccharomyces cerevisiae; Steden M et al.; The SSL1 gene of S . cerevisiae which is involved in the cell-type specific recovery of mating type alpha cells from cell cycle arrest by the mating hormone a-factor has been cloned as a 1 kb Sau3A fragment in a complementing plasmid from a library of S . cerevisae genomic DNA . Disruption of this gene in wild-type alpha-cells results in the ssl1- phenotype which is characterized by an extreme sensitivity to a-factor . Transcription analysis revealed that the SSL1 gene is transcribed only in MAT alpha cells but not in MAT alpha or diploid (MAT alpha/MAT alpha) cells defining it as a member of the set of alpha-specific genes.

Gene, 1990 Mar 15, 87(2), 167 - 75
Lanosterol 14 alpha-demethylase-encoding gene: systematic analysis of homologous overexpression in Saccharomyces cerevisiae using strong yeast promoters; Weber JM et al.; The Saccharomyces cerevisiae 14DM gene, encoding cytochrome P450 lanosterol 14 alpha-demethylase (14DM), was overexpressed in various S . cerevisiae strains under the control of three strong heterologous yeast transcription promoters (pADC1, pGPD, pPHO5) and under the control of its own promoter . Striking, strain-specific differences in 14DM transcription and in 14DM contents have been observed . The relative abundances of 14DM-specific mRNA and protein derived from a series of different expression plasmids were compared . It was found that the inducible PHO5 promoter in combination with the JL745 host led to the highest expression levels . 14DM-specific RNA reached up to 2% of the total cellular mRNA in this strain and approx . 3% of the total soluble yeast-cell protein was determined to be 14DM by quantitative Western blotting . By comparing the abundances of the different fusion transcripts with the transcript originating from the corresponding endogenous gene from which the promoter was derived, it could be concluded that the expression levels of the different 14DM fusion genes were far below the theoretically attainable values.

Biochemistry, 1990 Mar 13, 29(10), 2471 - 82
Localization of alpha 1----3-linked mannoses in the N-linked oligosaccharides of Saccharomyces cerevisiae mnn mutants; Alvarado E et al.; Neutral and phosphorylated N-linked oligosaccharides were isolated from Saccharomyces cerevisiae mnn9 and mnn9 gls1 mutant mannoproteins and separated into homologues that differed in the number of terminal alpha 1----3-linked mannoses . In each type of oligosaccharide, the addition of such mannose was shown to occur in an ordered rather than a random fashion . The results confirm and extend an earlier report that dealt with the N-linked oligosaccharides from yeast invertase {Trimble, R.B., & Atkinson, P.H . (1986) J . Biol . Chem . 261, 9815-9824}, and they suggest that the postulated processing pathway can be generalized to include phosphorylated and glucose-containing N-linked oligomannosides . We conclude that this processing pathway is identical for the analogous oligosaccharides from the mnn9 and wild-type strains of S . cerevisiae . Analysis of the mnn2 mnn10 mannoprotein revealed that a similar modification occurred at the branched terminus of the outer chain as well as in the core in this mutant.

Cell, 1990 Mar 9, 60(5), 803 - 7
S . cerevisiae genes IRA1 and IRA2 encode proteins that may be functionally equivalent to mammalian ras GTPase activating protein; Tanaka K et al.; The IRA1 and IRA2 genes of S . cerevisiae encode closely related proteins that also share homology with mammalian GAP (ras GTPase activating protein) . The RAS1 and RAS2 proteins overexpressed in ira mutants accumulated in the GTP-bound form, whereas in the wild-type strain the proteins were found mostly in the GDP-bound form, indicating that IRA1 and IRA2 negatively regulate the level of RAS-GTP . In contrast, the RAS2Val-19 or RAS2Thr-66 mutant protein was bound to GTP in high amounts irrespective of the IRA genotype . Overexpression of bovine GAP suppressed the phenotypes of ira mutants by reducing the level of RAS-GTP, suggesting that IRA proteins may be functionally analogous to mammalian GAP.

Cell, 1990 Mar 9, 60(5), 705 - 17
A putative ATP binding protein influences the fidelity of branchpoint recognition in yeast splicing; Burgess S et al.; We previously described a dominant suppressor of the splicing defect conferred by an A----C intron branchpoint mutation in S . cerevisiae . Suppression occurs by increasing the frequency with which the mutant branchpoint is utilized . We have now cloned the genomic region encoding the prp16-1 suppressor function and have demonstrated that PRP16 is essential for viability . A 1071 amino acid open reading frame contains sequence motifs characteristic of an NTP binding fold and further similarities to a superfamily of proteins that includes members with demonstrated RNA-dependent ATPase activity . A single nucleotide change necessary to confer the prp16-1 suppressor phenotype results in a Tyr----Asp substitution near the "A site" consensus for NTP binding proteins . We propose that PRP16 is an excellent candidate for mediating one of the many ATP-requiring steps of spliceosome assembly and that accuracy of branchpoint recognition may be coupled to ATP binding and/or hydrolysis.

Nature, 1990 Mar 8, 344(6262), 170 - 3
Gene targeting in normal and amplified cell lines; Zheng H et al.; Targeted recombination in mammalian cells is rare compared with non-homologous integration . In Saccharomyces cerevisiae the reverse is true . Differences in targeting efficiency could arise because a target of unique DNA is 200 times more dilute in mammalian genomes than it is in yeast . We tested this possibility by measuring gene targeting in normal CHO cells with two copies of the dihydrofolate reductase (DHFR) gene and in amplified CHOC 400 cells, which carry 800 copies . If the concentration of the target gene is critical, amplified cells should show an enhanced frequency of targeted recombination relative to non-homologous integration . Using a positive/negative selection protocol, we demonstrated that the efficiency of targeting into DHFR genes is indistinguishable in normal and amplified CHO cells . As targeting does not depend on the number of targets, the search for homology is not a rate-limiting step in the mammalian pathway of gene targeting . Thus, the difference in genome size is not the basis for the different outcomes of targeting experiments in S . cerevisiae and mammals.

Biochemistry, 1990 Mar 6, 29(9), 2349 - 56
Expression of UDP-glucuronosyltransferase cDNA in Saccharomyces cerevisiae as a membrane-bound and as a cytosolic form; Toghrol F et al.; The mouse clone UDPGTm-1 encodes a UDP-glucuronosyltransferase enzyme which was isolated from a lambda gt11 cDNA library constructed with phenobarbital-induced liver mRNA {Kimura, T., & Owens, I . S . (1987) Eur . J . Biochem . 168, 515-521} . In order to establish substrate specificity, UDPGTm-1 was inserted into the yeast vector pEVP11 and expressed in Saccharomyces cerevisiae strain AH22 . Cells transformed with the expression unit pUDPGTm-1c (insert in correct orientation with respect to promoter) stably transcribe the transferase cDNA . Consistent with the presence of mRNA, pUDPGTm-1c-transformed AH22 cells synthesize a transferase protein with Mr congruent to 51,000 by Western immunoblot analysis . The membrane-bound transferase expressed in yeast in glycosylated as indicated by its enhanced electrophoretic mobility in a SDS-polyacrylamide gel following endoglycosidase H treatment and detection by Western immunoblot analysis . A survey, using 12 aglycons in an assay with microsomes from cells which express the protein, shows preferential glucuronidation of naphthol and estrone followed by p-nitrophenol . Testosterone, phenolphthalein, dihydrotestosterone, androsterone, and 4-methylumbelliferone are conjugated at an intermediate level . There is barely detectable glucuronidation of 3-hydroxy- and 9-hydroxybenzo{a}pyrene and no detectable conversion of morphine or lithocholic acid . The truncated cDNA (lacking the putative membrane-insertion signal-peptide coding sequence, but with a newly adapted translation-start codon) is ligated into pAAH5 and is expressed as a cytosolic transferase form in the protease-deficient ZA521 strain of S . cerevisiae . The Mr congruent to 51,000-52,000 is similar to that seen in microsomes from AH22 cells where the protein is presumably processed as it is inserted into the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Cell Biol, 1990 Mar, 10(3), 1174 - 9
In vivo analysis of the Saccharomyces cerevisiae HO nuclease recognition site by site-directed mutagenesis; Nickoloff JA et al.; HO nuclease introduces a specific double-strand break in the mating-type locus (MAT) of Saccharomyces cerevisiae, initiating mating-type interconversion . To define the sequence recognized by HO nuclease, random mutations were produced in a 30-base-pair region homologous to either MAT alpha or MATa by a chemical synthesis procedure . The mutant sites were introduced into S . cerevisiae on a shuttle vector and tested for the ability to stimulate recombination in an assay that mimics mating-type interconversion . The results suggest that a core of 8 noncontiguous bases near the Y-Z junction of MAT is essential for HO nuclease to bind and cleave its recognition site . Other contacts must be required because substrates that contain several mutations outside an intact core reduce or eliminate cleavage in vivo . The results show that HO site recognition is a complex phenomenon, similar to promoter-polymerase interactions.

