Mol Cell Biol, 1996 Jun, 16(6), 3094 - 105 TEL2, an essential gene required for telomere length regulation and telomere position effect in Saccharomyces cerevisiae; Runge KW et al.; The DNA-protein complexes at the ends of linear eukaryotic chromosomes are called the telomeres . In Saccharomyces cerevisiae, telomeric DNA consists of a variable length of the short repeated sequence C1-3A . The length of yeast telomeres can be altered by mutation, by changing the levels of telomere binding proteins, or by increasing the amount of C1-3A DNA sequences . Cells bearing the tel1-1 or tel2-1 mutations, known previously to have short telomeres, did not respond to perturbations that caused telomere lengthening in wild-type cells . The transcription of genes placed near yeast telomeres is reversibly repressed, a phenomenon called the telomere position effect . The tel2-1 mutation reduced the position effect but did not affect transcriptional repression at the silent mating type cassettes, HMRa and HML alpha . The TEL2 gene was cloned, sequenced, and disrupted . Cells lacking TEL2 function died, with some cells arresting as large cells with three or four small protrusions or "blebs." Mol Cell Biol, 1996 Jun, 16(6), 2922 - 31 The REG2 gene of Saccharomyces cerevisiae encodes a type 1 protein phosphatase-binding protein that functions with Reg1p and the Snf1 protein kinase to regulate growth; Frederick DL et al.; The GLC7 gene of Saccharomyces cerevisiae encodes the catalytic subunit of type 1 protein phosphatase (PP1) and is essential for cell growth . We have isolated a previously uncharacterized gene, REG2, on the basis of its ability to interact with Glc7p in the two-hybrid system . Reg2p interacts with Glc7p in vivo, and epitope-tagged derivatives of Reg2p and Glc7p coimmunoprecipitate from cell extracts . The predicted protein product of the REG2 gene is similar to Reg1p, a protein believed to direct PP1 activity in the glucose repression pathway . Mutants with a deletion of reg1 display a mild slow-growth defect, while reg2 mutants exhibit a wild-type phenotype . However, mutants with deletions of both reg1 and reg2 exhibit a severe growth defect . Overexpression of REG2 complements the slow-growth defect of a reg1 mutant but does not complement defects in glycogen accumulation or glucose repression, two traits also associated with a reg1 deletion . These results indicate that REG1 has a unique role in the glucose repression pathway but acts together with REG2 to regulate some as yet uncharacterized function important for growth . The growth defect of a reg1 reg2 double mutant is alleviated by a loss-of-function mutation in the SNF1-encoded protein kinase . The snf1 mutation also suppresses the glucose repression defects of reg1 . Together, our data are consistent with a model in which Reg1p and Reg2p control the activity of PP1 toward substrates that are phosphorylated by the Snf1p kinase. Mol Cell Biol, 1996 Jun, 16(6), 2764 - 71 Inactivation of YME2/RNA12, which encodes an integral inner mitochondrial membrane protein, causes increased escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae; Hanekamp T et al.; Inactivation of the yeast nuclear gene YMe2 causes an increased rate of DNA escape from mitochondria to the nucleus . Mutations in yme2 also show genetic interactions with yme1, a second gene that affects DNA escape from mitochondria to the nucleus . The yme1 cold-sensitive growth phenotype is suppressed by yme2 mutations . In addition, yme1 yme2 double mutants exhibit a synthetic growth defect on ethanol-glycerol medium at 30 degrees C . YME2 was isolated by complementation of the synthetic growth defect of yme1 yme2 strains and was found to be identical with the previously cloned RNA12 gene . The dominant temperature-sensitive mutation RNA12-1 prevents growth of yeast cells at 37 degrees C . YME2 encodes a protein with a predicted molecular weight of 96,681 and is an integral inner mitochondrial membrane protein . The larger carboxyl-terminal domain of the YME2 gene product faces the intermembrane space . Null alleles of yme2 display the same genetic interactions with yme1 and high rate of DNA escape from mitochondria as do the originally isolated yme2 mutant strains . Disruption of yme2 causes a strain-dependent growth defect on nonfermentable carbon sources. Mol Cell Biol, 1996 Jun, 16(6), 2719 - 27 The immunosuppressant SR 31747 blocks cell proliferation by inhibiting a steroid isomerase in Saccharomyces cerevisiae; Silve S et al.; SR 31747 is a novel immunosuppressant agent that arrests cell proliferation in the yeast Saccharomyces cerevisiae, SR 31747-treated cells accumulate the same aberrant sterols as those found in a mutant impaired in delta 8- delta 7-sterol isomerase . Sterol isomerase activity is also inhibited by SR 31747 in in vitro assays . Overexpression of the sterol isomerase-encoding gene, ERG2, confers enhanced SR resistance . Cells growing anaerobically on ergosterol-containing medium are not sensitive to SR . Disruption of the sterol isomerase-encoding gene is lethal in cells growing in the absence of exogenous ergosterol, except in SR-resistant mutants lacking either the SUR4 or the FEN1 gene product . The results suggest that sterol isomerase is the target of SR 31747 and that both the SUR4 and FEN1 gene products are required to mediate the proliferation arrest induced by ergosterol depletion. Mol Cell Biol, 1996 Jun, 16(6), 2647 - 55 The Saccharomyces cerevisiae Start-specific transcription factor Swi4 interacts through the ankyrin repeats with the mitotic Clb2/Cdc28 kinase and through its conserved carboxy terminus with Swi6; Siegmund RF et al.; At a point in late G1 termed Start, yeast cells enter S phase, duplicate their spindle pole bodies, and form buds . These events require activation of Cdc28 kinase by G1 cyclins . Swi4 associates with Swi6 to form the SCB-binding factor complex which activates G1 cyclin genes CLN1 and CLN2 in late G1 . In G2 and M phases, the transcriptional activity of SCB-binding factor is repressed by the mitotic Clb2/Cdc28 kinase . Mbp1, a transcription factor related to Swi4, forms the MCB-binding factor complex with Swi6, which activates DNA synthesis genes and S-phase cyclin genes CLB5 and CLB6 in late G1 . Clb2/Cdc28 kinase is not required for the repression of MCB-binding factor transcriptional activity in G2 and M phase . We show here that the Swi4 carboxy terminus is sufficient for interaction with Swi6 in vitro . A carboxy-terminal domain of Swi6 is required and sufficient for interaction with Swi4 . The carboxy terminus of Mbp1 is sufficient for interaction with Swi6, and the carboxy terminus of Swi6 is required for interaction with Mbp1 . By coimmunoprecipitation, we show that Swi4 but not Mbp1 interacts with Clb2/Cdc28 kinase in vivo during the G2 and M phases of the cell cycle . We demonstrate that the ankyrin repeats of Swi4 mediate the interaction with Clb2/Cdc28 kinase . The ankyrin repeats constitute a domain by which a cell cycle-specific transcription factor can interact with cyclin-dependent kinase complexes, thus enabling it to link its transcriptional activity to cell cycle progression. Mol Cell Biol, 1996 Jun, 16(6), 2614 - 26 AKR1 encodes a candidate effector of the G beta gamma complex in the Saccharomyces cerevisiae pheromone response pathway and contributes to control of both cell shape and signal transduction; Pryciak PM et al.; Mating pheromones of Saccharomyces cerevisiae control both signal transduction events and changes in cell shape . The G beta gamma complex of the pheromone receptor-coupled G protein activates the signal transduction pathway, leading to transcriptional induction and cell cycle arrest, but how pheromone-dependent signalling leads to cell shape changes is unclear . We used a two-hybrid system to search for proteins that interact with the G beta gamma complex and that might be involved in cell shape changes . We identified the ankyrin repeat-containing protein Akr1p and show here that it interacts with the free G beta gamma complex . This interaction may be regulated by pheromone, since Akr1p is excluded from the G alpha beta gamma heterotrimer . Both haploid and diploid cells lacking Akr1p grow slowly and develop deformed buds or projections, suggesting that this protein participates in the control of cell shape . In addition, Akr1p has a negative influence on the pheromone response pathway . Epistasis analysis demonstrates that this negative effect does not act on the G beta gamma complex but instead affects the kinase cascade downstream of G beta gamma, so that the kinase Ste20p and components downstream of Ste20p (e.g., Ste11p and Ste7p) are partially activated in cells lacking Akr1p . Although the elevated signalling is eliminated by deletion of Ste20p (or components downstream of Ste20p), the growth and morphological abnormalities of cells lacking Akr1p are not rescued by deletion of any of the known pheromone response pathway components . We therefore propose that Akr1p negatively affects the activity of a protein that both controls cell shape and contributes to the pheromone response pathway upstream of Ste20p but downstream of G beta gamma . Specifically, because recent evidence suggests that Bem1p, Cdc24p, and Cdc42p can act in the pheromone response pathway, we suggest that Akr1p affects the functions of these proteins, by preventing them from activating mating-specific targets including the pheromone-responsive kinase cascade, until G beta gamma is activated by pheromone. Mol Cell Biol, 1996 Jun, 16(6), 2545 - 53 Functional analysis of histones H2A and H2B in transcriptional repression in Saccharomyces cerevisiae; Recht J et al.; The presence of H2A-H2B dimers in nucleosomes can inhibit the binding of transcription factors to chromatin templates . To study the roles of histones H2A and H2B in transcriptional repression in vivo, mutant forms of these histones were analyzed in two different assay systems . Two repression domains were identified in H2A . One domain includes residues that fall in the beginning of the H2A-H2B dimerization region, and the second is in the H2A N terminus, a region of potential interactions with nonhistone proteins . The function of H2A and H2B in one repression assay was found to be dependent on three SPT (suppressor of Ty) genes whose products are important for chromatin-mediated repression . These results suggest that repressive chromatin structure may be established through the interactions of the Spt proteins with these histones . In contrast, other proteins, the products of the HIR (histone regulation) genes, may function to direct H2A and H2B to specific promoters. J Virol, 1996 Jun, 70(6), 4086 - 9 Synthesis of Semliki Forest virus RNA polymerase components nsP1 through nsP4 in Saccharomyces cerevisiae by expression of cDNA encoding the nonstructural polyprotein; Russo P et al.; A Semliki Forest virus nonstructural polyprotein, P1234, expressed in the yeast Saccharomyces cerevisiae in the absence of a replicative RNA template appeared to be properly cleaved into nsP1 to nsP4 . All nsPs were membrane associated, and nsP2 was also transported to the nucleus . The membrane fraction containing nsPs showed guanine-7-methyltransferase and guanylyltransferase-like activities, typical for Semliki Forest virus nsP1. Genes Dev, 1996 Jun 1, 10(11), 1327 - 40 A search for proteins that interact genetically with histone H3 and H4 amino termini uncovers novel regulators of the Swe1 kinase in Saccharomyces cerevisiae; Ma XJ et al.; In a genetic screen for second-site mutations that are lethal in combination with a deletion of the amino terminus of histone H3, we have uncovered three new gene products that regulate the Saccharomyces cerevisiae Swe1 kinase . The Swe1 protein kinase phosphorylates tyrosine residue 19 of Cdc28 and inhibits its activity . One histone synthetic-lethal gene, HSL1, encodes a putative protein kinase that has high sequence and functional homology to fission yeast cdr1/nim1, an inhibitory kinase of wee1 . Another gene, HSL7, is a novel negative regulator of Swe1 function . Sequences similar to Hsl7 exist in Caenorhabditis elegans and humans . In addition, we have isolated a dosage-dependent suppressor, OSS1, of hsl1 and hsl7 . OSS1 is important for the transcriptional repression of SWE1 and CLN2 in G2 . Mutations in HSL1 and HSL7 therefore cause hyperactivity of the Swe1 kinase, which in turn decreases mitotic Cdc28 kinase activity . Moreover, HSL5 is identical to CDC28, further suggesting that it is the decreased Cdc28 kinase activity in these hsl mutants that causes lethality in the histone mutant background . Because neither HSL1 nor HSL7 is essential in yeast, and histone transcription is unaffected by the hsl5/cdc28 mutation, it is unlikely that synthetic lethality results from reduced transcription of HSL1 and HSL7 caused by histone mutations, or from reduced histone transcription when Cdc28 kinase activity is compromised . We suggest that these cell cycle regulators function in a pathway upstream of both histones H3 and H4, thereby modulating histone function in the cell cycle. Genes Dev, 1996 Jun 1, 10(11), 1310 - 26 A novel mechanism for telomere size control in Saccharomyces cerevisiae; Li B et al.; One of the central requirements for eukaryotic chromosome stability is the maintenance of the simple sequence tracts at telomeres . In this study, we use genetic and physical assays to reveal the nature of a novel mechanism by which telomere length is controlled . This mechanism, telomeric rapid deletion (TRD), is capable of reducing elongated telomeres to wild-type tract length in an apparently single-division process . The deletion of telomeres to wild-type lengths is stimulated by the hpr1 mutation, suggesting that TRD in these cells is the consequence of an intrachromatid pathway . Paradoxically, TRD is also dependent on the lengths of the majority of nonhomologous telomeres in the cell . Defects in the chromatin-organizing protein Sir3p increase the rate of hpr1-induced rapid deletion and specifically change the spectrum of rapid deletion events . We propose a model in which interactions among telosomes of nonhomologous chromosomes form higher order complexes that restrict the access of the intrachromatid recombination machinery to telomeres . This mechanism of size control is distinct from that mediated through telomerase and is likely to maintain telomere length within a narrow distribution. Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5208 - 12 Absolute mRNA levels and transcriptional initiation rates in Saccharomyces cerevisiae; Iyer V et al.; We quantitate the absolute levels of individual mRNAs per yeast cell by hybridizing total yeast RNA with an excess of gene-specific 32P-oligonucleotides, and digesting the resulting RNA-DNA hybrids with S1 nuclease . By comparing the his3 hybridization signal from a known amount of yeast cells to the signal generated by a known amount of his3 RNA synthesized in vitro, we determine that yeast strain KY114 growing in yeast extract/peptone/glucose medium at 30 degrees C contains seven molecules of his3 mRNA per cell . Using a galactose shut-off procedure, we determined that the half-life of his3 mRNA is approximately 11 min under these conditions . From these observations, we calculate that one his3 mRNA molecule is synthesized every 140 s . Analysis of other his3 promoter derivatives suggests that the maximal transcriptional initiation rate in yeast cells is one mRNA molecule every 6-8 s . Using his3 as an internal standard, the number of mRNA molecules per cell have been determined for ded1, trp3, rps4, and gall under a variety of growth conditions . From these results, the absolute mRNA level of any yeast gene can be determined in a single hybridization experiment . Moreover, the rate of transcriptional initiation can be determined for mRNAs whose decay rates are known. Mol Cell Biochem, 1996 May 24, 158(2), 121 - 4 Regulation of intracellular level of Na+, K+ and glycerol in Saccharomyces cerevisiae under osmotic stress; Sunder S et al.; The intracellular level of Na+ and K+ ofS . cerevisiae strain AB1375 revealed that under KCl as well as sorbitol stress, the cationic level was comparable to the level under no stress conditions . On the other hand, there was a sharp drop in the intracellular K+ content and increase in the Na+ content on addition of NaCl to the medium . However, the total cationic level was close to that under control conditions . In addition to changes in the cationic level, an enhanced production and accumulation of glycerol were also observed under osmotic stress . A regulatory mechanism co-ordinating the intracellular concentration of glycerol as well as Na+, K+ content under osmotic stress conditions has been proposed. Gene, 1996 May 24, 171(1), 41 - 7 A putative new membrane protein, Pho86p, in the inorganic phosphate uptake system of Saccharomyces cerevisiae; Yompakdee C et al.; The PHO84 gene in Saccharomyces cerevisiae encodes the P(i) transporter Pho84p . The other three genes, GTR1, PHO86 and PHO87, are also suggested to be involved in the P(i) uptake system . We cloned and sequenced PHO86 and found that it encodes a 34-kDa protein consisting of 311 amino acid residues with two strongly hydrophobic segments in its N-terminal half . Western blotting analysis of cell extracts revealed that Pho86p, tagged with c-Myc, was fractionated into a water-insoluble fraction . Disruption of PHO86 did not affect cell viability even in combination with the pho84 and/or pho87 disruptions . The triple disruptants showed high levels of constitutive rAPase synthesis and arsenate resistance similar to the pho84 mutant, but showed slower cell growth than the pho84 mutant . PHO86 has two putative binding sites for the transcriptional activator, Pho4p, at nucleotide positions -191 and -497 relative to the ATG start codon, and showed substantial levels of transcription under high-P(i) conditions and more enhanced levels in low-P(i) medium. Gene, 1996 May 24, 171(1), 33 - 40 A new Saccharomyces cerevisiae ankyrin repeat-encoding gene required for a normal rate of cell proliferation; Lycan DE et al.; We have characterized a new ankyrin (ANK) repeat-containing Saccharomyces cerevisiae gene, YAR1, located between the HSP82 and SUI3 genes on chromosome XVI . YAR1 encodes a 200-amino-acid (aa) protein with two ANK repeat motifs and an acidic C terminus rich in PEST-like sequences . The Yar1 ANK repeats are most similar to the conserved ANK repeats in the yeast cell cycle transcription factor, Swi6 . We show that YAR1 is transcribed as an 800-nucleotide (nt) poly(A)+ mRNA from a promoter lacking a consensus TATA sequence . YAR1 is transcribed in both haploid and diploid cells, and in haploid cells arrested in G1 with alpha-factor or in S phase with hydroxyurea . YAR1 shares an intergenic region with HSP82, and while HSP82 transcription is induced 15-fold by heat shock, transcription of YAR1 is transiently repressed by heat shock . We show that YAR1 is not an essential gene, but that haploid cells bearing a yar1 deletion grow significantly more slowly than do isogenic wild-type cells, especially at low temperature. Gene, 1996 May 24, 171(1), 27 - 32 A novel cross-phylum family of proteins comprises a KRR1 (YCL059c) gene which is essential for viability of Saccharomyces cerevisiae cells; Gromadka R et al.; We demonstrate here that the open reading frame (ORF) YCL059c, discovered during the systematic sequencing of chromosome III {Oliver et al., Nature 357 (1992) 38-46}, codes for a protein essential for yeast: neither spore germination nor cell division occur in strains deleted for this gene . We have cloned the wild-type (wt) gene and shown that it complements the deletion . A relatively abundant RNA transcript corresponds to the gene . The protein has no similarity to proteins of known function . Interestingly, however, it is homologous to several expressed sequence tags (EST) of unknown function from Caenorhabditis elegans, Oryza sativa and Homo sapiens . Thus, a novel family of proteins of presumably nuclear localization, with a characteristic highly basic motif, KRR-R, transcends various phyla, and plays an important role in cellular processes . We propose to call this essential gene KRR1. Gene, 1996 May 24, 171(1), 107 - 11 Structural and functional conservation of human and yeast HCP1 genes which can suppress the growth defect of the Saccharomyces cerevisiae ire15 mutant; Nikawa J et al.; The Saccharomyces cerevisiae ire15 mutant has a defect in the expression of the IN01 gene, showing an inositol auxotrophic phenotype . The growth defect of this mutant is suppressed by human cDNAs such as for the TGF-beta receptor-encoding gene (TGFR) {Nikawa, Gene 149 (1994) 367-372} . Here, we isolated a new human cDNA, HCP1, which suppresses the ire15 mutation by genetic complementation . Sequencing analysis revealed that HCP1 encodes 360 amino acid residues (40,515 Da) . The product of HCP1 is highly conserved among species and the yeast homolog was also found to suppress the ire15 mutation . Northern blot analysis revealed that multicopies of the yeast and human HCP1, as well as TGFR, resulted in an increase in the IN01 mRNA level in the yeast mutant . These results clearly indicate that the products of human and yeast HCP1 are structural and functional homologs, and are involved in expression of genes such as of IN01. Biochem Biophys Res Commun, 1996 May 24, 222(3), 827 - 32 Folding-dependent in vitro protein splicing of the Saccharomyces cerevisiae VMA1 protozyme; Kawasaki M et al.; VMA1 translational product undergoes excision of a 50-kDa intervening segment (VDE: VMA1-derived endonuclease) and religation of the flanking regions to create a 69-kDa catalytic subunit of vacuolar membrane H+-ATPase . VDEs conjugated with polypeptides at both N- and C-terminal ends were expressed in Escherichia coli and examined for their ability to catalyze self-splicing . Processed VDE was found in soluble pools, while unspliced precursors accumulated in insoluble pools, forming inclusion bodies . We demonstrate in vitro protein splicing by refolding of the denatured precursor molecules . The processing reaction efficiently occurs with the purified precursor peptide . VDE bracketed by only 6 proximal and 4 distal amino acids is autocatalytically processed. J Biol Chem, 1996 May 24, 271(21), 12585 - 94 The predominant protein-arginine methyltransferase from Saccharomyces cerevisiae; Gary JD et al.; We have identified the major enzymatic activity responsible for the S-adenosyl-L-methionine-dependent methylation of arginine residues (EC 2.1.1.23) in proteins of the yeast Saccharomyces cerevisiae . The RMT1 (protein-arginine methyltransferase), formerly ODP1, gene product encodes a 348-residue polypeptide of 39.8 kDa that catalyzes both the NG-mono- and NG, NG-asymmetric dimethylation of arginine residues in a variety of endogenous yeast polypeptides . A yeast strain in which the chromosomal RMT1 gene was disrupted is viable, but the level of NG,NG-{3H}dimethylarginine residues detected in intact cells incubated with S-adenosyl-L-{methyl-3H}methionine is reduced to less than 15% of the levels found in the parent strain, while the NG-{3H}monomethylarginine content is reduced to less than 30% . We show that soluble extract from parent cell, but not from mutant rmt1 cells, catalyzes the in vitro methylation of endogenous polypeptides of 55, 41, 38, 34, and 30 kDa . The hypomethylated form of these five polypeptides, as well as that of several others, can be mono- and asymmetrically dimethylated by incubating the mutant rmt1 extract with a purified, bacterially produced, glutathione S-transferase-RMT1 fusion protein and S-adenosyl-L-{methyl-3H}methionine . This glutathione S-transferase-RMT1 fusion protein is also able to methylate a number of mammalian polypeptides including histones, recombinant heterogeneous ribonucleoprotein A1, cytochrome c, and myoglobin, but cannot methylate myelin basic protein . RMT1 appears to be a yeast homolog of a recently characterized mammalian protein-arginine methyltransferase whose activity may be modulated by mitotic stimulation of cells. J Biol Chem, 1996 May 24, 271(21), 12275 - 80 Superoxide dismutase activity is essential for stationary phase survival in Saccharomyces cerevisiae . Mitochondrial production of toxic oxygen species in vivo; Longo VD et al.; Yeast lacking copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (SOD), catalase T, or metallothionein were studied using long-term stationary phase (10-45 days) as a simple model system to study the roles of antioxidant enzymes in aging . In well aerated cultures, the lack of either SOD resulted in dramatic loss of viability over the first few weeks of culture, with the CuZnSOD mutant showing the more severe defect . The double SOD mutant died within a few days . The severity reversed in low aeration; the CuZnSOD mutant remained viable longer than the manganese SOD mutant . To test whether reactive oxygen species generated during respiration play an important role in the observed cellular death, growth in nonfermentable carbon sources was measured . All strains grew under low aeration, indicating respiratory competence . High aeration caused much reduced growth in single SOD mutants, and the double mutant failed to grow . However, removal of respiration via another mutation dramatically increased short term survival and reversed the known air-dependent methionine and lysine auxotrophies . Our results suggest strongly that mitochondrial respiration is a major source of reactive oxygen species in vivo, as has been shown in vitro, and that these species are produced even under low aeration. Mol Gen Genet, 1996 May 23, 251(2), 211 - 9 Genetic evidence for the functional redundancy of the calcineurin- and Mpk1-mediated pathways in the regulation of cellular events important for growth in Saccharomyces cerevisiae; Nakamura T et al.; Saccharomyces cerevisiae mutants which exhibit phenotypes (calcium resistance and vanadate sensitivity) similar to those of calcineurin-deficient mutants were isolated . The mutants were classified into four complementation groups (crv1,2,3 and 4) . Crv1 was allelic to cnb1, a mutation in the regulatory subunit of calcineurin . The nucleotide sequences of CRV2 and CRV3 genes which complemented the crv2 and crv3 mutations, respectively, are identical to those of BCK1/SLK1/SKC1/SSP31 and MPK1/SLT2, respectively, which are both involved in the MAP kinase cascade . A calcineurin-deletion mutation (delta cnb1), which by itself has no detectable effect on growth and morphology, enhanced some phenotypes (slow growth and morphological abnormality) of crv2 and crv3 mutants . These phenotypes of crv2 and crv3 mutants were partially suppressed by Ca2+ or by overproduction of the calcineurin subunits (Cmp2 and Cnb1) . Like the calcineurin-deficient mutant, crv2 and crv3 mutants were defective in recovery from alpha-factor-induced growth arrest . The defect in recovery of the delta cnb1 mutant was suppressed by overexpression of MPK1 . These results indicated that the calcineurin-mediated and the Mpk1- (Bck1-) mediated signaling pathways act in parallel to regulate functionally redundant cellular events important for growth. Mol Gen Genet, 1996 May 23, 251(2), 146 - 52 A multicopy suppressor of nin1-1 of the yeast Saccharomyces cerevisiae is a counterpart of the Drosophila melanogaster diphenol oxidase A2 gene, Dox-A2; Kawamura M et al.; NIN1 is an essential gene for growth of the yeast Saccharomyces cerevisiae and was recently found to encode a component of the regulatory subunit of the 26S proteasome . The nin1-1 mutant is temperature