Gene, 1994 May 3, 142(1), 107 - 12 PAD1 encodes phenylacrylic acid decarboxylase which confers resistance to cinnamic acid in Saccharomyces cerevisiae; Clausen M et al.; The yeast enzyme phenylacrylic acid decarboxylase (PAD) confers resistance to phenylacrylic acids . Cinnamic acid (CA)-sensitive mutants lacking PAD activity were isolated and the PAD1 gene was cloned by phenotypic complementation . The nucleotide sequence of the smallest complementing fragment was determined . The predicted 242-amino-acid PAD polypeptide is 48.6% identical to the product of dedF of Escherichia coli . PAD activity and CA resistance, but not steady-state PAD1 mRNA levels, are influenced by mitochondrial genotype . PAD1 is a single-copy gene in the yeast genome and not essential for viability . The PAD1 locus was physically mapped to a position approx . 140 kb from the left end of chromosome IV. Biochemistry, 1994 May 3, 33(17), 5305 - 11 Purification of Rad1 protein from Saccharomyces cerevisiae and further characterization of the Rad1/Rad10 endonuclease complex; Tomkinson AE et al.; The yeast recombination and repair proteins Rad1 and Rad10 associate with a 1:1 stoichiometry to form a stable complex with a relative molecular mass of 190 kDa . This complex, which has previously been shown to degrade single-stranded DNA endonucleolytically, also cleaves supercoiled duplex DNA molecules . In this reaction, supercoiled (form I) molecules are rapidly converted to nicked, relaxed (form II) molecules, presumably as a result of nicking at transient single-stranded regions in the supercoiled DNA . At high enzyme concentrations, there is a slow conversion of the form II molecules to linear (form III) molecules . The Rad1/Rad10 endonuclease does not preferentially cleave UV-irradiated DNA and has no detectable exonuclease activity . The nuclease activity of the Rad1/Rad10 complex is consistent with the predicted roles of the RAD1 and RAD10 genes of Saccharomyces cerevisiae in both the incision events of nucleotide excision repair and the removal of nonhomologous 3' single strands during intrachromosomal recombination between repeated sequences . In these pathways, the specificity and reactivity of the Rad1/Rad10 endonuclease will probably be modulated by further protein-protein interactions. J Cell Biol, 1994 May, 125(4), 825 - 42 Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smy1p, to the same regions of polarized growth in Saccharomyces cerevisiae; Lillie SH et al.; Myo2 protein (Myo2p), an unconventional myosin in the budding yeast Saccharomyces cerevisiae, has been implicated in polarized growth and secretion by studies of the temperature-sensitive myo2-66 mutant . Overexpression of Smy1p, which by sequence is a kinesin-related protein, can partially compensate for defects in the myo2 mutant (Lillie, S . H . and S . S . Brown, 1992 . Nature (Lond.) . 356:358-361) . We have now immunolocalized Smy1p and Myo2p . Both are concentrated in regions of active growth, as caps at incipient bud sites and on small buds, at the mother-bud neck just before cell separation, and in mating cells as caps on shmoo tips and at the fusion bridge of zygotes . Double labeling of cells with either Myo2p or Smy1p antibody plus phalloidin was used to compare the localization of Smy1p and Myo2p to actin, and by extrapolation, to each other . These studies confirmed that Myo2p and Smy1p colocalize, and are concentrated in the same general regions of the cell as actin spots . However, neither colocalizes with actin . We noted a correlation in the behavior of Myo2p, Smy1p, and actin, but not microtubules, under a number of circumstances . In cdc4 and cdc11 mutants, which produce multiple buds, Myo2p and Smy1p caps were found only in the subset of buds that had accumulations of actin . Mutations in actin or secretory genes perturb actin, Smy1p and Myo2p localization . The rearrangements of Myo2p and Smy1p correlate temporally with those of actin spots during the cell cycle, and upon temperature and osmotic shift . In contrast, microtubules are not grossly affected by these perturbations . Although wild-type Myo2p localization does not require Smy1p, Myo2p staining is brighter when SMY1 is overexpressed . The myo2 mutant, when shifted to restrictive temperature, shows a permanent loss in Myo2p localization and actin polarization, both of which can be restored by SMY1 overexpression . However, the lethality of MYO2 deletion is not overcome by SMY1 overexpression . We noted that the myo2 mutant can recover from osmotic shift (unlike actin mutants; Novick, P., and D . Botstein . 1985 . Cell . 40:405-416) . We have also determined that the myo2-66 allele encodes a Lys instead of a Glu at position 511, which lies at an actin-binding face in the motor domain. Mol Cell Biol, 1994 May, 14(5), 3414 - 25 Mutations in XRS2 and RAD50 delay but do not prevent mating-type switching in Saccharomyces cerevisiae; Ivanov EL et al.; In Saccharomyces cerevisiae, a large number of genes in the RAD52 epistasis group has been implicated in the repair of chromosomal double-strand breaks and in both mitotic and meiotic homologous recombination . While most of these genes are essential for yeast mating-type (MAT) gene switching, neither RAD50 nor XRS2 is required to complete this specialized mitotic gene conversion process . Using a galactose-inducible HO endonuclease gene to initiate MAT switching, we have examined the effect of null mutations of RAD50 and of XRS2 on intermediate steps of this recombination event . Both rad50 and xrs2 mutants exhibit a marked delay in the completion of switching . Both mutations reduce the extent of 5'-to-3' degradation from the end of the HO-created double-strand break . The steps of initial strand invasion and new DNA synthesis are delayed by approximately 30 min in mutant cells . However, later events are still further delayed, suggesting that XRS2 and RAD50 affect more than one step in the process . In the rad50 xrs2 double mutant, the completion of MAT switching is delayed more than in either single mutant, without reducing the overall efficiency of the process . The XRS2 gene encodes an 854-amino-acid protein with no obvious similarity to the Rad50 protein or to any other protein in the database . Overexpression of RAD50 does not complement the defects in xrs2 or vice versa. Mol Cell Biol, 1994 May, 14(5), 3197 - 207 The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II; Caron PR et al.; A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo . The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins . Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation . Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form . In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo . The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results. Mol Cell Biol, 1994 May, 14(5), 3158 - 65 The Glc7 type 1 protein phosphatase of Saccharomyces cerevisiae is required for cell cycle progression in G2/M; Hisamoto N et al.; We isolated a mutant carrying a conditional mutation in the GLC7 gene, encoding the catalytic subunit of a type 1 protein phosphatase, by selection of suppressors that restored the growth defect of cdc24 mutants at high temperature and simultaneously conferred cold-sensitive growth . This cold sensitivity for growth is caused by a single mutation (glc7Y-170) at position 170 of the Glc7 protein, resulting in replacement of cysteine with tyrosine . Genetic analysis suggested that the glc7Y-170 allele is associated with a recessive negative phenotype, reducing the activity of Glc7 in the cell . The glc7Y-170 mutant missegregated chromosome III at the permissive temperature, arrested growth as large-budded cells at the restrictive temperature, exhibited a significant increase in the number of nuclei at or in the neck, and had a short spindle . Furthermore, the glc7Y-170 mutant exhibited a high level of CDC28-dependent protein kinase activity when incubated at the restrictive temperature . These findings suggest that the glc7Y-170 mutation is defective in the G2/M phase of the cell cycle . Thus, type 1 protein phosphatase in Saccharomyces cerevisiae is essential for the G2/M transition. Mol Cell Biol, 1994 May, 14(5), 3139 - 49 MOT2 encodes a negative regulator of gene expression that affects basal expression of pheromone-responsive genes in Saccharomyces cerevisiae; Cade RM et al.; Pheromones induce haploid cells of Saccharomyces cerevisiae to differentiate into a mating-competent state . Ste11p is one of several protein kinases required to transmit the pheromone-induced signal and to maintain basal expression of certain mating-specific genes in the absence of pheromone stimulation . To identify potential regulators of Ste11p, we screened for suppressors that restored mating and basal transcriptional competence to a strain with a conditionally functional Ste11p . This screen uncovered a novel gene we call MOT2, for modulator of transcription . A mot2 deletion mutation leads to modest increases in the basal amounts of mRNA for several pheromone-responsive genes . Yet mot2 deletion does not affect the signal transmission activity of the pathway in either the presence or absence of pheromone stimulation . Therefore, we propose that Mot2p, directly or indirectly, represses basal transcription of certain mating-specific genes . Because mot2 deletion mutants also have a conditional cell lysis phenotype, we expect that Mot2p regulatory effects may be more global than for mating-specific gene expression. Mol Cell Biol, 1994 May, 14(5), 3065 - 73 Two distinctly regulated genes are required for ferric reduction, the first step of iron uptake in Saccharomyces cerevisiae; Georgatsou E et al.; Iron uptake in Saccharomyces cerevisiae involves at least two steps: reduction of ferric to ferrous ions extracellularly and transport of the reduced ions through the plasma membrane . We have cloned and molecularly characterized FRE2, a gene which is shown to account, together with FRE1, for the total membrane-associated ferric reductase activity of the cell . Although not similar at the nucleotide level, the two genes encode proteins with significantly similar primary structures and very similar hydrophobicity profiles . The FRE1 and FRE2 proteins are functionally related, having comparable properties as ferric reductases . FRE2 expression, like FRE1 expression, is induced by iron deprivation, and at least part of this control takes place at the transcriptional level, since 156 nucleotides upstream of the initiator AUG conferred iron-dependent regulation when fused to a heterologous gene . However, the two gene products have distinct temporal regulation of their activities during cell growth. Curr Genet, 1994 May, 25(5), 472 - 4 The Escherichia coli recA gene increases UV-induced mitotic gene conversion in Saccharomyces cerevisiae; Vlckova V et al.; The effect of the Escherichia coli RecA protein on mitotic recombination in the diploid D7 strain of Saccharomyces cerevisiae damaged by UV radiation was investigated . The D7 strain was transformed by two modified versions of the pNF2 plasmid: one, containing the ADH-1 promoter, and the other containing the recA gene tandemly arranged behind the ADH-1 promoter region . Immunological analysis proved the presence of the 38-kDa RecA protein in D7/pNF2ADHrecA transformants . We observed a positive effect of recA gene expression on mitotic gene conversion, mainly at higher doses of UV radiation . The results indicate that a RecA-like activity could participate in steps preceding mitotic conversion events in yeast. Curr Genet, 1994 May, 25(5), 469 - 71 Overexpression of the SNQ3/YAP1 gene confers hyper-resistance to nitrosoguanidine in Saccharomyces cerevisiae via a glutathione-independent mechanism; Grey M et al.; The MNNG hyper-resistance of yeast transformants containing multiple copies of the SNQ3/YAP1 yeast gene is not caused by lowered MNNG activation due to depleted pools of glutathione . On the contrary, the SNQ3/YAP1-encoded protein stimulates production of GSH, apparently by promoter activation due to the AP-1 recognition element . Expression of at least one further gene, encoding a protein with a strong detoxifying activity, must also be stimulated to explain the MNNG hyper-resistance phenotype. Curr Genet, 1994 May, 25(5), 407 - 11 Isolation and characterization of three mutants with increased sensitivity to photoactivated 3-carbethoxypsoralen in Saccharomyces cerevisiae; Querol CB et al.; The complementation and genetical analysis of yeast mutants sensitive to photoactivated 3-carbethoxypsoralen define three novel recessive mutant alleles pso5-1, pso6-1, and pso7-1 . Their cross-sensitivity to UV254nm, radiomimetic mutagens, and to chemicals enhancing oxidative stress suggest that these mutants are either impaired in metabolic steps protecting from oxidative stress or in mechanisms of the repair of oxygen-dependent DNA lesions . None of the three novel mutant alleles block the induction of reverse mutation by photoactivated mono- and bi-functional psoralens, nitrogen mustards, or UV254nm. Curr Genet, 1994 May, 25(5), 396 - 406 Approaching the function of new genes by detection of their potential upstream activation sequences in Saccharomyces cerevisiae: application to chromosome III; Fondrat C et al.; The systematic sequencing of the yeast genome reveals the presence of many potential genes of unknown function . One way to approach their function is to define which regulatory system controls their transcription . This can also be accomplished by the detection of an upstream activation sequence (UAS) . Such a detection can be done by computer, provided that the definition of a UAS includes sufficient and precise rules . We have established such rules for the UASs of the GAL4, RAP1 (RPG box), GCN4, and the HAP2/HAP3/HAP4 regulatory proteins, as well as for a motif (PAC) frequently found upstream of the genes of the RNA polymerase A and C subunits . These rules were applied to the chromosome III DNA sequence, and gave precise predictions. Curr Genet, 1994 May, 25(5), 385 - 95 Polypeptide chain termination in Saccharomyces cerevisiae; Stansfield I et al.; The study of translational termination in yeast has been approached largely through the identification of a range of mutations which either increase or decrease the efficiency of stop-codon recognition . Subsequent cloning of the genes encoding these factors has identified a number of proteins important for maintaining the fidelity of termination, including at least three ribosomal proteins (S5, S13, S28) . Other non-ribosomal proteins have been identified by mutations which produce gross termination-accuracy defects, namely the SUP35 and SUP45 gene products which have closely-related higher eukaryote homologues (GST1-h and SUP45-h respectively) and which can complement the corresponding defective yeast proteins, implying that the yeast ribosome may be a good model for the termination apparatus existing in higher translation systems . While the yeast mitochondrial release factor has been cloned (Pel et al . 