Yeast, 1990 Mar-Apr, 6(2), 141 - 8
Cloning and sequencing of the ornithine carbamoyltransferase gene from Pachysolen tannophilus; Skrzypek M et al.; A fragment of DNA from a yeast Pachysolen tannophilus, bearing the ornithine carbamoyltransferase gene (OCTase, EC 2.1.3.3) has been cloned from a genomic library by functional complementation of the Escherichia coli OCT-negative mutant . The gene was located within the cloned segment of DNA and its coding sequence identified by DNA sequencing . This has indicated that P . tannophilus OCT gene encodes a 347 amino acid polypeptide, which shows 60% identity to the homologous Saccharomyces cerevisiae protein . The amino acid composition of its N-terminus indicates that this protein is translocated across the mitochondrial membrane . The gene can be expressed in E . coli as well as in S . cerevisiae . Comparison with other OCTases confirms a high degree of conservation among these proteins.

Mol Microbiol, 1990 Mar, 4(3), 329 - 36
Cis- and trans-acting factors involved in centromere function in Saccharomyces cerevisiae; Murphy M et al.; The function of centromeric DNA in the yeast Saccharomyces cerevisiae has been studied in detail . Twelve of the sixteen S . cerevisiae centromeres have been sequenced to date, and a consensus sequence has been identified . This sequence consists of a central region 78 to 86bp in length which is greater than 90% A + T, usually in runs of As and runs of Ts . The central region is flanked on one side by a highly conserved 8bp sequence and on the other side by a highly conserved 25bp sequence which contains partial dyad symmetry around a central C/G base pair . Mutational analyses have been used to determine the importance of each subset of the consensus sequence to centromere function . A protein which binds to the 8bp sequence and at least one that binds to the 25bp sequence have been identified . The roles of these proteins in centromere function in mitosis and meiosis are currently under investigation.

Yeast, 1990 Mar-Apr, 6(2), 127 - 37
A novel aspartyl protease allowing KEX2-independent MF alpha propheromone processing in yeast; Egel-Mitani M et al.; Mutants of Saccharomyces cerevisiae which lack the KEX2-encoded endopeptidase are unable to process proteolytically the mating factor alpha (MF alpha) propheromone produced from the chromosomal MF alpha 1 and MF alpha 2 genes (Julius et al., 1983) . Overproduction of pheromone precursor from multiple, plasmid-borne MF alpha genes did, however, lead to the production of active MF alpha peptides in the absence of the KEX2 gene product . S . cerevisiae therefore must possess an alternative processing enzyme . The cleavage site of this enzyme appeared identical to that of the KEX2-encoded endopeptidase . To identify the gene responsible for the alternative processing, we have isolated clones which allowed production of mature MF alpha in a kex2-disrupted strain even from the chromosomal MF alpha genes . The gene isolated in this way was shown also to be essential for the KEX2-independent processing of propheromone overproduced from plasmid-borne MF alpha 1 . The amino acid sequence deduced from the gene shows extensive homology to a number of aspartyl proteases including the PEP4 and BAR1 gene products from S . cerevisiae . In contrast to the BAR1 gene product, the novel aspartyl protease (YAP3 for Yeast Aspartyl Protease 3) contains a C-terminal serine/threonine-rich sequence and potential transmembrane domain similar to those found in the KEX2 gene product . The corresponding gene YAP3 was located to chromosome XII . The normal physiological role of the YAP3 gene product is not known . Strains disrupted in YAP3 are both viable and able to process the mating factor a precursor.

EMBO J, 1990 Mar, 9(3), 705 - 9
A yeast tRNA precursor containing a pre-mRNA intron is spliced via the pre-mRNA splicing mechanism; Kohrer K et al.; We have replaced the 14 nucleotide long intervening sequence of the Saccharomyces cerevisiae SUP6 (ochre) tRNA(Tyr) gene by the 52 nucleotide long second intron of the S . cerevisiae MATa1 gene . Yeast cells containing this modified pre-tRNA showed the typical suppressor phenotype indicating that the MATa1 pre-mRNA intron was exactly excised in vivo from the primary tRNA transcript and a mature and functional tRNA was formed . Several lines of evidence show that the splicing reaction proceeded via the pre-mRNA splicing mechanism: the reaction yielded a lariat shaped excised intron with a lariat shaped intron-exon 2 molecule as intermediate; point mutations in the conserved UAC-UAAC box of the intron impaired splicing of the precursor RNA; in a temperature sensitive rna2 strain splicing of this tRNA precursor was inhibited at the restrictive temperature . Our results imply that in yeast the excision of a pre-mRNA intron is not dependent on the transcription apparatus by which it was generated and that transcription and splicing are uncoupled processes in vivo, too . Furthermore these data demonstrate that recognition of an RNA as a substrate for a pre-mRNA splicing reaction is, at least qualitatively, only intron dependent.

EMBO J, 1990 Mar, 9(3), 653 - 61
Myoinositol gets incorporated into numerous membrane glycoproteins of Saccharomyces cerevisiae; incorporation is dependent on phosphomannomutase (sec53); Conzelmann A et al.; We recently described a 125 kd membrane glycoprotein in Saccharomyces cerevisiae which is anchored in the lipid bilayer by an inositol-containing phospholipid . We now find that when S . cerevisiae cells are metabolically labeled with {3H}myoinositol, many glycoproteins become labeled more strongly than the 125 kd protein . Myoinositol is attached to these glycoproteins as part of a phospholipid moiety which resembles glycophospholipid anchors of other organisms . Labeling of proteins with {3H}myoinositol for short times and in secretion mutants blocked at various stages of the secretory pathway shows that these phospholipid moieties can be added to proteins in the endoplasmic reticulum and that these proteins are transported to the Golgi by the regular secretory pathway . sec53, a mutant which cannot produce GDP-mannose at 37 degrees C, does not incorporate myoinositol or palmitic acid into membrane glycoproteins at this temperature, suggesting that GDP-mannose is required for the biosynthesis of these phospholipid moieties . All other secretion and glycosylation mutants tested add phospholipid moieties to proteins normally.