sensitive and its main defect is in G1/S progression and G2/M progression at non-permissive temperatures . One of the two multicopy suppressors of nin1-1, SUN2 (SUppressor of Nin1-1), was found to encode a protein of 523 amino acids whose sequence is similar to those of Drosophila melanogaster diphenol oxidase A2 and the mouse mast-cell Tum(-) transplantation antigen, P91A . The C-terminal half of Sun2p was found to be functional as Sun2p at 25 degrees C, 30 degrees C, and 34 degrees C but not at 37 degrees C . The open reading frame (ORF) of the Drosophila diphenol oxidase A2 gene (Dox-A2) was obtained from a lambda phage cDNA library using the polymerase chain reaction technique . The Dox-A2 ORF driven by the TDH3 promoter complemented the phenotype of a strain deleted for sun2 . This Dox-A2-dependent strain was temperature sensitive and accumulated dumb-bell-shaped cells, with an undivided nucleus at the isthmus, after temperature upshift . This morphology is similar to that of nin1-1 cells kept at a restrictive temperature . These results suggest that SUN2 is a functional counterpart of Dox-A2 and that these genes play a pivotal role in the cell cycle in each organism. Biochim Biophys Acta, 1996 May 21, 1290(1), 95 - 100 Characterisation of an acid trehalase of Saccharomyces cerevisiae present in trehalase-sucrase aggregate; Biswas N et al.; An acid trehalase-sucrase aggregate was purified (by 780-fold) from Saccharomyces cerevisiae, following conventional protein purification techniques, to an apparent yield of 18.5% . The aggregate was electrophoretically homogeneous but contained 175, 90, 68, 60, 40 molar mass (kDa) bands on SDS-electrophoresis . The purified aggregate had a specific activity (acid trehalase) of 22 U/mg; a Km value of 5.0 mM but contained 3-times more sucrase activity . Only sucrose and trehalose were hydrolysed by this aggregate and both activities were inhibited by acetate or phosphate . Temperature and pH optima for trehalose hydrolysis appeared to be 40-45 degrees C and 5.0, respectively . The purified aggregate appeared to be disaggregating spontaneously resulting in inactivation of both enzymes, which was enhanced either at pH 3.5 or at pH 7.0 . Separation of acid trehalase from the aggregate by hydrophobic interaction chromatography resulted in inactivation . Rechromatography (HPGPLC) of the purified aggregate also gave disaggregation as well as inactivation of both enzymes . Disaggregated acid trehalase and sucrase contained 20-fold and 13-fold lower specific activities, respectively, and appeared to be unstable . Based on these observations we suggest that acid trehalase is stabilised by aggregation with sucrase. FEBS Lett, 1996 May 20, 386(2-3), 235 - 8 Deletion of the ATH1 gene in Saccharomyces cerevisiae prevents growth on trehalose; Nwaka S et al.; The biological function of the yeast trehalases (EC 3.2.1.28) consists of down-regulation of the concentration of trehalose via glucose formation by trehalose hydrolysis . While it is generally accepted that the cytosolic neutral trehalase (encoded by the NTH1 gene) is responsible for trehalose hydrolysis in intact cells, very little is known about a role of the vacuolar acid trehalase and the product of the recently described neutral trehalase gene YBRO106 (NTH2) . We have analyzed the role of the acid trehalase in trehalose hydrolysis using the ATH1 deletion mutant (delta ath1) of Saccharomyces cerevisiae {M . Destruelle et al . (1995) Yeast 11, 1015-10251 deficient in acid trehalase activity under various nutritional conditions . In contrast to wild-type and a mutant deficient in the neutral trehalase (delta nth1), the delta ath1 mutant does not grow on trehalose as a carbon source . Experiments with diploid strains heterozygous for delta ath1 show a gene dosage effect for the ATH1 gene for growth on trehalose . The need for acid trehalase for growth on trehalose is supported by the finding that acid trehalase activity is induced during exponential growth of cells on trehalose while no such induction is measurable during growth on glucose . Our results show that the vacuolar acid trehalase Ath1p is necessary for the phenotype of growth on trehalose, i.e . trehalose utilization, in contrast to cytosolic neutral trehalase Nth1p which is necessary for intracellular degradation of trehalose . For explanation of the need for vacuolar acid trehalase and not cytosolic neutral trehalase for growth on trehalose, the participation of endocytosis for uptake of trehalose from medium to the vacuoles is discussed. J Chromatogr B Biomed Appl, 1996 May 17, 680(1-2), 55 - 63 Liquid-liquid partitioning of some enzymes, especially phosphofructokinase, from Saccharomyces cerevisiae at sub-zero temperature; Grimonprez B et al.; The effects of low temperature (-18 degrees C) on the stability and partitioning of some glycolytic enzymes within an aqueous two-phase system were studied . The enzymes were phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase present in a crude extract of bakers' yeast . The partitioning of pure phosphofructokinase, isolated from bakers' yeast, was also examined . The two-phase systems were composed of water, poly(ethylene glycol), dextran, and ethylene glycol and buffer . THe influence on the partitioning of the presence of ethylene glycol, phenylmethylsulfonyl fluoride and poly(ethylene glycol)-bound Cibacron Blue F3G-A was investigated at -18, 0 and (in some cases) 20 degrees C . The presence of ethylene glycol, phase polymers and low temperature stabilized all three enzyme activities . Cibacron Blue, an affinity ligand for phosphofructokinase, increased its partitioning into the upper phase with decreasing temperature . Depending on the conditions, various amounts of the enzymes were recovered at the interface, also in systems not containing ethylene glycol . The implications of the observed effects on the use of aqueous two-phase systems for the extraction and fractionation of proteins are discussed. J Biol Chem, 1996 May 17, 271(20), 11844 - 51 Intragenic suppressors of P-loop mutations in the beta-subunit of the mitochondrial ATPase in the yeast Saccharomyces cerevisiae; Shen H et al.; Three intragenic second-site suppressors, P353L, T237I, and L390F, were identified that suppressed two mutations in, and one adjacent to, the P-loop in the beta-subunit of the yeast F1-ATPase . The crystal structure of bovine F1-ATPase (Abrahams, J . P., Leslie, A . G . W., Lutter, R., and Walker, J . E . (1994) Nature 370, 621-628) shows that these suppressor residues are located in the nucleotide-binding domain . Specific hypotheses have been formulated that suggest the conformational coupling of the P-loop with the suppressor sites . P353L is in a "catch" region, which forms unique interactions with the gamma-subunit in the three different conformational states of the catalytic site . The identification of this suppressor mutation demonstrates genetically that the catch region is conformationally coupled to the P-loop . T237I is shown to interact with Lys-209, which occurs just after the P-loop . This suggests that this interaction changes the conformation of the P-loop to suppress the initial mutation . L390F interacts with Ala-181, which is adjacent to the P-loop . The mechanism of this suppression is suggested to occur through the interactions of L390F with Ala-181 . These results identify critical interactions that modulate the structure of the P-loop and thus the biochemistry of the enzyme. J Biol Chem, 1996 May 17, 271(20), 12103 - 10 Interaction of wild-type and truncated forms of transcription factor IIIA from Saccharomyces cerevisiae with the 5 S RNA gene; Rowland O et al.; Transcription factor (TF) IIIA, which contains nine zinc finger motifs, binds to the internal control region of the 5S RNA gene as the first step in the assembly of a multifactor complex that promotes accurate initiation of transcription by RNA polymerase III . We have monitored the interaction of wild-type and truncated forms of yeast TFIIIA with the 5 S RNA gene . The DNase I footprints obtained with full-length TFIIIA and a polypeptide containing the amino-terminal five zinc fingers (TF5) were indistinguishable, extending from nucleotides +64 to +99 of the 5 S RNA gene . This suggests that fingers 6 through 9 of yeast TFIIIA are not in tight association with DNA . The DNase I footprint obtained with a polypeptide containing the amino-terminal four zinc fingers (TF4) was 14 base pairs shorter than that of TF5, extending from nucleotides +78 to +99 on the nontranscribed strand and from nucleotides +79 to +98 on the transcribed strand of the 5 S RNA gene . Protection provided by a polypeptide containing the first three zinc fingers (TF3) was similar to that provided by TF4, with the exception that protection on the nontranscribed strand ended at nucleotide +80, rather than nucleotide +78 . Methylation protection analysis indicated that finger 5 makes major groove contacts with guanines +73 and +74 . The amino-terminal four zinc fingers make contacts that span the internal control region, which extends from nucleotides +81 to +94 of the 5 S RNA gene, with finger 4 appearing to contact guanine +82 . Measurements of the apparent Kd values of the TFIIIA.DNA complexes indicated that the amino-terminal three zinc fingers of TFIIIA have a binding energy that is similar to that of the full-length protein. J Biol Chem, 1996 May 17, 271(20), 11810 - 6 Probing the active-site residues in Saccharomyces cerevisiae ferrochelatase by directed mutagenesis . In vivo and in vitro analyses; Gora M et al.; Ferrochelatase is a mitochondrial inner membrane-bound enzyme that catalyzes the insertion of ferrous iron into protoporphyrin, the terminal step in protoheme biosynthesis . The functional/structural roles of 10 invariant amino acid residues were investigated by site-directed mutagenesis in the yeast Saccharomyces cerevisiae ferrochelatase . The mutant enzymes were expressed in a yeast strain lacking the ferrochelatase gene HEM15 and in Escherichia coli . The kinetic parameters of the mutant enzymes were determined for the enzymes associated with the yeast membranes and the enzymes in the bacterial soluble fraction . They were compared with the in vivo functioning of the mutant enzymes . The main conclusions are the following . Glu-314 is critical for catalysis, and we suggest that it is the base responsible for abstracting the N-pyrrole proton(s) . His-235 is essential for metal binding . Asp-246 and Tyr-248 are also involved in metal binding in a synergistic manner . The Km for protoporphyrin was also increased in the H235L, D246A, and Y248L mutants, suggesting that the binding sites of the two substrates are not independent of each other . The R87A, Y95L, Q111E, Q273E, W282L, and F308A mutants had 1.2-2-fold increased Vm and 4-10-fold increased Km values for protoporphyrin, but the amount of heme made in vivo was 10-100% of the normal value . These mutations probably affected the geometry of the active center, resulting in improper positioning of protoporphyrin. J Biol Chem, 1996 May 17, 271(20), 12068 - 75 Purification, identification, and properties of a Saccharomyces cerevisiae oleate-activated upstream activating sequence-binding protein that is involved in the activation of POX1; Luo Y et al.; Peroxisomes have a central function in lipid metabolism, and it is well established that these organelles are inducible by many compounds including fatty acids . Peroxisomes are the sole site for the beta-oxidation of fatty acids in yeast . The first and rate-limiting enzyme of this cycle is fatty acyl-CoA oxidase . The gene encoding this enzyme in Saccharomyces cerevisiae (POX1) undergoes a complex regulation that is dependent on the growth environment . When this yeast is grown in medium containing oleic acid as the main carbon source, peroxisomes are induced and POX1 expression is activated . When cells are grown in the presence of glucose, the expression of POX1 mRNA is repressed, whereas growth on a carbon source such as glycerol or raffinose causes derepression . This rigorous regulation is brought about by the complex interactions between trans-acting factors and cis-elements in the POX1 promoter . Previously, we characterized regulatory elements in the promoter region of POX1 that are involved in the repression and activation of this gene (Wang, T., Luo, Y., and Small, G . M . (1994) J . Biol . Chem . 269, 24480-24485) . In this study we have purified and identified an oleate-activated transcription factor (Oaf1p) that binds to the activating sequence (UAS1) in the POX1 gene . The protein has a predicted molecular mass of approximately 118 kDa. Biochem Biophys Res Commun, 1996 May 15, 222(2), 280 - 6 Requirement of multiple DNA-protein interactions for inducible expression of RNR3 gene in Saccharomyces cerevisiae in response to DNA damage; Endo-Ichikawa Y et al.; The RNR3 gene encodes the large subunit of ribonucleotide reductase . Transcription of this gene is induced 12-fold in response to DNA damage or by a DNA replication blocker . To investigate cis-acting regulation, deletion analysis of the promoter region of the RNR3 gene was performed and we identified two upstream-repressing sequences in the RNR3 regulatory region . An 18-base-pairs fragment, termed DNA-damage responsive element 1 (DRE1) located between -212 and -194 in this region was found to be essential for the induction of RNR3 . This fragment contained a negatively acting sequence where a protein factor bound to the region during normal growth but disappeared by exposure to 4-nitroquinoline-1-oxide . The other repressive element homologue to DRE1 was located at -263 to -254 . One possible upstream-activating sequence which regulates the basal expression of RNR3 was also found . These results show that at least three potential cis-elements are necessary for the inducible expression of yeast expression of yeast RNR3 in response to DNA damage. Eur J Biochem, 1996 May 15, 238(1), 77 - 87 Reactions of mitochondrial cruciform cutting endonuclease 1 (CCE1) of yeast Saccharomyces cerevisiae with branched DNAs in vitro; Kupfer C et al.; Cruciform-cutting endonuclease 1 (CCE1) is an X-solvase from yeast Saccharomyces cerevisiae {Kleff, S., Kemper, B . & Sternglanz, R . (1992) EMBO J . 