Mol Gen Genet, 1996 Feb 25, 250(3), 316 - 22 The CBP2 gene from Saccharomyces douglasii is a functional homologue of the Saccharomyces cerevisiae gene and is essential for respiratory growth in the presence of a wild-type (intron-containing) mitochondrial genome; Li GY et al.; In Saccharomyces cerevisiae the only known role of the CBP2 gene is the excision of the fifth intron of the mitochondrial cyt b gene (bI5) . We have cloned the CBP2 gene from Saccharomyces douglasii (a close relative of S . cerevisiae) . A comparison of the S . douglasii and S . cerevisiae sequences shows that there are 14% nucleotide substitutions in the coding region, with transitions being three times more frequent than transversions . At the protein level sequence identity is 87% . We have demonstrated that the S . douglasii CBP2 gene is essential for respiratory growth in the presence of a wild-type S . douglasii mitochondrial genome, but not in the presence of an intronless S . cerevisiae mitochondrial genome . Also the S . douglasii and S . cerevisiae CBP2 genes are completely interchangeable, even though the intron bI5 is absent from the S . douglasii mitochondrial genome. Cell, 1996 Feb 23, 84(4), 633 - 42 Loss of transcriptional silencing causes sterility in old mother cells of S . cerevisiae; Smeal T et al.; We show that sterility is an aging-specific phenotype in S . cerevisiae and, by genetic and physical means, demonstrate that this phenotype results from a loss of silencing in most old cells by the SIR complex at the HM loci . This loss of silencing is specific because transcription of genes, such as ME14 and DCM1, normally induced by sporulation, is not observed, while transcription of HMRa is observed . These findings pinpoint the molecular cause of an aging-specific phenotype in yeast . Further, they provide direct evidence for a breakdown of silencing in old cells, as predicted from earlier findings that SIR4 is a determinant of life span in this organism. Nucleic Acids Res, 1996 Feb 15, 24(4), 721 - 9 Recognition of DNA insertion/deletion mismatches by an activity in Saccharomyces cerevisiae; Miret JJ et al.; An activity in nuclear extracts of S.cerevisiae binds specifically to heteroduplexes containing four to nine extra bases in one strand . The specificity of this activity (IMR, for insertion mismatch recognition) in band shift assays was confirmed by competition experiments . IMR is biochemically and genetically distinct from the MSH2 dependent, single base mismatch binding activity . The two activities migrate differently during electrophoresis, they are differentially competable and their spectra of mispair binding are distinct . Furthermore, IMR activity is observed in extracts from an msh2- msh3- msh4- strain . IMR exhibits specificity for insertion mispairs in two different sequence contexts . Binding is influenced by the structure of the mismatch since an insertion with a hairpin configuration is not recognized by this activity . IMR does not result from single-strand binding because single-stranded probes to not yield IMR complex and single-stranded competitors are unable to displace insertion heteroduplexes from the complex . Similar results with intrinsically bent duplexes make it unlikely that recognition is conferred by a bend alone . Heteroduplexes bound by IMR do not contain any obvious damage . These findings are consistent with the idea that yeast contains a distinct recognition factor, IMR that is specific for insertion/deletion mismatches. Genes Dev, 1996 Feb 15, 10(4), 407 - 20 Redundancy of Saccharomyces cerevisiae MSH3 and MSH6 in MSH2-dependent mismatch repair; Marsischky GT et al.; Saccharomyces cerevisiae encodes six genes, MSH1-6, which encode proteins related to the bacterial MutS protein . In this study the role of MSH2, MSH3, and MSH6 in mismatch repair has been examined by measuring the rate of accumulating mutations and mutation spectrum in strains containing different combinations of msh2, msh3, and msh6 mutations and by studying the physical interaction between the MSH2 protein and the MSH3 and MSH6 proteins . The results indicate that S . cerevisiae has two pathways of MSH2-dependent mismatch repair: one that recognized single-base mispairs and requires MSH2 and MSH6, and a second that recognizes insertion/deletion mispairs and requires a combination of either MSH2 and MSH6 or MSH2 and MSH3 . The redundancy of MSH3 and MSH6 explains the greater prevalence of hmsh2 mutations in HNPCC families and suggests how the role of hmsh3 and hmsh6 mutations in cancer susceptibility could be analyzed. J Biol Chem, 1996 Feb 9, 271(6), 2914 - 20 ROD1, a novel gene conferring multiple resistance phenotypes in Saccharomyces cerevisiae; Wu AL et al.; Glutathione-dependent detoxification reactions are catalyzed by the enzyme glutathione S-transferase and are important in drug resistance in organisms ranging from bacteria to humans . The yeast Issatchenkia orientalis expresses a glutathione S-transferase (GST) protein that is induced when the GST substrate o-dinitrobenzene (o-DNB) is added to the culture . In this study, we show that overproduction of the I . orientalis GST in Saccharomyces cerevisiae leads to an increase in o-dinitrobenzene resistance in S . cerevisiae cells . To recover genes that influence o-DNB resistance in S . cerevisiae, a high copy plasmid library was screened for loci that elevate o-DNB tolerance . One gene was recovered and designated ROD1 (resistance to o-dinitrobenzene) . This locus was found to encode a novel protein with no significant sequence similarity with proteins of known function in the data base . An epitope-tagged version of Rod1p was produced in S . cerevisiae and shown to function properly . Subcellular fractionation experiments indicated that this factor was found in the particulate fraction by differential centrifugation . Overproduction of Rod1p leads to resistance to not only o-DNB but also zinc and calcium . Strains that lack the ROD1 gene are hypersensitive to these same compounds . Rod1p represents a new type of molecule influencing drug tolerance in eukaryotes. J Biol Chem, 1996 Feb 2, 271(5), 2717 - 23 Multimerization of Hsp42p, a novel heat shock protein of Saccharomyces cerevisiae, is dependent on a conserved carboxyl-terminal sequence; Wotton D et al.; Rap1p is a transcriptional regulator of Saccharomyces cerevisiae, which plays roles in both transcriptional activation and silencing . To identify proteins involved in Rap1p-dependent regulation of transcription, we used the two-hybrid system to screen for Rap1p-interacting proteins . Two of the clones isolated from this screen encode a truncated protein with homology to small heat shock proteins (HSPs) . Here we present an analysis of this novel S . cerevisiae HSP, which we name Hsp42p . Expression of HSP42 is regulated by a range of stress conditions similar to S . cerevisiae HSP26, with which Hsp42p shares most homology . However, HSP42 expression is more sensitive to increased salt concentration and to starvation and, in contrast to HSP26 is expressed in unstressed cells . Hsp42p interacts with itself in the two-hybrid assay . This interaction is dependent on a hydrophobic region which is conserved among small HSPs . Using bacterially expressed Hsp42p fusion proteins . we demonstrate that this is a direct interaction . Fractionation of yeast protein extracts by size demonstrates that all of the Hsp42p in these extracts is present in complexes with a molecular mass of greater than 200 kDa, suggesting that Hsp42p exists in high molecular mass complexes. Fungal Genet Biol, 1996 Feb, 21(1), 40 - 9 Cross-pathway and pathway-specific control of amino acid biosynthesis in Magnaporthe grisea; Shen WC et al.; The gene encoding the small subunit of the arginine-specific carbamoyl phosphate synthetase, ARG2, of Magnaporthe grisea was characterized to examine the basic regulation of biosynthetic genes in this plant pathogen . The transcript of the ARG2 gene contains an upstream open reading frame (uORF) that is similar to uORFs found in the homologous genes of Neurospora crassa (arg-2) and Saccharomyces cerevisiae (CPA1), suggesting that the M . grisea gene is translationally regulated by a mechanism that is conserved in these fungi . Amino acid imbalance leads to elevated levels of ARG2 mRNA, indicating that in addition to translational control, ARG2 is subject to cross-pathway transcriptional control . A DNA-binding activity that has properties similar to those of the global transcriptional regulator mediating cross-pathway control in N . crassa was detected in M . grisea cell extracts . Thus, it appears that both specific regulation of ARG2 by arginine and global regulation of amino acid biosynthesis are present in M . grisea and highly conserved among M . grisea, N . crassa, and S . cerevisiae. Mol Biol Cell, 1996 Feb, 7(2), 307 - 17 Biochemical and functional analysis of the YME1 gene product, an ATP and zinc-dependent mitochondrial protease from S . cerevisiae; Weber ER et al.; Inactivation of YME1 in yeast causes several distinct phenotypes: an increased rate of DNA escape from mitochondria, temperature-sensitive growth on nonfermentable carbon sources, extremely slow growth when mitochondrial DNA is completely absent from the cell, and altered morphology of the mitochondrial compartment . The protein encoded by YME1, Yme1p, contains two highly conserved sequence elements, one implicated in the binding and hydrolysis of ATP, and the second characteristic of active site residues found in neutral, zinc-dependent proteases . Both the putative ATPase and zinc-dependent protease elements are necessary for the function of Yme1p as genes having mutations in critical residues of either of these motifs are unable to suppress any of the phenotypes exhibited by yme1 deletion strains . Yme1p co-fractionates with proteins associated with the mitochondrial inner membrane, is tightly associated with this membrane, and is oriented with the bulk of the protein facing the matrix . Unassembled subunit II of cytochrome oxidase is stabilized in yme1 yeast strains . The data support a model in which Yme1p is an ATP and zinc-dependent protease associated with the matrix side of the inner mitochondrial membrane . Subunit II of cytochrome oxidase, when not assembled into a higher order complex, is a likely substrate of Yme1p. Yeast, 1996 Feb, 12(2), 129 - 34 pMPY-ZAP: a reusable polymerase chain reaction-directed gene disruption cassette for Saccharomyces cerevisiae; Schneider BL et al.; Gene disruption is an important method for genetic analysis in Saccharomyces cerevisiae . We have designed a polymerase chain reaction-directed gene disruption cassette that allows rapid disruption of genes in S . cerevisiae without previously cloning them . In addition, this cassette allows recycling of URA3, generating gene disruptions without the permanent loss of the ura3 marker . An indefinite number of disruptions can therefore be made in the same strain. J Cell Biol, 1996 Feb, 132(3), 399 - 411 Identification of a developmentally regulated septin and involvement of the septins in spore formation in Saccharomyces cerevisiae; Fares H et al.; The Saccharomyces cerevisiae CDC3, CDC10, CDC11, and CDC12 genes encode a family of related proteins, the septins, which are involved in cell division and the organization of the cell surface during vegetative growth . A search for additional S . cerevisiae septin genes using the polymerase chain reaction identified SPR3, a gene that had been identified previously on the basis of its sporulation-specific expression . The predicted SPR3 product shows 25-40% identity in amino acid sequence to the previously known septins from S . cerevisiae and other organisms . Immunoblots confirmed the sporulation-specific expression of Spr3p and showed that other septins are also present at substantial levels in sporulating cells . Consistent with the expression data, deletion of SPR3 in either of two genetic backgrounds had no detectable effect on exponentially growing cells . In one genetic background, deletion of SPR3 produced a threefold reduction in sporulation efficiency, although meiosis appeared to be completed normally . In this background, deletion of CDC10 had no detectable effect on sporulation . In the other genetic background tested, the consequences of the two deletions were reversed . Immunofluorescence observations suggest that Spr3p, Cdc3p, and Cdc11p are localized to the leading edges of the membrane sacs that form near the spindle-pole bodies and gradually extend to engulf the nuclear lobes that contain the haploid chromosome sets, thus forming the spores . Deletion of SPR3 does not prevent the localization of Cdc3p and Cdc11p, but these proteins appear to be less well organized, and the intensity of their staining is reduced . Taken together, the results suggest that the septins play important but partially redundant roles during the process of spore formation. Biochem J, 1996 Feb 1, 313 ( Pt 3), 729 - 35 Phospholipid and cation activation of chimaeric choline/ethanolamine phosphotransferases; McMaster CR et al.; The Saccharomyces cerevisiae CPT1 and EPT1 genes encode for a cholinephosphotransferase (CPT) and choline/ethanolaminephosphotransferase, respectively . Both Cpt1p and Ept1p activities display an absolute requirement for cations and phospholipids . A mixed-micelle assay was employed to determine cation and lipid activators of parental and chimaeric Cpt1p/Ept1p enzymes to gain insight into their mechanism(s) of activation . Mg2+, Mn2+ and Co2+ were the only cations capable of activating Cpt1p and Ept1p in vitro . Kinetic data revealed that only Mg2+ is present in appropriate amounts to activate CPT activity in vivo . Kinetic data revealed that only Mg2+ is present in appropriate amounts to activate CPT activity in vivo . The two enzymes displayed distinct activation profiles on the basis of their relative affinities for Mg2+, and Mn2+ and Co2+ . This allowed the use of chimaeric enzymes to determine the mechanism of cation