EMBO J, 1990 Mar, 9(3), 641 - 51
In vitro reconstitution of cdc25 regulated S . cerevisiae adenylyl cyclase and its kinetic properties; Engelberg D et al.; The attenuated GTP regulation adenylyl cyclase (CDC35) lysates or membranes prepared from cells of a cdc25ts strain is enhanced 2.5- to 6-fold by mixing these lysates or membranes with lysates or membranes from a cdc35ts strain harboring wild-type CDC25 . The kinetics of activation of the Saccharomyces cerevisiae adenylyl cyclase in vitro is first order, as is the activation of mammalian adenylyl cyclase . The rate of enzyme activation in the presence of non-hydrolysable analogs of GTP increases with the number of CDC25 gene copies present in the cell . When GppNHp was used the rate of activation of the cyclase in a strain harboring a multicopy plasmid of CDC25 was 7.0-fold higher than the rate in an isogenic strain with the cdc25-2 mutation . The rate of adenylyl cyclase activation from a strain with a disrupted CDC25 gene is 14.7-fold lower than the rate in an isogenic strain containing the CDC25 gene on a multicopy plasmid . The reconstitution experiments described provide direct biochemical evidence for the role of the CDC25 protein in regulating the RAS dependent adenylyl cyclase in S.cerevisiae . The reconstitution experiments and the kinetic experiments may also provide a biochemical assay for the CDC25 protein and can form the basis for its characterization . In this study we also show that adenylyl cyclase activity in ras1ras2byc1 cells is found in the soluble fraction, whereas in wild-type strain it is found in the membrane fraction . Overexpression of the gene CDC25 in the ras1ras2bcy1 strain relocalizes adenylyl cyclase activity to the membrane fraction . This finding suggests a biochemical link between CDC25 and CDC35 in the absence of RAS, in addition to its role in regulating RAS dependent adenylyl cyclase.

Mol Cell Biol, 1990 Mar, 10(3), 898 - 909
Vanadate-resistant mutants of Saccharomyces cerevisiae show alterations in protein phosphorylation and growth control; Kanik-Ennulat C et al.; This work describes two spontaneous vanadate-resistant mutants of Saccharomyces cerevisiae with constitutive alterations in protein phosphorylation, growth control, and sporulation . Vanadate has been shown by a number of studies to be an efficient competitor of phosphate in biochemical reactions, especially those that involve phosphoproteins as intermediates or substrates . Resistance to toxic concentrations of vanadate can arise in S . cerevisiae by both recessive and dominant spontaneous mutations in a large number of loci . Mutations in two of the recessive loci, van1-18 and van2-93, resulted in alterations in the phosphorylation of a number of proteins . The mutant van1-18 gene also showed an increase in plasma membrane ATPase activity in vitro and a lowered basal phosphatase activity under alkaline conditions . Cells containing the van2-93 mutant allele had normal levels of plasma membrane ATPase activity, but this activity was not inhibited by vanadate . Both of these mutants failed to enter stationary phase, were heat shock sensitive, showed lowered long-term viability, and sporulated on rich medium in the presence of 2% glucose . The wild-type VAN1 gene was isolated and sequenced . The open reading frame predicts a protein of 522 amino acids, with no significant homology to any genes that have been identified . Diploid cells that contained two mutant alleles of this gene demonstrated defects in spore viability . These data suggest that the VAN1 gene product is involved in regulation of the phosphorylation of a number of proteins, some of which appear to be important in cell growth control.

Mol Cell Biol, 1990 Mar, 10(3), 1066 - 75
Association of catalytic and regulatory subunits of cyclic AMP-dependent protein kinase requires a negatively charged side group at a conserved threonine; Levin LR et al.; In Saccharomyces cerevisiae, as in higher eucaryotes, cyclic AMP (cAMP)-dependent protein kinase is a tetramer composed of two catalytic (C) subunits and two regulatory (R) subunits . In the absence of cAMP, the phosphotransferase activity of the C subunit is inhibited by the tight association with R . Mutation of Thr-241 to Ala in the C1 subunit of S . cerevisiae reduces the affinity of this subunit for the R subunit approximately 30-fold and results in a monomeric cAMP-independent C subunit . The analogous residue in the mammalian C subunit is known to be phosphorylated . Peptide maps of in vivo 32P-labeled wild-type C1 and mutant C1(Ala241) suggest that Thr-241 is phosphorylated in yeast cells . Substituting Thr-241 with either aspartate or glutamate partially restored affinity for the R subunit . Uncharged and positively charged residues substituted at this site resulted in C subunits that failed to associate with the R subunit . Replacement with the phosphorylatable residue serine resulted in a C subunit with wild-type affinity for the R subunit . Analysis of this protein revealed that it appears to be phosphorylated on Ser-241 in vivo . These data suggest that the interaction between R and C involves a negatively charged phosphothreonine at position 241 of yeast C1, which can be mimicked by either aspartate, glutamate, or phosphoserine.

J Gen Microbiol, 1990 Mar, 136 ( Pt 3), 395 - 403
Physiology of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures; Verduyn C et al.; The physiology of Saccharomyces cerevisiae CBS 8066 was studied in anaerobic glucose-limited chemostat cultures in a mineral medium supplemented with ergosterol and Tween 80 . The organism had a mu max of 0.31 h-1 and a Ks for glucose of 0.55 mM . At a dilution rate of 0.10 h-1, a maximal yield of 0.10 g biomass (g glucose)-1 was observed . The yield steadily declined with increasing dilution rates, so a maintenance coefficient for anaerobic growth could not be estimated At a dilution rate of 0.10 h-1, the yield of the S . cerevisiae strain H1022 was considerably higher than for CBS 8066, despite a similar cell composition . The major difference between the two yeast strains was that S . cerevisiae H1022 did not produce acetate, suggesting that the observed difference in cell yield may be ascribed to an uncoupling effect of acetic acid . The absence of acetate formation in H1022 correlated with a relatively high level of acetyl-CoA synthetase . The uncoupling effect of weak acids on anaerobic growth was confirmed in experiments in which a weak acid (acetate or propionate) was added to the medium feed . This resulted in a reduction in yield and an increase in specific ethanol production . Both yeasts required approximately 35 mg oleic acid (g biomass)-1 for optimal growth . Lower or higher concentrations of this fatty acid, supplied as Tween 80, resulted in uncoupling of dissimilatory and assimilatory processes.

Biotechnology (N Y), 1990 Mar, 8(3), 223 - 7
High efficiency transformation of intact yeast cells by electric field pulses; Meilhoc E et al.; We have developed an efficient method that electrically introduces DNA into intact yeast cells . Saccharomyces cerevisiae was used as a model in order to optimize the transformation protocol . Transformation efficiencies of 10(7) transformants/micrograms of plasmid DNA were obtained with a square wave electric pulse of 2.7 kV/cm during 15 milliseconds . The technique is simple and rapid . Even small quantities of DNA (100 pg) can be used to transform 10(8) cells . Important parameters are the pulse field strength and duration . Pretreatment of the yeast cells in the early phase of exponential growth with dithiothreitol increases transformation efficiency . The method has been successfully applied to various strains of S . cerevisiae as well as to other types of yeast.

Biochim Biophys Acta, 1990 Feb 28, 1022(2), 211 - 4
Transport of 2-methyl-4-amino-5-hydroxymethylpyrimidine in Saccharomyces cerevisiae; Iwashima A et al.; The transport of 2-methyl-4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine) was studied in resting cells of Saccharomyces cerevisiae . Hydroxymethylpyrimidine uptake was an energy- and temperature-dependent process which has an optimal pH at 4.5 . The apparent Km for hydroxymethylpyrimidine uptake was 0.37 microM, and the uptake was inhibited by 2-methyl-4-amino-5-aminomethylpyrimidine, thiamin and pyrithiamin . Furthermore, hydroxymethylpyrimidine uptake was inhibited by 4-azido-2-nitrobenzoylthiamin, a specific and irreversible inhibitor of the yeast thiamin transport system and it was greatly impaired in the thiamin transport mutant of S . cerevisiae . Thus, hydroxymethylpyrimidine is taken up by a common transport system with thiamin in S . cerevisiae, but in contrast to thiamin transport, accumulated hydroxymethylpyrimidine is released from yeast cells showing an overshoot phenomenon.