1992), the corresponding cytosolic RF has not yet been identified . It seems likely, however, that the identification of the gene encoding eRF could be achieved using a multicopy antisuppressor screen such as that employed to clone the E . coli prfA gene (Weiss et al . 1984) . Identification of the yeast eRF and an investigation of its interaction with other components of the yeast translational machinery will no doubt further the definition of the translational termination process . While a large number of mutations have been isolated in which the efficiency of termination-codon recognition is impaired, it seems probable that a proportion of mutations within this class will comprise those where the accuracy of 'A' site codon-anticodon interaction is compromised: such defects would also have an effect on termination-codon suppression, allowing mis- or non-cognate tRNAs to bind stop-codons, causing nonsense suppression . The remainder of mutations affecting termination fidelity should represent mutations in genes coding for components of the termination apparatus, including the eRF: these mutations reduce the efficiency of termination, allowing nonsense suppression by low-efficiency natural suppressor tRNAs . Elucidation of the mechanism of termination in yeast will require discrimination between these two classes of mutations, thus allowing definition of termination-specific gene products. Genetics, 1994 May, 137(1), 55 - 65 Isolation and characterization of a mutant of Saccharomyces cerevisiae with pleiotropic deficiencies in transcriptional activation and repression; Lamping E et al.; The isolation of the dep1 mutant of Saccharomyces cerevisiae is reported . The mutant was identified by its disability to regulate expression of structural genes involved in phospholipid biosynthesis, INO1, CHO1 and OPI3, in response to supplementation with soluble lipid precursors . Expression of the INO1, CHO1 and OPI3 genes was not fully derepressed in the absence of soluble lipid precursors, inositol and choline in the dep1 mutant, as compared to wild type . The mutant also exhibited incomplete repression of these same genes in the presence of inositol and choline . Repression by phosphate of the PHO5 gene was reduced in the mutant, as was derepression of this gene in the absence of phosphate . In addition, we show that expression of INO1 and OPI3 structural genes is strongly dependent on the growth phase both in wild-type and dep1 mutant strains . However, in the mutant, elevated basal steady-state mRNA levels were reached in the late stationary growth phase, independent of supplementation conditions . The dep1 mutation represents a new complementation group with respect to phospholipid synthesis and was mapped to a position of about 12 cM distal from the centromere on the left arm of chromosome I . Deficiencies in transcription activation and repression of metabolically unrelated genes, as well as reduced mating efficiency and lack of sporulation of homozygous diploid dep1/dep1 mutants indicate a pleiotropic regulatory function of the DEP1 gene product . Thus, Dep1p appears to be a new member of a class of transcriptional modulators, including Rpd1p/Sin3p/Ume4p/Sdi1p/Gam3p, Rpd3p, Spt10p and Spt21p. Genetics, 1994 May, 137(1), 5 - 18 Analysis of a recombination hotspot for gene conversion occurring at the HIS2 gene of Saccharomyces cerevisiae; Malone RE et al.; The properties of gene conversion as measured in fungi that generate asci containing all the products of meiosis imply that meiotic recombination initiates at specific sites . The HIS2 gene of Saccharomyces cerevisiae displays a high frequency of gene conversion, indicating that it is a recombination hotspot . The HIS2 gene was cloned and sequenced, and the cloned DNA was used to make several different types of alterations in the yeast chromosome by transformation; these alterations were used to determine the location of the sequences necessary for the high levels of meiotic conversion observed at HIS2 . Previous work indicated that the gene conversion polarity gradient is high at the 3' end of the gene, and that the promoter of the gene is not necessary for the high frequency of conversion observed . Data presented here suggest that at least some of the sequences necessary for high levels of conversion at HIS2 are located over 700 bp downstream of the end of the coding region, extend over (at least) several hundred base pairs, and may be quite complex, perhaps involving chromatin structure . Additional data indicate that multiple single base heterologies within a 1-kb interval contribute little to the frequency of gene conversion . This contrasts with other reports about the role of heterologies at the MAT locus. Genetics, 1994 May, 137(1), 49 - 54 Synergistic release from glucose repression by mig1 and ssn mutations in Saccharomyces cerevisiae; Vallier LG et al.; In the yeast Saccharomyces cerevisiae, glucose repression of SUC2 transcription requires the SSN6-TUP1 repressor complex . It has been proposed that the DNA-binding protein MIG1 secures SSN6-TUP1 to the SUC2 promoter . Here we show that a mig1 deletion does not cause nearly as dramatic a loss of repression as ssn6: glucose-grown mig1 mutants display 20-fold lower SUC2 expression than ssn6 mutants . Thus, repression by SSN6-TUP1 is not mediated solely by MIG1, but also involves MIG1-independent mechanisms . We report that mig1 partially restores SUC2 expression in mutants lacking the SNF1 protein kinase and show that mig1 is allelic to ssn1, a mutation selected as a suppressor of snf1 . Other SSN genes identified in this selection were therefore candidates for a role in repression of SUC2 . We show that mig1 acts synergistically with ssn2 through ssn5, ssn7, and ssn8 to relieve glucose repression of SUC2 and to suppress the requirement for SNF1 . These findings indicate that the SSN proteins contribute to repression of SUC2, and the pleiotropic phenotypes of the ssn mutants suggest global roles in repression . Finally, the regulated SUC2 expression observed in snf1 mig1 mutants indicates that signals regarding glucose availability can be transmitted independently of the SNF1 protein kinase. Genetics, 1994 May, 137(1), 19 - 39 Interaction between mismatch repair and genetic recombination in Saccharomyces cerevisiae; Alani E et al.; The yeast Saccharomyces cerevisiae encodes a set of genes that show strong amino acid sequence similarity to MutS and MutL, proteins required for mismatch repair in Escherichia coli . We examined the role of MSH2 and PMS1, yeast homologs of mutS and mutL, respectively, in the repair of base pair mismatches formed during meiotic recombination . By using specifically marked HIS4 and ARG4 alleles, we showed that msh2 mutants displayed a severe defect in the repair of all base pair mismatches as well as 1-, 2- and 4-bp insertion/deletion mispairs . The msh2 and pms1 phenotypes were indistinguishable, suggesting that the wild-type gene products act in the same repair pathway . A comparison of gene conversion events in wild-type and msh2 mutants indicated that mismatch repair plays an important role in genetic recombination . (1) Tetrad analysis at five different loci revealed that, in msh2 mutants, the majority of aberrant segregants displayed a sectored phenotype, consistent with a failure to repair mismatches created during heteroduplex formation . In wild type, base pair mismatches were almost exclusively repaired toward conversion rather than restoration . (2) In msh2 strains 10-19% of the aberrant tetrads were Ab4:4 . (3) Polarity gradients at HIS4 and ARG4 were nearly abolished in msh2 mutants . The frequency of gene conversion at the 3' end of these genes was increased and was nearly the frequency observed at the 5' end . (4) Co-conversion studies were consistent with mismatch repair acting to regulate heteroduplex DNA tract length . We favor a model proposing that recombination events occur through the formation and resolution of heteroduplex intermediates and that mismatch repair proteins specifically interact with recombination enzymes to regulate the length of symmetric heteroduplex DNA. Plant Mol Biol, 1994 May, 25(2), 323 - 8 The plant transcription factor TGA1 stimulates expression of the CaMV 35S promoter in Saccharomyces cerevisiae; Ruth J et al.; We have previously shown that two CRE elements situated on a 31 bp region of the cauliflower mosaic virus (CaMV) 35S promoter activate gene expression in the yeast Saccharomyces cerevisiae and are regulated by cAMP . Studies with the yeast transcription factors GCN4, SKO1 and YAP1, which bind CRE-like sequences, showed no influence on expression of the 35S promoter indicating that a yet unknown factor is involved in activation . Band shift experiments with the 31 bp promoter region revealed binding of similar factors in yeast and plant protein extracts . In a previous study this promoter region was shown to confer tissue-specific expression in plants and to interact with the transcription factor TGA1 . To test whether expression of TGA1 in yeast also stimulates transcription of the 35S promoter, we co-transformed yeast cells with a cDNA clone of this transcription factor and a 35S promoter/reporter gene construct . Promoter activity studies revealed that TGA1 confers enhanced expression of a reporter gene under the control of the 35S promoter in yeast cells . Yeast cells that were transformed with a 35S promoter construct that containing a mutated TGA1-binding site showed that both TGA1 and the intact binding site are necessary for this activation . These results suggest that stimulation of the 35S promoter by TGA1 is mediated by competition with an endogenous down-regulating yeast factor that is modulated by the nutritional state of the cells. Biochem Mol Biol Int, 1994 May, 33(2), 211 - 20 On the hsp26 of Saccharomyces cerevisiae; Silva JT et al.; The function of the small size hsps in Saccharomyces cerevisiae has yet to be convincingly established . In this paper we present some aspects of the physiology of hsp26 . Several mutant strains were analyzed with respect to the expression of the HSP26 gene using anti-hsp26 antibody for identification . The bcy1 mutant which lacks the regulating subunit of protein kinase A failed to produce full expression of HSP26 under heat shock whereas a ras2 mutation which lowers significantly the level of cAMP, produced no detectable effect . During normal growth hsp26 protein is induced during diauxie and its synthesis continues during the second exponential phase . Both BCY1 and CYR1 genes seen to be required for induction during the transition phase albeit not directly but rather interacting with some other regulatory component . The structure of hsp26 is discussed by homology with other small hsps. Yeast, 1994 May, 10(5), 697 - 9 RPL44 and RPL44', encoding acidic ribosomal phosphoproteins YP2 alpha(L44) and YP1 beta(L44'), are adjacent to rig and STF1 on Saccharomyces cerevisiae chromosomes XV and IV respectively; Rodriguez-Gabriel MA et al.; The RPL44' gene from Saccharomyces cerevisiae encoding the ribosomal protein YP1 beta(L44') has been found to be linked to the STF1 gene, encoding a stabilizing factor of the F1F0-ATPase inhibitor protein from mitochondria . Evidence of this linkage comes from results obtained from Northern hybridization using a DNA probe that contains a complementary region to the 5' end of the mRNA of RPL44' . Similarly, a data bank search has shown that RPL44, encoding ribosomal protein YP2 alpha (L44) is linked to the rig gene that encodes ribosomal protein S21. Yeast, 1994 May, 10(5), 687 - 91 The MAG1* 3-methyladenine DNA glycosylase gene is closely linked to the SPT15 TATA-binding TFIID gene on chromosome V-R in Saccharomyces cerevisiae; Xiao W et al.; The MAG1 gene encodes a 3-methyladenine DNA glycoslyase, which is involved in DNA alkylation repair in Saccharomyces cerevisiae . The mag1 mutant is deficient in 3-methyladenine DNA glycosylase activity and shows enhanced sensitivity to several monofunctional alkylating agents . MAG1 is allelic to MMS5 . This gene has been previously located on chromosome V by chromosomal hybridization . We present physical and genetic mapping data here showing that the MAG1 gene is located on chromosome V-R, proximal to and about 10 kilobase pairs away from the SPT15 gene coding for the yeast TATA-binding protein TFIID. Yeast, 1994 May, 10(5), 579 - 94 Saccharomyces cerevisiae MATa mutant cells defective in pointed projection formation in response to alpha-factor at high concentrations; Yorihuzi T et al.; We have isolated Saccharomyces cerevisiae MATa mutant cells that do not form a pointed projection but elongate in response to alpha-factor at high concentrations . Complementation tests defined three genes, PPF1, PPF2, and PPF3 (for pointed projection formation), necessary for pointed projection formation . Allelism tests with genes known to be needed for projection formation revealed that PPF1 is identical to SPA2, while PPF2 and PPF3 are not allelic to SST2, STE2, SPA2, BEM1 or SLK1/SSP31/BCK1 . The morphology of MATa ppf mutants treated with high concentrations of alpha-factor is similar to that of MATa PPF cells treated with alpha-factor at low concentrations . Quantitative mating tests showed that PPF2 and PPF3 are not essential for mating in either MATa or MAT alpha background . Monitoring of division arrest and expression of an alpha-factor-inducible gene revealed that mutations in the PPF genes do not affect the responses of MATa cells to low concentrations of alpha-factor . Unlike wild-type cells, the ppf mutants exhibited early recovery from alpha-factor-induced division arrest . Furthermore, vegetatively growing ppf3-1 cells are slightly defective in cell separation of mother and daughter cells and in selection of the correct bud sites in all cell types . These results indicate that PPF2 and PPF3 are involved in the response to alpha-factor at high concentrations and that PPF3 is also required for proper establishment of polarity in vegetative growth. Genes Dev, 1994 May 1, 8(9), 1087 - 105 Large-scale analysis of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae; Burns N et al.; We have developed a large-scale screen to identify genes expressed at different times during the life cycle of Saccharomyces cerevisiae and to determine the subcellular locations of many of the encoded gene products . Diploid yeast strains containing random lacZ insertions throughout the genome have been constructed by transformation with a mutagenized genomic library . Twenty-eight hundred transformants containing fusion genes expressed during vegetative growth and 55 transformants containing meiotically induced fusion genes have been identified . Based on the frequency of transformed strains producing beta-galactosidase, we estimate that 80-86% of the yeast genome (excluding the rDNA) contains open reading frames expressed in vegetative cells and that there are 93-135 meiotically induced genes . Indirect immunofluorescence analysis of 2373 strains carrying fusion genes expressed in vegetative cells has identified 245 fusion proteins that localize to discrete locations in the cell, including the nucleus, mitochondria, endoplasmic reticulum, cytoplasmic dots, spindle pole body, and microtubules . The DNA sequence adjacent to the lacZ gene has been determined for 91 vegetative fusion genes whose products have been localized and for 43 meiotically induced fusions . Although most fusions represent genes unidentified previously, many correspond to known genes, including some whose expression has not been studied previously and whose products have not been localized . For example, Sec21-beta-gal fusion proteins yield a Golgi-like staining pattern, Ty1-beta-gal fusion proteins localize to cytoplasmic dots, and the meiosis-specific Mek1/Mre4-beta-gal and Spo11-beta-gal fusion proteins reside in the nucleus . The phenotypes in haploid cells have been analyzed for 59 strains containing chromosomal fusion genes expressed during vegetative growth; 9 strains fail to form colonies indicating that the disrupted genes are essential . Fifteen additional strains display slow growth or are impaired for growth on specific media or in the presence of inhibitors . Of 39 meiotically induced fusion genes examined, 14 disruptions confer defects in spore formation or spore viability in homozygous diploids . Our results will allow researchers who identify a yeast gene to determine immediately whether that gene is expressed at a specific time during the life cycle and whether its gene product localizes to a specific subcellular location. Mol Cell Biol, 1994 May, 14(5), 3350 - 63 A novel mammalian protein, p55CDC, present in dividing cells is associated with protein kinase activity and has homology to the Saccharomyces cerevisiae cell division cycle proteins Cdc20 and Cdc4; Weinstein J et al.; A novel protein, p55CDC, has been identified in cycling mammalian cells . This transcript is readily detectable in all exponentially growing cell lines but disappears when cells are chemically induced to fall out of the cell cycle and differentiate . The p55CDC protein appears to be essential for cell division, since transfection of antisense p55CDC cDNA into CHO cells resulted in isolation of only those cells which exhibited a compensatory increase in p55CDC transcripts in the sense orientation . Immunoprecipitation of p55CDC yielded protein complexes with kinase activity which fluctuated during the cell cycle . Since p55CDC does not have the conserved protein kinase domains, this activity must be due to one or more of the associated proteins in the immune complex . The highest levels of protein kinase activity were seen with alpha-casein and myelin basic protein as substrates and demonstrated a pattern of activity distinct from that described for the known cyclin-dependent cell division kinases . The p55CDC protein was also phosphorylated in dividing cells . The amino acid sequence of p55CDC contains seven repeats homologous to the beta subunit of G proteins, and the highest degree of homology in these repeats was found with the Saccharomyces cerevisiae Cdc20 and Cdc4 proteins, which have been proposed to be involved in the formation of a functional bipolar mitotic spindle in yeast cells . The G beta repeat has been postulated to mediate protein-protein interactions and, in p55CDC, may modulate its association with a unique cell cycle protein kinase . These findings suggest that p55CDC is a component of the mammalian cell cycle mechanism. J Biol Chem, 1994 Apr 29, 269(17), 12947 - 53 Isolation and characterization of QCR10, the nuclear gene encoding the 8.5-kDa subunit 10 of the Saccharomyces cerevisiae cytochrome bc1 complex; Brandt U et al.; We have cloned and sequenced QCR10, the nuclear gene encoding an 8.5-kDa protein proposed to be a subunit of the cytochrome bc1 complex of Saccharomyces cerevisiae . QCR10 includes a 231-base pair open reading frame capable of encoding a protein of 77 amino acids with a predicted molecular mass of 8492 Da . The codons for the amino-terminal methionine and alanine are separated from the remainder of the open reading frame by a 63-base pair intron that contains 5'-donor, 3'-acceptor, and TACTAAC sequences known to be necessary for splicing . The deduced amino acid sequence of the 8.5-kDa yeast protein is 28% identical to that of the 6.4-kDa subunit 11 from the bovine heart cytochrome bc1 complex, and the predicted secondary structures of the two proteins are very similar . Deletion of the chromosomal copy of QCR10 did not affect the growth of yeast on nonfermentable carbon sources . However, deletion of QCR10 synergistically contributes to the temperature-dependent phenotype resulting from deletion of QCR6, the gene for subunit 6 of the cytochrome bc1 complex . In addition, ubiquinol-cytochrome c oxidoreductase activity was reduced 40% in mitochondrial membranes from the QCR10 deletion strain, and the Rieske iron-sulfur protein was lost when the cytochrome bc1 complex lacking the 8.5-kDa protein was purified . We conclude that the 8.5-kDa protein encoded by QCR10 is a subunit of the yeast cytochrome bc1 complex and that the presence of this subunit during assembly is required for stable association of the iron-sulfur protein with the complex. J Biol Chem, 1994 Apr 29, 269(17), 12908 - 17 The Saccharomyces cerevisiae oligosaccharyltransferase is a protein complex composed of Wbp1p, Swp1p, and four additional polypeptides; Kelleher DJ et al.; Asparagine-linked glycosylation of proteins in the lumen of the endoplasmic reticulum is catalyzed by the oligosaccharyltransferase . Previously, the mammalian oligosaccharyltransferase was shown to co-purify with a protein complex consisting of three integral membrane proteins: ribophorin I and ribophorin II and a nonglycosylated 48-kDa polypeptide designated OST48 . Here, we describe the purification of the oligosaccharyltransferase from Saccharomyces cerevisiae . The yeast oligosaccharyltransferase complex is composed of six subunits (alpha, beta, gamma, delta, epsilon, and zeta) . The alpha subunit of the yeast oligosaccharyltransferase complex is a heterogeneously glycosylated protein with three glycoforms of 64, 62, and 60 kDa that contain, respectively, four, three, and two asparagine-linked oligosaccharide chains . The beta and delta subunits were shown to correspond to the 45-kDa Wbp1 glycoprotein and the 30-kDa Swp1 protein, respectively . The Wbp1 and Swp1 proteins were previously shown to be essential for asparagine-linked glycosylation in vivo . The nonglycosylated gamma, epsilon, and zeta subunits have apparent molecular masses of 34, 16, and 9 kDa . Homology between the yeast and mammalian oligosaccharyltransferase complexes first became evident when the 48-kDa subunit of the mammalian enzyme was found to be 25% identical in sequence with the Wbp1 protein . Here we present an alignment between the Swp1 protein and the carboxyl-terminal half of human ribophorin II that reveals that these two proteins are related gene products. FEMS Microbiol Lett, 1994 Apr 15, 117(3), 311 - 8 Several compartments of Saccharomyces cerevisiae are equipped with Ca(2+)-ATPase(s) Okorokov LA. Sucrose density fractionation of yeast membranes revealed two major and two minor peaks of 45Ca2+ transport activity which all co-migrate with marker enzymes of the endoplasmic reticulum, Golgi and membranes associated with these compartments as well as with ATPase activity measured when all other known ATPase are inhibited . Co-migration of 45Ca2+ transport and ATPase activities was also found after removal of plasma membranes by concanavalin A treatment . SDS-PAGE at pH 6.3 shows the Ca(2+)-dependent formation of acyl phosphate polypeptides of about 110 and 200 kDa . It is concluded that several compartments or sub-compartments of yeast are equipped with Ca(2+)-ATPase(s) . It is proposed that these compartments are derived from the protein secretory apparatus of yeast. Biochem Biophys Res Commun, 1994 Apr 15, 200(1), 647 - 53 Molecular characterization of the USO1 gene product which is essential for vesicular transport in Saccharomyces cerevisiae; Seog DH et al.; We have previously shown that USO1 gene required in the protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus encodes a hydrophilic protein of 1790 amino acids . The sequence of carboxyl-terminal 1010 amino acids was predicted to have an alpha-helical structure characteristic of the coiled-coil rod region of the cytoskeleton-related proteins . Antibodies raised against partial sequences of the Uso1 polypeptide reacted with a 200 kDa protein in Western blots of the wild-type yeast proteins . The Uso1 protein was found predominantly in the soluble fraction and displayed a molecular mass of 800-900 kDa in gel filtration when globular protein were used as molecular mass standards . In sucrose density gradient centrifugation, however, the Uso1 protein cosedimented with a globular 6S marker protein, horseradish peroxidase (44 kDa) . These results suggest that, in its native state, the Uso1 protein forms a nonglobular oligomer. Biochem Biophys Res Commun, 1994 Apr 15, 200(1), 45 - 51 Identification of the maltose transport protein of Saccharomyces cerevisiae; Van den Broek PJ et al.; Identification of the maltose transport protein of Saccharomyces cerevisiae was attempted by searching for maltose-inducible proteins in isolated plasma membranes . Membranes from maltose-grown cells contained two proteins that were absent in glucose-grown cells . The proteins differed in size, but peptide sequence analysis indicated a high degree of homology . The amino-terminal and internal sequences of the largest protein, with an apparent molecular mass of 64 kDa, were determined . These sequences were identical to predicted amino acid sequences in the MAL61 gene product . It is concluded that this protein is the inducible maltose permease of Saccharomyces cerevisiae. J Biol Chem, 1994 Apr 15, 269(15), 11045 - 53 Isothermal titration calorimetric studies of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase . Determinants of binding energy and catalytic discrimination among acyl-CoA and peptide ligands; Bhatnagar RS et al.; Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential, monomeric enzyme that catalyzes the transfer of myristate from CoA to the amino-terminal Gly residue of cellular proteins . Product inhibition studies indicate that Nmt1p has an ordered Bi Bi reaction mechanism with myristoyl-CoA binding to the apo-enzyme to form a high affinity binary complex followed by binding of peptide with subsequent release of CoA and then the myristoylpeptide product . We have used isothermal titration calorimetry to quantify the effects of varying acyl chain length and removing the 3'-phosphate group of CoA on the energetics of interaction between Nmt1p and acyl-CoA ligands . Myristoyl-CoA binds to apo-Nmt1p with an affinity of 15 nM, corresponding to a binding free energy of -10.9 kcal/mol . This free energy is composed of a large favorable enthalpy of -24 kcal/mol and a large unfavorable entropic term . This large negative delta H degrees is consistent with a conformational change in the enzyme upon ligation, allowing synthesis of a functional peptide binding site . Binding of palmitoyl-CoA and lauroyl-CoA is driven by an exothermic enthalpy change which is much smaller than the corresponding parameter for myristoyl-CoA binding . The large differences in binding enthalpy and entropy (delta delta H degrees and T delta delta S degrees = 8-9 kcal/mol) demonstrate that the "off-length" acyl-CoAs bind to Nmt1p in a significantly different energetic fashion from myristoyl-CoA, even though the enzyme does not have a great deal of specificity among these ligands in terms of binding free energy (delta delta G degrees < or = 1 kcal/mol) . The effect of removing the CoA 3'-phosphate group from myristoyl-CoA is similar to the effect of a two-carbon change in acyl chain length: i.e . an enthalpy dominated reduction in binding affinity . However, kinetic studies reveal that removing the 3'-phosphate from myristoyl-CoA has little effect on Nmt1p's catalytic efficiency, indicating that the 3'-phosphate group contributes binding free energy but little catalytic destabilization . The greater delta delta G degrees, with smaller delta delta H degrees and delta delta S degrees components, produced by removing the 3'-phosphate compared to increasing chain length suggests that it is not primarily the interactions of the 3'-phosphate which are disrupted when palmitoyl-CoA is substituted for myristoyl-CoA . No detectable interactions were noted between apo-Nmt1p and the substrate peptide, GAAPSKIV-NH2, providing additional support for the preferred ordered reaction mechanism.