activation . Cations do not activate Cpt1p or Ept1p by complexing with the substrate, CDP-choline, but instead bind to disparate regions within the enzymes themselves . Cpt1p and Ept1p also displayed distinct phospholipid activation profiles . Phospholipid activation required a phosphate and/or glycero-phosphoester linkage, with the phospho-head group moiety positioned at the surface of the micelle . Assays with parental and chimaeric Cpt1p/Ept1p constructs revealed that the phospholipid binding/activation domains are not located within linear segments of the protein, but instead are contained within distinct and separate regions of the proteins that require an intact tertiary structure for formation . Phosphatidylcholine (and its structural analogue sphingomyelin) were the best lipid activators of Cpt1p, the main biologically relevant CPT activity in S . cerevisiae . Hence CPT displays product activation . Because phosphatidylcholine is an efficient activator of CPT activity (and hence Cpt1p is not subject to feedback inhibition by its product), Cpt1p is incapable of functioning as a direct monitor of membrane phosphatidylcholine composition. RNA, 1996 Feb, 2(2), 183 - 97 The yeast Hansenula wingei U3 snoRNA gene contains an intron and its coding sequence co-evolved with the 5' ETS region of the pre-ribosomal RNA; Brule F et al.; The 5' external transcribed spacer (ETS) region of the pre-rRNA in Saccharomyces cerevisiae contains a sequence with 10 bp of perfect complementarity to the U3 snoRNA . Base pairing between these sequences has been shown to be required for 18S rRNA synthesis, although interaction over the full 10 bp of complementarity is not required . We have identified the homologous sequence in the 5' ETS from the evolutionarily distant yeast Hansenula wingei; unexpectedly, this shows two sequence changes in the region predicted to base pair to U3 . By PCR amplification and direct RNA sequencing, a single type of U3 snoRNA coding sequence was identified in H . wingei . As in the S . cerevisiae U3 snoRNA genes, it is interrupted by an intron with features characteristic of introns spliced in a spliceosome . Consequently, this unusual property is not restricted to the yeast genus Saccharomyces . The introns of the H . wingei and S . cerevisiae U3 genes show strong differences in length and sequence, but are located at the same position in the U3 sequence, immediately upstream of the phylogenetically conserved Box A region . The 3' domains of the H . wingei and S . cerevisiae U3 snoRNAs diverge strongly in primary sequence, but have very similar predicted secondary structures . The 5' domains, expected to play a direct role in pre-ribosomal RNA maturation, are more conserved . The sequence predicted to base pair to the pre-rRNA contains two nucleotide substitutions in H . wingei that restore 10 bp of perfect complementarity to the 5' ETS . This is a strong phylogenetic evidence for the importance of the U3/pre-rRNA interaction. Curr Genet, 1996 Feb, 29(3), 219 - 26 Construction of the complete rat fatty acid synthase cDNA and its expression in Saccharomyces cerevisiae; Kupfer R et al.; The 272 647-dalton polypeptide of fatty acid synthase (FAS) from Rattus norvegicus has been expressed in a proteinase-deficient strain of Saccharomyces cerevisiae . The seven overlapping cDNA clones for rat FAS spanning the entire coding region were the starting material for this undertaking . In a series of cloning steps an expression plasmid was constructed in which the cDNA was placed under the control of the yeast ADH1 promoter . Northern blotting of total RNA isolated from yeast transformed with this expression plasmid demonstrated a high rate of transcription of the 7.4-kb cDNA . However, a successful translation required further manipulation of the sequence immediately upstream of the rat FAS translational start codon . This was obtained when the 86 bp of the rat FAS cDNA immediately 5' to the start codon were replaced by a nonamer corresponding to the immediate 5'-vicinity of the translational start codon of the yeast ADH1 gene . Nevertheless, the translation product could be detected only by Western blotting . The FAS proteins of S . cerevisiae and rat are not functionally interchangeable . Using the purification protocol of rat FAS the heterologously expressed FAS could be enriched by at least one order of magnitude. Mol Cell Biol, 1996 Feb, 16(2), 669 - 76 Paf1p, an RNA polymerase II-associated factor in Saccharomyces cerevisiae, may have both positive and negative roles in transcription; Shi X et al.; Regulated transcription initiation requires, in addition to RNA polymerase II and the general transcription factors, accessory factors termed mediators or adapters . We have used affinity chromatography to identify a collection of factors that associate with Saccharomyces cerevisiae RNA polymerase II (P . A . Wade, W . Werel, R . C . Fentzke, N . E . Thompson, J . F . Leykam, R . R . Burgess, J . A . Jaehning, and Z . F . Burton, submitted for publication) . Here we report identification and characterization of a gene encoding one of these factors, PAF1 (for RNA polymerase-associated factor 1) . PAF1 encodes a novel, highly charged protein of 445 amino acids . Disruption of PAF1 in S . cerevisiae leads to pleiotropic phenotypic traits, including slow growth, temperature sensitivity, and abnormal cell morphology . Consistent with a possible role in transcription, Paf1p is localized to the nucleus . By comparing the abundances of many yeast transcripts in isogenic wild-type and paf1 mutant strains, we have identified genes whose expression is affected by PAF1 . In particular, disruption of PAF1 decreases the induction of the galactose-regulated genes three- to fivefold . In contrast, the transcript level of MAK16, an essential gene involved in cell cycle regulation, is greatly increased in the paf1 mutant strain . Paf1p may therefore be required for both positive and negative regulation of subsets of yeast genes . Like Paf1p, the GAL11 gene product is found associated with RNA polymerase II and is required for regulated expression of many yeast genes including those controlled by galactose . We have found that a gal11 paf1 double mutant has a much more severe growth defect than either of the single mutants, indicating that these two proteins may function in parallel pathways to communicate signals from regulatory factors to RNA polymerase II. J Bacteriol, 1996 Feb, 178(3), 656 - 61 Dominant negative rat DNA polymerase beta mutants interfere with base excision repair in Saccharomyces cerevisiae; Clairmont CA et al.; DNA polymerase beta is one of the smallest known eukaryotic DNA polymerases . This polymerase has been very well characterized in vitro, but its functional role in vivo has yet to be determined . Using a novel competition assay in Escherichia coli, we isolated two DNA polymerase beta dominant negative mutants . When we overexpressed the dominant negative mutant proteins in Saccharomyces cerevisiae, the cells became sensitive to methyl methanesulfonate . Interestingly, overexpression of the same polymerase beta mutant proteins did not confer sensitivity to UV damage, strongly suggesting that the mutant proteins interfere with the process of base excision repair but not nucleotide excision repair in S . cerevisiae . Our data implicate a role for polymerase IV, the S . cerevisiae polymerase beta homolog, in base excision repair in S . cerevisiae. J Biol Chem, 1996 Jan 26, 271(4), 1868 - 76 Purification and characterization of diacylglycerol pyrophosphate phosphatase from Saccharomyces cerevisiae; Wu WI et al.; Diacylglycerol pyrophosphate (DGPP) phosphatase is a novel membrane-associated enzyme that catalyzes the dephosphorylation of the beta phosphate of DGPP to yield phosphatidate and Pi . DGPP phosphatase was purified 33,333-fold from Saccharomyces cerevisiae by a procedure that included Triton X-100 solubilization of microsomal membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, and Mono Q . The procedure resulted in the isolation of an apparent homogeneous protein with a subunit molecular mass of 34 kDa . DGPP phosphatase activity was associated with the 34-kDa protein . DGPP phosphatase had a broad pH optimum between 6.0 and 8.5 and was dependent on Triton X-100 for maximum activity . The enzyme was inhibited by divalent cations, NaF, and pyrophosphate and was relatively insensitive to thioreactive agents . The turnover number (molecular activity) for the enzyme was 5.8 x 10(3) min-1 at pH 6.5 and 30 degrees C . DGPP phosphatase exhibited typical saturation kinetics with respect to DGPP (Km = 0.55 mol %) . The Km value for DGPP was 3-fold greater than its cellular concentration (0.18 mol %) . DGPP phosphatase also catalyzed the dephosphorylation of phosphatidate, but this dephosphorylation was subsequent to the dephosphorylation of the beta phosphate of DGPP . The dependence of activity on phosphatidate (Km = 2.2 mol %) was cooperative (Hill number = 2.0) . DGPP was the preferred substrate for the enzyme with a specificity constant (Vmax/Km) 10-fold greater than that for phosphatidate . In addition, DGPP potently inhibited (Ki = 0.35 mol %) the dephosphorylation of phosphatidate by a competitive mechanism whereas phosphatidate did not inhibit the dephosphorylation of DGPP . DGPP was neither a substrate nor an inhibitor of pure phosphatidate phosphatase from S . cerevisiae . DGPP was synthesized from phosphatidate via the phosphatidate kinase reaction. Mol Gen Genet, 1996 Jan 15, 250(1), 69 - 80 AgTHR4, a new selection marker for transformation of the filamentous fungus Ashbya gossypii, maps in a four-gene cluster that is conserved between A . gossypii and Saccharomyces cerevisiae; Altmann-Johl R et al.; Single-read sequence analysis of the termini of eight randomly picked clones of Ashbya gossypii genomic DNA revealed seven sequences with homology to Saccharomyces cerevisiae genes (15% to 69% on the amino acid level) . One of these sequences appeared to code for the carboxy-terminus of threonine synthase, the product of the S . cerevisiae THR4 gene (52.4% identity over 82 amino acids) . We cloned and sequenced the complete putative AgTHR4 gene of A . gossypii . It comprises 512 codons, two less than the S . cerevisiae THR4 gene . Overall identity at the amino acid sequence level is 67.4% . A continuous stretch of 32 amino acids displaying complete identity between these two fungal threonine synthases presumably contains the pyridoxal phosphate attachment site . Disruption of the A . gossypii gene led to threonine auxotrophy, which could be complemented by transformation with replicating plasmids carrying the AgTHR4 gene and various S . cerevisiae ARS elements . Using these plasmids only very weak complementation of a S . cerevisiae thr4 mutation was observed . Investigation of sequences adjacent to the AgTHR4 gene identified three additional ORFs . Surprisingly, the order and orientation of these four ORFs is conserved in A . gossypii and S . cerevisiae. FEBS Lett, 1996 Jan 15, 378(3), 207 - 12 Stress signal, mediated by a Hog1-like MAP kinase, controls sexual development in fission yeast; Kato T Jr et al.; We identified the phh1+ gene that encodes a MAP kinase as the effector of Wis1 MAP kinase kinase in fission yeast, which is highly homologous with HOG1 of S . cerevisiae . Heterothalic phh1 dsiruptant is phenotypically indistinguishable from wis1 deletion mutant, both displaying the same extent of partial sterility and enhanced sensitivity to a variety of stress . In phh1 disruptant, nitrogen starvation-induced expression of ste11+, a key controller of sexual differentiation, is markedly diminished . Ectopic expression of ste11+ effectively restores fertility, but not stress resistance, to the phh1 disruptant . These data show that stress signal, mediated by a MAP kinase, is required for efficient start of sexual differentiation. Exp Cell Res, 1996 Jan 10, 222(1), 157 - 62 Metabolic fluxes regulate the success of sporulation in Saccharomyces cerevisiae; Aon JC et al.; In this work we investigated to what extent cellular metabolism and energetics regulate sporulation in Saccharomyces cerevisiae and which metabolic pathways are involved in such regulation . Sporulation, meiosis, and associated metabolic fluxes in S . cerevisiae strain CH1211 were studied in several experimental protocols involving changes of carbon source (acetate, lactate, or pyruvate) or cell density in sporulation medium, or changing the phase of batch growth at which cells were harvested before transfer to sporulation medium . In acetate-based sporulation medium, the rate at which cells utilized glyoxylate and gluconeogenic pathways correlated positively with the percentage of asci per cell at 72 h . In contrast, in lactate sporulation medium the frequency of sporulation correlated negatively with both the rate of lactate consumption and the fluxes through gluconeogenesis and the pyruvate-carboxylase catalyzed step . In the presence of lactate, the respiratory capacity did correlate positively with the percentage of asci per cell . The experimental data suggest that acetate limits fluxes to anabolic precursors during sporulation . In contrast, sporulation on lactate appears to be influenced by catabolic processes or, even more precisely, by the respiratory capacity of yeast cells . The results obtained are discussed in terms of the hypothesis that an imbalance between anabolic and catabolic fluxes may be required for an efficient sporulation. Biochem Biophys Res Commun, 1996 Jan 5, 218(1), 234 - 7 The pleiotropic effect of the GTS1 gene product on heat tolerance, sporulation and the life span of Saccharomyces cerevisiae; Yaguchi S et al.; We investigated whether or not the potential clock gene, GTS1, of the yeast Saccharomyces cerevisiae, shows pleiotropic effects on the yeast cellular processes . We tested the efect of the Gts1 protein on heat tolerance, sporulation and life-span, by characterizing the phenotypes of transformants with different copy numbers