Cell, 1990 Feb 23, 60(4), 629 - 36
Integration of group II intron bI1 into a foreign RNA by reversal of the self-splicing reaction in vitro; Morl M et al.; Group II intron bI1, the first intron of the COB gene in the mitochondria of S . cerevisiae, is able to self-splice in vitro with the basic pathway similar to nuclear pre-mRNA splicing . We show that incubation of the intron lariat with ligated exons bE1 and bE2 leads to a complete reversal of the splicing reaction . The integration of the intron into the ligated exons is correct; the reconstituted preRNA of the reverse reaction can undergo a self-splicing reaction anew . When incubated with a foreign RNA species bearing a sequence motif that is complementary to exon binding site 1, the lariat can integrate into this RNA with the position of insertion immediately downstream of this sequence . This result implies that transposition of group II introns on the RNA level by reversal of the splicing reaction is, in principle, conceivable.

Biochim Biophys Acta, 1990 Feb 9, 1037(2), 147 - 54
High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates; Nosaka K; The enzymatic properties of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) encoded by PHO3 gene in Saccharomyces cerevisiae, which is repressed by thiamin and has thiamin-binding activity at pH 5.0, were investigated to study physiological functions . The following results led to the conclusion that thiamin-repressible acid phosphatase physiologically catalyzes the hydrolysis of thiamin phosphates in the periplasmic space of S . cerevisiae, thus participating in utilization of the thiamin moiety of the phosphates by yeast cells: (a) thiamin-repressible acid phosphatase showed Km values of 1.6 and 1.7 microM at pH 5.0 for thiamin monophosphate and thiamin pyrophosphate, respectively . These Km values were 2-3 orders of magnitude lower than those (0.61 and 1.7 mM) for p-nitrophenyl phosphate; (b) thiamin exerted remarkable competitive inhibition in the hydrolysis of thiamin monophosphate (Ki 2.2 microM at pH 5.0), whereas the activity for p-nitrophenyl phosphate was slightly affected by thiamin; (c) the inhibitory effect of inorganic phosphate, which does not repress the thiamin-repressible enzyme, on the hydrolysis of thiamin monophosphate was much smaller than that of p-nitrophenyl phosphate . Moreover, the modification of thiamin-repressible acid phosphatase of S . cerevisiae with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in the complete loss of thiamin-binding activity and the Km value of the modified enzyme for thiamin monophosphate increased nearly to the value of the native enzyme for p-nitrophenyl phosphate . These results also indicate that the high affinity of the thiamin-repressible acid phosphatase for thiamin phosphates is due to the thiamin-binding properties of this enzyme.

J Bacteriol, 1990 Feb, 172(2), 678 - 85
pdc1(0) mutants of Saccharomyces cerevisiae give evidence for an additional structural PDC gene: cloning of PDC5, a gene homologous to PDC1; Seeboth PG et al.; The PDC1 gene coding for a pyruvate decarboxylase (PDC; EC 4.1.1.1) was deleted from the Saccharomyces cerevisiae genome . The resulting pdc1(0) mutants were able to grow on glucose and still contained 60 to 70% of the wild-type PDC activity . Two DNA fragments with sequences homologous to that of the PDC1 gene were cloned from the yeast genome . One of the cloned genes (PDC5) was expressed at high rates predominantly in pdc1(0) strains and probably encodes the remaining PDC activity in these strains . Expression from the PDC1 promoter in PDC1 wild-type and pdc1(0) strains was examined by the use of two reporter genes . Deletion of PDC1 led to increased expression of the two reporter genes regardless of whether the fusions were integrated into the genome or present on autonomously replicating plasmids . The results suggested that this effect was due to feedback regulation of the PDC1 promoter-driven expression in S . cerevisiae pdc1(0) strains . The yeast PDC1 gene was expressed in Escherichia coli, leading to an active PDC . This result shows that the PDC1-encoded subunit alone can form an active tetramer without yeast-specific processing steps.

J Mol Evol, 1990 Feb, 30(2), 109 - 15
Doublet preference and gene evolution; Hanai R et al.; Doublet preference analysis was carried out on coding and noncoding regions of Escherichia coli, Saccharomyces cerevisiae, and human mitochondrial and nuclear DNA . The preference pattern in 1-2 and 2-3 doublets in E . coli and S . cerevisiae correlated with that in noncoding regions . The 3-1 doublet preference in E . coli genes with low optimal codon frequency and in S . cerevisiae genes also showed a correlation with each of their noncoding doublet preference . A mechanism to explain these double preference correlations in doublet preference is presented: mutational biases, the origin of the noncoding region doublet preference, evolved so as to maintain the 1-2 and 2-3 doublet preference, which is determined by codon usage . These biases then acted on the 3-1 doublet, which was almost free of coding constraints, resulting in a similar preference in this doublet.

Mutat Res, 1990 Feb, 228(2), 141 - 8
Adaptive resistance of Saccharomyces cerevisiae to chronic treatment with mutagens being due to a dominant mutation; Fahrig R et al.; The exposure of mammalian cells or tumors for weeks or months to low non-lethal doses of cytostatic drugs may induce multi-drug resistance, which can be enhanced by a variety of DNA-damaging agents . In yeast multi-drug resistance to a variety of drugs has been observed . DNA-damaging agents have not yet been tested . As the appearance of resistance is the result of long-term exposure, we decided to extend the application of test substances to a period of up to 400 days . In such long-term experiments S . cerevisiae MP1 adapted to treatment with low doses of mutagens . Consistent results were obtained for both alkylating and non-alkylating mutagenic substances . Furthermore, the adaptive resistance to the alkylating agent also adapted cells to the non-alkylating agent, which implies that there may be a single pathway for mutagens with different modes of action . Random spore analysis of adapted yeast cells and the back-cross to the parental wild type indicates that a single dominant mutation is responsible for the adaptive resistance.

Cell, 1990 Jan 26, 60(2), 307 - 17
A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis; Sikorski RS et al.; We have identified and characterized a novel, repeating 34 amino acid motif (the TPR motif) that is reiterated several times within the CDC23 gene product of S . cerevisiae . Multiple copies of this motif were discovered in five other proteins, three encoded by cell division cycle genes required to complete mitosis and two involved in RNA synthesis . Quantitative sequence analyses suggest the existence of a common underlying structure in each TPR unit that consists of amphipathic alpha-helical regions punctuated by proline-induced turns . The TPR motif defines a new family of genes and an important structural unit common to several proteins whose functions are required for mitosis and RNA synthesis.

Cell, 1990 Jan 26, 60(2), 235 - 45
S . cerevisiae TFIIIB is the transcription initiation factor proper of RNA polymerase III, while TFIIIA and TFIIIC are assembly factors; Kassavetis GA et al.; The S . cerevisiae RNA polymerase III (pol III) transcription factor TFIIIB binds to DNA upstream of the transcription start site of the SUP4 tRNA(Tyr) gene in a TFIIIC-dependent reaction and to the major 5S rRNA gene in a reaction requiring TFIIIC and TFIIIA . It is shown here that TFIIIB alone correctly positions pol III for repeated cycles of transcription on both genes, with the same efficiency as fully assembled transcription complexes . Thus, TFIIIB is the sole transcription initiation factor of S . cerevisiae pol III; TFIIIC and TFIIIA are assembly factors for TFIIIB . The TFIIIB-dependent binding of pol III to the SUP4 tRNA and 5S rRNA genes has been analyzed in binary (protein and DNA only) and precisely arrested ternary (protein, DNA, and RNA) transcription complexes . Pol III unwinds at least 14 bp of DNA at the SUP4 transcription start in a temperature-dependent process . The unwound DNA segment moves downstream with nascent RNA as a transcription bubble of approximately the same size.