(ABSTRACT TRUNCATED AT 400 WORDS) J Biol Chem, 1994 Apr 15, 269(15), 11018 - 24 Regulation of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae by CDP-diacylglycerol; Nickels JT Jr et al.; Regulation of the 45- and 55-kDa forms of Saccharomyces cerevisiae membrane-associated phosphatidylinositol (PI) 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase) by phospholipids was examined using Triton X-100/phospholipid-mixed micelles . CDP-diacylglycerol and phosphatidylglycerol inhibited 45-kDa PI 4-kinase activity in a dose-dependent manner . Kinetic analyses of the 45-kDa PI 4-kinase showed that phosphatidylglycerol was a competitive inhibitor with respect to PI (Ki = 2 mol %), and CDP-diacylglycerol was a mixed type of inhibitor with respect to PI (Ki = 4 mol %) and MgATP (Ki = 5 mol %) . 55-kDa PI 4-kinase activity was not significantly affected by phospholipids . The physiological relevance of CDP-diacylglycerol inhibition of 45-kDa PI 4-kinase activity was examined using plasma membranes from inositol auxotrophic (ino1) cells . Immunoblot analysis showed that 45-kDa PI 4-kinase expression in plasma membranes was not affected by inositol starvation of ino1 cells . However, both 45-kDa PI 4-kinase activity and its product PI 4-phosphate were reduced in plasma membranes from inositol-starved ino1 cells . The CDP-diacylglycerol concentration (9.6 mol %) in plasma membranes of inositol-starved ino1 cells was 12-fold higher than its concentration (0.8 mol %) in plasma membranes of inositol-supplemented cells . Plasma membranes of inositol-starved ino1 cells also had increased levels of phosphatidate, phosphatidylserine, phosphatidylethanolamine, and cardiolipin . However, these phospholipids did not affect pure 45-kDa PI 4-kinase activity . The concentration of CDP-diacylglycerol in plasma membranes of inositol-starved ino1 cells was in the range of the inhibitor constants determined for CDP-diacylglycerol by kinetic analyses using pure 45-kDa PI 4-kinase . These results raised the suggestion that 45-kDa PI 4-kinase activity may be regulated in vivo by CDP-diacylglycerol. J Biol Chem, 1994 Apr 15, 269(15), 10996 - 1001 A soluble exopolyphosphatase of Saccharomyces cerevisiae . Purification and characterization; Wurst H et al.; A soluble polyphosphatase of Saccharomyces cerevisiae, purified to apparent homogeneity, is monomeric with a molecular mass of 40 kDa . It acts as an exoenzyme in a processive mode releasing orthophosphate residues from long polyphosphate chains until pyrophosphate is reached . Polyphosphates of all the lengths examined are used as substrates with a preference for those of about 250 residues . These are degraded with a kcat/Km near the limit for diffusion-controlled reactions . At 37 degrees C, the enzyme releases about 500 phosphate residues/s . It does not act on pyrophosphate, ATP, or the cyclic form of tripolyphosphate . For optimal activity the enzyme requires magnesium, manganese, or cobalt. Mutat Res, 1994 Apr 15, 306(2), 187 - 96 A test for uniparental disomy in Saccharomyces cerevisiae; Zimmermann FK; Uniparental disomy is a condition in a diploid organisms where one parental chromosome is absent and its homolog from the other parent duplicated . It can be a cause of genetic somatic disease in mammals because of imprinting . Imprinting creates a sex-specific pattern of epigenetic gene inactivation at least in mammals and, consequently, a complete set of both maternal and paternal chromosomes is required for normal development . Moreover, it has been shown for several types of tumors that recessive tumor alleles originally present in a heterozygous condition in normal somatic tissue have become homozygous in the tumor cells . Homozygosity is frequently caused by uniparental disomy . A similar situation is found in Saccharomyces cerevisiae where the spontaneous or induced expression of linked recessive alleles flanking a common centromere is preponderantly due to isodisomy where one of the homologs is lost and the retained homolog duplicated . In contrast to the situation in Aspergillus nidulans, isodisomy does not appear to be caused by two sequential and independent events of malsegregation resulting first in an unstable trisomic condition from which a normal disomic condition is restored through segregational loss of one supernumerary chromosome . Rather, an as yet unknown mechanism seems to directly generate isodisomy and thus Saccharomyces cerevisiae could provide a short-term test for the detection of this type of genetic change. Appl Biochem Biotechnol, 1994 Apr, 47(1), 65 - 72 L-malic acid production by entrapped Saccharomyces cerevisiae into polyacrylamide gel beads; Oliveira EA et al.; The yeast Saccharomyces cerevisiae was entrapped within polyacrylamide gel beads by employing a procedure that uses sodium dodecylsulfate as a detergent to improve the spherical configuration of the beads . The resulting preparation showed a rate of fumarate bio-conversion to L-malic acid about 60 times higher than that found for the free cells . Almost all fumarate was converted in 30 min of incubation . The thermal stability of the immobilized cells did not significantly differ from the free cells . An optimal pH of 5.7 was found for the immobilized preparation and no succinic acid was detected as a byproduct in the incubation mixture. Mol Gen Genet, 1994 Apr, 243(1), 63 - 70 Mating type regulates the radiation-associated stimulation of reciprocal translocation events in Saccharomyces cerevisiae; Fasullo M et al.; Both ultraviolet (UV) and ionizing radiation were observed to stimulate mitotic, ectopic recombination between his3 recombinational substrates, generating reciprocal translocations in Saccharomyces cerevisiae (yeast) . The stimulation was greatest in diploid strains competent for sporulation and depends upon both the ploidy of the strain and heterozygosity at the MATlocus . The difference in levels of stimulation between MATa/MAT alpha diploid and MAT alpha haploid strains increases when cells are exposed to higher levels of UV radiation (sevenfold at 150 J/m2), whereas when cells are exposed to higher levels of ionizing radiation (23.4 krad), only a twofold difference is observed . When the MAT alpha gene was introduced by DNA transformation into a MATa/mat alpha::LEU2+ diploid, the levels of radiation-induced ectopic recombination approach those obtained in a strain that is heterozygous at MAT . Conversely, when the MATa gene was introduced by DNA transformation into a MAT alpha haploid, no enhanced stimulation of ectopic recombination was observed when cells were irradiated with ionizing radiation but a threefold enhancement was observed when cells were irradiated with UV . The increase in radiation-stimulated ectopic recombination resulting from heterozygosity at MAT correlated with greater spontaneous ectopic recombination and higher levels of viability after irradiation . We suggest that MAT functions that have been previously shown to control the level of mitotic, allelic recombination (homolog recombination) also control the level of mitotic, radiation-stimulated ectopic recombination between short dispersed repetitive sequences on non-homologous chromosomes. Mol Gen Genet, 1994 Apr, 243(2), 158 - 65 Increases in cell size at START caused by hyperactivation of the cAMP pathway in Saccharomyces cerevisiae; Mitsuzawa H; In the budding yeast Saccharomyces cerevisiae, passage through START, which commits cells to a new round of cell division, requires growth to a critical size . To examine the effect of hyperactivation of the cAMP pathway on cell size at START, a strain was constructed that is able to respond to exogenously added cAMP . In the presence of cAMP, this strain showed increased cell volume at bud emergence, suggesting that the critical cell size necessary for START is increased . In addition, a mutation that results in unregulated cAMP-dependent protein kinase (bcy1) caused increased cell size at START . These results indicate that hyperactivation of the cAMP pathway causes increases in cell size through cAMP-dependent protein kinase . Cells carrying a hyperactive allele of CLN3 (CLN3-2) also showed increased size at START in the presence of cAMP . These cells retained resistance to alpha factor, however, suggesting that increases in cell size by cAMP are not due to a reduction of Cln3 activity . The observed increases in cell size due to hyperactivation of the cAMP pathway suggest that cell size modulation by nutrient conditions may be associated with a change of the activity of the cAMP pathway. EMBO J, 1994 Apr 1, 13(7), 1706 - 17 The Saccharomyces cerevisiae APS1 gene encodes a homolog of the small subunit of the mammalian clathrin AP-1 complex: evidence for functional interaction with clathrin at the Golgi complex; Phan HL et al.; Clathrin-associated protein (AP) complexes have been implicated in the assembly of clathrin coats and the selectivity of clathrin-mediated protein transport processes . We have identified a yeast gene, APS1, encoding a homolog of the small (referred to herein as sigma) subunits of the mammalian AP-1 complex . Sequence comparisons have shown that Aps1p is more similar to the sigma subunit of the Golgi-localized mammalian AP-1 complex than Aps2p, which is more related to the plasma membrane AP-2 sigma subunit . Like their mammalian counterparts, Aps1p and Aps2p are components of distinct, large (> 200 kDa) complexes and a significant portion of the Aps proteins co-fractionate with clathrin-coated vesicles during gel filtration chromatography . Unexpectedly, even though the evolutionary conservation of AP small subunits is substantial (50% identity between mammalian and yeast proteins), disruptions of APS1 (aps1 delta) and APS2 (aps2 delta), individually or in combination, elicit no detectable mutant phenotypes . These data indicate that the Aps proteins are not absolutely required for clathrin-mediated selective protein transport in cells expressing wild type clathrin . However, aps1 delta accentuated the slow growth and alpha-factor pheromone maturation defect of cells carrying a temperature-sensitive allele of clathrin heavy chain (Chc) (chc1-ts) . In contrast, aps1 delta did not influence the effects of chc1-ts on vacuolar protein sorting or receptor-mediated endocytosis . The aps2 delta mutation resulted in a slight effect on chc1-ts cell growth but had no additional effects . The growth defect of cells completely lacking Chc was compounded by aps1 delta but not aps2 delta . These results comprise evidence that Aps1p is involved in a subset of clathrin functions at the Golgi apparatus . The effect of aps1 delta on cells devoid of clathrin function suggests that Aps1p also participates in clathrin-independent processes. J Bacteriol, 1994 Apr, 176(7), 1872 - 80 Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: analysis of the genes in the FUN38-MAK16-SPO7 region; Barton AB et al.; Transcribed regions on a 42-kb segment of chromosome I from Saccharomyces cerevisiae were mapped . Polyadenylated transcripts corresponding to eight previously characterized genes (MAK16, LTE1, CCR4, FUN30, FUN31, TPD3, DEP1, and CYS3) and eight new genes were identified . All transcripts were present at one to four copies per cell except for one which was significantly less abundant . This region has been sequenced, and the sizes, locations, and orientations of the transcripts were in nearly perfect agreement with the open reading frames . Disruptions in eight genes identified solely on the basis of a transcribed region, FUN38, FUN25, FUN26, FUN28, FUN30, FUN31, FUN33, and FUN34, indicated that all were nonessential for growth on rich medium at 30 degrees C . Disruption of FUN30, a gene closely related to RAD16 and RAD54, surprisingly resulted in increased resistance to UV irradiation . No additional phenotypes, other than slow growth, were observed for all other mutants . The distribution of essential genes on chromosome I is discussed. Exp Cell Res, 1994 Apr, 211(2), 203 - 11 Characterization of the function of the NIN1 gene product of Saccharomyces