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