FEMS Microbiol Lett, 1990 Jan 15, 55(1-2), 89 - 91
Induction of sexual agglutinability by unsaturated fatty acids in Saccharomyces cerevisiae; Doi S et al.; Agglutinins in S . cerevisiae are necessary for mating, for recognition between cells of opposite mating type . The mode of agglutinin synthesis is altered by the growth temperature and by the carbon source, from constitutive to inducible synthesis and vice versa . Some of the unsaturated fatty acids tested induced synthesis of agglutinins in cells grown at an elevated temperature, even in the absence of the pheromone . However, synthesis of agglutinins in glycerol-grown cells, that are inducible by the pheromone, was not induced by linolenic acid . Hence, the change in the mechanism of regulation of agglutinability produced by differences in temperature probably differs from that produced by differences in carbon source.

J Biol Chem, 1990 Jan 5, 265(1), 20 - 5
Identification and preliminary characterization of an O6-methylguanine DNA repair methyltransferase in the yeast Saccharomyces cerevisiae; Sassanfar M et al.; Saccharomyces cerevisiae contains a DNA repair methyltransferase (MTase) that repairs O6-methylguanine . Methyl groups are irreversibly transferred from O6-methylguanine in DNA to a 25-kilodalton protein in S . cerevisiae cell extracts, and methyl transfer is accompanied by the formation of S-methylcysteine . The yeast MTase is expressed at approximately 150 molecules/cell in exponentially growing yeast cultures but is not detectable in stationary phase cells . Unlike mammalian and bacterial MTases, the yeast MTase is very temperature-sensitive, having a half-life of about 4 min at 37 degrees C, which may explain why others have failed to detect it . Like other DNA repair MTases, the S . cerevisiae MTase repairs O6-methylguanine more efficiently in double-stranded DNA than in single-stranded DNA . Synthesis of the yeast DNA MTase is apparently not inducible by sublethal exposures to alkylating agent, but rather MTase activity is depleted in cells exposed to low doses of alkylating agent . Judging from its molecular weight and substrate specificity, the yeast DNA MTase is more closely related to mammalian MTases than to Escherichia coli MTases.

Proc Natl Acad Sci U S A, 1990 Jan, 87(2), 851 - 5
Saccharomyces cerevisiae U1 small nuclear RNA secondary structure contains both universal and yeast-specific domains; Kretzner L et al.; The five small nuclear RNAs (snRNAs) involved in mammalian pre-mRNA splicing (U1, U2, U4, U5, and U6) are well conserved in length, sequence, and especially secondary structure . These five snRNAs from Saccharomyces cerevisiae show notable size and sequence differences from their metazoan counterparts . This is most striking for the large S . cerevisiae U1 and U2 snRNAs, for which no secondary structure models currently exist . Because of the importance of U1 snRNA in the early steps of "spliceosome" assembly, we wanted to compare the highly conserved secondary structure of metazoan U1 snRNA (approximately 165 nucleotides) with that of S . cerevisiae U1 snRNA (568 nucleotides) . To this end, we have cloned and sequenced the U1 gene from two other yeast species possessing large U1 RNAs . Using computer-derived structure predictions, phylogenetic comparisons, and structure probing, we have arrived at a secondary structure model for S . cerevisiae U1 snRNA . The results show that most elements of higher eukaryotic U1 snRNA secondary structure are conserved in S . cerevisiae . The hundreds of "extra" nucleotides of yeast U1 RNA, also highly structured, suggest that large insertions and/or deletions have occurred during the evolution of the U1 gene.

J Cell Biol, 1990 Jan, 110(1), 105 - 14
Purification of profilin from Saccharomyces cerevisiae and analysis of profilin-deficient cells; Haarer BK et al.; We have isolated profilin from yeast (Saccharomyces cerevisiae) and have microsequenced a portion of the protein to confirm its identity; the region microsequenced agrees with the predicted amino acid sequence from a profilin gene recently isolated from S . cerevisiae (Magdolen, V., U . Oechsner, G . Muller, and W . Bandlow . 1988 . Mol . Cell . Biol . 8:5108-5115) . Yeast profilin resembles profilins from other organisms in molecular mass and in the ability to bind to polyproline, retard the rate of actin polymerization, and inhibit hydrolysis of ATP by monomeric actin . Using strains that carry disruptions or deletions of the profilin gene, we have found that, under appropriate conditions, cells can survive without detectable profilin . Such cells grow slowly, are temperature sensitive, lose the normal ellipsoidal shape of yeast cells, often become multinucleate, and generally grow much larger than wild-type cells . In addition, these cells exhibit delocalized deposition of cell wall chitin and have dramatically altered actin distributions.

Biosystems, 1990, 24(2), 127 - 33
Does a cell perform isoelectric focusing?
Flegr J.
A model of intracellular electrical sorting of enzymes and organelles in the cytosol, based on isoelectric focusing, is proposed . The focusing is suggested to take place over a centrally symmetric pH gradient which in the cytosol of the yeast Saccharomyces cerevisiae is known to be 7.2-6.4 . From published data on the energetic capacity and from the computed electric resistance of the S . cerevisiae cell, the maximum value of the electric field that can be maintained in the cytosol was estimated . The results showed that the strength of a centrally symmetric intracytosolic electric field could be as high as 90 mV/cm, which is sufficient to account for sorting of cytosolic proteins according to their isoelectric points . Although direct experimental evidence for intracellular isoelectric focusing is still missing, several phenomena of physiological importance can be understood on the assumption of its real existence.

Int Arch Allergy Appl Immunol, 1990, 92(1), 9 - 15
Serum antibodies reactive with Saccharomyces cerevisiae in inflammatory bowel disease: is IgA antibody a marker for Crohn's disease?
Barnes RM, Allan S, Taylor-Robinson CH, Finn R, Johnson PM.
Sera from patients with Crohn's disease (CD) and ulcerative colitis (UC) have been evaluated for antibodies reactive with Saccharomyces cerevisiae (anti-Sacc antibodies) using an enzyme-linked immunoassay (ELISA) . IgG anti-Sacc antibodies were detected in 63% (25/40) of CD patients, compared with 15% (4/27) of UC patients (p less than 0.001) and 8% (5/60) healthy adult controls (p less than 0.001) . Furthermore, the prevalence of detectable IgG anti-Sacc antibodies in adult patients with coeliac disease, dermatitis herpetiformis, irritable bowel syndrome or atopic eczema was not significantly different to controls . In comparison, the prevalence of detectable IgG anti-Escherichia coli antibodies was not significantly different between CD (75%) or UC (79%) patients . More particularly, elevated levels of serum IgA anti-Sacc antibodies were detected in 17/40 CD patients, but in none of the 27 UC patients . These data confirm that serum antibodies reactive with S . cerevisiae are strongly associated with CD and further show that serum IgA anti-Sacc antibodies may be specific for this disorder.