11, 699-704} . We report here the purification of the cloned enzyme CCE1 to near homogeneity from over-expressing Escherichia coli cells . The purified protein has a globular shape and an apparent molecular mass of 38 kDa . CCE1 reacts specifically with branched DNAs, preferably with four-armed cruciforms . The enzyme linearizes native supercoiled DNA by cutting at the base of cruciform structures as they occur in derivatives of phage M13 . Supercoiling was not required for cleavage per se and a relaxed circular DNA hybrid with a stable cruciform was linearized with the same relative cleavage efficiency . Fully synthetic cruciforms (four-armed X-junctions) were also good substrates for CCE1, provided a symmetric 6-bp sequence (in our case an EcoRI restriction site) was maintained at the junction . Consequently, a synthetic cruciform made from fully randomized oligonucleotide sequences was not a substrate for CCE1 . In general, cleavage sites were found clustered in a characteristic pattern in each arm of a cruciform structure . A synthetic three-armed Y-junction was also cleaved by CCE1, but with a lower efficiency than the related four-armed construct . CCE1 resolves efficiently branched synthetic DNAs in vitro . The function is consistent with the idea that CCE1 is responsible for a timely reversal of branched recombination intermediates preceding petite formation in mitochondrial DNA. Ann N Y Acad Sci, 1996 May 15, 782, 202 - 7 A URA3-promoter deletion in a pYES vector increases the expression level of a fungal lipase in Saccharomyces cerevisiae; Okkels JS; A simple deletion of the URA3 promoter from a Saccharomyces cerevisiae expression plasmid was performed . The promoter-deleted plasmid is shown to have an increased expression level of a fungal lipase gene . The deletion probably causes a poor expression of the URA3 selection marker, probably resulting in a higher copy number per cell of the plasmid . This higher copy number can increase the transcript level per cell and there by the expression level . In the case of the fungal lipase gene, the expression level with defined inoculum is increased at least three times . The principle is most likely similar to the LEU2d plasmids described previously . A part of the 2-micron origin of the pYES type plasmid was also deleted by the URA3 promoter deletion without affecting transformation frequency . The URA3 promoter can easily be deleted from most pYES type plasmids by the described method. Nucleic Acids Res, 1996 May 15, 24(10), 1822 - 8 Identification and characterisation of two transcriptional repressor elements within the coding sequence of the Saccharomyces cerevisiae HXK2 gene; Herrero P et al.; A well-defined set of isogenic yeast strains has been constructed whereby each strain contains a different HXK2::lacZ gene fusion integrated at the URA3 locus . These HXK2::lacZ fusions differ in the amount of the HXK2 gene (encoding hexokinase 2 isoenzyme) that is fused to the lacZ reporter gene . Comparison of the beta-galactosidase activities of each strain during growth on glucose or ethanol revealed that some part of the coding region between +39 and +404 bp is involved in repressing gene expression in a carbon source dependent manner . A series of deletions of this HXK2 coding region were constructed and fused upstream of a minimal CYC1::lacZ promoter . beta-Galactosidase activities on glucose or ethanol growth yeast calls revealed that two different regulatory elements are present in this DNA region . Gel mobility shift analysis and in vitro DNase I footprinting have shown that proteins bind specifically to two downstream repressor sequences (DRS1 located from +140 to +163 and DRS2 located between +231 and +251) that influence the rate of HXK2 transcription when ethanol is used as carbon source by Saccharomyces cerevisiae . We identified and partially purified a 18 kDa protein that binds specifically to synthetic double-stranded oligonucleotides containing the (A/C)(A/G)GAAAT box sequence . Our data suggest that p18 synthesis is under the control of genes involved in glucose repression (MIG1 = CAT4) and glucose derepression (SNF1 = CAT1). Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 5116 - 21 Oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae; Davidson JF et al.; The cause for death after lethal heat shock is not well understood . A shift from low to intermediate temperature causes the induction of heat-shock proteins in most organisms . However, except for HSP104, a convincing involvement of heat-shock proteins in the development of stress resistance has not been established in Saccharomyces cerevisiae . This paper shows that oxidative stress and antioxidant enzymes play a major role in heat-induced cell death in yeast . Mutants deleted for the antioxidant genes catalase, superoxide dismutase, and cytochrome c peroxidase were more sensitive to the lethal effect of heat than isogenic wild-type cells . Overexpression of catalase and superoxide dismutase genes caused an increase in thermotolerance . Anaerobic conditions caused a 500- to 20,000-fold increase in thermotolerance . The thermotolerance of cells in anaerobic conditions was immediately abolished upon oxygen exposure . HSP104 is not responsible for the increased resistance of anaerobically grown cells . The thermotolerance of anaerobically grown cells is not due to expression of heat-shock proteins . By using an oxidation-dependent fluorescent molecular probe a 2- to 3-fold increase in fluorescence was found upon heating . Thus, we conclude that oxidative stress is involved in heat-induced cell death. Mol Cell Biochem, 1996 May 10, 158(1), 65 - 75 Saccharomyces cerevisiae DNA repair processes: an update; Ramotar D et al.; The budding yeast Saccharomyces cerevisiae plays a central role in contributing to the understanding of one of the most important biological process, DNA repair, that maintains genuine copies of the cellular chromosomes . DNA lesions produce either spontaneously or by DNA damaging agents are efficiently repaired by one or more DNA repair proteins . While some DNA repair proteins function independently as in the case of base excision repair, others belong into three separate DNA repair pathways, nucleotide excision, mismatch, and recombinational . Of these pathways, nucleotide excision and mismatch repair show the greatest functional conservation between yeast and human cells . Because of this high degree of conservation, yeast has been regarded as one of the best model system to study DNA repair . This report therefore updates current knowledge of the major yeast DNA repair processes. J Biol Chem, 1996 May 10, 271(19), 11400 - 9 Requirement for the carboxyl-terminal domain of Saccharomyces cerevisiae carbamoyl-phosphate synthetase; Lim AL et al.; The arginine-specific carbamoyl phosphate synthetase of Saccharomyces cerevisiae is a heterodimeric enzyme, with a 45-kDa CPA1 subunit binding and cleaving glutamine, and a 124-kDa CPA2 subunit accepting the ammonia moiety cleaved from glutamine, binding all of the remaining substrates and carrying out all of the other catalytic events . CPA2 is composed of two apparently duplicated amino acid sequences involved in binding the two ATP molecules needed for carbamoyl phosphate synthesis and a carboxyl-terminal domain which appears to be less tightly folded than the remainder of the protein . Using deletion mutagenesis, we have established that essentially all of the carboxyl-terminal domain of CPA2 is required for catalytic function and that even small truncations lead to significant changes in the CPA2 conformation . In addition, we have demonstrated that the C-terminal region of CPA2 can be expressed as an autonomously folded unit which is stabilized by specific interactions with the remainder of CPA2 . We also made the unexpected finding that, even when ammonia is used as the substrate and there is no catalytic role for CPA1, interaction with CPA1 led to an increase in the Vmax of CPA2 in crude extracts. Gene, 1996 May 8, 170(2), 243 - 8 A lea-class gene of tomato confers salt and freezing tolerance when expressed in Saccharomyces cerevisiae; Imai R et al.; During periods of water deficit, plants accumulate late embryogenesis-abundant (LEA) proteins which are thought to protect cells from stresses associated with dehydration . One of these genes, le25, is expressed in tomato leaves and roots in response to water deficit and abscisic acid accumulation . To study the function of this protein and to test the effect of overproduction of the LE25 protein in Saccharomyces cerevisiae (Sc), a recombinant plasmid in which le25 is expressed under the control of the GAL1 promoter was constructed . The content of LE25 was high in Sc cells transformed with the recombinant plasmid . The transformant exhibited several stress-tolerant phenotypes . Growth of the transformant in a medium with 1.2 M NaCl was improved, as compared to a control strain . While the control strain showed a long lag phase of 40 h, le25-expressing cells showed a shortened lag phase of 10 h . However, no growth improvement was observed in a medium with 2 M sorbitol . In addition, the transformant had an increased survival rate after freezing stress, but not after high-temperature stress . These results, together with its predicted secondary structure, may indicate that LE25 functions as an ion scavenger. Biochemistry, 1996 May 7, 35(18), 5879 - 82 Stoichiometry of binding of mature and truncated forms of the dihydrolipoamide dehydrogenase-binding protein to the dihydrolipoamide acetyltransferase core of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae; Maeng CY et al.; The dihydrolipoamide dehydrogenase-binding protein (E3BP), a component of the Saccharomyces cerevisiae and mammalian pyruvate dehydrogenase (PDH) complexes, anchors an E3 homodimer inside each of the 12 pentagonal faces of the 60-mer dihydrolipoamide acetyltransferase (E2) . To gain further insight into the number and localization of binding sites for E3BP on the 60-mer E2, truncated forms of the E3BP lacking the lipoyl and E3-binding domains were engineered by deletion mutagenesis . The recombinant proteins contained a polyhistidine extension on the amino terminus to facilitate purification to near-homogeneity . The stoichiometry of binding of the truncation mutants to a truncated form (inner core) of E2 (tE2, residues 181-454), lacking the lipoyl domain and the E1-binding domain, was determined . Mixtures containing tE2 and excess intact or truncated forms of E3BP were subjected to ultracentrifugation to separate the large complexes from unbound E3BP or tE3BP, and the complexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis . After staining with Coomassie brilliant blue and destaining, the gels were analyzed with a video area densitometer . The results showed that tE2 binds about 20 copies of intact E3BP-H, about 24 copies of tE3BP-H144 (residues 144-380), lacking the lipoyl domain, and about 31 copies of tE3BP-H218 (residues 218-380), lacking both the lipoyl and E3-binding domains . The results indicate that there apparently is a binding site for E3BP on each E2 subunit and that steric hindrance by segments of E3BP prevents full stoichiometric binding of E3BP to the pentagonal dodecahedron-like E2. J Biol Chem, 1996 May 3, 271(18), 10859 - 65 Ribosomal association of poly(A)-binding protein in poly(A)-deficient Saccharomyces cerevisiae; Proweller A et al.; Poly(A)-binding protein, the most abundant eukaryotic mRNP protein, is known primarily for its association with polyadenylate tails of mRNA . In the yeast, Saccharomyces cerevisiae, this protein (Pabp) was found to be essential for viability and has been implicated in models featuring roles in mRNA stability and as an enhancer of translation initiation . Although the mechanism of action is unknown, it is thought to require an activity to bind poly(A) tails and an additional capacity for an interaction with 60 S ribosomal subunits, perhaps via ribosomal protein L46 (Rpl46) . We have found that a significant amount of Pabp in wild-type cells is not associated with polyribosome complexes . The remaining majority, which is found in these complexes, maintains its association even in yeast cells deficient in polyadenylated mRNA and/or Rpl46 . These observations suggest that Pabp may not require interaction with poly(A) tails during translation . Further treatment of polyribosome lysates with agents known to differentially disrupt components of polyribosomes indicated that Pabp may require contact with some RNA component of the polyribosome, which could be either non-poly(A)-rich sequences of the translated mRNA or possibly a component of the ribosome . These findings suggest that Pabp may possess the ability to bind to ribosomes independently of its interaction with poly(A) . We discuss these conclusions with respect to current models suggesting a multifunctional binding capacity of Pabp. Mikrobiologiia, 1996 May-Jun, 65(3), 345 - 7 {NAD-metabolizing capacity of Saccharomyces cerevisiae mutants}; Khalmuradov AG et al.; The activity of the key enzymes of the salvage synthesis and degradation of NAD (nicotinic acid mononucleotide pyrophosphorylase and NAD glycohydrolase) was studied in Saccharomyces cerevisiae mutants selected earlier as potential producers of NAD . An enhanced NAD-producing ability of the mutants was shown to be related to their reduced NAD-degrading activity: thus, NAD