of the gene . We found that the Gts1 protein affects the capacity of heat tolerance in the stationary phase and the speed leading to sporulation in a gene-dose dependent manner, and that both inactivation and overexpression of the gene shortened the life-span of yeast . These results supported the notion that GTS1 affects the biological clock of the yeast S . cerevisiae, although this cannot be definitively concluded because the strain cannot be synchronized with circadian or ultradian rhythms. FEBS Lett, 1996 Jan 2, 378(1), 48 - 50 A novel yeast protein showing specific association with the cyclin-dependent kinase 5; Huang QQ et al.; The present study describes a significant amino acid sequence homology between neuronal Cdk5 activator (nck5a) and an open reading frame of an unknown gene on the yeast S . cerevisiae chromosome III . A cDNA encoding a 25 kDa fragment of this yeast protein, the region containing homologous sequence to nck5a was cloned and expressed in E . coli as a glutathine-S-transferase fusion protein (GST-p25Y) . GST-p25Y was found to block the in vitro activation of Cdk5 by nck5a and to affinity precipitate Cdk5 from bovine brain extract . The observations indicate that the yeast protein is capable of specific and high affinity association with Cdk5. Folia Microbiol (Praha), 1996, 41(4), 347 - 52 Characterization of yeasts isolated from red wine surface film; Vollekova A et al.; We have isolated 6 morphologically different axenic yeast cultures from the film surface of red wine . Based on morphological, physiological and biochemical characteristics we have identified the strains as follows: isolates 1-4 are morphologically different strains of the anamorph basidiomycetous film-forming yeast Candida humicola (DASZEWSKA) DIDDENS et LODDER, syn . Apiotrichum humicola (DASZEWSKA) VON ARX WEISMAN . Isolates 5 and 6 belong to the genus Saccharomyces of the associated species S . cerevisiae (isolate 5 originally S . bayanus, isolate 6 S . capensis) . These do not participate in the surface film formation. Cytobios, 1996, 86(345), 123 - 35 The possible role of yeast cell walls in modifying cellular response to chlorhexidine diacetate; Hiom SJ et al.; The relative porosity (RP) and thickness of cell walls of Saccharomyces cerevisiae depended upon the age of culture, the RP decreasing and the cell wall thickness increasing in older cultures . Chlorhexidine diacetate (CHA) produced cytological changes in S . cerevisiae cells, involving dense and granular cytoplasmic constituents, withdrawal of the interior from the cell wall and a general loss of the typical cellular organization . Cell wall mannan is unlikely to play a role in limiting the entry of CHA into yeast cells but changes in glucan composition, wall thickness and RP might be involved in determining the sensitivity of cells to the biguanide. Neoplasma, 1996, 43(5), 315 - 9 Biological consequences of E.coli RecA protein expression in the repair defective pso4-1 and rad51::URA3 mutants of S . cerevisiae after treatment with N-methyl-N'-nitro-N-nitrosoguanidine; Slaninova M et al.; RecA protein of E.coli is a multifunctional protein participating in genetic recombination, recombinational repair and mutagenesis . We used E.coli recA gene as a probe for complementation of repair defects after treatment of N-methyl-N'-nitro-N-nitrosoguanidine in the pso4-1 and rad51::URA3 mutants of S . cerevisiae . We tried to find the role of the RecA protein in S . cerevisiae mutants defective in different repair pathways . The RecA protein had no effect on survival of haploid and diploid pso4-1 mutants, but it had a significant effect on MNNG induced mutagenesis, which was increased to the wild type level . No effect of the RecA protein on survival was observed in rad51::URA3 mutant after MNNG treatment . However, in this case the RecA protein decreased the induced mutagenesis . We can suppose that the RecA protein, with its multifunctional enzymatic activity, can bind to special intermediates and initiate function of different repair pathways depending on repair defects of the mutants studied. Annu Rev Cell Dev Biol, 1996, 12, 129 - 60 ACTIN: general principles from studies in yeast; Ayscough KR et al.; Three of the most important questions concerning actin function are: (a) How does actin structure relate to actin function? (b) How does each of the numerous proteins that interact with actin contribute to actin cytoskeleton function in vivo? (c) How are the activities of these proteins regulated? Powerful molecular genetics combined with well-established biochemical techniques make the yeast Saccharomyces cerevisiae an ideal organism for studies aimed at answering these questions . The protein sequences and biochemical properties of actin and its interacting proteins and the pathways that regulate these interactions all appear to be conserved, indicating that principles elucidated from studies in yeast will apply to all eukaryotes . In this review, we highlight advances in our general understanding of actin properties, interactions with other proteins, and regulation of the actin cytoskeleton, derived from studies in the budding yeast S . cerevisiae. Biochimie, 1996, 78(5), 311 - 4 Searching for a family of orphan sequences with SAMBA, a parallel hardware dedicated to biological applications; Guerdoux-Jamet P et al.; A significant proportion of coding sequences or open reading frames discovered in the course of sequencing projects do not show any similarity with other sequences deposited with the protein databanks . In such cases the search for similarities must be performed with as many comparison algorithms as possible, so as to increase the chance of finding weak relationships . A specialised parallel hardware (SAMBA) implementing the Smith & Waterman algorithm has been developed at the 'Institut de Recherche en Informatique et Systemes Aleatoires' (IRISA) . It makes it possible to scan protein databanks at a speed comparable with that of BLAST or FASTA . We report here a study performed with SAMBA on 814 orphan sequences from S cerevisiae and compare the results with those from BLAST and FASTA. Adv Enzyme Regul, 1996, 36, 115 - 40 The phosphatidylinositol 4-phosphate 5-kinase family; Loijens JC et al.; The existence of a PIP5K family of enzymes has been suggested by Western blotting and purification of numerous PIP5Ks from various tissues and cell types . The erythrocyte has at least two PIP5Ks, named PIP5KI and PIP5KII, while the brain appears to have even more isoforms . The cloning of the first PIP5K, the PIP5KII alpha, is just the beginning of the molecular classification of this protein family . The PIP5KII alpha sequence has shown that these enzymes lack obvious homology to protein, sugar and other lipid kinases . The identification of two S . cerevisiae homologues, Mss4p and Fab1p, confirms that this family of kinases is widely distributed in eukaryotes . Not surprisingly, cloning experiments have identified additional isoforms . By cloning additional isoforms, insights into the structure and functions of this family of enzymes will be gained . One reason for a large family of PIP5Ks is that many forms of regulation and cellular functions have been ascribed to PIP5Ks, as summarized in Figure 10 . Some of these functional links result from PtdIns{4,5}P2 being required for a given process, but the direct involvement of specific PIP5Ks is not well defined . Which PIP5K isoforms are regulated by a specific mechanism or are involved in a cellular process often is not clear . For example, which PIP5Ks produce PtdIns{4,5}P2 that is hydrolyzed by PLC or phosphorylated by the PI 3-kinase is not known . A few exceptions are PIP5KII not being able to phosphorylate PtdIns{4,5}P2 in native membranes, and PIP5KIs being stimulated by PtdA, required for secretion, and possibly regulated by G proteins of the Rho subfamily . The multiplicity of regulation and functions of each PIP5K isoform remains to be elucidated . Another factor governing the number of isoforms may be presence of multiple pools of polyphosphoinositides and the localizing of PIP5K function within cells . The polyphosphoinositides appear to be compartmentalized within cells and each pool appears to be sensitive to specific signals . These polyphosphoinositide pools may include those in the plasma membrane that are used by PLC, nuclear pools that appear to turn over separately from cytoplasmic pools and a small pool at sites of vesicle fusion with the plasma membrane . Each pool may be controlled by a specific PIP5K isoform . This would explain the diversity of PIP5K cellular roles . Another possibility is that the PIP5Ks are localized to certain areas of the cell by being part of a protein or proteolipid complex . Furthermore, the presence of PITP or PLC in the complex would potentially impart specificity and speed on the use of PtdIns{4}P and PtdIns{4,5}P2 because these lipids could be channeled quickly from one enzyme to the next . The concept of localized complexes containing particular PIP5K isoforms that control the composition of different polyphosphoinositide pools will likely be important as the family of PIP5K isoforms grows. Acta Biochim Pol, 1996, 43(2), 397 - 401 Solubilization and one-step purification of mannosylphosphodolichol synthase from Trichoderma reesei; Kruszewska JS et al.; Mannosylphosphodolichol synthase (MPD-synthase) (EC 2.4.1.830) catalyzing formation of MPD from GDPMan and dolichylphosphate (PD) has been purified from T . reesei cellular membranes almost to homogeneity . Selective solubilization of the enzyme was followed by one step purification on Phenyl-Sepharose column . SDS/ PAGE of the purified enzyme fraction revealed the presence of a protein band of 31 kDa corresponding to the apparent molecular mass of the MPD-synthase purified from S . cerevisiae {Babczinski, P . et al . (1980) Eur . J . Biochem . 105, 509-515; Haselbeck A . (1989) Eur . J . Biochem . 181, 663-668} . During solubilization, the enzyme was stabilized by the presence of a lipophilic substrate dolichylphosphate and phospholipids as well as by protease inhibitors . The Phenyl-Sepharose purified enzyme had an absolute requirement for dolichylphosphate and was activated by cAMP dependent protein kinase. Biosci Biotechnol Biochem, 1996 Jan, 60(1), 161 - 3 Molecular cloning of a genomic DNA for enolase from Aspergillus oryzae; Machida M et al.; We have isolated an enolase gene (enoA) from Aspergillus oryzae by heterologous hybridization using the corresponding Saccharomyces cerevisiae ENO2 gene as a probe . A 2.9-kb BglII-fragment contained the entire structural gene enoA including 5'- and 3'- flanking regions . The homology between A . oryzae enoA and S . cerevisiae ENO2 genes is 66.9% when introns are removed . Genomic Southern analysis indicated that there is only one enolase gene in A . oryzae. Curr Genet, 1996 Jan, 29(2), 122 - 9 The cloning and sequencing of the alcB gene, coding for alcohol dehydrogenase II, in Aspergillus nidulans; Hunter GD et al.; Alcohol dehydrogenase II (ADH II, structural gene alcB) was purified from a strain H1035, biA1; alcE1; alc500 alcD1, which produces 100-times more ADH II activity than the alcAalcR deletion strain (alc500) . Antibodies were raised against this ADH, and were used to screen a cDNA library in lambda gt11 . We have isolated the gene for an ADH which is over-expressed in H1035, and which we believe to be the alcB gene: cDNA and genomic clones were sequenced . The sequence contains three introns and encodes a protein of 367 amino acids . This protein shows a clear level of identity to a range of alcohol dehydrogenases, but is no more closely related to the ADH I and ADH III previously described in A . nidulans than to the ADHs of S . pombe and S . cerevisiae . The significance of consensus sequences found in the 5' region of the gene is discussed in relation to the regulation of the gene. Curr Genet, 1996 Jan, 29(2), 106 - 13 Over-expression of S . cerevisiae G1 cyclins restores the viability of alg1 N-glycosylation mutants; Benton BK et al.; In budding yeast, one of three G1 cyclins is required for progression though START, when cells commit to a further round of cell division . We have identified mutations in ALG1 (ERC14), a gene required for N-glycosylation, which are inviable in a cln1 cln2 background but are rescued by over-expression of CLNs . CLN1 and CLN2 are much more efficient than CLN3 in rescuing the erc14-1 allele . The erc14-1 allele results in a significant N-glycosylation defect, and no rescue of this defect by CLN1 over-expression was detected . These data suggest that CLN over-expression could be allowing cells to live with lower levels of N-glycosylation, possibly by overcoming a checkpoint sensitive to N-glycosylation capacity . A plasmid suppressor of alg1, PSA1, encodes a 361 amino-acid protein with homology to NDP-hexose pyrophosphorylases, the enzymes that catalyze the formation of activated sugar nucleotides . PSA1 is an essential gene, and PSA1 transcription is nearly co-ordinately regulated with CLN2 transcription, peaking near START . Co-ordinate regulation of glycosylation, sugar nucleotide metabolism, and cell-cycle progression through G1 may be a feature that ensures adequate cell-wall precursors are present before bud emergence. Biochimie, 1996, 78(2), 111 - 6 Products of S cerevisiae cis-prenyltransferase activity in vitro; Szkopinska A et al.; Products of cis-prenyltransferase activity, the first committed enzyme of the dolichol biosynthetic pathway, have been characterized in Saccharomyces cerevisiae . The evidence based on the results of ion exchange, HPTLC chromatography and acid phosphatase digestion has been presented indicating that the final product of the enzyme action in vitro is free polyprenol and not polyprenol mono- or diphosphate . On the other hand, the results of HPLC analysis confirmed that in vivo yeast accumulate alpha-saturated polyprenols (dolichols) . Phosphorylation of endogenous dolichols by cytidine triphosphate (CTP)-dependent kinase