Folia Microbiol (Praha), 1990, 35(3), 209 - 17
Transport of L-tryptophan in Saccharomyces cerevisiae; Kotyk A et al.; In addition to the general amino acid transport system (GAP) of S . cerevisiae L-tryptophan is transported by another system with approximately 25% capacity of GAP, with a KT of 0.41 +/- 0.08 mmol/L and with a similar specificity as GAP (lower inhibition by Met, Pro, Ser, Thr and 2-aminoisobutyric acid; greater inhibition by Glu and His) . The pH optimum of this system is at 5.0-5.5, activation energy above the transition point (20 degrees C) was 20 kJ/mol, below the transition point 55 kJ/mol . The transport by this system was virtually unidirectional, efflux amounting to at most 10% into a tryptophan-free medium . The transport itself was blocked by 2,4-dinitrophenol, antimycin A and uranyl nitrate . The system was synthesized de novo during preincubation with glucose = fructose greater than trehalose greater than ethanol within 30 min, and was degraded with a half-time of 15 min in the absence of further synthesis . The accumulation ratios of L-tryptophan in gap1 mutants were concentration-dependent (200:1 at 1 mumol L-Trp/L, 4:1 at 2.5 mmol L-Trp/L) and decreased with increasing suspension density from 200:1 to 5:1 (for 10 mumol L-Trp/L) . The involvement of hydrogen ions in the uptake was clearly demonstrated by the effect of D2O even if it could not be established by either shifts of pHout or membrane depolarization.

Biochem Int, 1990, 21(2), 199 - 207
Proton magnetic resonance study of cycloheximide-ribosome interactions; Mandiyan V et al.; Cycloheximide-ribosome interactions from sensitive and resistant organisms were studied by proton magnetic resonance spectroscopic techniques . The two methyl resonances of cycloheximide upon interaction with ribosomes from Saccharomyces cerevisiae showed preferential broadening . Comparison of cycloheximide line broadening as effected by ribosomes from S . cerevisiae (sensitive) and Microsporum canis (resistant) revealed that less cycloheximide is bound to the M . canis ribosomes . From the decrease in line broadening observed with increasing temperature it may be concluded that cycloheximide-ribosome interaction is a fast exchange reaction . Tetracycline did not compete with cycloheximide for binding site(s) on the ribosomes of S . cerevisiae.

FEMS Microbiol Lett, 1990 Jan 1, 54(1-3), 333 - 8
A possible role of histidine in a nickel resistant mechanism of Saccharomyces cerevisiae; Joho M et al.; When a nickel resistant strain N08 of S . cerevisiae was grown in a Ni-supplemented medium, approximately 70% of the nickel is distributed in the soluble fraction . The soluble fraction was chromatographed on Sephadex G-10 and the fraction contained both nickel and large amounts of histidine . When cells were grown in medium containing various combinations of nickel and magnesium and which exhibited approximately 50% growth inhibition, a molar ratio of intracellular histidine and nickel contents remained constant at 1.2-1.4, indicating that the increase in histidine content is correlated with nickel accumulation . The wild type strain 0605-S6, however, exhibits no increase in histidine content when grown in a Ni-supplemented medium, and, therefore, a nickel-resistant mechanism of yeast appears to be the formation of histidine-nickel complexes.

DNA Cell Biol, 1990 Jan-Feb, 9(1), 27 - 36
Genetically engineered P450 monooxygenases: construction of bovine P450c17/yeast reductase fused enzymes; Shibata M et al.; Seven P450/reductase fused enzymes were produced in Saccharomyces cerevisiae by expressing fused cDNAs consisting of bovine cytochrome P450c17 (P450c17) and yeast NADPH-cytochrome P450 reductase (reductase) . These fused enzymes differed in the length and amino acid sequence of the hinge region between the P450 and reductase moieties . Expression of the fused constructs under the control of the yeast alcohol dehydrogenase I promoter and terminator of expression vector pAAH5 in S . cerevisiae AH22 cells resulted in the production of about 2-8 X 10(4) molecules per cell of the seven corresponding fused enzymes . Six of the fused enzymes incorporated a protoheme, as confirmed by reduced CO-difference spectra . Recombinant yeast strains producing each of the fused hemoproteins showed P450c17-dependent 17 alpha-hydroxylase activity toward progesterone . The most active fused enzyme, delta N23FE, which lacked the amino-terminal 23 amino acids of the reductase, showed about 10 times higher 17 alpha-hydroxylase activity than bovine P450c17, although the fused enzyme (delta N23FE)' with an amino acid sequence in the hinge region different from delta N23FE was less active than delta N23FE . The fused enzyme delta N0FE, consisting of P450c17 and whole reductase, showed about 1.8 times higher activity than bovine P450c17 . No activity was found with delta N84FE lacking the amino-terminal 84 amino acids of the reductase moiety . P450c17-dependent C17,(20)-lyase activity toward 17 alpha-hydroxyprogesterone was detected to lesser extents in the recombinant yeast . Fused bovine P450c17/yeast reductase enzymes show enhanced 17 alpha-hydroxylase activity, and the length and amino acid sequence in the hinge region between the P450c17 and yeast reductase moieties can be important for efficient intramolecular electron transfer in the fused enzymes.

Curr Genet, 1990 Jan, 17(1), 85 - 8
The OGD1 gene, affecting 2-oxoglutarate dehydrogenase in S . cerevisiae, is closely linked to HIS5 on chromosome IX; Ruttkay-Nedecky B et al.; Ogd1 mutants of Saccharomyces cerevisiae are deficient in mitochondrial 2-oxoglutarate dehydrogenase activity; they cannot grow on glycerol and produce an increased amount of organic acids during growth on glucose as substrate . Using gamma ray-induced rad52-mediated chromosome loss the ogd1 mutation can be assigned to chromosome IX . Tetrad analysis of crosses between ogd1 and other markers on chromosome IX revealed that the OGD1 gene maps on the left arm of this chromosome 1.9 cM from his5.

J Bacteriol, 1990 Jan, 172(1), 507 - 10
Neurospora endo-exonuclease is immunochemically related to the recC gene product of Escherichia coli; Fraser MJ et al.; Immunochemical cross-reaction between the endo-exonuclease of Neurospora crassa, an enzyme previously implicated in recombination and recombinational DNA repair, and the recC-encoded polypeptide of Escherichia coli has been detected by immunoblotting extracts of strains of E . coli having a deletion that includes the recBCD genes but carrying multicopy plasmids bearing all three of the recBCD genes or only one or two of these genes . It was predicted that homology would also be found at the amino acid sequence level between the recC polypeptide and both nuclear and mitochondrial endo-exonucleases of Saccharomyces cerevisiae, which cross-react with antibodies raised to the N . crassa endo-exonuclease . Since the gene for the S . cerevisiae mitochondrial enzyme, NUC1, has been cloned and sequenced and the predicted amino acid sequence is known, this sequence was aligned with the predicted amino acid sequence of the recC polypeptide . Extensive homology was found by aligning 306 of the 329 amino acids of the yeast mitochondrial nuclease sequence with the carboxy-terminal one-quarter of the amino acid sequence of the recC polypeptide.

Biomed Biochim Acta, 1990, 49(8-9), 733 - 6
Mathematical model of the cell cycle regulation in budding yeasts; Prikrylova D et al.; A mathematical model of the cell cycle regulation in S . cerevisiae is proposed . The model is based on the assumption of the G1----S phase transition control mediated by two signals . One of them is correlated with the cellular energy level--its messenger could be cAMP; the second one depends on the change of the cellular growth rate (reaching the critical size) and remains hypothetical.