glycohydrolase in mutants 110, 118, and 206 was inactive in all growth phases regardless of the level of NAD biosynthesis. Prikl Biokhim Mikrobiol, 1996 May-Jun, 32(3), 311 - 4 {Cloning the Saccharomycopsis fibuligera alpha-amylase gene and its expression in Saccharomyces cerevisiae}; Baeva LF et al.; The alpha-amylase gene of yeast Saccharomycopsis fibuligera was cloned, partially sequenced, and expressed in Saccharomyces cerevisiae . Four amino acid substitutions were identified in the enzyme structure as compared to the earlier cloned gene . The Saccharomycopsis fibuligera alpha-amylase gene was expressed in Saccharomyces cerevisiae under the control of its own promoter . The native alpha-amylase signal peptide provided an efficient secretion of the enzyme by Saccharomyces cerevisiae . The efficiency of the alpha-amylase secretion was 98% . The pH optimum of the enzyme secreted by Saccharomyces cerevisiae was 4.0. Protein Expr Purif, 1996 May, 7(3), 299 - 308 Functional and structural properties of the homogeneous beta-glycosidase from the extreme thermoacidophilic archaeon sulfolobus solfataricus expressed in Saccharomyces cerevisiae; D'Auria S et al.; A protein with beta-glycosidase activity from Sulfolobus solfataricus (S beta gly) was expressed in the yeast Saccharomyces cerevisiae . The purification procedure was made fast and easy by employing a single chromatographic step . After 5.8-fold purification, the cell extract gave a homogeneous enzyme at 166 U/mg . The recombinant enzyme was functionally and structurally similar to the wild-type enzyme . Kinetic experiments showed the same wide substrate specificity; in fact, the expressed enzyme hydrolyzed beta-D-gluco-, fuco-, and galactosides and a large number of glucoside dimers and oligomers, linked beta 1 -> 4 . Moreover, the molecular mass of the enzyme was estimated to be 60 kDa by SDS-PAGE and 240 kDa by gel filtration, glycerol gradient, and ultracentrifugation analyses, indicating that the enzyme has a tetrameric structure . The N-terminal amino acid sequence, the temperature dependent activity, and content of secondary structure were similar to those of the wild-type enzyme . CD spectral and kinetic analyses showed that the only differences from the wild-type enzyme consist of the absence of lysine methylation, the presence of some glycosylated amino acid residues, and lower thermostability . Furthermore, calorimetric analyses on the expressed protein indicated values of delta dH = 5072 kJ/ mol and delta (d)C(p)= 100 kJ/mol, appreciably lower than those of the wild-type protein. Biol Chem Hoppe Seyler, 1996 May, 377(5), 313 - 7 Purification and characterisation of the pyruvate decarboxylase from a haploid strain of Saccharomyces cerevisiae; Killenberg-Jabs M et al.; A novel purification procedure was developed for pyruvate decarboxylase (PDC, E.C . 1.1.1.4) from the haploid yeast strain YSH 4.127-1A expressing only one (PDC1) of the three structural genes for PDC . The purified enzyme is homotetrameric with a molecular mass of about 240,000 whereas PDC from brewer's yeast is a dimer of dimers composed of subunits of different size (alpha 2 beta 2) with the same molecular mass as the tetramer . Despite these structural variations there are no significant differences in the kinetic behaviour of the two enzyme species . PDC purified from the haploid yeast mutants shows a sigmoid dependence of the reaction rate from the substrate concentration due to the substrate activation . In the presence of the substrate surrogate pyruvamide the shape of the v/S plot is transformed into a hyperbolic one . As expected, polyclonal antibodies react with both the enzyme from haploid yeast strain mutants and that from brewer's yeast. Yeast, 1996 May, 12(6), 609 - 13 ERG1, encoding squalene epoxidase, is located on the right arm of chromosome VII of Saccharomyces cerevisiae; Landl KM et al.; The ERG1 gene of Saccharomyces cerevisiae encodes squalene epoxidase, a key enzyme in the ergosterol pathway . ERG1 is an essential gene . Disruption of the gene with URA3 results in a lethal phenotype when cells are grown under aerobic conditions, even in the presence of ergosterol . However, cells are viable in the presence of ergosterol under anaerobic growth conditions during which ergosterol is taken up by cells . Physical and genetic mapping data reveal that ERG1 is located on the right arm of chromosome VII proximal to QCR9 at a distance of 14.6 cM from ADE3. Yeast, 1996 May, 12(6), 599 - 608 Sequence analysis of a 14.2 kb fragment of Saccharomyces cerevisiae chromosome XIV that includes the ypt53, tRNALeu and gsr m2 genes and four new open reading frames; Garcia-Cantalejo JM et al.; As part of the EU yeast genome program, a fragment of 14,262 bp from the left arm of Saccharomyces cerevisiae chromosome XIV has been sequenced . This fragment corresponds to cosmid 14-14b and is located roughly 130 kb from the centromere . It contains four new open reading frames which encode potential proteins of more than 99 amino acids, as well as the ypt53, tRNALeu and gsr moffenes . The putative protein N2212 is similar to the ribosomal protein S7 from humans . N2215 contains several predicted transmembrane elements . N2231 contains regions which are rich in acidic, as well as basic, residues which could from alpha-helical structures . Similar regions are found in a variety of proteins including glutamic acid rich protein, trichohyalin, caldesmon, Tb-29 and several cytoskeleton-interacting proteins. Mol Biol Cell, 1996 May, 7(5), 769 - 89 Different subcellular localization of Saccharomyces cerevisiae HMG-CoA reductase isozymes at elevated levels corresponds to distinct endoplasmic reticulum membrane proliferations; Koning AJ et al.; In all eucaryotic cell types analyzed, proliferations of the endoplasmic reticulum (ER) can be induced by increasing the levels of certain integral ER proteins . One of the best characterized of these proteins is HMG-CoA reductase, which catalyzes the rate-limiting step in sterol biosynthesis . We have investigated the subcellular distributions of the two HMG-CoA reductase isozymes in Saccharomyces cerevisiae and the types of ER proliferations that arise in response to elevated levels of each isozyme . At endogenous expression levels, Hmg1p and Hmg2p were both primarily localized in the nuclear envelope . However, at increased levels, the isozymes displayed distinct subcellular localization patterns in which each isozyme was predominantly localized in a different region of the ER . Specifically, increased levels of Hmg1p were concentrated in the nuclear envelope, whereas increased levels of Hmg2p were concentrated in the peripheral ER . In addition, an Hmg2p chimeric protein containing a 77-amino acid lumenal segment from Hmg1p was localized in a pattern that resembled that of Hmg1p when expressed at increased levels . Reflecting their different subcellular distributions, elevated levels of Hmg1p and Hmg2p induced sets of ER membrane proliferations with distinct morphologies . The ER membrane protein, Sec61p, was localized in the membranes induced by both Hmg1p and Hmg2p green fluorescent protein (GFP) fusions . In contrast, the lumenal ER protein, Kar2p, was present in Hmg1p:GFP membranes, but only rarely in Hmg2p:GFP membranes . These results indicated that the membranes synthesized in response to Hmg1p and Hmg2p were derived from the ER, but that the membranes were not identical in protein composition . We determined that the different types of ER proliferations were not simply due to quantitative differences in protein amounts or to the different half-lives of the two isozymes . It is possible that the specific distributions of the two yeast HMG-CoA reductase isozymes and their corresponding membrane proliferations may reveal regions of the ER that are specialized for certain branches of the sterol biosynthetic pathway. Biochem Mol Biol Int, 1996 May, 38(6), 1271 - 6 Lack of evidence of oxidative damage in antioxidant-deficient strains of Saccharomyces cerevisiae; Fortuniak A et al.; The content of reactive carbonyls and of glutathione-protein mixed disulfides, two indices of oxidative stress, were compared in wild-type Saccharomyces cerevisiae and in strains deficient in superoxide dismutase and catalase, and of decreased glutathione level . Both indices were higher in stationary than in logarithmic cultures and were not increased in antioxidant-deficient strains . Oxidation of dichlorofluorescin, an estimate of peroxide production, measured in the presence of exogenous peroxidase, was higher in antioxidant-deficient strains . These results corroborate our previous results on compensatory antioxidant mechanisms in the mutant yeast strains. Genetics, 1996 May, 143(1), 119 - 27 Genetic interactions between REG1/HEX2 and GLC7, the gene encoding the protein phosphatase type 1 catalytic subunit in Saccharomyces cerevisiae; Huang D et al.; Mutations in GLC7, the gene encoding the type 1 protein phosphatase catalytic subunit, cause a variety of abberrant phenotypes in yeast, such as impaired glycogen synthesis and relief of glucose repression of the expression of some genes . Loss of function of the REG1/HEX2 gene, necessary for glucose repression of several genes, was found to suppress the glycogen-deficient phenotype of the glc7-1 allele . Deletion of REG1 in a wild-type background led to overaccumulation of glycogen as well as slow growth and an enlarged cell size . However, loss of REG1 did not suppress other phenotypes associated with GLC7 mutations, such as inability to sporulate or, in cells bearing the glc7Y-170 allele, lack of growth at 14 degrees . The effect of REG1 deletion on glycogen accumulation is not simply due to derepression of glucose-repressed genes, although it does require the presence of SNF1, which encodes a protein kinase essential for expression of glucose-repressed genes and for glycogen accumulation . We propose that REG1 has a role in controlling glycogen accumulation. Genetics, 1996 May, 143(1), 81 - 93 Genetic analysis of Rap1p/Sir3p interactions in telomeric and HML silencing in Saccharomyces cerevisiae; Liu C et al.; We have identified three SIR3 suppressors of the telomeric silencing defects conferred by missense mutations within the Rap1p C-terminal tail domain (aa 800-827) . Each SIR3 suppressor was also capable of suppressing a rap1 allele (rap1-21), which deletes the 28 aa C-terminal tail domain, but none of the suppressors restored telometric silencing to a 165 amino acid truncation allele . These data suggest a Rap1p site for Sir3p association between the two truncation points (aa 664-799) . In SIR3 suppressor strains lacking the Rap1p C-terminal tail domain, the presence of a second intragenic mutation within the rap1s domain (aa 727-747), enhanced silencing 30-300-fold . These data suggest a competition between Sir3p and factors that interfere with silencing for association in the rap1s domain . Rap1-21 strains containing both wild-type Sir3p and either of the Sir3 suppressor proteins displayed a 400-4000-fold increase in telomeric silencing over rap1-21 strains carrying either Sir3p suppressor in the absence of wild-type Sir3p . We propose that this telomere-specific synergism is mediated in part through stabilization of Rap1p/Sir3p telometric complexes by Sir3p-Sir3p interactions. Genetics, 1996 May, 143(1), 57 - 66 Deletion of the gene encoding the cyclin-dependent protein kinase Pho85 alters glycogen metabolism in Saccharomyces cerevisiae; Timblin BK et al.; Pho85, a protein kinase with significant homology to the cyclin-dependent kinase, Cdc28, has been shown to function in repression of transcription of acid phosphatase (APase, encoded by PHO5) in high phosphate (Pi) medium, as well as in regulation of the cell cycle at G1/S . We described several unique phenotypes associated with the deletion of the PHO85 gene including growth defects on a variety of carbon sources and hyperaccumulation of glycogen in rich medium high in Pi . Hyperaccumulation of glycogen in the pho85 strains is independent of other APase regulatory molecules and is not signaled through Snfl kinase . However, constitutive activation of cAPK suppresses the hyperaccumulation of glycogen in a pho85 mutant . Mutation of the type-1 protein phosphatase encoded by GLC7 only partially suppresses the glycogen phenotype of the pho85 mutant . Additionally, strains containing a deletion of the PHO85 gene show an increase in expression of GSY2 . This work provides evidence that Pho85 has functions in addition to transcriptional regulation of APase and cell-cycle progression including the regulation of glycogen levels in the cell and may provide a link between the nutritional state of the cell and these growth related responses. Genetics, 1996 May, 143(1), 45 - 55 Genetic studies of the PRP17 gene of Saccharomyces cerevisiae: a domain essential for function maps to a nonconserved region of the protein; Seshadri V et al.; The PRP17 gene product is required for the second step of pre-mRNA splicing reactions . The C-terminal half of this protein bears four repeat units with homology to the beta transducin repeat . Missense mutations in three temperature-sensitive prp17 mutants map to a region in the N-terminal half of the protein . We have generated, in vitro, 11 missense alleles at the beta transducin repeat units and find that only one affects function in vivo . A phenotypically silent missense allele at the fourth repeat unit enhances the slow-growing phenotype conferred by an allele at the third repeat, suggesting an interaction between these domains . Although many missense mutations in highly conserved amino acids lack phenotypic effects, deletion analysis suggests an essential role for these units . Only mutations in the N-terminal nonconserved domain of PRP17 are synthetically lethal in combination with mutations