is demonstrated . The hypothesis is put forth that in S cerevisiae free polyprenol is the substrate for the alpha-reductase responsible for its conversion to dolichol which in turn is phosphorylated into its active form: dolichyl phosphate. Biochimie, 1996, 78(2), 95 - 102 Maintenance of the peroxisomal compartment in glucose-repressed and anaerobically grown Saccharomyces cerevisiae cells; Skoneczny M et al.; According to the current model of peroxisome biogenesis, the inheritance of this compartment requires the growth and division of pre-existing organelles followed by their distribution between mother and daughter cells . However, no known peroxisomal functions are present nor required for Saccharomyces cerevisiae cells grown under glucose repression and in anaerobiosis and the peroxisomal compartment becomes virtually indistinguishable under such conditions . This raised the question of the fate of this compartment in such cells . Is it maintained throughout prolonged growth under glucose repression or does it disappear from the cell and then reassemble on demand? To study the maintenance of putatively functional peroxisomes in S cerevisiae cells grown under conditions of glucose repression and anaerobiosis, we applied the vector-mediated overexpression of peroxisome matrix enzyme's catalase A and acyl-CoA oxidase . Evidence is presented that in S cerevisiae the peroxisomal import machinery responsible for targeting of matrix enzymes into this compartment is preserved under glucose repression and in the absence of oxygen. Acta Microbiol Immunol Hung, 1996, 43(1), 33 - 8 Glutathione-glutathione reductase system and lipid peroxidation in Saccharomyces cerevisiae under alcohol stress; Gupta S et al.; The content of malondialdehyde (MDA) as well as diene conjugates increased in Saccharomyces cerevisiae under alcoholic conditions . These cells had lower content of water soluble antioxidant, i.e., glutathione . Ethanol-treated S . cerevisiae also exhibited lower activity of glutathione reductase (GR) . The decrease in reduced glutathione (GSH) content cannot be attributed completely to lower GR activity . Not only the GSH content was lowered, but oxidized glutathione (GSSG) was also less in ethanol-treated yeast cells . The decrease in oxidized glutathione was much more (84%) as compared to that of reduced glutathione (40%). J Basic Microbiol, 1996, 36(4), 283 - 8 Studies on an amylolytic strain of Saccharomyces cerevisiae isolated from yam tuber; Olasupo NA et al.; An amylolytic yeast strain identified as Saccharomyces cerevisiae was isolated from yam tuber . Studies on the effect of physical agents such as pH and temperature on the activity of the amylase showed that the optimum pH and temperature for the amylase produced by the S . cerevisiae were 5.0 and 60 degrees C, respectively . Heavy metal resistance study on the amylolytic yeast strain indicated different levels of resistance to copper (6.0 mM), zinc (3.0 mM) and manganese (15.0 mM) ions . The results are discussed in relation to the potential use of an amylolytic yeast in the brewing industry in Nigeria. Receptors Channels, 1996, 4(1), 51 - 62 The S . cerevisiae outwardly-rectifying potassium channel (DUK1) identifies a new family of channels with duplicated pore domains; Reid JD et al.; Potassium channel subunits have six or two transmembrane segments in addition to a conserved pore-forming (P) domain; four subunits come together to form a channel . A gene was identified in S . cerevisiae (J0911) encoding a protein with eight probable membrane-spanning segments and two such P regions . This protein (Duk1p) is a potassium channel because Xenopus oocytes injected with the corresponding RNA express potassium currents activated by depolarization that are not seen in control oocytes . Similar potassium currents were recorded from wildtype S . cerevisiae spheroplasts, but not from those in which the DUK1 locus had been disrupted . Cells carrying the duk1 delta 1::HIS disruption in addition to a chimeric gene comprising DUK1 behind the GAL1 promoter showed outward currents when grown in galactose, but not when grown in glucose . Additional sequences with the duplicate pore motif were found in C . elegans, suggesting that these proteins represent a novel structural family of potassium channel proteins. Appl Microbiol Biotechnol, 1996 Jan, 44(5), 624 - 8 Recombinant outer-surface protein A (des-Cys1-OspA) from the Lyme disease spirochete Borrelia burgdorferi: high production levels in Saccharomyces cerevisiae yeast cultures; Mendoza-Vega O et al.; The recombinant outer-surface protein A with an N-terminally truncated form (des-Cys1-OspA) from the Lyme disease spirochete Borrelia burgdorferi was expressed in Saccharomyces cerevisiae at high production levels . Since the recombinant vaccine candidate expressed in Escherichia coli exhibits low production yields and the purification of lipoproteins appears to be difficult, we have investigated the secretion of a soluble recombinant OspA in the yeast S . cerevisiae . In this way, a Leu+ derivative of S . cerevisiae cI3ABYS86 was used as the host strain transformed with an expression plasmid containing the gene encoding des-Cys1-OspA and driven by the MF alpha 1 promoter . The fed-batch culture results revealed that an efficient secretion of des-Cys1-OspA is obtained with a high production level of about 2.1 g l-1 at a cell density of 101 g l-1 cell dry weight . The accumulation of recombinant protein in the supernatant exceeds 6% of the total yeast proteins when estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis . Moreover, des-Cys1-OspA showed lower solubilities at high cell densities and, as a consequence, a fraction of the recombinant protein precipitated . An internal cleavage of the MF alpha 1 pro::des-Cys1-OspA precursor was also detected . However, in this case the cleavage occurred at a frequency such that the large amounts of the secreted des-Cys1-OspA could be employed for the evaluation of an immunogenic effect on animal immunization . These studies will extend the knowledge of the usefulness of OspA as a vaccine for Lyme borreliosis. J Comput Biol, 1996 Spring, 3(1), 1 - 17 A Bayesian evolutionary distance for parametrically aligned sequences; Agarwal P et al.; There is an inherent relationship between the process of pairwise sequence alignment and the estimation of evolutionary distance . This relationship is explored and made explicit . Assuming an evolutionary model and given a specific pattern of observed base mismatches, the relative probabilities of evolution at each evolutionary distance are computed using a Bayesian framework . The mean or the median of this probability distribution provides a robust estimate of the central value . The evolutionary distance has traditionally been computed as zero for an observed homology of 20 bases with no mismatches; we prove that it is highly probable that the distance is greater than 0.01 . The mean of the distribution is 0.047, which is a better estimate of the evolutionary distance . Bayesian estimates of the evolutionary distance incorporate arbitrary prior information about variable mutation rates both over time and along sequence position, thus requiring only a weak form of the molecular-clock hypothesis . The endpoints of the similarity between genomic DNA sequences are often ambiguous . The probability of evolution at each evolutionary distance can be estimated over the entire set of alignments by choosing the best alignment at each distance and the corresponding probability of duplication at that evolutionary distance . A central value of this distribution provides a robust evolutionary distance estimate . We provide an efficient algorithm for computing the parametric alignment, considering evolutionary distance as the only parameter . These techniques and estimates are used to infer the duplication history of the genomic sequence in C . elegans and in S . cerevisiae . Our results indicate that repeats discovered using a single scoring matrix show a considerable bias in subsequent evolutionary distance estimates. Yi Chuan Xue Bao, 1996, 23(2), 142 - 8 {Studies on the cloning, expression and function of the yeast PHO 80 gene}; Zhao Y et al.; Through in situ hybridization, a 4.2 kb Pst I-BamH I fragment was obtained from S . cerevisiae gene library . The cloned fragment contained 1100 bp upstream sequence and 879 bp coding sequence of the PHO80 gene . Coding region of PHO80 gene was substituted with URA3 gene and used as donor to transform YPH499 to URA3 . A pho80 mutant resulted from deletion of the chromosomal counterpart in PHO80 was obtained . In vivo functional study of the PHO80 gene indicated that PHO80 was a negative regulator in the Pi-repressible acid phosphatase system including the structural genes PHO5 and PHO11 and the regulatory gene PHO81, whereas the expression of PHO4 or PHO85 was independent of PHO80 . The coding region of PHO80 was fused in frame with LacZ and beta-galactosidase activities in various cells was determined . The results Showed that the PHO80 gene was expressed at a low level and suppressed by itself and PHO85. Appl Biochem Biotechnol, 1996 Spring, 57-58, 407 - 12 Hexokinase production from S . cerevisiae . Culture conditions; Abrahao-Neto J et al.; The effects of pH (4.0, 4.5, or 5.0), temperature (T) (30, 35, or 40 degrees C) and dissolved oxygen (DO) (0.2, 2.0, 4.0,or 6.0 mg O2/L) on hexokinase and invertase formation by yeast were studied . The highest enzyme activities were attained at pH 4.0, DO = 4.0 mg O2/L, and T = 35 or 40 degrees C. Biometals, 1996 Jan, 9(1), 91 - 7 Electron paramagnetic resonance studies and effects of vanadium in Saccharomyces cerevisiae; Zoroddu MA et al.; Vanadium uptake by whole cells and isolated cell walls of the yeast Saccharomyces cerevisiae was studied . When orthovanadate was added to wild-type S . cerevisiae cells growing in rich medium, growth was inhibited as a function of the VO4(3-) concentration and the growth was completely arrested at a concentration of 20 mM of VO4(3-) in YEPD . Electron paramagnetic resonance (EPR) spectroscopy was used to obtain structural and dynamic information about the cell-associated paramagnetic vanadyl ion . The presence of EPR signals indicated that vanadate was reduced by whole cells to the vanadyl ion . On the contrary, no EPR signals were detected after interaction of vanadate with isolated cell walls . A 'mobile' and an 'immobile' species associated in cells with small chelates and with macromolecular sites, respectively, were identified . The value of rational correlation time tau r indicated the relative motional freedom at the macromolecular site . A strongly 'immobilized' vanadyl species bound to polar sites mainly through coulombic attractions was detected after interaction of VO2+ ions with isolated cell walls. Appl Environ Microbiol, 1996 Jan, 62(1), 209 - 13 Expression of Aureobasidium pullulans xynA in, and secretion of the xylanase from, Saccharomyces cerevisiae; Li XL et al.; A previous report dealt with the cloning in Escherichia coli and sequencing of both the cDNA and genomic DNA encoding a highly active xylanase (XynA) of Aureobasidium pullulans (X.-L . Li and L . G . Ljungdahl, Appl . Environ . Microbiol . 60:3160-3166, 1994) . Now we show that the gene was expressed in Saccharomyces cerevisiae under the GAL1 promoter in pYES2 and that its product was secreted into the culture medium . S . cerevisiae clone pCE4 with the whole open reading frame of xynA, including the part coding for the signal peptide, had xylanase activity levels of 6.7 U ml-1 in the cell-associated fraction and 26.2 U ml-1 in the culture medium 4 h after galactose induction . Two protein bands with sizes of 25 and 27 kDa and N-terminal amino acid sequences identical to that of APX-II accounted for 82% of the total proteins in the culture medium of pCE4 . These proteins were recognized by anti-APX-II antibody . The results suggest that the XynA signal peptide supported the posttranslational processing of xynA product and the efficient secretion of the active xylanase from S . cerevisiae . Clones pCE3 and pGE3 with inserts of cDNA and genomic DNA, respectively, containing only the mature enzyme region attached by a Met codon had low levels of xylanase activity in the cell-associated fractions (1.6 U ml-1) but no activity in the culture media . No xylanase activity was detected in clone pGE4, which was the same as pCE4, except that pGE4 had a 59-bp intron in the signal peptide region . A comparison of the A . pullulans and S . cerevisiae signal peptides demonstrated that the XynA signal peptide was at least three times more efficient than those of S . cerevisiae invertase or mating alpha-factor pheromone in secreting the heterologous xylanase from S . cerevisiae cells. J Cell Biol, 1996 Jan, 132(1-2), 137 - 51 BED1, a gene encoding a galactosyltransferase homologue, is required for polarized growth and efficient bud emergence in Saccharomyces cerevisiae; Mondesert G et al.; The ellipsoidal shape of the yeast Saccharomyces cerevisiae is the result of successive isotropic/apical growth switches that are regulated in a cell cycle-dependent manner . It is thought that growth polarity is governed by the remodeling of the actin cytoskeleton that is itself under the control of the cell cycle machinery . The cell cycle and the morphogenesis cycle are tightly coupled and it has been recently suggested that a morphogenesis/polarity checkpoint control monitors bud emergence in order to maintain the coupling of these two events (Lew, D . J., and S . I . Reed . 1995 . J . Cell Biol . 