cerevisiae; Kominami K et al.; The nin1-1 mutant has been isolated as a temperature-sensitive mutant whose nucleus arrested at G2 phase and eventually disintegrated upon temperature upshift . In this study, a genetic event occurring in the nin1-1 mutant was found to be a frameshift mutation, resulting in a truncated protein smaller than the wild-type Nin1 protein . We found new phenotypes associated with the nin1-1 mutation: (i) rates of mitotic recombination and chromosome/plasmid loss in the nin1-1 strain were higher than those in the wild-type strain, and (ii) the mutant was more sensitive to UV irradiation than the wild-type strain . We found dotted structures in the cytoplasm of the wild-type cells by indirect immunofluorescence microscopy using the anti-Nin1 antibody . Similar results were obtained when we analyzed the localization of Nin1-beta-galactosidase fusion protein formed in the cells expressing the NIN-lacZ fusion gene, which is active as NIN1, with anti-beta-galactosidase antibody . The subcellular fractionation method revealed that Nin1 protein was not localized in a particular fraction of the cell lysate. Mol Cell Biol, 1994 Apr, 14(4), 2664 - 74 His-154 is involved in the linkage of the Saccharomyces cerevisiae L-A double-stranded RNA virus Gag protein to the cap structure of mRNAs and is essential for M1 satellite virus expression; Blanc A et al.; The coat protein (Gag) of the double-stranded RNA virus L-A was previously shown to form a covalent bond with the cap structure of eukaryotic mRNAs . Here, we identify the linkage as a phosphoroimidazole bond between the alpha phosphate of the cap structure and a nitrogen in the Gag protein His-154 imidazole side chain . Mutations of His-154 abrogate the ability of Gag to bind to the cap structure, without affecting cap recognition, in vivo virus particle formation from an L-A cDNA clone, or in vitro specific binding and replication of plus-stranded single-stranded RNA . However, genetic analyses demonstrate that His-154 is essential for M1 satellite virus expression. J Cell Biol, 1994 Apr, 125(1), 143 - 58 The JNM1 gene in the yeast Saccharomyces cerevisiae is required for nuclear migration and spindle orientation during the mitotic cell cycle; McMillan JN et al.; JNM1, a novel gene on chromosome XIII in the yeast Saccharomyces cerevisiae, is required for proper nuclear migration . jnm1 null mutants have a temperature-dependent defect in nuclear migration and an accompanying alteration in astral microtubules . At 30 degrees C, a significant proportion of the mitotic spindles is not properly located at the neck between the mother cell and the bud . This defect is more severe at low temperature . At 11 degrees C, 60% of the cells accumulate with large buds, most of which have two DAPI staining regions in the mother cell . Although mitosis is delayed and nuclear migration is defective in jnm1 mutant, we rarely observe more than two nuclei in a cell, nor do we frequently observe anuclear cells . No loss of viability is observed at 11 degrees C and cells continue to grow exponentially with increased doubling time . At low temperature the large budded cells of jnm1 mutants exhibit extremely long astral microtubules that often wind around the periphery of the cell . jnm1 mutants are not defective in chromosome segregation during mitosis, as assayed by the rate of chromosome loss, or nuclear migration during conjugation, as assayed by the rate of mating and cytoduction . The phenotype of a jnm1 mutant is strikingly similar to that for mutants in the dynein heavy chain gene (Eshel, D., L . A . Urrestarazu, S . Vissers, J.-C . Jauniaux, J . C . van Vliet-Reedijk, R . J . Plants, and I . R . Gibbons . 1993 . Proc . Natl . Acad . Sci . USA . 90:11172-11176; Li, Y . Y., E . Yeh, T . Hays, and K . Bloom . 1993 . Proc . Natl . Acad . Sci . USA . 90:10096-10100) . The JNM1 gene product is predicted to encode a 44-kD protein containing three coiled coil domains . A JNM1:lacZ gene fusion is able to complement the cold sensitivity and microtubule phenotype of a jnm1 deletion strain . This hybrid protein localizes to a single spot in the cell, most often near the spindle pole body in unbudded cells and in the bud in large budded cells . Together these results point to a specific role for Jnm1p in spindle migration, possibly as a subunit or accessory protein for yeast dynein. Yeast, 1994 Apr, 10 Suppl A, S47 - 62 The sequence of a 32,420 bp segment located on the right arm of chromosome II from Saccharomyces cerevisiae; Holmstrom K et al.; The sequence of a 32,420 bp segment of Saccharomyces cerevisiae chromosome II has been deduced . The sequence data revealed 19 potential new genes covering 83.5% of the sequence . Four genes had already been cloned and sequenced: part of RIF1, DPB3, MRP-L27 and SNF5 . Besides these four genes, 15 open reading frames (ORFs) of at least 100 amino acids encoding potential new genes were identified . Two of these ORFs are overlapping and a third is located within another ORF . The putative gene product of ORF YBR2039 was homologous to the group of uncoupling proteins involved in the mitochondrial energy transfer system . We propose a remapping of the MRP-L27 gene encoding the mitoribosomal protein YmL27 as it previously has been mapped on chromosome X . The ORF YBR2020 has a strong homology with a 31.9% identity in a 473 amino acid region to the yeast gene SEC61, suggesting that YBR2020 is a new gene encoding a protein involved in translocation of proteins in the yeast cell . Six of the potential genes do not exhibit any significant homology to previously sequenced genes as predicted in the Fast A analysis. Yeast, 1994 Apr, 10 Suppl A, S41 - 6 Sequence around the centromere of Saccharomyces cerevisiae chromosome II: similarity of CEN2 to CEN4; Wolfe KH et al.; We report the sequence of a 12 kilobase region spanning the centromere of Saccharomyces cerevisiae chromosome II . The sequence from the left arm includes genes for histones H2A and H2B . The sequence from the right arm includes a gene that probably encodes a novel trehalase, as well as the COQ1 gene (for an enzyme involved in coenzyme Q biosynthesis), and an open reading frame with significant similarity to bacterial genes of unknown function . The trehalase gene (YBR0106) on chromosome II is located beside the centromere and transcribed towards it . This is identical to the arrangement of the neutral trehalase gene (NTH1) beside the centromere of chromosome IV . The centromere regions of chromosomes II and IV may therefore have arisen through a duplication of the centromere region of an ancestral chromosome . The YBR0106 and NTH1 proteins are 77% identical in predicted amino acid sequence, but there is no pronounced sequence similarity between the two centromeres (CEN2 and CEN4) outside of the universally conserved CDE I and CDE III elements . The genes flanking the centromere and trehalase genes differ between the two chromosomes, so the similarity between chromosomes II and IV may be less extensive than that recently reported between chromosomes III and XIV. Yeast, 1994 Apr, 10 Suppl A, S35 - 40 DNA sequencing of a 36.2 kb fragment located between the FAS1 and LAP loci of chromosome XI of Saccharomyces cerevisiae; Vandenbol M et al.; We have completely sequenced on both strands a continuous DNA segment of 36.2 kb located on the left arm of Saccharomyces cerevisiae chromosome XI . Sequence analysis reveals the presence of 20 open reading frames (ORFs) at least 100 amino acids long . Five of these ORFs correspond to known genes; five others show homology with known proteins; the ten remaining ORFs identified show no detectable homology with other protein sequences contained in data banks and may represent new biological functions. Yeast, 1994 Apr, 10 Suppl A, S13 - 24 The sequence of an 8.8 kb segment on the left arm of chromosome II from Saccharomyces cerevisiae reveals four new open reading frames including homologs of animal DNA polymerase alpha-primases and bacterial GTP cyclohydrolase II; Skala J et al.; The DNA sequence of two contiguous 7648 bp and 1194 bp BamHI fragments from the cosmid alpha 1201 located about 60 kb from the centromere on the left arm of chromosome II from Saccharomyces cerevisiae has been determined . Sequence analysis reveals four new open reading frames longer than 300 bp: YBL0415 (309 bp), YBL0416 (4539 bp), YBL0417 (1035 bp) and YBL0414 (2115 bp), which extends into the neighbouring 5.2 kb BamHI fragment . The YBL0414 shows homologies to the mouse 68 kDa and Drosophila melanogaster 76 kDa subunits of the DNA polymerase alpha-primase complex . The YBL0417 is homologous to bacterial GTP cyclohydrolase II (EC 3.5.4.25). J Biochem (Tokyo), 1994 Apr, 115(4), 762 - 6 Characterization of multiple molecular forms of Mg(2+)-dependent protein phosphatase from Saccharomyces cerevisiae; Murakami T et al.; Three molecular species of Mg(2+)-dependent protein phosphatase (MPPs-1, -2, and -3) were isolated by DEAE cellulose column chromatography and gel filtration from an extract of Saccharomyces cerevisiae . MPP-1 was further purified 150-fold by chromatography using thio-phosphorylated myosin light chain-agarose . MPPs-1, -2, and -3 were distinct from the major acid and alkaline phosphatases, and their activities were not affected by okadaic acid, microcystin-LR or Ca2+, and calmodulin, resembling the enzymatic properties of type 2C protein phosphatase of mammalian cells . The apparent molecular masses of MPPs-1, -2, and -3 on gel filtration were 53, 112, and 128 kDa, respectively . It was demonstrated that MPP-1 is a globular protein of 53-55 kDa and that MPPs-2 and -3 are oligomeric proteins that dissociate upon sucrose density gradient centrifugation, generating catalytic proteins of about 50 kDa . Since the substrate specificities of MPPs-1, -2, and -3 differed from each other both before and after sucrose density gradient centrifugation, it was suggested that the catalytic proteins of these three enzymes are distinct molecular species. Curr Genet, 1994 Apr, 25(4), 299 - 304 PET112, a Saccharomyces cerevisiae nuclear gene required to maintain rho+ mitochondrial DNA; Mulero JJ et al.; The nuclear gene PET112 was originally identified by a mutation (pet112-1) that specifically blocked accumulation of cytochrome c oxidase subunit II . The mutation causes a post-transcriptional defect since the level of COX2 mRNA in the mutant is the same as in the wild-type . However, PET112 does not have a function similar to that of PET111, a COX2 mRNA-specific translational activator: while pet111 mutations are suppressed by chimeric COX2 mRNAs bearing 5' leaders of other mitochondrial mRNAs, pet112-1 is not . The PET112 gene was isolated and shown to code a protein of 541 residues (62 kDa) with no significant homology to known amino-acid sequences . By hybridization to defined genomic clones the gene was mapped to chromosome II between cdc25 and ils1 . Disruption of the PET112 open reading frame destabilized the mitochondrial genome, causing cells to become rho- . This finding suggests that PET112 has an important general function in mitochondrial gene expression, probably in translation. Curr Genet, 1994 Apr, 25(4), 291 - 8 Expression of the Saccharomyces cerevisiae CYT2 gene, encoding cytochrome c1 heme lyase; Zollner A et al.; In this paper we examine the expression of the Saccharomyces cerevisiae CYT2 gene, which encodes cytochrome c1 heme lyase . This enzyme is required for covalent attachment of heme to apocytochrome c1, a subunit of the mitochondrial respiratory chain . Transcription of the 1-kb CYT2 mRNA initiates at four prominent sites at a distance of 52-225 bp in front of the AUG start codon . The level of CYT2 mRNA is not influenced by the presence or absence of oxygen or of heme, but it is subject to carbon-source control . The concentration of the CYT2 mRNA is significantly reduced in glucose-grown cells as compared to cells grown under non-repressing conditions . Neither the HAPp activator proteins nor MIG1p, a repressor protein involved in glucose repression, seem to mediate this effect. Chromosoma, 1994 Apr, 103(2), 129 - 41 Saccharomyces cerevisiae cells lacking the homologous pairing protein p175SEP1 arrest at pachytene during meiotic prophase; Bahler J et al.; Saccharomyces cerevisiae cells containing null mutations in the SEP1 gene, which encodes the homologous pairing and strand exchange protein p175SEP1, enter pachytene with a delay . They arrest uniformly at this stage of meiotic prophase, probably revealing a checkpoint in the transition from pachytene to meiosis I . At the arrest point, the cells remain largely viable and are cytologically characterized by the duplicated but unseparated spindle pole bodies of equal size and by the persistence of the synaptonemal complex, a cytological marker for pachytene . In addition, fluorescence in situ hybridization revealed that in arrested mutant cells maximal chromatin condensation and normal homolog pairing is achieved, typical for pachytene in wild type . A hallmark of meiosis is the high level of homologous recombination, which was analyzed both genetically and physically . Formation and processing of the double-strand break intermediate in meiotic recombination is achieved prior to arrest . Physical intragenic (conversion) and intergenic (crossover) products are formed just prior to, or directly at, the arrest point . Structural deficits in synaptonemal complex morphology, failure to separate spindle pole bodies, and/or defects in prophase DNA metabolism might be responsible for triggering the observed arrest . The pachytene arrest in sep1 cells is likely to be regulatory, but is clearly different from the RAD9 checkpoint in meiotic prophase, which