in PRP16 and PRP18, two other gene products required for the second splicing reaction . A mutually allele-specific interaction between Prp17 and snr7, with mutations in U5 snRNA, was observed . We therefore suggest that the functional region of Prp17p that interacts with Prp18p, Prp16p, and U5 snRNA is the N terminal region of the protein. Biosci Biotechnol Biochem, 1996 May, 60(5), 798 - 801 Isolation and genetic characterization of pGKL killer-insensitive mutants (iki) from Saccharomyces cerevisiae; Kishida M et al.; The linear double stranded DNA plasmid pGKL1 encodes the yeast killer toxin complex (Gunge et al., 1981) of which the killing mechanism is not understood . We isolated and characterized eight mutants in Saccharomyces cerevisiae that were insensitive to both the intracellularly expressed 28-kDa killer subunit and the native killer toxin complex . These mutations (iki1 through iki5) were all recessive, and classified into five complementation groups . The iki2 mutation was mapped to a position near the centromere on chromosome XIII . We developed a novel screening system to isolate the DNA fragments complementing the iki mutations from a Saccharomyces gene library, and isolated three DNA fragments that complement the iki1, iki3, and iki4 mutations, respectively. Plant Physiol, 1996 May, 111(1), 223 - 6 Functional expression of the extraplastidial Arabidopsis thaliana oleate desaturase gene (FAD2) in Saccharomyces cerevisiae; Covello PS et al.; The functional expression in yeast of the Arabidopsis thaliana FAD2 gene, encoding the extraplastidial oleate desaturase (1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase) is reported . Dienoic fatty acids constituted up to 11% (w/w) of the total fatty acids in transformed Saccharomyces cerevisiae cells and were confirmed to be linoleic acid and delta 9, delta 12-hexadecadienoic acid by gas chromatography-mass spectrometry. RNA, 1996 May, 2(5), 441 - 51 Mutational analysis of Saccharomyces cerevisiae nuclear RNase P: randomization of universally conserved positions in the RNA subunit; Pagan-Ramos E et al.; Three regions in the Saccharomyces cerevisiae RNase P RNA have been identified, at positions Sce 87-94, Sce 309-316, and Sce 339-349, that contain nucleotides that are invariant in identity and position among all the known RNase P RNAs . To study the importance of these conserved RPR1 RNA regions in enzyme function, three independent mutational libraries were created in which the positions of invariant nucleotides were randomized simultaneously . Screening in vivo was used to identify viable RPR1 variants when reconstituted into holoenzyme in cells . Despite the universal evolutionary conservation, most of these positions tolerate certain sequence changes without severely affecting function . Most changes, however, produced subtle defects in cell growth and RNase P function, supporting the importance of these conserved regions . Isolation of conditional growth mutants allowed the characterization of the effects of mutations on cell growth, RPR1 RNA maturation, and activity of the holoenzyme in vitro . Kinetic analysis showed that viable variants were usually more defective in catalytic rate (Kcat) than in substrate recognition (Km). Curr Genet, 1996 May, 29(6), 594 - 6 Complementation of a pgk deletion mutation in Saccharomyces cerevisiae with expression of the phosphoglycerate-kinase gene from the hyperthermophilic Archaeon Sulfolobus solfataricus; Piper PW et al.; The gene encoding phosphoglycerate kinase (PGK) from the Archaeon Sulfolobus solfataricus, an organism growing optimally at 87 degrees C, was inserted into a yeast expression vector under the control of the galactose-inducible GAL1 yeast promoter . This vector was then transformed into a pgk::TRP1 yeast mutant, a strain inhibited for growth on galactose or glucose due to its lack of PGK enzyme . Slow-growing transformants were obtained on galactose plates at 37 degrees C, but not 28 degrees C . These transformants contained low levels of transcripts of the heterologous gene and low amounts of thermostable PGK activity . Weak expression of the hyperthermophile gene in yeast, a mesophile, therefore enabled complementation of the yeast pgk defect at 37 degrees C but not at 28 degrees C. Curr Genet, 1996 May, 29(6), 511 - 5 Glutathione is an essential metabolite required for resistance to oxidative stress in the yeast Saccharomyces cerevisiae; Grant CM et al.; Glutathione (GSH) is an abundant cellular thiol which has been implicated in numerous cellular processes and in protection against stress caused by xenobiotics, carcinogens and radiation . Our experiments address the requirement for GSH in yeast, and its role in protection against oxidative stress . Mutants which are unable to synthesis GSH due to a gene disruption in GSH 1, encoding the enzyme for the first step in the biosynthesis of GSH, require exogenous GSH for growth under non-stress conditions . Growth can also be restored with reducing agents containing a sulphydryl group, including dithiothreitol, beta-mercaptoethanol and cysteine, indicating that GSH is essential only as a reductant during normal cellular processes . In addition, the GSH 1-disruption strain is sensitive to oxidative stress caused by H2O2 and tert-butyl hydroperoxide . The requirement for GSH in protection against oxidative stress is analogous to that in higher eukaryotes, but unlike the situation in bacteria where it is dispensable for growth during both normal and oxidative stress conditions. Curr Genet, 1996 May, 29(6), 503 - 10 Expression of functional HIV-1 integrase in the yeast Saccharomyces cerevisiae leads to the emergence of a lethal phenotype: potential use for inhibitor screening; Caumont AB et al.; The integrase of the human immunodeficiency virus type 1 (HIV-1) has been expressed in yeast in order to investigate its potential lethal effect mediated by DNA damage . To this end, we have constructed an expression plasmid containing the retroviral integrase gene under the control of the inducible promotor ADH2/GAPDH which is regulated by the glucose concentration of the medium . Haploid yeast strain W303-1A did not appear to be clearly sensitive to HIV-1 integrase expression . However, disruption of the RAD 52 gene, which is involved in the repair of double-strand DNA breaks, strongly increased the deleterious effects of the retroviral enzyme in this yeast strain . The diploid strain constructed with W303-1A and an isogenic strain of the opposite mating type also showed a strong sensitivity to the HIV-1 integrase . Under yeast culture conditions allowing moderate integrase synthesis, the deleterious effect was totally abolished by missense integrase mutations, which are known to abolish HIV-1 integrase activities in vitro . We conclude that the lethal phenotype due to HIV-1 integrase expression in yeast may be closely related to the HIV-1 integration reaction in infected human cells, and that yeast may be a useful tool to study the HIV-1 integration process and to screen drugs capable of inhibiting HIV-1 integration in vivo. EMBO J, 1996 May 1, 15(9), 2227 - 35 The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE); Martinez-Pastor MT et al.; The MSN2 and MSN4 genes encode homologous and functionally redundant Cys2His2 zinc finger proteins . A disruption of both MSN2 and MSN4 genes results in a higher sensitivity to different stresses, including carbon source starvation, heat shock and severe osmotic and oxidative stresses . We show that MSN2 and MSN4 are required for activation of several yeast genes such as CTT1, DDR2 and HSP12, whose induction is mediated through stress-response elements (STREs) . Msn2p and Msn4p are important factors for the stress-induced activation of STRE dependent promoters and bind specifically to STRE-containing oligonucleotides . Our results suggest that MSN2 and MSN4 encode a DNA-binding component of the stress responsive system and it is likely that they act as positive transcription factors. J Bacteriol, 1996 May, 178(10), 2978 - 81 A halotolerant mutant of Saccharomyces cerevisiae; Gaxiola R et al.; FRD, a nuclear and dominant spontaneous mutant of Saccharomyces cerevisiae capable of growing in up to 2 M NaCl, was isolated . Compared with parental cells, the mutant cells have a lower intracellular Na+/K+ ratio, shorter generation times in the presence of 1 M NaCl, and alterations in gene expression. Mol Cell Biol, 1996 May, 16(5), 2518 - 26 Induction of meiosis in Saccharomyces cerevisiae depends on conversion of the transcriptional represssor Ume6 to a positive regulator by its regulated association with the transcriptional activator Ime1; Rubin-Bejerano I et al.; The transcription of meiosis-specific genes, as well as the initiation of meiosis, in the budding yeast Saccharomyces cerevisiae depends on IME1 . IME1 encodes a transcriptional activator which lacks known DNA binding motifs . In this study we have determined the mode by which Ime1 specifically activates the transcription of meiotic genes . We demonstrate that Ime1 is recruited to the promoters of meiotic genes by interacting with a DNA-binding protein, Ume6 . This association between Ime1 and Ume6 depends on both starvation and the activity of a protein kinase, encoded by RIM11 In the absence of Ime1, Ume6 represses the transcription of meiotic genes . However, in the presence of Ime1, or when Ume6 is fused in frame to the Gal4 activation domain, Ume6 is converted from a repressor to an activator, resulting in the transcription of meiosis-specific genes and the formation of asci. Mol Cell Biol, 1996 May, 16(5), 2504 - 8 Analysis of the galactose signal transduction pathway in Saccharomyces cerevisiae: interaction between Gal3p and Gal80p; Suzuki-Fujimoto T et al.; The GAL3 gene plays a critical role in galactose induction of the GAL genes that encode galactose- metabolizing enzymes in Saccharomyces cerevisiae . Defects in GAL3 result in a long delay in GAL gene induction, and overproduction of Gal3p causes constitutive expression of GAL . Here we demonstrate that concomitant overproduction of the negative regulator, Gal80p, and Gal3p suppresses this constitutive GAL expression . This interplay between Gal80p and Gal3p is direct, as tagged Gal3p coimmunoprecipitated with Gal80p . The amount of coprecipitated Gal80p increased when GAL80 yeast cells were grown in the presence of galactose . When both GAL80 and GAL3 were overexpressed, the amount of coprecipitated Gal80p was not affected by galactose . Tagged gal3 mutant proteins bound to purified Gal80p, but only poorly in comparison with the wild type, suggesting that formation of the Gal80p-Gal3p complex depends on the normal function of Gal3p . Gal3p appeared larger in Western blots (immunoblots) than predicted by the published nucleic acid sequence . Reexamination of the DNA sequence of GAL3 revealed several mistakes, including an extension at the 3' end of another predicted 97 amino acids. Mol Cell Biol, 1996 May, 16(5), 2483 - 95 Tethered Sir3p nucleates silencing at telomeres and internal loci in Saccharomyces cerevisiae; Lustig AJ et al.; Rap1p binds to sites embedded within the Saccharomyces cerevisiae telomeric TG1-3 tract . Previous studies have led to the hypothesis that Rap1p may recruit Sir3p and Sir3p-associating factors to the telomere . To test this, we tethered Sir3p adjacent to the telomere via LexA binding sites in the rap1-17 mutant that truncates the Rap1p C-terminal 165 amino acids thought to contain sites for Sir3p association . Tethering of LexA-Sir3p adjacent to the telomere is sufficient to restore telomeric silencing, indicating that Sir3p can nucleate silencing at the telomere . Tethering of LexA-Sir3p or the LexA-Sir3p(N2O5) gain-of-function protein to a telomeric LexA site hyperrepresses an adjacent ADE2 gene in wild-type cells . Hence, Sir3p recruitment to the telomere is limiting in telomeric silencing . In addition, LexA-Sir3p(N2O5) hyperrepresses telomeric silencing when tethered to a subtelomeric site 3.6 kb from the telomeric tract . This hyperrepression is dependent on the C terminus of Rap1p, suggesting that subtelomeric LexA-Sir3p(N205) can interact with Rap1p-associated factors at the telomere . We also demonstrate that LexA-Sir3p or LexA-Sir3p(N205) tethered in cis with a short tract of telomeric TG1-3 sequences is sufficient to confer silencing at an internal chromosomal position . Internal silencing is enhanced in rap1-17 strains . We propose that sequestration of silencing factors at the telomere limits the efficiency of internal silencing. Mol Cell Biol, 1996 May, 16(5), 2164 - 73 Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae; Moore JK et al.; In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break can be repaired by at least two pathways of nonhomologous end joining (NHEJ) that closely resemble events in mammalian cells . In one pathway the chromosome ends are degraded to yield deletions with different sizes whose endpoints have 1 to 6 bp of homology . Alternatively, the 4-bp overhanging 3' ends of HO-cut DNA (5'-AACA-3') are not degraded but can be base paired in misalignment to produce +CA and +ACA insertions . When HO was expressed throughout the cell cycle, the efficiency of NHEJ repair was 30 times higher than when HO was expressed only in G1 . The types of repair events were also very different when HO was expressed throughout the cell cycle; 78% of survivors had small insertions, while almost none had large deletions . When HO expression was confined to the G1 phase, only 21% were insertions and 38% had large deletions . These results suggest that there are distinct mechanisms of NHEJ repair producing either insertions or deletions and that these two pathways are differently affected by the time in the cell cycle when HO is expressed . The frequency of NHEJ is unaltered in strains from which