129:739-749) . During a screen based on the inability of cells impaired in the budding process to survive when the morphogenesis checkpoint control is abolished, we identified and characterized BED1, a new gene that is required for efficient budding . Cells carrying a disrupted allele of BED1 no longer have the wild-type ellipsoidal shape characteristic of S . cerevisiae, are larger than wild-type cells, are deficient in bud emergence, and depend upon an intact morphogenesis checkpoint control to survive . These cells show defects in polarized growth despite the fact that the actin cytoskeleton appears normal . Our results suggest that Bed1 is a type II membrane protein localized in the endoplasmic reticulum . BED1 is significantly homologous to gma12+, a S . pombe gene coding for an alpha-1,2,-galactosyltransferase, suggesting that glycosylation of specific proteins or lipids could be important for signaling in the switch to polarized growth and in bud emergence. J Bacteriol, 1996 Jan, 178(2), 477 - 83 HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern in the yeast Saccharomyces cerevisiae; Yabe T et al.; We previously isolated the Saccharomyces cerevisiae HKR1 gene that confers on S . cerevisiae cells resistance to HM-1 killer toxin secreted by Hansenula mrakii (S . Kasahara, H . Yamada, T . Mio, Y . Shiratori, C . Miyamoto, T . Yabe, T . Nakajima, E . Ichishima, and Y . Furuichi, J . Bacteriol . 176:1488-1499, 1994) . HKR1 encodes a type 1 membrane protein that contains a calcium-binding consensus sequence (EF hand motif) in the cytoplasmic domain . Although the null mutation of HKR1 is lethal, disruption of the 3' part of the coding region, which would result in deletion of the cytoplasmic domain of Hkr1p, did not affect the viability of yeast cells . This partial disruption of HKR1 significantly reduced beta-1,3-glucan synthase activity and the amount of beta-1,3-glucan in the cell wall and altered the axial budding pattern of haploid cells . Neither chitin synthase activity nor chitin content was significantly affected in the cells harboring the partially disrupted HKR1 allele . Immunofluorescence microscopy with an antibody raised against Hkr1p expressed in Escherichia coli revealed that Hkr1p was predominantly localized on the cell surface . The cell surface localization of Hkr1p required the N-terminal signal sequence because the C-terminal half of Hkr1p was detected uniformly in the cells . These results demonstrate that HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern and suggest that bud site assembly is somehow related to beta-glucan synthesis in S . cerevisiae. Mol Cell Biol, 1996 Jan, 16(1), 121 - 9 The AD1 transactivation domain of E2A contains a highly conserved helix which is required for its activity in both Saccharomyces cerevisiae and mammalian cells; Massari ME et al.; A conserved region, designated the AD1 domain, is present in a class of helix-loop-helix (HLH) proteins, E proteins, that includes E12, E47, HEB, E2-2, and a Xenopus laevis HLH protein closely related to E12 . We demonstrate that the AD1 domain in E2A and the conserved region of E2-2 activate transcription in both yeast and mammalian cells . The AD1 domain contains a highly conserved putative helix that is crucial for its transactivation properties . Circular dichroism spectroscopy data show that AD1 is structured and contains distinctive helical properties . In addition, we show that a synthetic peptide corresponding to the conserved region is unstructured in aqueous solution at neutral pH but can adopt an alpha-helical conformation in the presence of the hydrophobic solvent trifluoroethanol . Amino acid substitutions that destabilize the helix abolish the transactivation ability of the AD1 domain . Both structural and functional analyses of AD1 reveal striking similarities to the acidic class of activators . Remarkably, when wild-type and mutant proteins are expressed in mammalian cells and Saccharomyces cerevisiae, identical patterns of transactivation are observed, suggesting that the target molecule is conserved between S . cerevisiae and mammals . These data show that transactivation by E proteins is mediated, in part, by a strikingly conserved peptide that has the ability to form a helix in a hydrophobic solvent . We propose that the unstructured domain may become helical upon interaction with its cellular target molecule. FEBS Lett, 1995 Dec 18, 377(2), 189 - 92 Immunological evidence that HMG-CoA reductase kinase-A is the cauliflower homologue of the RKIN1 subfamily of plant protein kinases; Ball KL et al.; Three different antibodies against the RKIN1 and BKIN12 gene products from rye and barley recognized the 58 kDa subunit of HMG-CoA reductase kinase-A (HRK-A) from Brassica oleracea on Western blots . HRK-A was also detected by an antipeptide antibody in enzyme-linked immunoassays, and this was competed by the peptide antigen . HRK-A was not recognized by antibodies against plant, mammalian and Saccharomyces cerevisiae relatives of RKIN1, i.e . wheat PKABA1, rat AMP-activated protein kinase and S . cerevisiae Snf1p . RKIN1/HMG-CoA reductase kinase-A are now among the first protein kinases in plants to be well characterized at both the molecular and biochemical levels. Biochem J, 1995 Dec 15, 312 ( Pt 3), 817 - 25 Polymorphism of the yeast pyruvate carboxylase 2 gene and protein: effects on protein biotinylation; Val DL et al.; In Saccharomyces cerevisiae there are two isoenzymes of pyruvate carboxylase (Pyc) encoded by separate genes designated PYC1 and PYC2 . We report the isolation and sequencing of a PYC2 gene, and the localization of both genes on the physical map of S . cerevisiae . Comparison with the previously reported sequence {Stucka, Dequin, Salmon and Gancedo (1991) Mol . Gen . Genet . 229, 307-315} revealed significant differences within the open reading frame . The most notable difference was near the 3' end, where we found a single base deletion reducing the open reading frame by 15 bases . We have confirmed the C-terminus of Pyc2 encoded by the gene isolated here by expressing and purifying an 86-amino-acid biotin-domain peptide . In addition, we investigated the effects of the two changes in the Pyc2 biotin domain (K1155R substitution and Q1178P/five-amino-acid extension) on the extent of biotinylation in vivo by Escherichia coli biotin ligase, and compared the biotinylation of peptides containing these changes with that of two different-length Pyc1 biotin-domain peptides . The K1155R substitution had very little effect on biotinylation, but the five-amino-acid C-terminal extension to Pyc2 and the N-terminal extension to Pycl both improved biotinylation in vivo. Cell Struct Funct, 1995 Dec, 20(6), 465 - 71 Analysis of the membrane structures involved in autophagy in yeast by freeze-replica method; Baba M et al.; Under starvation conditions, the yeast S . cerevisiae sequesters its own cytoplasmic components by forming autophagosomes with double membrane in the cytoplasm . The autophagosome then fuses with the vacuolar membrane and delivers its own cytoplasmic components into the vacuole in the form of an autophagic body with a single membrane (Baba, M., Takeshige, K., Baba, N., and Ohsumi, Y . 1994 . J . Cell Biol., 124: 903-913) . We examined membrane structures involved in the autophagy induced by nitrogen-starvation by using freeze-replica method . The most conspicuous characteristic of the autophagic body is that the intramembrane particles were rarely detected on either the protoplasmic or exoplasmic face of its fracture membrane . This morphological feature of the fractured face was clearly different from other intracellular organelles . Next we examined the autophagosomal membrane . The inner membrane of autophagosome was also intramembrane particle-free, and its morphological feature was identical to the membrane of autophagic body . At the fusion site between autophagosome and vacuole we obtained direct evidence that two different membranes, the outer membrane of autophagosome and vacuolar membrane, became continuous by using freeze-etching technique . From these results we concluded that the autophagic body originated from the inner membrane of the autophagosome, and its membrane reflects an intrinsic feature of autophagosomal membrane . The outer membrane of autophagosome had only a few intramembrane particles and may be differentiated from the inner membrane . In cells under nitrogen-starvation condition, the density of intramembrane particles of vacuolar membrane decreased beyond that in control cells. Yeast, 1995 Dec, 11(15), 1493 - 9 Detection method for polygalacturonase-producing strains of Saccharomyces cerevisiae; Gainvors A et al.; In the presence of glycerol or ethanol, SCPP (a strain of Saccharomyces cerevisiae that expresses pectinolytic activity) is capable of utilizing galacturonic acid or pectins for growth purposes . We now establish a relationship between the pectinolytic power of various strains of S . cerevisiae and their ability to grow on a pectin/glycerol-based medium . This property is further exploited for the detection of polygalacturonase-producing strains of S . cerevisiae. Bioessays, 1995 Dec, 17(12), 1013 - 5 Histone H4, the cell cycle and a question of integrity; Turner BM; The N-terminal domain of histone H4 has been implicated in various nuclear functions, including gene silencing and activation and replication-linked chromatin assembly . Many of these have been identified by using h4 mutants in the yeast S . cerevisiae . In a recent paper, Megee et al . use this approach to show that mutants in which all four N-terminal H4 lysines are substituted with glutamines accumulate increased levels of DNA damage . A single lysine, but not an arginine, anywhere in the N-terminal domain suppresses this phenotype . It is suggested that histone H4 plays a role in maintaining genome integrity through the cell cycle, possibly by a mechanism involving lysine acetylation. Curr Genet, 1995 Dec, 29(1), 10 - 7 Different respiratory-defective phenotypes of Neurospora crassa and Saccharomyces cerevisiae after inactivation of the gene encoding the mitochondrial acyl carrier protein; Schneider R et al.; The nuclear genes (acp-1, ACP1) encoding the mitochondrial acyl carrier protein were disrupted in Neurospora crassa and Saccharomyces cerevisiae . In n . crassa acp-1 is a peripheral subunit of the respiratory NADH : ubiquinone oxidoreductase (complex I) . S . cerevisiae lacks complex I and its ACP1 appears to be located in the mitochondrial matrix . The loss of acp-1 in N . crassa causes two biochemical lesions . Firstly, the peripheral part of complex I is not assembled, and the membrane part is not properly assembled . The respiratory ubiquinol : cytochrome c oxidoreductase (complex III) and cytochrome c oxidase (complex IV) are made in normal amounts . Secondly, the lysophospholipid content of mitochondrial membranes is increased four-fold . In S . cerevisiae, the loss of ACP1 leads to a pleiotropic respiratory deficient phenotype. Antimicrob Agents Chemother, 1995 Dec, 39(12), 2765 - 9 The AUR1 gene in Saccharomyces cerevisiae encodes dominant resistance to the antifungal agent aureobasidin A (LY295337); Heidler SA et al.; Aureobasidin A (LY295337) is a cyclic depsipeptide antifungal agent with activity against Candida spp . The mechanism of action of LY295337 remains unknown . LY295337 also shows activity against the yeast Saccharomyces cerevisiae . Generation of a mutant of S . cerevisiae resistant to LY295337 is reported . Resistance was found to reside in a dominant mutation of a single gene which has been named AUR1 (aureobasidin resistance) . This gene was cloned and sequenced . A search for homologous sequences in GenBank and by BLAST did not elucidate the function of this gene, although sequence homology too an open reading frame from the Saccharomyces genome sequencing project and several other adjacent loci was noted . Deletion of aur1 was accomplished in a diploid S . cerevisiae strain . Subsequent sporulation and dissection of the aur1/aur1 delta diploid resulted in tetrads demonstrating 2:2 segregation of viable and nonviable spores, indicating that deletion of aur1 is lethal . As LY295337 is fungicidal and deletion of aur1 is lethal, aur1 represents a potential candidate for the target of LY295337. Mol Biol Cell, 1995 Dec, 6(12), 1843 - 59 Localization and possible functions of Drosophila septins; Fares H et al.; The septins are a family of homologous proteins that were originally identified in Saccharomyces cerevisiae, where they are associated with the "neck filaments" and are involved in cytokinesis and other aspects of the organization of the cell surface . We report here the identification of Sep1, a Drosophila melanogaster septin, based on its homology to the yeast septins . The predicted Sep1 amino acid sequence is 35-42% identical to the known S . cerevisiae septins; 52% identical to Pnut, a second D . melanogaster septin; and 53-73% identical to the known mammalian septins . Sep1-specific antibodies have been used to characterize its expression and localization . The protein is concentrated at the leading edge of the cleavage furrows of dividing cells and cellularizing embryos, suggesting a role in furrow formation . Other aspects of Sep1 localization suggest roles not directly related to cytokinesis . For example, Sep1 exhibits orderly, cell-cycle-coordinated rearrangements within the cortex of syncytial blastoderm embryos and in the cells of post-gastrulation embryos; Sep1 is also concentrated at the leading edge of the epithelium during dorsal closure in the embryo, in the neurons of the embryonic nervous system, and at the baso-lateral surfaces of ovarian follicle cells . The distribution of Sep1 typically overlaps, but is distinct from, that of actin . Both immunolocalization and biochemical experiments show that Sep1 is intimately associated with Pnut, suggesting that the Drosophila septins, like those in yeast, function as part of a complex. J Cell Biol, 1995 Dec, 131(6 Pt 2), 1775 - 88 A highly divergent gamma-tubulin gene is essential for cell growth and proper microtubule organization in Saccharomyces cerevisiae; Sobel SG et al.; A Saccharomyces cerevisiae gamma-tubulin-related gene, TUB4, has been characterized . The predicted amino acid sequence of the Tub4 protein (Tub4p) is 29-38% identical to members of the gamma-tubulin family . Indirect immunofluorescence experiments