occurs prior to the pachytene stage. Curr Opin Genet Dev, 1994 Apr, 4(2), 212 - 20 Repair of UV-damaged DNA by mammalian cells and Saccharomyces cerevisiae; Aboussekhra A et al.; Cells use many strategies to repair genomic damage caused by environmental agents and arising from the natural instability of the polynucleotide structure . Nucleotide excision repair is the most versatile DNA repair pathway and is the main defense of mammalian cells against UV-induced DNA damage . Defects in proteins involved in this pathway can lead to inherited disorders (such as xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy) that are associated with hypersensitivity to sunlight . Most of the proteins and genes involved in these syndromes have now been identified . Study of UV-sensitive yeast RAD mutants has greatly aided this process and has revealed strong conservation of the components of nucleotide excision repair in eukaryotes . It has recently become clear that some of the proteins involved in the DNA repair process have dual functions and also participate in basal transcription and DNA replication. Bioessays, 1994 Apr, 16(4), 253 - 8 The RAD6 DNA repair pathway in Saccharomyces cerevisiae: what does it do, and how does it do it? Lawrence C. The RAD6 pathway of budding yeast, Saccharomyces cerevisiae, is responsible for a substantial fraction of this organism's resistance to DNA damage, and also for induced mutagenesis . The pathway appears to incorporate two different recovery processes, both regulated by RAD6 . The error-prone recovery process accounts for only a small amount of RAD6-dependent resistance, but probably all induced mutagenesis . The underlying mechanism for error-prone recovery is very likely to be translesion synthesis . The error-free recovery process accounts for most of RAD6-dependent resistance, but its mechanism is less clear; it may entail error-free bypass by template switching and/or DNA gap filling by recombination . RAD6 regulates these activities by ubiquitinating target proteins, but the identities of these target proteins, and the roles they play in error-free and error-prone recovery, have not yet been established. Genetics, 1994 Apr, 136(4), 1287 - 96 Identification of genes required for normal pheromone-induced cell polarization in Saccharomyces cerevisiae; Chenevert J et al.; In response to mating pheromones, cells of the yeast Saccharomyces cerevisiae adopt a polarized "shmoo" morphology, in which the cytoskeleton and proteins involved in mating are localized to a cell-surface projection . This polarization is presumed to reflect the oriented morphogenesis that occurs between mating partners to facilitate cell and nuclear fusion . To identify genes involved in pheromone-induced cell polarization, we have isolated mutants defective in mating to an enfeebled partner and studied a subset of these mutants . The 34 mutants of interest are proficient for pheromone production, arrest in response to pheromone, mate to wild-type strains, and exhibit normal cell polarity during vegetative growth . The mutants were divided into classes based on their morphological responses to mating pheromone . One class is unable to localize cell-surface growth in response to mating factor and instead enlarges in a uniform manner . These mutants harbor special alleles of genes required for cell polarization during vegetative growth, BEM1 and CDC24 . Another class of mutants forms bilobed, peanut-like shapes when treated with pheromone and defines two genes, PEA1 and PEA2 . PEA1 is identical to SPA2 . A third class forms normally shaped but tiny shmoos and defines the gene TNY1 . A final group of mutants exhibits apparently normal shmoo morphology . The nature of their mating defect is yet to be determined . We discuss the possible roles of these gene products in establishing cell polarity during mating. Genetics, 1994 Apr, 136(4), 1279 - 85 Altered regulatory responses to glucose are associated with a glucose transport defect in grr1 mutants of Saccharomyces cerevisiae; Vallier LG et al.; The GRR1 gene of Saccharomyces cerevisiae affects glucose repression, cell morphology, divalent cation transport and other processes . We present a kinetic analysis showing that the grr1 mutant is also defective in high affinity glucose transport . In combination with a mutation in SNF3, a member of the glucose transporter gene family, grr1 strikingly impairs growth on glucose . These findings suggest that GRR1 and SNF3 affect glucose transport by distinct pathways . The mutation rgt1-1, a suppressor of snf3, restores both glucose transport and glucose repression to a grr1 mutant, but does not remedy the morphological defect . We suggest that GRR1 affects the glucose sensing process and that the association between transport and regulation may reflect the involvement of a transporter in glucose sensing. Genetics, 1994 Apr, 136(4), 1271 - 8 Suppressors reveal two classes of glucose repression genes in the yeast Saccharomyces cerevisiae; Erickson JR et al.; We selected and analyzed extragenic suppressors of mutations in four genes--GRR1, REG1, GAL82 and GAL83-required for glucose repression of the GAL genes in the yeast Saccharomyces cerevisiae . The suppressors restore normal or nearly normal glucose repression of GAL1 expression in these glucose repression mutants . Tests of the ability of each suppressor to cross-suppress mutations in the other glucose repression genes revealed two groups of mutually cross-suppressed genes: (1) REG1, GAL82 and GAL83 and (2) GRR1 . Mutations of a single gene, SRG1, were found as suppressors of reg1, GAL83-2000 and GAL82-1, suggesting that these three gene products act at a similar point in the glucose repression pathway . Mutations in SRG1 do not cross-suppress grr1 or hxk2 mutations . Conversely, suppressors of grr1 (rgt1) do not cross-suppress any other glucose repression mutation tested . These results, together with what was previously known about these genes, lead us to propose a model for glucose repression in which Grr1p acts early in the glucose repression pathway, perhaps affecting the generation of the signal for glucose repression . We suggest that Reg1p, Gal82p and Gal83p act after the step(s) executed by Grr1p, possibly transmitting the signal for repression to the Snf1p protein kinase. Genetics, 1994 Apr, 136(4), 1245 - 59 Heterogeneous functional Ty1 elements are abundant in the Saccharomyces cerevisiae genome; Curcio MJ et al.; Despite the abundance of Ty1 RNA in Saccharomyces cerevisiae, Ty1 retrotransposition is a rare event . To determine whether transpositional dormancy is the result of defective Ty1 elements, functional and defective alleles of the retrotransposon in the yeast genome were quantitated . Genomic Ty1 elements were isolated by gap repair-mediated recombination of pGTy1-H3(delta 475-3944) HIS3, a multicopy plasmid containing a GAL1/Ty1-H3 fusion element lacking most of the gag domain (TYA) and the protease (PR) and integrase (IN) domains . Of 39 independent gap repaired pGTyHIS3 elements isolated, 29 (74%) transposed at high levels following galactose induction . The presence of restriction site polymorphisms within the gap repaired region of the 29 functional pGTyHIS3 elements indicated that they were derived from at least eight different genomic Ty1 elements and one Ty2 element . Of the 10 defective pGTyHIS3 elements, one was a partial gap repair event while the other nine were derived from at least six different genomic Ty1 elements . These results suggest that most genomic Ty1 elements encode functional TYA, PR and IN proteins . To understand how functional Ty1 elements are regulated, we tested the hypothesis that a TYB protein associates preferentially in cis with the RNA template that encodes it, thereby promoting transposition of its own element . A genomic Ty1 mhis3AI element containing either an in-frame insertion in PR or a deletion in TYB transposed at the same rate as a wild-type Ty1mhis3AI allele, indicating that TYB proteins act efficiently in trans . This result suggests in principle that defective genomic Ty1 elements could encode trans-acting repressors of transposition; however, expression of only one of the nine defective pGTy1 isolates had a negative effect on genomic Ty1 mhis3AI element transposition in trans, and this effect was modest . Therefore, the few defective Ty1 elements in the genome are not responsible for transpositional dormancy. J Mol Evol, 1994 Apr, 38(4), 363 - 8 Chimeric evolution of the 2-microns genome in Saccharomyces cerevisiae; Xie Y et al.; We compared the nucleotide substitution pattern over the entire genome of two unique variants of the 6,300-bp selfish DNA (2 microns) plasmid in Saccharomyces cerevisiae . The DNA sequence of the left-unique region is identical among 2-microns variants, while the right-unique region shows substantial divergence . This chimeric pattern cannot be explained by neutral or Darwinian selection models . We propose that horizontal transmission of the 2-microns plasmid coupled with a directed, polarized gene conversion maintains the DNA sequence of the left-unique region, whereas the right-unique region is subject to random drift and Darwinian selection. Yeast, 1994 Apr, 10(4), 535 - 41 Sequencing of chromosome I of Saccharomyces cerevisiae: analysis of the 42 kbp SPO7-CENI-CDC15 region; Clark MW et al.; Determination of the DNA sequence and preliminary functional analysis of a 42 kbp centromeric section of chromosome I have been completed . The section spans the SPO7-CEN1-CDC15 loci and contains 19 open reading frames (ORFs) . They include an apparently inactive Ty1 retrotransposon and eight new ORFs with no known homologs or function . The remaining ten genes have been previously characterized since this part of the yeast genome has been studied in an unusually intensive manner . Our directed sequencing allows a complete ordering of the region. Yeast, 1994 Apr, 10(4), 523 - 33 Organization of the centromeric region of chromosome XIV in Saccharomyces cerevisiae; Lalo D et al.; A 15.1 kb fragment of the yeast genome was allocated to the centromeric region of chromosome XIV by genetic mapping . It contained six bona fide genes, RPC34, FUN34, CIT1 (Suissa et al., 1984), RLP7, PET8 and MRP7 (Fearon and Mason, 1988) and two large open reading frames, DOM34 and TOM34 . RPC34 and RLP7 define strictly essential functions, whereas CIT1, PET8 and MRP7 encode mitochondrial proteins . The PET8 product belongs to a family of mitochondrial carrier proteins . FUN34 encodes a putative transmembraneous protein that is non-essential as judged from the normal growth of the fun34-::LUK18(URA3) allele even on respirable substrates . TOM34 codes for a putative RNA binding protein, and DOM34 defines a hypothetical polypeptide of 35 kDa, with no significant homology to known proteins . The region under study also contains two divergently transcribed tDNAs, separated only by a chimeric transposable element . This tight tDNA linkage pattern is commonly encountered in yeast, and a general hypothesis is proposed for its emergence on the Saccharomyces cerevisiae genome . RPC34, RLP7, PET8 and MRP7 are unique on the yeast genome, but the remaining genes belong to an extant centromeric duplication between chromosome III and XIV. Yeast, 1994 Apr, 10(4), 491 - 6 A search for an essential function of the replication origin ARS1 in the life cycle of Saccharomyces cerevisiae; Kirpekar F et al.; We have investigated the significance of the chromosomal replication origin, ARS1, during the entire life cycle of yeast . This was done by substituting the chromosomal copy with a series of ars1 deletion mutants . It was shown that the ARS1 replication origin is not essential for mitotic or premeiotic DNA replication since no effect on growth, chromosomal loss rate and spore viability was observed in the ars1 mutant strains . We conclude that replication origins are abundantly, present in the yeast genome and that the removal of a single replication origin is compensated for by replication forks emanating from neighbouring origins. Yeast, 1994 Apr, 10(4), 481 - 90 Regulation of THI4 (MOL1), a thiamine-biosynthetic gene of Saccharomyces cerevisiae; Praekelt UM et al.; THI4, a Saccharomyces cerevisiae gene originally identified as a result of transient expression in molasses medium and named MOL1 is regulated by thiamine . Using a THI4 promoter-lacZ fusion on a centromeric yeast vector, we have shown that the THI4 is completely repressed throughout batch culture by thiamine at a concentration around 1 microM, but shows high level constitutive expression in thiamine-free medium . The transient expression pattern observed in molasses medium can be mimicked by the addition of 0.15 microM-thiamine to defined minimal medium . Cells grown in thiamine-free medium have an intracellular thiamine concentration of around 9 pmol/10(7) cells . A low level (1 microM) of exogenous thiamine is completely sequestered from the medium within 30 min; intracellular thiamine concentrations rise rapidly, followed by a gradual decrease as a result of dilution during growth . A saturating extracellular level of thiamine leads to a maximal intracellular concentration of around 1600 pmol/10(7) cells, at which point the transport system is shut down . After transfer from repressing to non-repressing medium, THI4 becomes induced when the intracellular concentration of thiamine falls to 20 pmol/10(7) cells . A thi4::URA3 disruption strain is auxotrophic for thiamine, but can grow in the presence of hydroxyethyl thiazole, indicating that the gene product is involved in the biosynthetic pathway leading to the formation of the thiazole precursor of thiamine. Yeast, 1994 Apr, 10(4), 463 - 74 Genetic