RAD1, RAD2, RAD51, RAD52, RAD54, or RAD57 is deleted; however, deletions of RAD50, XRS2, or MRE11 reduced NHEJ by more than 70-fold when HO was not cell cycle regulated . Moreover, mutations in these three genes markedly reduced +CA insertions, while significantly increasing the proportion of both small (-ACA) and larger deletion events . In contrast, the rad5O mutation had little effect on the viability of G1-induced cells but significantly reduced the frequency of both +CA insertions and -ACA deletions in favor of larger deletions . Thus, RAD50 (and by extension XRS2 and MRE11) exerts a much more important role in the insertion-producing pathway of NHEJ repair found in S and/or G2 than in the less frequent deletion events that predominate when HO is expressed only in G1. Mol Cell Biol, 1996 May, 16(5), 2091 - 100 The Saccharomyces cerevisiae IMP2 gene encodes a transcriptional activator that mediates protection against DNA damage caused by bleomycin and other oxidants; Masson JY et al.; Bleomycin belongs to a class of antitumor drugs that damage cellular DNA through the production of free radicals . The molecular basis by which eukaryotic cells provide resistance to the lethal effects of bleomycin is not clear . Using the yeast Saccharomyces cerevisiae as a model with which to study the effect of bleomycin damage on cellular DNA, we isolated several mutants that display hypersensitivity to bleomycin . A DNA clone containing the IMP2 gene that complemented the most sensitive bleomycin mutant was identified . A role for IMP2 in defense against the toxic effects of bleomycin has not been previously reported . imp2 null mutants were constructed and were found to be 15-fold more sensitive to bleomycin than wild-type strains . The imp2 null mutants were also hypersensitive to several oxidants but displayed parental resistance to UV light and methyl methane sulfonate . Exposure of mutants to either bleomycin or hydrogen peroxide resulted in the accumulation of strand breaks in the chromosomal DNA, which remained even after 6 h postchallenge, but not in the wild type . These results suggest that the oxidant hypersensitivity of the imp2 mutant results from a defect in the repair of oxidative DNA lesions . Molecular analysis of IMP2 indicates that it encodes a transcriptional activator that can activate a reporter gene via an acidic domain located at the N terminus . Imp2 lacks a DNA binding motif, but it possesses a C-terminal leucine-rich repeat . With these data taken together, we propose that Imp2 prevents oxidative damage by regulating the expression of genes that are directly required to repair DNA damage. J Bacteriol, 1996 May, 178(9), 2721 - 4 Sensitivity of polyamine-deficient Saccharomyces cerevisiae to elevated temperatures; Balasundaram D et al.; Saccharomyces cerevisiae cells that cannot synthesize spermidine or spermine because of a deletion in the gene coding for S-adenosylmethionine decarboxylase are very sensitive to elevated temperatures when incubated in a polyamine-deficient medium; i.e., growth is inhibited and the cells are killed . This sensitivity is very pronounced at 39 degrees C, but a moderate effect is noted even at 33 to 34 degrees C . These findings support findings from other studies from our laboratory on the importance of polyamines in protecting cell components against damage . The sensitivity of spermidine-deficient cells to the temperature 39 degrees C provides a useful method for screening for polyamine auxotrophs. FEBS Lett, 1996 Apr 29, 385(1-2), 7 - 10 Site-directed mutagenesis of Saccharomyces cerevisiae beta-tubulin: interaction between residue 167 and benzimidazole compounds; Li J et al.; Benzimidazoles are widely used as anthelmintic agents and systemic fungicides . In susceptible organisms, benzimidazoles bind to beta-tubulin and block microtubule polymerization . To further characterize this interaction, site-directed mutagenesis followed by gene replacement was used to change Saccharomyces cerevisiae beta-tubulin residue Phe-167 to Tyr . Consistent with previous studies, this mutation resulted in at least 3-4-fold decreased sensitivity to the benzimidazole derivatives carbendazim and nocodazole . The Tyr-167 mutant was cold sensitive, implying a direct effect on benzimidazole binding rather than a nonspecific increase in microtubule stability . Surprisingly, the mutant had 8-fold increased sensitivity to the derivative benomyl, which is structurally identical to carbendazim except at position 1 . This suggests that residue 167 interacts with benzimidazoles in the vicinity of the 1-position. FEBS Lett, 1996 Apr 29, 385(1-2), 43 - 6 Glucose-induced inactivation of isocitrate lyase in Saccharomyces cerevisiae is mediated by the cAMP-dependent protein kinase catalytic subunits Tpk1 and Tpk2; Ordiz I et al.; Glucose-induced inactivation of isocitrate lyase (Icl) has been related to protein phosphorylation . Moreover, since rapid reversible inactivation preceded irreversible inactivation of the enzyme, phosphorylation was proposed as the triggering reaction that makes the enzyme accessible to the proteolytic machinery . The protein kinase involved in the process is unknown at the moment . In this work we demonstrate that Tpk1 and Tpk2, the catalytic subunits of cAMP-dependent protein kinase, are involved in the signalling of short-term and long-term inactivation processes of Icl . We also demonstrate that threonine 53 is involved in a regulatory mechanism necessary for short-term reversible inactivation of Icl, probably mediated through its phosphorylation . Other, as yet unidentified, residues are likely to be the target of distinct protein kinases mediating the irreversible long-term inactivation of Icl. Biochem Pharmacol, 1996 Apr 26, 51(8), 1041 - 50 Multiple forms of human P450 expressed in Saccharomyces cerevisiae . Systematic characterization and comparison with those of the rat; Imaoka S et al.; We systematically characterized the levels and substrate specificity of P450s from humans and rats to extrapolate drug metabolism data from experimental animals to humans . Human P450s (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, and 3A4) were expressed in Saccharomyces cerevisiae and purified . Rat P450s were purified from hepatic microsomes of rats . We investigated the catalytic activities of purified P450s in a reconstituted system . Human CYP2B6 and rat CYP2B1 had high lidocaine N-deethylation activity . Human and rat CYP2D forms had high debrisoquine 4-hydroxylation activity . Human CYP3A4 and rat CYP3A2 had high testosterone 2 beta- and 6 beta-hydroxylation activities in a modified reconstituted system with a lipid mixture . The hydroxylation site of testosterone by CYP2B6 (16 alpha- and 16 beta-positions) agreed with that by rat CYP2B1 . Human CYP2E1 had the highest lauric acid (omega-1)-hydroxylation activity and also had catalytic properties similar to those of rat CYP2E1 . Human CYP2A and 2C forms had catalytic properties in testosterone metabolism different from those of rats . Antibodies raised against purified P450s were used to measure the levels of hepatic P450s . The level of CYP3A4 was the highest in human hepatic microsomes, comprising 30-40% of the total P450 . CYP2C9 comprised 10-20% of the total . The levels of CYP1A2, 2A6, 2C8, 2D6, and 2E1 were moderate (5-15% of total P450) . CYP2B6 content was very low . The information of this study is useful for drug metabolism and toxicological studies. Biochem Biophys Res Commun, 1996 Apr 25, 221(3), 515 - 20 In vivo phosphorylation of type II myosin in Saccharomyces cerevisiae; Negron JA et al.; Phosphorylation of the myosin heavy chain has been shown to be a key regulatory mechanism of several non-muscle myosins . In this study we present evidence demonstrating that the yeast type II myosin heavy chain is phosphorylated in vivo . Phosphorylation of serine residues was confirmed by direct metabolic labeling with {32P} and by indirect immunostaining of phosphoserine with a specific monoclonal antibody . Loss of immunoreactivity in a targeted deletion of the 26 amino acid carboxyl terminal segment of the type II myosin heavy chain suggests that the phosphorylation occurs at one or more serine residues located between residues 1903 and 1928. Mol Gen Genet, 1996 Apr 24, 251(1), 38 - 43 Molecular and genetic characterization of SLC1, a putative Saccharomyces cerevisiae homolog of the metazoan cytoplasmic dynein light chain 1; Dick T et al.; Cytoplasmic dynein is a multisubunit, microtubule-dependent motor enzyme that has been proposed to function in a variety of intracellular movements . As part of an effort to understand the evolution and the biological roles of cytoplasmic dynein, we have identified the first non-metazoan dynein light chain 1, SLC1, in the yeast Saccharomyces cerevisiae . The amino acid sequence of the SLC1 protein is similar to those of the human, Drosophila and Caenorhabditis cytoplasmic dynein light chains 1 . The SLC1 gene lies adjacent to the YAP2 (= CAD1) transcription unit . The SLC1 coding sequence is split by two introns and its mRNA is detectable throughout the cell cycle . Tetrad analysis of heterozygotes harboring a TRP insertion in the SLC1 coding region indicate that SLC1 function is not essential for cell viability . Furthermore, we demonstrate that double mutants, defective for SLC1 and the kinesin-related CIN8 genes are non-lethal . The redundancy of SLC1 function in yeast contrasts with the cell death caused by loss-of-function mutations in the dynein light chain 1 gene in Drosophila melanogaster. J Biol Chem, 1996 Apr 19, 271(16), 9801 - 8 Identification of a novel membrane transporter associated with intracellular membranes by phenotypic complementation in the yeast Saccharomyces cerevisiae; Hogue DL et al.; A partial mouse cDNA was isolated by its ability to functionally complement a thymidine transport deficiency in plasma membranes of the yeast, Saccharomyces cerevisiae . The full-length cDNA encoded a previously unidentified 27-kDa protein (mouse transporter protein (MTP)) with four predicted transmembrane-spanning domains . MTP mRNA was detected in cells of several mammalian species, and its predicted protein sequence exhibited near identity (98%) with that of a human cDNA (HUMORF13) . MTP and its homologs evidently reside in an intracellular membrane compartment because a protein (about 24 kDa) that was recognized by MTP-specific antibodies was observed in a subcellular fraction of rat hepatocytes enriched for Golgi membranes . Deletion of the hydrophilic C terminus of MTP, which encompassed two putative signal motifs for intracellular localization (Tyr-X-X-hydrophobic amino acid), allowed expression of recombinant protein (MTP deltaC) in plasma membranes of Xenopus laevis oocytes . MTP deltaC-expressing oocytes exhibited greater fragility than nonexpressing oocytes, and those that survived the experimental manipulations were capable of mediated uptake of thymidine, uridine, and adenosine . Thymidine uptake by MTP deltaC-expressing oocytes was inhibited by thymine and dTMP . MTP may function in the transport of nucleosides and/or nucleoside derivatives between the cytosol and the lumen of an intracellular membrane-bound compartment. J Biotechnol, 1996 Apr 18, 46(1), 43 - 54 Expression of the major bean proteins from Theobroma cacao (cocoa) in the yeasts Hansenula polymorpha and Saccharomyces cerevisiae; Yavuz MO et al.; The production in two yeast expression systems of recombinant forms of the major proteins from the cocoa bean is described . Three major protein species are found in the cocoa bean: an albumin of molecular mass 21 kDa (p21) and two insoluble vicilin-like proteins of molecular mass 31 kDa and 47 kDa (p31 and p47, respectively) . The p31 and p47 species are known to be derived from a common 67-kDa precursor (p67) by post-translational processing that includes the deletion of a hydrophilic domain located immediately after an N-terminal signal sequence . All three proteins appear to be targeted to membrane-bound storage organelles by N-terminal signal sequences . The p21 and p67 coding sequences were expressed in Hansenula polymorpha using the powerful methanol oxidase (MOX) promoter and in Saccharomyces cerevisiae using the promoter of the pyruvate kinase (PYK) gene . The expression constructs contained the native plant signal sequence, or various yeast signals . The p21 protein was successfully expressed and secreted from both yeasts . The insoluble p67 protein proved more difficult . Species of the correct molecular mass were recovered internally and small amounts of a p47 species were secreted using a yeast leader sequence . However, proteolytic cleavage, probably due to Kex2p-like processing, led to the appearance of other protein species. J Biotechnol, 1996 Apr 18, 46(1), 33 - 42 Dynamics of ammonia uptake in nitrogen limited anaerobic cultures of Saccharomyces cerevisiae; Schulze U et al.; Dynamics of the ammonia uptake by Saccharomyces cerevisiae under anaerobic conditions was studied in ammonia limited continuous cultures . A large number of pulse additions of ammonia (25-100 mg 1(-1)) were made at different dilution rates (0.05-0.20 h-1) . The response was followed by on-line monitoring of the carbon dioxide evolution rate (CER), optical density, and by frequent analysis of extra- and intracellular metabolites . The uptake of a pulse of ammonia proceeded in a qualitatively highly reproducible pattern . Initially, a rapid and growth rate dependent uptake of ammonia was observed (lasting for about 10-15 min) . Next followed a phase with little uptake (approx . 