using a strain containing an epitope-tagged Tub4p indicate that Tub4p resides at the spindle pole body throughout the yeast cell cycle . Deletion of the TUB4 gene indicates that Tub4p is essential for yeast cell growth . Tub4p-depleted cells arrest during nuclear division; most arrested cells contain a large bud, replicated DNA, and a single nucleus . Immunofluorescence and nuclear staining experiments indicate that cells depleted of Tub4p contain defects in the organization of both cytoplasmic and nuclear microtubule arrays; such cells exhibit nuclear migration failure, defects in spindle formation, and/or aberrantly long cytoplasmic microtubule arrays . These data indicate that the S . cerevisiae gamma-tubulin protein is an important SPB component that organizes both cytoplasmic and nuclear microtubule arrays. Mutat Res, 1995 Dec, 345(3-4), 111 - 25 Genotoxic evaluation of three heterocyclic N-methylcarbamate pesticides using the mouse bone marrow micronucleus assay and the Saccharomyces cerevisiae strains D7 and D61.M; Stehrer-Schmid P et al.; The carbamate insecticides benfuracarb, carbosulfan and furathiocarb were investigated in the mouse bone marrow micronucleus assay to establish whether they show cytogenetic activity in vivo . Two doses of each substance were administered intraperitoneally to NMRI mice . All of the three substances led to a positive micronucleus response in polychromatic erythrocytes of the bone marrow at different expression times . While furathiocarb and carbosulfan showed similar patterns of the time-dependence of the micronucleus formation with maximum values after 72 h, benfuracarb exhibited a different behaviour with the maximum increase taking place within 24 h after substance application . In furathiocarb-treated animals the ratio of normochromatic to polychromatic erythrocytes showed a dose and time depending increase with the highest value obtained after 72 h in animals treated with the upper dose . The two yeast test systems Saccharomyces cerevisiae strains D7 and D61.M were applied in order to evaluate the genetic endpoints gene mutation, gene conversion and aneuploidy induction . None of the three insecticides had an influence on the frequencies of gene conversion and reverse mutation in the yeast S . cerevisiae D7 when tested with and without metabolic activation . In strain D61.M however benfuracarb and furathiocarb led to an increase of chromosome loss in the presence of the S9 metabolizing system. Appl Environ Microbiol, 1995 Dec, 61(12), 4296 - 303 Direct detection of recombinant gene expression by two genetically engineered yeasts in soil on the transcriptional and translational levels; Tebbe CC et al.; The expression of a recombinant gene by yeasts seeded into soil samples was directly measured by analyzing transcripts and gene product occurrences in soil extracts . Two yeast species, Saccharomyces cerevisiae WHL292 and Hansenula polymorpha LR9-Apr4, both engineered by a synthetic gene sequence encoding the mammalian peptide aprotinin, produced and secreted this peptide in batch cultures at concentrations of 90 and 64 ng ml-1, respectively . In S . cerevisiae, the aprotinin gene was located on plasmid p707 and expressed constitutively . H . polymorpha carried the gene chromosomally integrated, and its expression was inducible by methanol . To detect aprotinin transcripts, cells were directly lysed in the soil samples and the crude lysates were hybridized to oligo(dT)-coated magnetized polystyrene beads (Dynabeads) . After separation and purification in a magnetic field, aprotinin mRNA was detected by reverse transcriptase PCR with aprotinin gene-specific primers . Transcripts from 10 cells g of soil-1 were sufficient for detection . When 10(7) cells of S . cerevisiae were inoculated into soil, aprotinin mRNA was detectable during the first 4 days . Addition of methanol and a combined nutrient solution was necessary to induce aprotinin gene expression of H . polymorpha in soil . Aprotinin could be detected directly in soil extracts by an indirect enzyme-linked immunosorbent assay with monoclonal aprotinin-specific antibodies . The detection threshold was 45 pg g of soil-1 . In presterilized soil inoculated with S . cerevisiae (10(6) CFU g-1), aprotinin accumulated during the first 10 days to 12 ng g of soil-1 and then remained constant.(ABSTRACT TRUNCATED AT 250 WORDS) Mutat Res, 1995 Dec, 335(3), 285 - 92 Monitoring of induced chromosomal aberrations in S . cerevisiae in agarose gels by pulsed field gel electrophoresis; Steinkamp-Zucht A et al.; Pulsed field gel electrophoresis (PFGE) has been used to detect aberrations of the chromosomal banding pattern referred to as chromosomal aberrations arising after treatment of yeast strain S . cerevisiae MP1 with the three different genotoxic substances 4-nitroquinoline-N-oxide (4-NQO), methotrexate (MTX) and 2-amino-6-mercaptopurine (AMP) . Different chromosomal aberrations were detectable directly in the pulsed field gel when growing yeast cells were incubated with a genotoxin for 6 h at 26 degrees C followed by treatment with the genotoxin for another twelve days at 4 degrees C . In the present study, clones of yeast cells were examined . In this way, distinct chromosomal aberrations and not only DNA smear could be detected . Moreover, this method allows selection for yeast strains with specific and rare chromosomal rearrangements. Mol Cell Biol, 1995 Dec, 15(12), 6884 - 94 Mutations altering the mitochondrial-cytoplasmic distribution of Mod5p implicate the actin cytoskeleton and mRNA 3' ends and/or protein synthesis in mitochondrial delivery; Zoladek T et al.; The Saccharomyces cerevisiae MOD5 gene encodes proteins that function in three subcellular locations: mitochondria, the cytoplasm, and nuclei (M . Boguta, L.A . Hunter, W.-C . Shen, E . C . Gillman, N . C . Martin, and A . K . Hopper, Mol . Cell . Biol . 14:2298-2306, 1994; E . C . Gillman, L . B . Slusher, N . C . Martin, and A . K . Hopper, Mol . Cell . Biol . 11:2382-2390, 1991) . A mutant allele of MOD5 encoding a protein (Mod5p-I,KR6) located predominantly in mitochondria was constructed . Mutants defective in delivering Mod5p-I,KR6 to mitochondria were sought by selecting cells with increased cytosolic activity of this protein . Twenty-five mutants defining four complementation groups, mdp1, mdp2, mdp3, and mdp4, were found . They are unable to respire at 34 degrees C or to grow on glucose medium at 38 degrees C . Cell fractionation studies showed that mdp1, mdp2, and mdp3 mutants have an altered mitochondrial-cytoplasmic distribution of Mod5p . mdp2 can be suppressed by ACT1, the actin-encoding gene . The actin cytoskeleton organization is also aberrant in mdp2 cells . MDP2 is the same as VRP1 (S . F . H . Donnelly, M . J . Picklington, D . Pallotta, and E . Orr, Mol . Microbiol . 10:585-596, 1993) . MDP3 is identical to PAN1, which encodes a protein that interacts with mRNA 3' ends and affects initiation of protein synthesis (A . B . Sachs and J . A . Deardoff, Cell 70:961-973, 1992) . These results implicate the actin cytoskeleton and mRNA 3' ends and/or protein synthesis as being as important for protein distribution in S . cerevisiae as they are for distribution of cytosolic proteins in higher eukaryotes . This provides the potential to apply genetic and molecular approaches to study gene products and mechanisms involved in this type of protein distribution . The selection strategy also offers a new approach for identifying gene products involved in the distribution of proteins to their subscellular destinations. J Bacteriol, 1995 Dec, 177(23), 6773 - 81 The Candida boidinii peroxisomal membrane protein Pmp30 has a role in peroxisomal proliferation and is functionally homologous to Pmp27 from Saccharomyces cerevisiae; Sakai Y et al.; The mechanism of peroxisome proliferation is poorly understood . Candida boidinii is a methylotrophic yeast that undergoes rapid and massive peroxisome proliferation and serves as a good model system for this process . Pmp30A and Pmp30B (formerly designated Pmp31 and Pmp32, respectively) are two closely related proteins in a polyploid strain of this yeast that are strongly induced by diverse peroxisome proliferators such as methanol, oleate, and D-alanine . The function of these proteins is not understood . To study this issue, we used a recently described haploid strain (S2) of C . boidinii that can be manipulated genetically . We now report that strain S2 contains a single PMP30 gene very similar in sequence (greater than 93% identity at the DNA level) to PMP30A and PMP30B . When PMP30 was disrupted, cell growth on methanol was greatly inhibited, and cells grown in both methanol and oleate had fewer, larger, and more spherical peroxisomes than wild-type cells . A similar phenotype was recently described for Saccharomyces cerevisiae cultured on oleate in which PMP27, which encodes a protein of related sequence that is important for peroxisome proliferation, was disrupted . To determine whether Pmp27 is a functional homolog of Pmp30, gentle complementation was performed . PMP30A was expressed in the PMP27 disruptant of S . cerevisiae, and PMP27 was expressed in the PMP30 disruptant of C . boidinii S2 . Complementation, in terms of both cell growth and organelle size, shape, and number, was successful in both directions, although reversion to a wild-type phenotype was only partial for the PMP30 disruptant . We conclude that these proteins are functional homologs and that both Pmp30 and Pmp27 have a direct role in proliferation and organelle size rather than a role in a specific peroxisomal metabolic pathway of substrate utilization. Cell, 1995 Nov 17, 83(4), 563 - 8 The multidomain structure of Orc1p reveals similarity to regulators of DNA replication and transcriptional silencing; Bell SP et al.; The origin recognition complex (ORC) is a six protein assembly that binds S . cerevisiae origins of replication and directs DNA replication throughout the genome and transcriptional silencing at the yeast mating-type loci . Here we report the cloning of the genes encoding the 120 kDa (ORC1), 62 kDa (ORC3), and 56 kDa (ORC4) subunits of ORC and the reconstitution of the complete complex after expression of all six subunits in insect cells . Orc1p is related to Cdc6p and Cdc18p, which regulate DNA replication and mitosis, and to Sir3p, a regulator of transcriptional silencing . The N-terminal region of Orc1p is highly related to Sir3p, and studies of Orc1p/Sir3p chimeric proteins indicate that this domain is dedicated to the transcriptional silencing function of ORC. Cell, 1995 Nov 17, 83(4), 539 - 45 The stabilization of repetitive tracts of DNA by variant repeats requires a functional DNA mismatch repair system; Heale SM et al.; Simple repetitive tracts of DNA are unstable in all organisms thus far examined . In the yeast S . cerevisiae, we show that a 51 bp poly(GT) tract alters length at a rate of about 10(-5) per cell division . Insertion of a single variant repeat (either AT or CT) into the middle of the poly(GT) tract results in 100-fold stabilization . This stabilization requires the DNA mismatch repair system . Alterations within tracts with variant repeats occur more frequently on one side of the interruption than on the other . The stabilizing effects of variant repeats and polarity of repeat alterations have also been observed in trinucleotide repeats associated with certain human diseases. Science, 1995 Nov 17, 270(5239), 1213 - 5 Requirement of Saccharomyces cerevisiae Ras for completion of mitosis; Morishita T et al.; In the yeast Saccharomyces cerevisiae, Ras regulates adenylate cyclase, which is essential for progression through the G1 phase of the cell cycle . However, even when the adenosine 3',5'-monophosphate (cAMP) pathway was bypassed, the double disruption of RAS1 and RAS2 resulted in defects in growth at both low and high temperatures . Furthermore, the simultaneous disruption of RAS1, RAS2, and the RAS-related gene RSR1 was lethal at any temperature . The triple-disrupted cells were arrested late in the mitotic (M) phase, which was accompanied by an accumulation of cells with divided chromosomes and sustained histone H1 kinase activity . The lethality of the triple disruption was suppressed by the multicopies of CDC5, CDC15, DBF2, SPO12, and TEM1, all of which function in the completion of the M phase . Mammalian ras also suppressed the lethality, which suggests that a similar signaling pathway exists in higher eukaryotes . These results demonstrate that S . cerevisiae Ras functions in the completion of the M phase in a manner independent of the Ras-cAMP pathway. EMBO J, 1995 Nov 15, 14(22), 5626 - 37 The higher plant Arabidopsis thaliana encodes a functional CDC48 homologue which is highly expressed in dividing and expanding cells; Feiler HS et al.; We have identified an Arabidopsis thaliana CDC48 gene which, unlike the putative mammalian homologue vasolin-containing protein (VCP), functionally complements Saccharomyces cerevisiae cdc48 mutants . CDC48 is an essential gene in S . cerevisiae and genetic studies suggest a role in spindle pole body separation . Biochemical studies link VCP function to membrane trafficking and signal transduction . We have described the AtCDC48 expression pattern in a multicellular eukaryote; the zones of cell division, expansion and differentiation are physically separated in higher plants, thus allowing the analysis of in situ expression patterns with respect to the state of cell proliferation . AtCDC48 is highly expressed in the proliferating cells of the vegetative shoot, root, floral inflorescence and flowers, and in rapidly growing cells . AtCDC48 mRNA and the encoded protein are up-regulated in the developing microspores and ovules . AtCDC48 expression is down-regulated in most differentiated cell types . AtCDC48p was primarily localized to the nucleus and, during cytokinesis, to the phragmoplast, a site where membrane vesicles are targeted in the deposition of new cell wall materials . This study shows