evidence for a functional interaction between Saccharomyces cerevisiae CDC24 and CDC42; Ziman M et al.; Cdc24p and Cdc42p are involved in the control of cell polarity during the Saccharomyces cerevisiae cell cycle . Cdc42p is a member of the Ras superfamily of GTPases and Cdc24p displays limited amino-acid sequence similarity with the Dbl proto-oncoprotein, which acts to stimulate guanine-nucleotide exchange on human Cdc42p . We have performed several genetic experiments to test whether Cdc24p and Cdc42p interact within the cell . First, overexpression of Cdc24p suppressed the dominant-negative cdc42D118A allele . Second, overexpression of wild-type CDC24 and CDC42 genes together was a lethal event resulting in a morphological phenotype of large, round, unbudded cells, indicating a loss of cell polarity . Third, a cdc24ts cdc42ts double mutant exhibited a synthetic-lethal phenotype at the semi-permissive temperature of 30 degrees C . These data suggest that Cdc24p and Cdc42p interact within the cell and that Cdc24p may be involved in the regulation of Cdc42p activity. Biosci Biotechnol Biochem, 1994 Apr, 58(4), 621 - 6 Cloning and nucleotide sequence of the alkaline protease gene from Fusarium sp . S-19-5 and expression in Saccharomyces cerevisiae; Morita S et al.; We have cloned a genomic DNA encoding the alkaline protease (Alp) of Fusarium sp . S-19-5 from a genomic DNA library and sequenced the nucleotides . Complementary DNA encoding Alp was also isolated from the cDNA library after amplifying the gene by PCR using partial sequences of the Alp genomic DNA as primers . The Alp gene has an open reading frame of 1137 nucleotides containing three introns . A TATA box (TAAATA) was observed 112 base pairs upstream from the translation initiation codon in the 5'-non coding region . The Alp protein has a pre region consisting of 14 amino acids and a pro region of 85 amino acids preceding the mature region, which consists of 280 amino acids . The amino acid sequence of Fusarium Alp has 52% homology with that of Aspergillus oryzae and 51% homology with that of Acremonium chrysogenum . The entire cDNA encoding Fusarium Alp was introduced into Saccharomyces cerevisiae, which then secreted enzymatically active Alp into the culture medium. Proc Natl Acad Sci U S A, 1994 Mar 29, 91(7), 2723 - 7 Cloning and analysis of the Saccharomyces cerevisiae MNN9 and MNN1 genes required for complex glycosylation of secreted proteins; Yip CL et al.; Proteins secreted by the yeast Saccharomyces cerevisiae are usually modified by the addition at asparagine-linked glycosylation sites of large heterogeneous mannan units that are highly immunogenic . Secreted proteins from mnn1 mnn9 mutant strains, in contrast, have homogeneous Man10GlcNAc2 oligosaccharides that lack the immunogenic alpha 1,3-mannose linkages . We have cloned and sequenced the MNN9 and MNN1 genes, both of which encode proteins with the characteristics of type II membrane proteins . Mnn9p is a membrane-associated protein with unknown function that is required for the addition of the long alpha 1,6-mannose backbone of the complex mannan, whereas Mnn1p is most likely the alpha 1,3-mannosyltransferase located in the Golgi apparatus. J Biol Chem, 1994 Mar 25, 269(12), 9205 - 12 The Saccharomyces cerevisiae MSI4 gene encodes the yeast counterpart of component A of Rab geranylgeranyltransferase; Fujimura K et al.; We describe the isolation of MSI4 as a multicopy suppressor of ira1 (inhibitory regulator of Ras) . We find it encodes a 66-kDa protein with three regions similar to component A of Rab geranylgeranyltransferase and Rab guanine nucleotide dissociation inhibitor . A gene disruption experiment showed that MSI4 is essential for cell growth . To investigate its functions further, we constructed a strain whose MSI4 is driven by the GAL1 promoter . This strain accumulated the endoplasmic reticulum precursor form of a vacuolar enzyme, carboxypeptidase Y, under the restrictive conditions . Moreover, we showed that the activity of geranylgeranyl-transferase for Ypt1p was very low in the mutant shifted to glucose medium . We propose that the MSI4 gene product is a yeast counterpart of component A of Rab geranylgeranyltransferase that is essential for Ypt1p to localize on membranes. J Biol Chem, 1994 Mar 25, 269(12), 8792 - 6 The protein phosphatase calcineurin is essential for NaCl tolerance of Saccharomyces cerevisiae; Mendoza I et al.; NaCl-sensitive yeast mutants were isolated to identify genes essential for NaCl tolerance . Complementation of a mutant highly sensitive to Na+ and Li+ led to the isolation of the CNB1 gene . This gene encodes the regulatory subunit (CNB) of the Ca2+/calmodulin-dependent protein phosphatase calcineurin . Cells deficient in CNB accumulated Li+ due to reduced expression of ENA1, a gene encoding a P-type ATPase involved in Na+ and Li+ efflux . In addition, the K+ transport system of cnb1 delta cells was not converted to the high affinity state that facilitates better discrimination of K+ over Na+ . Thus the cnb1 delta strain resembled a trk1 mutant . These results indicate that adaptation to NaCl stress in Saccharomyces cerevisiae requires a signal transduction pathway involving Ca2+ and protein phosphorylation-dephosphorylation . In this pathway, calcineurin would coordinate gene expression and activity of ion transporters to facilitate ion homeostasis. J Biol Chem, 1994 Mar 18, 269(11), 8341 - 7 Thymidylate synthase is localized to the nuclear periphery in the yeast Saccharomyces cerevisiae; Poon PP et al.; To date, the organization of DNA precursor synthesis within eukaryotic cells remains unresolved . Previous studies have suggested the existence of a multienzyme complex that is responsible for DNA precursor synthesis and is associated with sites of replication within the nucleus . Contrasting this, other studies have proposed that DNA precursor synthesis occurs outside the nucleus . To further these studies, we have addressed the location where thymidylate synthase resides in yeast . Subcellular fractionation experiments indicate thymidylate synthase is associated with purified nuclei . Consistent with this, immunofluorescence analysis suggests that thymidylate synthase is situated at the nuclear periphery. Eur J Biochem, 1994 Mar 15, 220(3), 997 - 1003 Analysis of genes encoding highly conserved lysine-rich proteins in Aplysia californica and Saccharomyces cerevisiae; Auclair D et al.; To isolate a gene that can be used as an internal control in studies on gene expression in Aplysia californica neurons, we have characterized a cDNA clone (pKRP-A) isolated on the basis of its high expression in A . californica neurons . This cDNA is of 850 nucleotides and codes for a putative 29-kDa lysine-rich protein . Blotting experiments revealed that the gene is expressed in all tested A . californica tissues, and in individually identified neurons of the abdominal ganglion, suggesting that this gene can be efficiently used as internal control in studies of gene expression . We have also isolated one cDNA and two different genomic clones from yeast libraries that show 59% identity with pKRP-A . Sequence comparison of genomic clones, as well as PCR and Southern blotting experiments, revealed that at least two homologous genes are present in yeast . Northern blotting experiments revealed that the expression of the gene is strongly repressed at 39 degrees C. Nucleic Acids Res, 1994 Mar 11, 22(5), 869 - 77 RNP1, a new ribonucleoprotein gene of the yeast Saccharomyces cerevisiae; Cusick ME; A previously unidentified ribonucleoprotein (RNP) gene of yeast has been cloned and sequenced . The gene, named RNP1, was found adjacent to a previously sequenced gene encoding the second gene for ribosomal protein L4 . RNP1 contains two RNA Recognition Motifs (RRM), {alternatively known as RNA binding Domains (RBD)}, but unlike most RNP genes does not contain any auxiliary simple sequence domains . The first RRM (RRM1) most resembles RRM domains found in the hnRNP A/B class of RNP proteins . The second RRM (RRM2) most resembles a RRM so far seen only in the single RRM of the yeast SSB1 gene . Two null mutants of RNP1 that were created, a frameshift disruption and a complete deletion of the gene, were viable, demonstrating that the gene is not essential for cell growth . Two double null mutants of yeast RNP genes that were created (delta RNP1/delta SSB1 and delta SSB1/delta NPL3) were also viable . A fragment identical in size to the RRM1 domain could be amplified by PCR from the DNA of fungi, plants, and animals, using primers matching the ends of this domain, indicating that the structure of RRM1 is conserved . Another potential open reading frame on the same cloned fragment of DNA encodes a gene product whose structure resembles that of a seven-transmembrane-segment membrane receptor protein. J Biol Chem, 1994 Mar 11, 269(10), 7402 - 11 Functional characterization of the INO2 gene of Saccharomyces cerevisiae . A positive regulator of phospholipid biosynthesis; Nikoloff DM et al.; The INO2 locus encodes a novel product showing structural similarity to the basic helix-loop-helix (b-HLH) family of regulatory proteins (Nikoloff, D.M., McGraw, P., and Henry, S.A . (1992) Nucleic Acids Res . 20, 3253) . The ino2 mutants exhibit pleiotropic defects in phospholipid metabolism including inability to derepress the biosynthetic enzyme inositol-1-phosphate synthase . Localization of mutations in ino2 strains has demonstrated that the b-HLH domain is required for biological activity and is sensitive to perturbation, thereby establishing a correlation between the structure and function of Ino2p . Defects in the b-HLH domain of Ino2p resulted in reduced DNA binding activity . In addition, the absence of a specific DNA-protein complex correlated with a reduction or loss of INO1 transcription . Studies using Ino2p-specific antibody revealed that Ino2p participates in the formation of specific DNA-protein complexes . Ino2p-dependent binding activity overlapped with a region of the INO1 promoter that contains two potential HLH consensus binding sites . Furthermore, Ino2p showed single base pair discrimination in a putative binding site, establishing a relationship between Ino2p and its target binding site. J Biol Chem, 1994 Mar 11, 269(10), 7279 - 84 A novel protein, CSG2p, is required for Ca2+ regulation in Saccharomyces cerevisiae; Beeler T et al.; Nineteen mutants that lost the ability to grow in 100 mM Ca2+ (but remained insensitive to 50 mM Sr2+) were identified in a screen of approximately 60,000 mutagenized yeast colonies . Cells carrying mutations in the CSG2 gene grow normally in low Ca2+ medium but have decreased growth rates when the Ca2+ concentration is above 10 mM . The csg2 mutant cells accumulate much higher levels of Ca2+ in a compartment that is exchangeable with extracellular Ca2+ but the nonexchangeable Ca2+ pool which predominates in wild-type cells is not influenced . Sr2+ influx is not increased in the csg2 mutant cells . Mg2+ decreases the amount of Ca2+ in the non-exchangeable pool without influencing the csg2-induced exchangeable Ca2+ pool . The data indicate that the csg2 mutation causes a selective increase in Ca2+ accumulation into a pool which is distinct from the vacuolar pool . The CSG2 protein consists of 410 amino acids, contains nine putative transmembrane segments, four potential sites for N-linked glycosylation, and a sequence with homology to the EF-hand Ca(2+)-binding site. Gene, 1994 Mar 11, 140(1), 121 - 6 Cloning and sequence determination of the gene encoding sorbitol dehydrogenase from Saccharomyces cerevisiae; Sarthy AV et al.; The identification of a sorbitol-induced sorbitol dehydrogenase (SDH) activity from Saccharomyces cerevisiae is described . The SDH1 structural gene was isolated from a lambda gt11 yeast genomic library using an antibody to a 40-kDa protein induced in yeast cells growing in medium containing sorbitol . The gene encodes a 357-amino-acid (aa) protein deduced from the nucleotide sequence . Comparison of the aa sequence of the yeast SDH1 with that of sheep liver SDH reveals a 63% overall similarity . Yeast transformants containing the cloned gene carried on a multicopy plasmid express high levels of SDH1 only when grown on sorbitol, suggesting that the cloned gene contains both regulatory and coding sequences. J Biol Chem, 1994 Mar 4, 269(9), 6543 - 9 Isolation and characterization of the Saccharomyces cerevisiae SDH4 gene encoding a membrane anchor subunit of succinate dehydrogenase; Bullis BL et al.; Succinate dehydrogenase (EC 1.3.99.1) is an intrinsic bacterial or inner mitochondrial membrane protein that catalyses the oxidation of succinate and donates electrons to the respiratory chain via quinone acceptors . It is a heterotetramer composed of a flavoprotein, an iron-sulfur, and two hydrophobic subunits . We purified succinate dehydrogenase by blue native gel electrophoresis, determined the amino-terminal sequence of the Sdh4p subunit and used this information to clone the SDH4 gene . It encodes a precursor protein of 181 amino acids that is converted to the 150-amino acid mature Sdh4p protein with a mass of 16,638 Da . Hydrophobicity analysis predicts that Sdh4p forms three transmembrane alpha-helices . We have constructed an SDH4 mutant by targeted gene disruption; it retains the ability to grow on rich glycerol medium . Western blot analysis of SDH4 disruption mutant membrane fractions indicates that membrane attachment of the flavoprotein and iron-sulfur subunits is impaired but not abolished . This membrane-bound enzyme is able to reduce ubiquinone, although less efficiently than the wild-type enzyme . These findings indicate that Sdh4p contributes both