5 min) . Finally, the rest of the ammonia pulse was taken up at a somewhat smaller rate which also depended on the growth rate . The first phase coincided with an increase in CER caused by mobilization of the intracellular carbohydrate trehalose and subsequently of glycogen . Regardless of dilution rate and the amount of ammonia added, the initial high uptake rate of ammonia was maintained until approximately the same amount of ammonia had been taken up . Transition from the first to the second uptake phase was associated with an increased glycerol production, indicating an elevated anabolic activity. Gene, 1996 Apr 17, 170(1), 23 - 30 Expression of the mitochondrial RNase P RNA subunit-encoding gene from a variant promoter sequence in Saccharomyces cerevisiae; Biswas TK; Ribonuclease P (RNase P) is a common tRNA processing enzyme that removes the 5' leader sequence of precursor tRNAs . This activity is identified in yeast mitochondria as a separate enzyme from the nuclear RNase P . Like other RNase P enzymes, the mitochondrial (mt) RNase P is also a ribonucleoprotein composed of both RNA and protein subunits . The RNA subunit is encoded by a mt gene and the protein subunit is supplied by a nuclear gene . Earlier studies described one active promoter (FP1) located 5' to the mt tRNA(fMet)-RNase P RNA-tRNA(Pro) gene cluster, so that the mitochondrially encoded RNA subunit was thought to be co-transcribed with two of its substrate tRNAs . However, the results of in vitro transcription and primer extension experiments presented here demonstrate that the mt RNase P RNA subunit-encoding gene (RPM1) is transcribed from a new promoter (SP)which is located between the tRNA(fMet) and RPM1 genes . The sequence {5'-TATAAGAA(+1)} of the new promoter varies from the conserved promoter sequence {5'-TATAAGTA(+1)}, but is one of the sequences that is active in the in vitro transcription assay to determine the consensus promoter sequence {5'-T A T/a A A/g/c G T/a/c N(+1)} . This result demonstrates that a naturally occurring variant promoter is used by RPM1 . Identification of the novel SP promoter suggests that the synthesis of the mt RNase P RNA subunit might be uncoupled from the expression of upstream tRNA(fMet) gene, and that RPM1 might be independently transcribed in Saccharomyces cerevisiae. Biochim Biophys Acta, 1996 Apr 17, 1289(3), 336 - 42 Preferential transfer to truncated oligosaccharides to the first sequon of yeast exoglucanase in Saccharomyces cerevisiae alg3 cells; Cueva R et al.; In addition to the exoglucanases (Exg) secreted into the culture medium by wild type cells, ExgIa and ExgIb, which have oligosaccharides attached to both potential N-glycosylation sites, Saccharomyces cerevisiae alg3 mutant secreted substantial amounts (35--44%) of underglycosylated and unglycosylated forms . Quantification of these forms indicated that no more than 78% of the available N-sites were occupied . About 50% of the transferred oligosaccharides were endo H sensitive, indicating that the lipid-linked precursor had completed its synthesis to Glc3-Man9-GlcNAc2 . The other 50% remained endo H-resistant and, accordingly, it should be derived from the precursor oligosaccharide Man5-GlcNAc2 synthesized by this mutant . A closer analysis of forms that have received two oligosaccharides (ExgIb) showed that the first sequon was enriched in truncated residues, whereas the second one was enriched in regular counterparts . Similarly, analysis of the individual underglycosylated glycoforms indicated that 38% of the oligosaccharides attached to the second site were regular . This percentage dropped to 20% for glycoforms carrying the oligosaccharide in the first sequon . The preferential transfer of truncated oligosaccharides to the first glycosylation site seems to be a consequence of (1) the low percentage of truncated lipid linked oligosaccharides that receives the glucotriose unit, and (2) the effect of the glucotriose unit on the selection of N-sites to be glycosylated. Biochemistry, 1996 Apr 16, 35(15), 4878 - 85 Modulated growth of Saccharomyces cerevisiae by altering the driving force of the reactions of cytochrome c: Marcus' theory in vitro and in vivo; Komar-Panicucci S et al.; According to Marcus' theory, rates of electron transfer reactions depend parabolically on the free energy of reaction . Amino acid replacements in the electron transport protein cytochrome c produced a series of proteins which changed the free energy of reaction for cytochrome c in oxidative phosphorylation . This study shows that Marcus' theory of electron transfer can be applied to the reactions of redox-altered cytochromes c with cytochrome c1 both in vitro and in vivo . In vitro, isolation of physiologically relevant partners of cytochrome c suggests that a change in free energy of reaction of cytochrome c changes the rate of electron transfer with cytochrome bc1 complex as would be predicted by Marcus' theory of electron transfer . Furthermore, the reactivity pattern observed in vitro is paralleled in in vivo studies . In vivo the rates of growth of Saccharomyces cerevisiae, in which these alternatives have been incorporated, also are consistent with the change in free energy of the reactions of cytochrome c with cytochrome bc1 complex . This study suggests that Marcus' theory of electron transport can predict rates not only in vitro, in isolated protein-protein systems, but also in vivo, where the relative growth rates of yeast may be predicted from the in vitro results. Biochim Biophys Acta, 1996 Apr 16, 1293(2), 213 - 21 RAP kinase, a new enzyme phosphorylating the acidic P proteins from Saccharomyces cerevisiae; Szyszka R et al.; A new protein kinase, showing a high specificity for the ribosomal acidic P proteins (RAP kinase) has been purified and characterized from Saccharomyces cerevisiae extracts . Purification was carried out by four chromatographic steps, including DEAE-cellulose, phosphocellulose, heparin-Sepharose and P protein-Sepharose . The purified enzyme preparation contains only one polypeptide of around 55 kDa as determined by SDS gel electrophoresis and gradient centrifugation . RAP kinase is different from all previous well-characterized kinases and does not show cross-reaction with antibodies to the 71 kDa 60S ribosomal subunit-specific kinase PK60 previously reported . The enzyme uses ATP as a better phosphate donor and is less sensitive to heparin than casein kinase II but is moderately affected by salt . Among the different substrates tested, ribosomal acidic proteins are preferentially modified by RAP kinase, which phosphorylates only serine residues in the four P proteins as well as the related ribosomal protein P0 . Casein is phosphorylated at a much lower level . All the data indicate that RAP kinase might be the enzyme responsible for the phosphorylation of the P proteins, and in this way may also participate in a possible translational regulatory mechanism. Biochem J, 1996 Apr 15, 315 ( Pt 2), 393 - 9 The purification and some properties of the Mg(2+)-activated cytosolic aldehyde dehydrogenase of Saccharomyces cerevisiae; Dickinson FM; A purification procedure has been developed for the cytosolic aldehyde dehydrogenase of Saccharomyces cerevisiae that yields homogeneous enzyme . The enzyme seems to be a tetramer of identical 58 kDa subunits . The enzyme reaction is strongly stimulated by Mg2+ at low NADP+ concentrations but there is no absolute requirement for bivalent cations . The kinetics of the reaction have been studied in the presence and absence of MgCl2 . NADP+ binding studies of the quenching of protein fluorescence in the presence and absence of MgCl2 show that the effect of Mg2+ is to increase the affinity of the enzyme for NADP+ by approx . 100-fold . NADP+ binding causes a slow conformational change in the enzyme and converts the enzyme from the inactive or low-activity form in which it is isolated into the fully active form . This conformational change seems to explain the marked lag-phases seen in enzyme assays . The enzyme is strongly inhibited by disulfiram and pyridoxal 5-phosphate. FEBS Lett, 1996 Apr 15, 384(2), 151 - 4 Deoxyhypusine synthase gene is essential for cell viability in the yeast Saccharomyces cerevisiae; Sasaki K et al.; Deoxyhypusine synthase catalyzes the first of two steps in the biosynthesis of hypusine, a modification of a specific lysine residue in the precursor of eukaryotic translation initiation factor 5A . We have purified deoxyhypusine synthase from yeast, and cloned and sequenced the corresponding gene encoding a 387-amino acid protein from Saccharomyces cerevisiae . Gene disruption experiments indicated that the deoxyhypusine synthase gene is essential for cell growth in yeast . This gene was shown to be an intron-free, single-copy gene, and its product can catalyze the synthesis of deoxyhypusine equally in two precursor forms of eIF-5A, derived from two distinct genes of yeast. FEMS Microbiol Lett, 1996 Apr 1, 137(2-3), 165 - 8 Molecular cloning of a novel allele of SMR1 which determines sulfometuron methyl resistance in Saccharomyces cerevisiae; Xie Q et al.; A different mutation (SMR1B) to SMR1-410 (ilv2), which determines resistance to sulfometuron methyl in Saccharomyces cerevisiae, was cloned by PCR . Sequence analysis indicated a C to T change at position 575 of the ILV2 coding sequence, which results in a proline transition to leucine at position 192 . Similarly to SMR1-410, the SMR1B gene was confirmed as a convenient dominant selective marker for yeast transformation including industrial strains. Lett Appl Microbiol, 1996 Apr, 22(4), 299 - 302 Determination of 2,3-butanediol in high and low acetoin producers of Saccharomyces cerevisiae wine yeasts by automated multiple development (AMD); Romano P et al.; High performance thin layer chromatography with automated multiple development was used to determine 2,3-butanediol levels in wine produced by high and low acetoin-forming strains of Saccharomyces cerevisiae . An inverse correlation between acetoin and 2,3-butanediol content was found suggesting a leaky mutation in acetoin reductase of the low 2,3-butanediol producing strains. Mol Microbiol, 1996 Apr, 20(1), 109 - 17 The ATP-binding cassette multidrug transporter Snq2 of Saccharomyces cerevisiae: a novel target for the transcription factors Pdr1 and Pdr3; Mahe Y et al.; Pleiotropic drug resistance (PDR) in the yeast Saccharomyces cerevisiae can arise from overexpression of ATP-binding cassette (ABC) efflux pumps such as Pdr5 and Snq2 . Mutations in the transcription factor genes PDR1 and PDR3 are also associated with PDR . We show here that a pdr1-3 mutant exhibits a PDR phenotype, including elevated resistance to the mutagen 4-nitroquinoline-N-oxide, a known substrate for Snq2 but not for Pdr5 . Northern analysis and immunoblotting demonstrated that the SNQ2 gene is 10-fold overexpressed in a pdr1-3 gain-of-function mutant strain, whereas Snq2 expression is severely reduced in a delta pdr1 deletion strain, and almost abolished in a delta pdr1 delta pdr3 double disruptant when compared to the PDR1 strain . However, expression of the Ste6 a-factor pheromone transporter, another yeast ABC transporter not associated with PDR, is unaffected in pdr1-3 mutant cells and in strains carrying delta pdr1, delta pdr3, or delta pdr1 delta pdr3 deletions . Finally, DNA footprint analysis revealed that the SNQ2 promoter contains three binding sites for Pdr3 . Our results identify SNQ2 as a novel target for both Pdr1 and Pdr3, and demonstrate that the PDR phenotype of a pdr1-3 mutant strain results from overexpression of more than one ABC drug-efflux pump. Genetics, 1996 Apr, 142(4), 1095 - 103 Mutations in GCR3, a gene involved in the expression of glycolytic genes in Saccharomyces cerevisiae, suppress the temperature-sensitive growth of hpr1 mutants; Uemura H et al.; To study the functions of DNA topoisomerase I and Hpr1 protein, a suppressor mutant of the temperature-sensitive growth of an hpr1 top1-5ts double mutant was isolated . The isolated triple mutant showed cold-sensitive growth . By complementation of this phenotype, the suppressor gene was cloned . DNA sequencing showed it to be GCR3, a gene involved in the expression of glycolytic genes . Further analysis showed that gcr3 mutations also suppressed the temperature-sensitive growth of hpr1 single mutants . Experiments with gcr3 truncation mutants also suggested a genetic interaction between GCR3 and HPR1 . The fact that top1 suppressed the growth defect of gcr3 suggested an interaction between those two genes also . Plasmid DNA isolated from gcr3 mutants was significantly more negatively supercoiled than normal, suggesting that Gcr3 protein, like topoisomerase I and Hpr1p, affects chromatin structure, perhaps during transcription. Methods, 1996 Apr, 9(2), 165 - 76 Immunophilins in the Yeast Saccharomyces cerevisiae: A Different Spin on Proline Rotamases Dhillon N, Thorner J. The clinically used immunosuppressant compounds-FK506, rapamycin, and cyclophilin A-are all natural products that were originally detected because of their antifungal action, not because of their fortuitous effects on the human immune system . Genetic and biochemical approaches have been used to identify binding proteins that serve as the receptors for these antibiotics in cells of the budding yeast Saccharomyces cerevisiae . Three FK506/rapamycin-binding proteins (FKBPs) and six cyclosporin-A-binding proteins (cyclophilins) have been characterized in some detail, but there is evidence that additional members of both families exist in this organism . Cloning of the corresponding genes has shown that the yeast gene products are strikingly similar to their mammalian counterparts and possess peptidylprolyl-cis, trans-isomerase (proline rotamase) activity in vitro . Genetic analysis in yeast has confirmed, and significantly extended, complementary research in animal cell systems that has shed light on the roles that the FKBPs and the cyclophilins play in the mechanism of action of the immunosuppressant drugs . The application of genetic methods in yeast is also be