Biochem Biophys Res Commun, 1989 Dec 29, 165(3), 1091 - 5
Sexual response in Saccharomyces cerevisiae: alteration of enzyme activity in the glyoxalase system by mating factor; Inoue Y et al.; Glyoxalase I activity in alpha-type budding yeast of the Saccharomyces cerevisiae strain was increased by exposure of alpha-type cells to supernatant of a culture of a-type yeast cells, although glyoxalase II activity was decreased by the same treatment . The alteration of enzyme activity in the glyoxalase system occurred during the 30-60 min period after exposure of alpha-type cells to a-type culture supernatant . No change of glyoxalase I and II activities was found in the case of the alpha-type strain, S . cerevisiae VQ3 (alpha ste3-1), which is deficient in a-factor receptors.

Cell, 1989 Dec 22, 59(6), 1127 - 33
An essential G1 function for cyclin-like proteins in yeast; Richardson HE et al.; Cyclins were discovered in marine invertebrates based on their dramatic cell cycle periodicity . Recently, the products of three genes associated with cell cycle progression in S . cerevisiae were found to share limited homology with cyclins . Mutational elimination of the CLN1, CLN2, and DAF1/WHI1 products leads to cell cycle arrest independent of cell type, while expression of any one of the genes allows cell proliferation . Using strains where CLN1 was expressed conditionally, the essential function of Cln proteins was found to be limited to the G1 phase . Furthermore, the ability of the Cln proteins to carry out this function was found to decay rapidly upon cessation of Cln biosynthesis . The data are consistent with the hypothesis that Cln proteins activate the Cdc28 protein kinase, shown to be essential for the G1 to S phase transition in S . cerevisiae . Because of the apparent functional redundancy of these genes, DAF1/WHI1 has been renamed CLN3.

Nature, 1989 Dec 21-28, 342(6252), 953 - 5
Sensitivity to cyclosporin A is mediated by cyclophilin in Neurospora crassa and Saccharomyces cerevisiae; Tropschug M et al.; Cyclosporin A, a cyclic fungal undecapeptide produced by Tolypocladium inflatum, is a potent immunosuppressive drug originally isolated as an antifungal antibiotic . Cyclosporin A (CsA) is widely used in humans to prevent rejection of transplanted organs such as kidney, heart, bone marrow and liver . The biochemical basis of CsA action is not known: its primary cellular target has been suggested to be calmodulin, the prolactin receptor or cyclophilin, a CsA-binding protein originally isolated from the cytosol of bovine thymocytes . Cyclophilin has been shown to be a highly conserved protein present in all eukaryotic cells tested and to be identical to peptidyl-prolyl cis-trans isomerase, a novel type of enzyme that accelerates the slow refolding phase of certain proteins in vitro . We demonstrate that in the lower eukaryotes N . crassa and S . cerevisiae, cyclo philin mediates the cytotoxic CsA effect . In CsA-resistant mutants of both organisms, the cyclophilin protein is either lost completely or, if present, has lost its ability to bind CsA.

Gene, 1989 Dec 14, 84(2), 335 - 43
Characterization of a leuA gene and an ARS element from Mucor circinelloides; Roncero MI et al.; A 4.4-kb PstI restriction endonuclease fragment of Mucor circinelloides DNA has previously been shown to both complement a leuA- mutation, and to enable the autonomous replication of plasmids within this organism . The complete nucleotide (nt) sequence of this fragment has been determined and an open reading frame of 1935 bp with no introns has been identified, which exhibits significant similarity (75% at the nt level) with 114 bp of the 5' coding region of the Saccharomyces cerevisiae LEU1 gene . Based on this and on the fact that the fragment weakly complements a leu1 auxotroph of S . cerevisiae, we concluded that the Mucor leu gene encodes alpha-isopropylmalate (alpha-IPM) isomerase and designated it leuA+ accordingly . Primer extension analysis of leuA mRNA and Northern-blot hybridization, indicated the leuA transcript to be approx . 2.3 kb in size, with 5'- and 3'-untranslated regions of 16-20 nt and approx . 450 nt, respectively . Specific Mucor ARS sequence(s) were not identified, although the general location of ARS was indicated by subcloning experiments . Nucleotide sequences are present within this region, which show some similarity with the core consensus of the S . cerevisiae ARS; however, any functional homology is doubtful, since insertion of the 4.4-kb PstI fragment into YIp5 did not increase the transformation frequency of S . cerevisiae with such a vector.

Eur J Biochem, 1989 Dec 8, 186(1-2), 249 - 53
Evolutionary relationships among aminotransferases . Tyrosine aminotransferase, histidinol-phosphate aminotransferase, and aspartate aminotransferase are homologous proteins; Mehta PK et al.; A data base was compiled containing the amino acid sequences of 12 aspartate aminotransferases and 11 other aminotransferases . A comparison of these sequences by a standard alignment method confirmed the previously reported homology of all aspartate aminotransferases and Escherichia coli tyrosine aminotransferase . However, no significant similarity between these proteins and any of the other aminotransferases was detected . A more rigorous analysis, focusing on short sequence segments rather than the total polypeptide chain, revealed that rat tyrosine aminotransferase and Saccharomyces cerevisiae and Escherichia coli histidinol-phosphate aminotransferase share several homologous sequence segments with aspartate aminotransferases . For comparison of the complete sequences, a multiple sequence editor was developed to display the whole set of amino acid sequences in parallel on a single work-sheet . The editor allows gaps in individual sequences or a set of sequences to be introduced and thus facilitates their parallel analysis and alignment . Several clusters of invariant residues at corresponding positions in the amino acid sequences became evident, clearly establishing that the cytosolic and the mitochondrial isoenzyme of vertebrate aspartate aminotransferase, E . coli aspartate aminotransferase, rat and E . coli tyrosine aminotransferase, and S . cerevisiae and E . coli histidinol-phosphate aminotransferase are homologous proteins . Only 12 amino acid residues out of a total of about 400 proved to be invariant in all sequences compared; they are either involved in the binding of pyridoxal 5'-phosphate and the substrate, or appear to be essential for the conformation of the enzymes.

Appl Environ Microbiol, 1989 Dec, 55(12), 3214 - 20
Competition for glucose between the yeasts Saccharomyces cerevisiae and Candida utilis; Postma E et al.; The competition between the yeasts Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 for glucose was studied in sugar-limited chemostat cultures . Under aerobic conditions, C . utilis always successfully completed against S . cerevisiae . Only under anaerobic conditions did S . cerevisiae become the dominant species . The rationale behind these observations probably is that under aerobic glucose-limited conditions, high-affinity glucose/proton symporters are present in C . utilis, whereas in S . cerevisiae, glucose transport occurs via facilitated diffusion with low-affinity carriers . Our results explain the frequent occurrence of infections by Crabtree-negative yeasts during bakers' yeast production.

J Bacteriol, 1989 Dec, 171(12), 6776 - 81
Isolation and characterization of a Saccharomyces cerevisiae mutant with impaired glutamate synthase activity; Folch JL et al.; A mutant of Saccharomyces cerevisiae that lacks glutamate synthase (GOGAT) activity has been isolated . This mutant was obtained after chemical mutagenesis of a NADP-glutamate dehydrogenase-less mutant strain . The gdh gus mutant is a glutamate auxotroph . The genetic analysis of the gus mutant showed that the GOGAT-less phenotype is due to the presence of two loosely linked mutations . Evidence is presented which suggests the possibility that S . cerevisiae has two GOGAT activities, designated GOGAT A and GOGAT B . These activities can be distinguished by their pH optima and by their regulation by glutamate . Furthermore, one of the mutations responsible for the GOGAT-less phenotype affected GOGAT A activity, while the other mutation affected GOGAT B activity.