that the essential cell division function of CDC48 has been conserved by, at least, some multicellular eukaryotes and suggests that in higher plants, CDC48 functions in cell division and growth processes. Mol Gen Genet, 1995 Nov 15, 249(2), 209 - 16 Yeast Kre1p is a cell surface O-glycoprotein; Roemer T et al.; The Saccharomyces cerevisiae KRE1 gene encodes a secretory protein required for the production of the cell wall polymer (1-->6)-beta-glucan . Here we report further characterization of the KRE1 gene product, Kre1p . A functional, epitope-tagged Kre1p is shown to be highly modified in a SEC53-dependent manner . Kre1p is O-glycosylated, but the basis for the majority of its post-translational modification is unknown . Fractionation of Kre1p reveals a cell wall-associated form and a less abundant membrane-associated species . Indirect immunoflurorescence demonstrates that Kre1p localizes to the cell surface, where it becomes concentrated at the surface of mother cells . Such a localization of Kre1p seems to parallel the CAL1/CSD2-dependent cell wall deposition of chitin found in S . cerevisiae, and is consistent with evidence from Schizophyllum commune that (1-->6)-beta-glucan accumulates during maturation of the subapical region of the wall distal to the hyphal tip. Mol Gen Genet, 1995 Nov 15, 249(2), 127 - 38 Cloning and characterization of seven cDNAs for hyperosmolarity-responsive (HOR) genes of Saccharomyces cerevisiae; Hirayama T et al.; Yeast cells can respond and adapt to osmotic stress . In our attempt to clarify the molecular mechanisms of cellular responses to osmotic stress, we cloned seven cDNAs for hyperosmolarity-responsive (HOR) genes from Saccharomyces cerevisiae by a differential screening method . Structural analysis of the clones revealed that those designated HOR1, HOR3, HOR4, HOR5 and HOR6 encoded glycerol-3-phosphate dehydrogenase (Gpd1p), glucokinase (Glk1p), hexose transporter (Hxt1p), heat-shock protein 12 (Hsp12p) and Na+, K+, Li(+)-ATPase (Ena1p), respectively . HOR2 and HOR7 corresponded to novel genes . Gpd1p is a key enzyme in the synthesis of glycerol, which is a major osmoprotectant in S . cerevisiae . Cloning of HOR1/GPD1 as a HOR gene indicates that the accumulation of glycerol in yeast cells under hyperosmotic stress is, at least in part, caused by an increase in the level of GPDH protein . We performed a series of Northern blot analyses using HOR cDNAs as probes and RNAs prepared from cells grown under various conditions and from various mutant cells . The results suggested that all the HOR genes are regulated by common signal transduction pathways . However, the fact that they exhibited certain distinct responses indicated that they might also be regulated by specific pathways in addition to the common pathways . Ca2+ seemed to be involved in the signaling systems . In addition, Hog1p, one of the MAP kinases in yeast, appeared to be involved in the regulation of expression of HOR genes, although its function seemed to be insufficient for the overall regulation of expression of these genes. Curr Genet, 1995 Nov, 28(6), 526 - 33 Characterization of a novel alpha-amylase from Lipomyces kononenkoae and expression of its gene (LKA1) in Saccharomyces cerevisiae; Steyn AJ et al.; A highly active alpha-amylase (76,250 Da) secreted by the raw starch-degrading yeast Lipomyces kononenkoae strain IGC4052B was purified and characterized . Using high performance liquid chromatography (HPLC), end-product analysis indicated that the L . kononenkoae alpha-amylase acted by endo-hydrolysis on glucose polymers containing alpha-1,4 and alpha-1,6 bonds, producing mainly maltose, maltotriose and maltotetraose . The following NH2-terminal amino acids were determined for the purified enzyme: Asp-Cys-Thr-Thr-Val-Thr-Val-Leu-Ser-Ser-Pro- Glu-Ser-Val-Thr-Gly . The L . kononenkoae alpha-amylase-encoding gene (LKA1), previously cloned as a cDNA fragment, was expressed in Saccharomyces cerevisiae under the control of the PGK1 promoter . The native signal sequence efficiently directed the secretion of the glycosylated protein in S . cerevisiae . De-glycosylation of the enzyme indicated that post-translational glycosylation is different in S . cerevisiae from that in L . kononenkoae . Zymogram analysis indicated that glycosylation of the protein in S . cerevisiae had a negative effect on enzyme activity . Southern-blot analysis revealed that there is only a single LKA1 gene present in the genome of L . kononenkoae. Biokhimiia, 1995 Nov, 60(11), 1911 - 6 {Detection of polyphosphatase activity in isolated Saccharomyces cerevisiae nuclei}; Lichko LP et al.; Intact nuclei have been isolated from cells of a diploid strain of Saccharomyces cerevisiae . The isolated nuclei were free from cytoplasmic, mitochondrial and vacuolar marker enzymes . The protein to DNA ratio (w/w) was 11 . Pyrophosphatase, tripolyphosphatase and exopolyphosphatase activities have been found in S . cerevisiae nuclei for the first time and were equal to 400, 130 and 55 mU/mg of protein, respectively. Yeast, 1995 Nov, 11(14), 1413 - 9 DNA sequence analysis of a 35 kb segment from Saccharomyces cerevisiae chromosome VII reveals 19 open reading frames including RAD54, ACE1/CUP2, PMR1, RCK1, AMS1 and CAL1/CDC43; James CM et al.; We present DNA sequence data from a 35,364 bp region on the left arm of chromosome VII of Saccharomyces cerevisiae . This region contains 19 open reading frames (ORFs) . ORF G1821 corresponds to the RAD54 gene involved in repair and recombination (Emery et al., 1991) . G1810 is identical to the ACE1 gene sequenced by Szczypka and Thiele (1989), required for copper-inducible transcription of the CUP1 gene . The first 693 bp on the minus strand represent part of the 3' non-coding region from the P-type ATPase gene PMR1, previously sequenced by Rudolph et al . (1989), which is identical to the SSC1 gene (Smith et al., 1988) . G1845 corresponds to the RCK1 protein kinase gene from S . cerevisiae (Dahlkvist and Sunnerhagen, 1994) . G1861 is almost identical to the alpha-mannosidase gene AMS1 reported by Yoshihisa and Anraku (1989) and G1864 has 100% identity with the yeast CAL1 gene (Ohya et al., 1989)/CDC43 gene (Johnson et al., 1990) which is involved in control of cell polarity . This region also contains a gene specifying a Leu-tRNA precursor and a remnant of a tau element . ORF G1880 shows some similarity to the S . cerevisiae SNF2, STH1 and NPS1 genes and to the human ERCC1 gene . A 93 bp region shows similarity to yeast EST sequenced by Burns et al . (1994) . None of the remaining ORFs has similarity to any sequence within the databases screened. Yeast, 1995 Nov, 11(14), 1381 - 91 Structural features of a polypeptide carrier promoting secretion of a beta-lactamase fusion protein in yeast; Jamsa E et al.; Escherichia coli beta-lactamase was secreted into the culture medium of Saccharomyces cerevisiae in biologically active form, when fused to the C-terminus of the hsp150 delta-carrier . The hsp150 delta-carrier is an N-terminal fragment of the yeast hsp150 protein, having a signal peptide and consisting mostly of a 19 amino acid peptide repeated 11 times in tandem . Here we expressed the hsp150 delta-carrier fragment alone in S . cerevisiae . Apparently due to a positional effect of the gene insertion, large amounts of the hsp150 delta-carrier were synthesized . About half of the de novo synthesized carrier molecules were secreted into the culture medium, the rest remaining mostly in the pre-Golgi compartment . The extensively O-glycosylated carrier fragment was purified from the culture medium under non-denaturing conditions . Circular dichroism spectroscopy showed that it had no regular secondary structure . Nuclear magnetic resonance spectroscopy showed that a non-glycosylated synthetic peptide, the consensus sequence of the repetitive 19 amino acid peptide, also lacked secondary structure . The unstructured carrier polypeptide may facilitate proper folding and secretion of heterologous proteins attached to it. Curr Biol, 1995 Nov 1, 5(11), 1257 - 69 S-phase-promoting cyclin-dependent kinases prevent re-replication by inhibiting the transition of replication origins to a pre-replicative state; Dahmann C et al.; BACKGROUND: DNA replication and mitosis are triggered by activation of kinase complexes, each made up of a cyclin and a cyclin-dependent kinase (Cdk) . It had seemed possible that the association of Cdks with different classes of cyclins specifies whether S phase (replication) or M phase (mitosis) will occur . The recent finding that individual B-type cyclins (encoded by the genes CLB1-CLB6) can have functions in both processes in the budding yeast Saccharomyces cerevisiae casts doubt on this notion . RESULTS: S . cerevisiae strains lacking C1b1-C1b4 undergo DNA replication once but fail to enter mitosis . We have isolated mutations in two genes, SIM1 and SIM2 (SIM2 is identical to SEC72), which allow such cells to undergo an extra round of DNA replication without mitosis . The Clb5 kinase, which promotes S phase, remains active during the G2-phase arrest of cells of the parental strain, but its activity declines rapidly in sim mutants . Increased expression of the CLB5 gene prevents re-replication . Thus, a cyclin B-kinase that promotes DNA replication in G1-phase cells can prevent re-replication in G2-phase cells . Inactivation of C1b kinases by expression of the specific C1b-Cdk1 inhibitor p40SIC1 is sufficient to induce a prereplicative state at origins of replication in cells blocked in G2/M phase by nocodazole . Re-activation of C1b-Cdk1 kinases induces a second round of DNA replication . CONCLUSIONS: We propose that S-phase-promoting cyclin B--Cdk complexes prevent re-replication during S, G2 and M phases by inhibiting the transition of replication origins to a pre-replicative state . This model can explain both why origins 'fire' only once per S phase and why S phase is dependent on completion of the preceding M phase. Can J Microbiol, 1995 Nov, 41(11), 1035 - 7 Calcium ion influx during sporulation in the yeast Saccharomyces cerevisiae; Suizu T et al.; The changes in intra- and (or) extra-cellular concentrations of Ca2+, Mg2+, K+, and Na+ during sporulation of a MATa/MAT alpha diploid yeast of Saccharomyces cerevisiae were examined in a nutrition-deprived medium with potassium acetate . Among these, Ca2+ in external medium was preferentially incorporated into cells, and sporulation was induced when the magnitude of free Ca2+ gradient between cytosol {Ca2+}i and external medium {Ca2+}o reached more than 3 x 10(3) ({Ca2+}i/{Ca2+}o = 3.5 x 10(3)) . The result indicated that the meiosis and (or) sporulation signal of the yeast S . cerevisiae was generated through elevated Ca2+ influx rather than release from the internal Ca2+ stores. J Cell Biol, 1995 Nov, 131(4), 845 - 61 Saccharomyces cerevisiae cells execute a default pathway to select a mate in the absence of pheromone gradients; Dorer R et al.; During conjugation, haploid S . cerevisiae cells find one another by polarizing their growth toward each other along gradients of pheromone (chemotropism) . We demonstrate that yeast cells exhibit a second mating behavior: when their receptors are saturated with pheromone, wild-type a cells execute a default pathway and select a mate at random . These matings are less efficient than chemotropic matings, are induced by the same dose of pheromone that induces shmoo formation, and appear to use a site near the incipient bud site for polarization . We show that the SPA2 gene is specifically required for the default pathway: spa2 delta mutants cannot mate if pheromone concentrations are high and gradients are absent, but can mate if gradients are present . ste2 delta, sst2 delta, and far1 delta mutants are chemotropism-defective and therefore must choose a mate by using a default pathway; consistent with this deduction, these strains require SPA2 to mate . In addition, our results suggest that far1 mutants are chemotropism-defective because their mating polarity is fixed at the incipient bud site, suggesting that the FAR1 gene is required for inhibiting the use of the incipient bud site during chemotropic mating . These observations reveal a molecular relationship between the mating and budding polarity pathways. Biochemistry, 1995 Oct 24, 34(42), 13776 - 83 Properties and regulation of the catalytic domain of Ira2p, a Saccharomyces cerevisiae GTPase-activating protein of Ras2p; Parrini MC et al.; This work describes the biochemical characterization of the catalytic domain of Ira2p, a Saccharomyces cerevisiae GTPase-activating protein (GAP) regulating the RAS gene products . A fragment of 383 residues (amino acids 1644-2026) was produced in Escherichia coli as glutathione S-transferase fusion protein (GST-Ira2p-383) and highly purified (> 90%) by affinity chromatography . The affinity of Ras2p for the GST-fused Ira2p-383 was 18 microM and the maximal stimulation of the Ras2p GTPase activity 6,000 times . The Ira2p activity was confirmed to be strictly specific for Ras2p, no stimulatory effect on human c-H-ras p21 GTPase being detectable . Comparison with the GAP-like domain of mammalian p120-GAP and neurofibromin using yeast Ras2p as substrate showed that Ira2p-383 has an affinity and turnover intermediary between GAP-334 and NF1-414 . The activity of Ira2p-383 was strongly inhibited by monovalent and divalent salts . The simultaneous presence of the catalytic domains of Ira2p and the yeast GDP/GTP exchange factor Cdc25p induced on Ras2p a multiple-round reaction of GTP hydrolysis and GDP/GTP exchange, showing that it is possible to reconstitute in vitro a S . cerevisiae system suitable for the study of the regulation of the Ras2p GDP/GTP cycle . The tubulin partially inhibited (25%) the GAP activity of the Ira2p-383 . A larger Ira2p catalytic fragment, Ira2p-505 (amino acids 1549-2053), that showed the same Km for Ras2p as Ira2p-383, was also inhibited by tubulin to the same extent but with a higher affinity than Ira2p-383.