to the membrane attachment of the catalytic flavoprotein and iron-sulfur subunits and to electron transfer to ubiquinone. Biochim Biophys Acta, 1994 Mar 2, 1199(2), 137 - 42 Evidence for production of platelet-activating factor by yeast Saccharomyces cerevisiae cells; Nakayama R et al.; Various yeast strains were screened for platelet-activating factor (PAF) production . High PAF production was found mainly in the strains of Saccharomyces genus . Yeast PAF showed a typical platelet aggregation pattern, which was inhibited by specific PAF antagonists, such as CV-3988, CV-6209 and L-652731 . The main molecular species of yeast PAF were identified as 1-palmitoleoyl-, -palmitoyl-, -oleoyl- and -stearoyl-2-acetyl-sn-glycero-3-phosphocholines (16:1, 16:0, 18:1 and 18:0 acylPAFs) and 1-hexadecenyl- and hexadecyl-2-acetyl-sn-glycero-3-phosphocholines (16:1 and 16:0 PAFs), by mass spectrometry . PAF formation in yeast cells increased at the middle stationary phase of growth. Prikl Biokhim Mikrobiol, 1994 Mar-Apr, 30(2), 275 - 8 {The effect of culture media on the protein level in Saccharomyces cerevisiae yeasts}; Abramov ShA et al.; A culture medium containing molasses, geothermal water and only one mineral addition--ammonium hydroorthophosphate is proposed for obtaining the biomass with a high protein content (about 40%) . It is recommended to use the Coomassie reagent for quantification of the total protein in the yeast biomass. Mol Gen Genet, 1994 Mar, 242(6), 708 - 16 Characterization of the Saccharomyces cerevisiae nuclear gene CYB3 encoding a cytochrome b polypeptide of respiratory complex II; Abraham PR et al.; Computer-assisted structural analysis of the predicted product of the previously described open reading frame (ORF) YKL4 located on the left arm of chromosome XI of Saccharomyces cerevisiae revealed a high degree of similarity (> 50%) to bovine cytochrome b560, the sdhC polypeptide of the Escherichia coli succinate dehydrogenase (SDH) complex and the protein specified by ORF137 located on the chloroplast DNA of Marchantia polymorpha . Disruption of the yeast gene severely impaired mitochondrial function, while Northern analysis showed it to be subject to catabolite repression . Deletion analysis of the CYB3 promoter identified a single HAP2/3/4-binding element that is necessary and sufficient for carbon source-dependent transcriptional regulation . These experiments also suggested the presence of additional, as yet unidentified, transcriptional control elements, both negative and positive . Taken together, these data lead us to conclude that the CYB3 gene encodes the yeast homolog of the bovine cytochrome b560 component of complex II of the mitochondrial electron transport chain. Mol Gen Genet, 1994 Mar, 242(6), 641 - 8 The hypo-osmolarity-sensitive phenotype of the Saccharomyces cerevisiae hpo2 mutant is due to a mutation in PKC1, which regulates expression of beta-glucanase; Shimizu J et al.; To obtain more information about the cell wall organization of Saccharomyces cerevisiae, we have developed a novel screening system to obtain cell wall-defective mutants, using a density gradient centrifugation method . Nine hypo-osmolarity-sensitive mutants were classified into two complementation groups, hpo1 and hpo2 . Phase contrast microscopic observation showed that mutant cells bearing lesions at either locus became abnormally large . A gene that complemented the mutant phenotype of hpo2 was cloned and sequenced . This gene turned out to be identical to PKC1, which encodes the yeast homologue of mammalian protein kinase C . Complementation tests with pkc1 delta showed that hpo2 is allelic to pkc1 . To study the reason for the fragility of hpo2 cells, cell wall was isolated and the glucan was analyzed . The amount of alkali, acid-insoluble glucan, which is responsible for the rigidity of the cell wall, was reduced to about 30% that of the wild-type cell and this may be the major cause of the fragility of the hpo2 mutant cell . Analysis of total wall proteins in hpo2 mutant cells on SDS-polyacrylamide gels revealed that a 33 kDa protein was overproduced two- to threefold relative to the wild-type level . This 33 kDa protein was identified as a beta-glucanase, encoded by BGL2 . Disruption of BGL2 in the hpo2 mutant partially rescued the growth rate defect . This suggests that the PKC1 kinase cascade regulates BGL2 expression negatively and overproduction of the beta-glucanase is partially responsible for the growth defect.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Gen Genet, 1994 Mar, 242(6), 631 - 40 Genetic interactions among genes involved in the STT4-PKC1 pathway of Saccharomyces cerevisiae; Yoshida S et al.; Loss of yeast protein kinase C function results in three distinct phenotypes: staurosporine sensitivity, cell lysis and blockage of cell cycle progression at the G2/M boundary . Genetic analysis of the PKC1/STT1 protein kinase C gene and its interactions with STT4, encoding an upstream phosphatidylinositol 4-kinase, and BCK1, encoding a downstream protein kinase, reveal that they form part of a single pathway . However, the BCK1-20 mutation (a gain-of-function mutation of BCK1) or overexpression of PKC1 cannot suppress all of the phenotypes caused by the loss of STT4 function, strongly suggesting the existence of a branch point between STT4 and PKC1 . We also describe the MSS4 gene, a multicopy suppressor of the temperature-sensitive stt4-1 mutation . MSS4 is predicted to encode a hydrophilic protein of 779 amino acid residues and is essential for cell growth . Based on genetic and biochemical data, we suggest that MSS4 acts downstream of STT4, but in a pathway that does not involve PKC1. Arch Biochem Biophys, 1994 Mar, 309(2), 231 - 8 Resonance energy transfer determination of the distance between the four cysteine-364 residues in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase; Alvear M et al.; Each of the four subunits of the Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase has one cysteine residue (Cys-364) that is protected against alkylation by MnATP and that is thought to be located at (or close to) the active site (M . Alvear, M . V . Encinas, S . Latshaw, R . G . Kemp, and E . Cardemil, 1992, Biochim . Biophys . Acta 1119, 35-38) . To determine the distance relationships between these residues within this tetrameric enzyme, we have derivatized one of these reactive thiols with N-acetyl-N'-(5-sulfo-1-naphthyl) ethylenediamine (AEDANS) and the others progressively with 4-{N-{(acetoxy)ethyl}-N-methylamino}-7-nitrobenz-2-oxa-1,3-diazole (ANBD) . In the doubly labeled protein nonradiative singlet-singlet energy transfer between AEDANS (donor) and ANBD (acceptor) was observed . The efficiency of the energy transfer is proportional to the number of occupied acceptor sites . From these data it has been determined that one of the acceptor sites is 33 A from the donor, and the remaining two sites are 44-46 A from the donor . Cross-linking experiments revealed that mainly cross-linked dimers were produced upon reaction of the enzyme with o-phthalaldehyde and dithiobissuccinimidylpropionate . We interpret these results as an indication that this tetrameric enzyme is most likely composed of an association of two dimers. Proc Natl Acad Sci U S A, 1994 Mar 1, 91(5), 1843 - 7 Expression and partial purification of enzymatically active recombinant Ty1 integrase in Saccharomyces cerevisiae; Moore SP et al.; Integration of the Saccharomyces cerevisiae retrotransposon Ty1 into the genome requires Ty1 integrase (IN) . Apparent functions of Ty1 IN are target-site determination, cleavage, and joining of donor strands . To further study the mechanism of Ty1 integration, an IN expression plasmid has been constructed for use in yeast . The recombinant IN coding sequence differs from mature Ty1 IN associated with Ty1 virus-like particles only in that it has several additional N-terminal amino acid codons . Inclusion of a polyhistidine tag facilitates purification of recombinant IN by metal chelate chromatography . Recombinant Ty1 IN is active in an in vitro assay with short double-stranded oligonucleotide substrates and has biochemical properties similar to those observed with Ty1 virus-like particles . The full-length Ty1 IN produced in yeast should be useful for further biochemical, genetic, and structural analyses of Ty1 integration and for comparative analyses with retroviral IN proteins. Eur J Biochem, 1994 Mar 1, 220(2), 569 - 76 The C-terminus of the 14-kDa subunit of ubiquinol-cytochrome-c oxidoreductase of the yeast Saccharomyces cerevisiae is involved in the assembly of a functional enzyme; Hemrika W et al.; Disruption of QCR7, the gene encoding the 14-kDa subunit of ubiquinol-cytochrome-c oxido-reductase of the yeast Saccharomyces cerevisiae, results in an inactive enzyme which lacks holo-cytochrome b and has severely reduced levels of apo-cytochrome b, the Rieske Fe-S protein and the 11-kDa subunit {Schoppink, P . J., Berden, J . A . & Grivell, L . A . (1989) Eur . J . Biochem . 181, 475-483} . An episomal system was developed to study the effect on complex III of transformation of in vitro mutagenised QCR7 genes to a QCR7(0) mutant . Transformation of a gene (TNT1) in which the 12 C-terminal residues are replaced by 3 amino acids encoded by an oligonucleotide containing a stop codon in all three reading frames (STOP-oligonucleotide), only leads to partial complementation of the respiratory capacity of the yeast strain . The amounts of apo-cytochrome b, the Rieske Fe-S protein and the 11-kDa subunit are reduced and enzymic activity, together with the amount of holo-cytochrome b, is lowered to about 40% of that of the wild type, indicating a normal turnover number of the mutant enzyme . Transformation of the QCR7(0) mutant with another gene (TNT2) encoding the first 96 residues of the 14-kDa subunit fused to 9 amino acids encoded by the STOP-oligonucleotide, leads to a phenotype almost indistinguishable from that of the QCR7(0) mutant . The role of the charged C-terminus of the 14-kDa (and the 11-kDa) subunit in the assembly of a functional complex III is discussed. Mol Gen Genet, 1994 Mar, 242(5), 517 - 27 Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae; Kimura Y et al.; The budding yeast Saccharomyces cerevisiae has two HSP90-related genes per haploid genome, HSP82 and HSC82 . Random mutations were induced in vitro in the HSP82 gene by treatment of the plasmid with hydroxylamine . Four temperature-sensitive (ts) mutants and one simultaneously ts and cold-sensitive (cs) mutant were then selected in a yeast strain in which HSC82 had previously been disrupted . The mutants were found to have single base changes in the coding region, which caused single amino acid substitutions in the HSP82 protein . All of these mutations occurred in amino acid residues that are well conserved among HSP90-related proteins of various species from Escherichia coli to human . Various properties including cell morphology, macromolecular syntheses and thermosensitivity were examined in each mutant at both the permissive and nonpermissive temperatures . The mutations in HSP82 caused pleiotropic effects on these properties although the phenotypes exhibited at the nonpermissive temperature varied among the mutants. Mol Cell Biol, 1994 Mar, 14(3), 2041 - 7 Genes that can bypass the CLN requirement for Saccharomyces cerevisiae cell cycle START; Epstein CB et al.; Cell cycle START in Saccharomyces cerevisiae requires at least one of the three CLN genes (CLN1, CLN2, or CLN3) . A total of 12 mutations bypassing this requirement were found to be dominant mutations in a single gene that we named BYC1 (for bypass of CLN requirement) . We also isolated a plasmid that had cln bypass activity at a low copy number; the gene responsible was distinct from BYC1 and was identical to the recently described BCK2 gene . Strains carrying bck2::ARG4 disruption alleles were fully viable, but bck2::ARG4 completely suppressed the cln bypass activity of BYC1 . swi4 and swi6 deletion alleles also efficiently suppressed BYC1 cln bypass activity; Swi4 and Swi6 are components of a transcription factor previously implicated in control of CLN1 and CLN2 expression . bck2::ARG4 was synthetically lethal with cln3 deletion, suggesting that CLN1 and CLN2 cannot function in the simultaneous absence of BCK2 and CLN3; this observation correlates with low expression of CLN1 and CLN2 in bck2 strains deprived of CLN3 function . Thus, factors implicated in CLN1 and CLN2 expression and/or function are also required for BYC1 function in the absence of all three CLN genes; this may suggest the involvement of other targets of Swi4, Swi6, and Bck2 in START. Mol Cell Biol, 1994 Mar, 14(3), 1972 - 8 Dosage-dependent modulation of glucose repression by MSN3 (STD1) in Saccharomyces cerevisiae; Hubbard EJ et al.; The SNF1 protein kinase of Saccharomyces cerevisiae is required to relieve glucose repression of transcription . To identify components of the SNF1 pathway, we isolated multicopy suppressors of defects caused by loss of SNF4, an activator of the SNF1 kinase . Increased dosage of the MSN3 gene restored invertase expression in snf4 mutants and also relieved glucose repression in the wild type . Deletion of MSN3 caused no substantial phenotype, and we identified a homolog, MTH1, encoding a protein 61% identical to MSN3 . Both are also homologous to chicken fimbrin, human plastin, and yeast SAC6 over a 43-residue region . Deletion of MSN3 and MTH1 together impaired derepression of invertase in response to glucose limitation . Finally, MSN3 physically interacts with the SNF1 protein kinase, as assayed by a two-hybrid system and by in vitro binding studies . MSN3 is the same gene as STD1,