J Bacteriol, 1989 Dec, 171(12), 6409 - 13
Nutritional regulation of yeast delta-9 fatty acid desaturase activity; Bossie MA et al.; The addition of unsaturated fatty acids to cultures of Saccharomyces cerevisiae significantly altered the microsomal lipid composition . Supplementation with either of the naturally occurring palmitoleic (16:1) or oleic (18:1) acids caused increased levels in membrane phospholipids and reduced levels of the complementary acid . Growth in the presence of equimolar quantities of 16:1 and 18:1 acids, however, produced a fatty acid composition similar to that found in unsupplemented cell membranes . Linoleic acid (18:2) was not found in S . cerevisiae grown under normal conditions . It was preferentially internalized and incorporated into microsomes, however, at levels exceeding 50% of the total fatty acid species . This resulted in an almost total loss of 16:1 and a reduction of 18:1 to 25% of its normal level . The delta-9 fatty acid desaturase, a microsomal enzyme that forms 16:1 and 18:1 from saturated acyl coenzyme A precursors, was affected by the presence of exogenous fatty acids . Enzyme activity toward the 16:0 coenzyme A substrate was elevated in microsomes from saturated-fatty-acid-supplemented cultures and sharply repressed following the addition of unsaturated fatty acids, including 18:2 . Northern (RNA blot) and slot-blot analyses of mRNA encoded by the OLE1 gene, which appears to be the structural gene for the delta-9 desaturase, indicated that it was sharply reduced in unsaturated-fatty-acid-fed cells . These data suggest that a significant part of the regulation involves modulation of available transcripts.

Cell, 1989 Dec 1, 59(5), 807 - 13
Trimerization of a yeast transcriptional activator via a coiled-coil motif; Sorger PK et al.; The transcriptional induction of heat shock genes is mediated by the heat shock transcription factor (HSF) . We have investigated the oligomerization state of HSF from S . cerevisiae and find that it forms a trimer in solution and when bound to DNA . Trimerization is mediated by a region of HSF that, like the leucine zipper, is characterized by the occurrence of hydrophobic amino acids every 7 residues . We propose that it forms a three-stranded coiled coil.

Experientia, 1989 Dec 1, 45(11-12), 1013 - 8
Further evidence for the existence of a bottleneck in the metabolism of Saccharomyces cerevisiae; Auberson LC et al.; The growth physiology of Saccharomyces cerevisiae strains H1022 and Whi2+ has been studied in aerobic batch and continuous (chemostat) cultures . Results from the measurement of biomass and medium components (off-line) together with oxygen, carbon dioxide and heat measurements (on-line) have been used in an attempt to explore the existence of 'overflow' or 'bottleneck' metabolism as opposed to catabolite repression (Crabtree effect) in these strains . Chemostat experiments indicated that specific oxygen uptake rate (qO2) was linearly related to the dilution rate (D) at values below the critical dilution rate (D crit), becoming constant above D crit, which is in agreement with the bottleneck theory . However, batch culture experiments indicated negligible oxygen consumption during the initial glucose growth phase, the culture exhibiting purely anaerobic metabolism . The bottleneck theory would propose that qO2 has a constant (maximum) value under these conditions . The results presented here suggest that while the bottleneck theory can be adequately used to describe chemostat growth of S . cerevisiae, some other control mechanism must be operating under conditions of high glucose concentrations, such as those initially prevailing in the batch culture experiments.

Gene, 1989 Nov 30, 83(2), 381 - 5
Cloning of chromosome I DNA from Saccharomyces cerevisiae: mutational analysis of the FUN2 transcribed region; Crowley JC et al.; To increase the number of mutationally defined genes on chromosome I from Saccharomyces cerevisiae, most of the FUN2 (Function Unknown Now) transcribed region was deleted by gene replacement . Strains containing the deletion were viable, but grew with a 20% longer generation time . The mutation was recessive . Mutant haploids were able to mate, and homozygous mutant diploids were able to sporulate, giving asci containing four viable ascospores . These results indicate FUN2 is dispensable for the life cycle of S . cerevisiae, but required for an optimal growth rate.

Biochem Biophys Res Commun, 1989 Nov 30, 165(1), 464 - 73
Primary structure of the S . cerevisiae gene encoding uridine monophosphokinase; Liljelund P et al.; In the yeast Saccharomyces cerevisiae, the biosynthesis of both pyrimidine nucleoside triphosphates UTP and CTP is dependent on the activity of the uridine monophosphokinase step . We have determined the sequence of the uridine monophosphokinase gene . The coding region is 615 base pairs long and encodes 205 amino acids (22,500 daltons) . The 5' terminus is comprised of a 17 amino acid-long hydrophobic leader sequence which is not present in genes encoding adenylate kinases . The coding region shows a strong degree of homology with the cytosolic adenylate kinases of vertebrates, and a lesser degree of homology with yeast and E . coli adenylate kinases.

Nucleic Acids Res, 1989 Nov 25, 17(22), 9319 - 31
Structure of a ribosomal protein gene in Mucor racemosus; Sosa L et al.; As an extension of our analysis of the translational apparatus of Mucor racemosus we have isolated a gene encoding a ribosomal protein of Mucor . Based on a method developed for S . cerevisiae, we identified by hybrid selection and in vitro translation a lambda-Charon 4A clone containing the genomic copy of a Mucor ribosomal protein . The gene consisted of two exons of 57 and 387 nucleotides . The two exons were separated by an 131 nucleotide intron . The processed transcript was 714 nucleotides in length and contained a 25 nucleotide untranscribed leader and an 114 nucleotide untranscribed 3'-end . The protein predicted from the nucleotide sequence contained 148 amino acids and exhibited 61% identity with the S19 ribosomal protein of Xenopus laevis . The promoter region of the gene contained sequences highly homologous to the RPG and Homol1 promoter elements found in S . cerevisiae . Southern blot analysis indicated that the Mucor genome contains three copies of this gene.

Nucleic Acids Res, 1989 Nov 25, 17(22), 9113 - 26
H2A.X . a histone isoprotein with a conserved C-terminal sequence, is encoded by a novel mRNA with both DNA replication type and polyA 3' processing signals; Mannironi C et al.; A full length cDNA clone that directs the in vitro synthesis of human histone H2A isoprotein H2A.X has been isolated and sequenced . H2A.X contains 142 amino acid residues, 13 more than human H2A.1 . The sequence of the first 120 residues of H2A.X is almost identical to that of human H2A.1 . The sequence of the carboxy-terminal 22 residues of H2A.X is unrelated to any known sequence in vertebrate histone H2A; however, it contains a sequence homologous with those of several lower eukaryotes . This homology centers on the carboxy-terminal tetrapeptide which in H2A.X is SerGlnGluTyr . Homologous sequences are found in H2As of three types of yeasts, in Tetrahymena and Drosophila . Seven of the nine carboxy-terminal amino acids of H2A.X are identical with those of S . cerevisiae H2A.1 . It is suggested that this H2A carboxy-terminal motif may be present in all eukaryotes . The H2A.X cDNA is 1585 bases long followed by a polyA tail . There are 73 nucleotides in the 5' UTR, 432 in the coding region, and 1080 in the 3' UTR . Even though H2A.X is considered a basal histone, being synthesized in G1 as well as in S-phase, and its mRNA contains polyA addition motifs and a polyA tail, its mRNA also contains the conserved stem-loop and U7 binding sequences involved in the processing and stability of replication type histone mRNAs . Two forms of H2A.X mRNA, consistent with the two sets of processing signals were found in proliferating cell cultures . One, about 1600 bases long, contains polyA; the other, about 575 bases long, lacks polyA . The short form behaves as a replication type histone mRNA, decreasing in amount when cell cultures are incubated with inhibitors of DNA synthesis, while the longer behaves as a basal type histone mRNA.

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