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1995 Oct 20, 270(42), 25185 - 93 The yeast immunophilin Fpr3 is a physiological substrate of the tyrosine-specific phosphoprotein phosphatase Ptp1; Wilson LK et al.; The tyrosine-specific phosphoprotein phosphatase encoded by the Saccharomyces cerevisiae PTP1 gene dephosphorylates artificial substrates in vitro, but little is known about its functions and substrates in vivo . The presence of Ptp1 resulted in dephosphorylation of multiple tyrosine-phosphorylated proteins in yeast expressing a heterologous tyrosine-specific protein kinase, indicating that Ptp1 can dephosphorylate a broad range of substrates in vivo . Correspondingly, several proteins phosphorylated at tyrosine by endogenous protein kinases exhibited a marked increase in tyrosine phosphorylation in ptp1 mutant cells . One of these phosphotyrosyl proteins (p70) was also dephosphorylated in vitro when incubated with recombinant Ptp1 . p70 was purified to homogeneity; analysis of four tryptic peptides revealed that p70 is identical to the recently described FPR3 gene product, a nucleolarly localized proline rotamase of the FK506- and rapamycin-binding family . The identity of p70 with Fpr3 was confirmed in the demonstration that the abundance of tyrosine-phosphorylated p70 in ptp1 mutants was strictly correlated with the level of FPR3 expression; immobilized phosphotyrosyl Fpr3 was directly dephosphorylated by recombinant Ptp1 . Site-directed mutagenesis demonstrated that the site of tyrosine phosphorylation is Tyr-184, which resides within the nucleolin-like amino-terminal domain of Fpr3 . Protein kinase activities from yeast cell extracts can bind to and phosphorylate the immobilized amino-terminal domain of Fpr3 on serine, threonine, and tyrosine . Fpr3 represents the first phosphotyrosyl protein identified in S . cerevisiae that is not itself a protein kinase and is as yet the only known physiological substrate of Ptp1. J Biol Chem, 1995 Oct 6, 270(40), 23828 - 37 Functional complementation of a null mutation of the yeast Saccharomyces cerevisiae plasma membrane H(+)-ATPase by a plant H(+)-ATPase gene; de Kerchove d'Exaerde A et al.; In plants, the proton pump-ATPase (H(+)-ATPase) of the plasma membrane is encoded by a multigene family . The presence within an organ of several isoforms prevents a detailed enzymatic characterization of individual H(+)-ATPases . We therefore used the yeast Saccharomyces cerevisiae as a heterologous host for the expression of PMA2, an H(+)-ATPase isoform of Nicotiana plumbaginifolia . Yeast transformed by the plant pma2 was still able to grow under conditions where the yeast ATPase gene (PMA1) was either repressed or deleted . The transformed yeast strain was resistant to hygromycin, and its growth was prevented when the medium pH was lowered to 5.0 . The N . plumbaginifolia PMA2 expressed in S . cerevisiae has unusual low Km for ATP (23 microM) and high pH optimum (6.8) . Electron microscopic examination revealed PMA2 in internal structures of the karmellae type which proliferated when cell growth was arrested, either at a nonpermissive pH or at the stationary phase in a minimal medium . Under the latter conditions, subcellular fractionation on sucrose gradients revealed, in addition to the expected plant PMA2 peak linked to the plasma membrane fraction, low density peak containing PMA2 and KAR2, an endoplasmic reticulum marker . These observations suggest that the partial internal accumulation of PMA2 occurs in membranes derived from the endoplasmic reticulum and largely depends on growth conditions. FEBS Lett, 1995 Oct 2, 373(1), 51 - 5 Interaction of p85 subunit of PI 3-kinase with insulin and IGF-1 receptors analysed by using the two-hybrid system; Lamothe B et al.; Interaction of the p85 subunit of PI 3-kinase with the insulin receptor (IR) and the IGF-1 receptor (IGF-1R) was investigated using the two-hybrid system by assessing for his3 and lacZ activation in S . cerevisiae . The experiments were performed with the cytoplasmic beta domain (wild type or mutated) of IR and IGF-1R and p85 or its subdomains (N + C-SH2, N-SH2, C-SH2, SH3 + N-SH2) . The results of his3 activation indicated that p85, N + C-SH2 and C-SH2 interact with both IR beta and IGF-1R beta, whereas N-SH2 and SH3 + N-SH2 interact only with IR beta . Interaction of p85 and N+C-SH2 with IR beta (delta C-43) or IGF-1R beta(delta C-43) in which the C-terminal 43 amino acids (including the YXXM motif) were deleted, persisted . The internal binding site thus revealed was not altered by further mutating Y960/F for IR or Y950/F for IGF-1R . Activation of lacZ upon interaction of p85 with IR beta(delta C-43) was 4-fold less as compared to IR beta . This activation with p85 and IGF-1R beta was 4-fold less as compared to IR beta and was somewhat increased (2-fold) for IGF-1R beta (delta C-43) . Thus, the C-terminal domain in IGF-1R appears to exert a negative control on binding of p85 thereby providing a possible regulatory mechanism for direct activation of the PI 3-kinase pathway. J Bioenerg Biomembr, 1995 Oct, 27(5), 527 - 39 Decoupling of the bc1 complex in S . cerevisiae; point mutations affecting the cytochrome b gene bring new information about the structural aspect of the proton translocation; Bruel C et al.; Four mutations in the mitochondrial cytochrome b of S . cerevisiae have been characterized with respect to growth capacities, catalytic properties, ATP/2e- ratio, and transmembrane potential . The respiratory-deficient mutant G137E and the three pseudo-wild type revertants E137 + I147F, E137 + C133S, and E137 + N256K were described previously (Tron and Lemesle-Meunier, 1990; Di Rago et al., 1990a) . The mutant G137E is unable to grow on respiratory substrates but its electron transfer activity is partly conserved and totally inhibited by antimycin A . The secondary mutations restore the respiratory growth at variable degree, with a phosphorylation efficiency of 12-42% as regards the parental wild type strain, and result in a slight increase in the various electron transfer activities at the level of the whole respiratory chain . The catalytic efficiency for ubiquinol was slightly (G137E) or not affected (E137 + I147F, E137 + C133S, and E137 + N256K) in these mutants . Mutation G137E induces a decrease in the ATP/2e- ratio (50% of the W.T . value) and transmembrane potential (60% of the W.T . value) at the bc1 level, whereas the energetic capacity of the cytochrome oxidase is conserved . Secondary mutations I147F, C133S, and N256K partly restore the ATP/2e- ratio and the transmembrane potential at the bc1 complex level . The results suggest that a partial decoupling of the bc1 complex is induced by the cytochrome b point mutation G137E . In the framework of the protonmotive Q cycle, this decoupling can be explained by the existence of a proton wire connecting centers P and N in the wild type bc1 complex which may be amplified or uncovered by the G137E mutation when the bc1 complex is functioning. Genetics, 1995 Oct, 141(2), 619 - 27 The Drosophila meiotic recombination gene mei-9 encodes a homologue of the yeast excision repair protein Rad1; Sekelsky JJ et al.; Meiotic recombination and DNA repair are mediated by overlapping sets of genes . In the yeast Saccharomyces cerevisiae, many genes required to repair DNA double-strand breaks are also required for meiotic recombination . In contrast, mutations in genes required for nucleotide excision repair (NER) have no detectable effects on meiotic recombination in S . cerevisiae . The Drosophila melanogaster mei-9 gene is unique among known recombination genes in that it is required for both meiotic recombination and NER . We have analyzed the mei-9 gene at the molecular level and found that it encodes a homologue of the S . cerevisiae excision repair protein Rad1, the probable homologue of mammalian XPF/ERCC4 . Hence, the predominant process of meiotic recombination in Drosophila proceeds through a pathway that is at least partially distinct from that of S . cerevisiae, in that it requires an NER protein . The biochemical properties of the Rad1 protein allow us to explain the observation that mei-9 mutants suppress reciprocal exchange without suppressing the frequency of gene conversion. Antimicrob Agents Chemother, 1995 Oct, 39(10), 2204 - 9 Growth impairment resulting from expression of influenza virus M2 protein in Saccharomyces cerevisiae: identification of a novel inhibitor of influenza virus; Kurtz S et al.; The gene encoding M2, the ion channel-forming protein of influenza virus A, was expressed under the control of an inducible promoter in Saccharomyces cerevisiae . By using single and multicopy plasmids containing GAL promoter-M2 fusions, a correlation was observed between plasmid copy number and growth in medium inducing M2 expression . Cells expressing M2 from multicopy plasmids have reduced growth rates, suggesting that high levels of M2 are toxic to growth . The addition of amantadine, a compound known to block the ion channel activity of certain M2 alleles, restores the growth rates to wild-type levels in cells expressing an amantadine-susceptible allele of M2 but not an amantadine-resistant allele of M2, suggesting that M2 expression in S . cerevisiae results in the formation of functional M2 ion channels . Measurements of extracellular acidification by microphysiometry suggest that proton efflux in M2-expressing cells is altered and that the addition of amantadine permits the reestablishment of the proton gradient . The growth impairment phenotype resulting from M2 expression was used to develop a high-capacity screening assay which identified a novel inhibitor possessing an antiviral profile similar to that of amantadine. Environ Health Perspect, 1995 Oct, 103 Suppl 7, 129 - 33 Estrogens in unexpected places: possible implications for researchers and consumers; Feldman D et al.; Estrogenic activity originating in unexpected places was encountered on three occasions during an investigation of whether Saccharomyces cerevisiae synthesized estrogens . In each instance, estradiol found in the conditioned yeast culture medium originated from an exogenous source and was not synthesized by the yeast . In the first instance, yeast grown in the laboratory showed a time-dependent increase in estradiol in the conditioned medium . However, the culture medium supplement Bacto-peptone was found to contain large amounts of estrone . When added to yeast cultures in the form of YPD medium (yeast extract, Bacto-peptone, and dextrose), S . cerevisiae converted the estrone to estradiol leading to the accumulation of estradiol over time . In the second instance, commercially purchased S . cerevisiae grown in a molasses medium exhibited substantial amounts of estradiol . However, corn and beet molasses contained sufficient estrone and estradiol to account for the findings . As in the first instance, the yeast converted the estrone into estradiol . In the third instance, autoclaving culture medium in polycarbonate plastic flasks was found to cause an estrogenic substance to be added to the medium, whether yeast were present or not . It was determined that the autoclaving process leached bisphenol-A (BPA) out of the polycarbonate plastic . BPA was shown to bind to estrogen receptors and to induce estrogenic activity, including stimulation of MCF-7 breast cancer-cell proliferation and induction of the expression of progesterone receptors . The three instances highlight potential problems for investigators who might inadvertently add estrogens to experimental systems confounding their results . The BPA findings raise concerns about the possible addition of this estrogenic molecule to the food supply since polycarbonate plastic is used in myriad applications in the packaging of food and beverages . Although we are unaware of the substantial contamination of food products with BPA, we believe this possibility should be carefully investigated. Curr Genet, 1995 Oct, 28(5), 454 - 7 Localization of lipase genes on Candida rugosa chromosomes; Brocca S et al.; In the yeast Candida rugosa the lipase isozymes are encoded by a family of genes, five of which have been cloned and sequenced in our laboratory . In this paper we report on the identification and preliminary characterization of two new related sequences, thus extending this multigene family to seven members . The total DNA content of Candida cells was estimated by laser flow-cytometry at about 20 Mb . Eight chromosomes with sizes ranging between 100 kb and 2.1 Mb, as determined by comparison with S . cerevisiae chromosomal bands, were resolved by pulsed-field gel electrophoresis . The lipase-encoding genes were localized on chromosome I, therefore suggesting that they have originated through multiple duplication events of an ancestral gene. Curr Genet, 1995 Oct, 28(5), 437 - 40 Isolation of a Chlamydomonas reinhardtii telomere by functional complementation in yeast; Hails T et al.; We attempted to determine whether Chlamydomonas reinhardtii telomeres, which do not form G-quartet structures readily in vitro, are able to nucleate telomere addition in Saccharomyces cerevisiae . Restricted C . reinhardtii genomic DNA was ligated to a linear S . cerevisiae vector lacking a telomere . A C . reinhardtii telomere ligated to this unprotected end allowed vector replication as a linear DNA molecule in S . cerevisiae . DNA sequencing revealed common {T4AG3}n and variant T6AG3 and T5AG3 C . reinhardtii telomere repeats capped by S . cerevisiae telomere repeat units . The recognition of a C . reinhardtii telomere by the telomere maintenance machinery of S . cerevisiae is consistent with a common theme for telomere structure in organisms with divergent telomere repeats. Protein Expr Purif, 1995 Oct, 6(5), 700 - 6 Production of rat protein disulfide isomerase in Saccharomyces cerevisiae; Laboissiere MC et al.; Protein disulfide isomerase (PDI) is an abundant protein of the endoplasmic reticulum that catalyzes the oxidation of protein sulfhydryl groups and the isomerization and r