S. cerevisiae

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 reduction of protein disulfide bonds . Saccharomyces cerevisiae cells lacking PDI are inviable . PDI is a component of many different protein processing complexes, and the actual activity of PDI that is required for cell viability is unclear . A cDNA that codes for rat PDI fused to the alpha-factor pre-pro segment was expressed in a protease-deficient strain of S . cerevisiae under the control of an ADH2-GAPDH hybrid promoter . The cells processed the resulting protein and secreted it into the medium as a monomer, despite having a KDEL or HDEL sequence at its C-terminus . The typical yield of isolated protein was 2 mg per liter of culture . The catalytic activity of the PDI from S . cerevisiae was indistinguishable from that of PDI isolated from bovine liver . This expression system is unique in allowing the same plasmid to be used both to complement pdi1 delta S . cerevisiae and to produce PDI for detailed in vitro analyses . Correlations of the in vivo behavior and in vitro properties of PDI are likely to reveal structure-function relationships of biological importance.

FEMS Microbiol Lett, 1995 Oct 1, 132(1-2), 159 - 63
Effects of glucose, tetrapyrroles and protein kinase C activators on cell proliferation in cultures of Saccharomyces cerevisiae; Overgaard AK et al.; Saccharomyces cerevisiae was inoculated into a yeast nitrogen base with either glycerol or glucose as carbon source . Cell proliferation was followed by colony counts on agar medium . Cells in the glycerol-supplemented medium divided less than once in 10 days . When glucose, 6-deoxy-glucose or protoporphyrin IX was added, the cells had doubling times of about 24 h and increased in number to about 0.5 x 10(6) cells ml-1 . Addition of either of the protein kinase C activators oleoyl-acetyl-glycerol or phorbol-12-myristate-13-acetate did not activate cell proliferation in the glycerol medium . However, when (i) glucose was combined with either protoporphyrin IX or chlorophyllin, or (ii) either protoporphyrin IX or chlorophyllin was combined with either of the protein kinase C activators, the cells had doubling times of about 12 h . Hence, (i) glucose can act as both a carbon source and a signalling molecule for proliferation, and (ii) two systems are involved in activating cell proliferation in S . cerevisiae: one operating through a protein kinase C system and another through a guanylate cyclase system.

Microbiology, 1995 Oct, 141 ( Pt 10), 2693 - 7
Mapping of beta-1,2-linked oligomannosidic epitopes among glycoconjugates of Candida species; Cantelli C et al.; The distribution of beta-1,2-linked oligomannosides among glycoconjugates of various Candida species was investigated by Western blotting, using monoclonal and polyclonal antibodies which react with these epitopes . Expression of beta-1,2-linked oligomannosidic epitopes on a 14-18 kDa polydisperse antigen nonreactive with concanavalin A (ConA), previously identified as a C . albicans serotype A phospholipomannan (PLM), appeared to be restricted to C . albicans serotypes A and B (including var . C . stellatoidea types I and II) and C . tropicalis . In C . albicans, beta-1,2-linked oligomannosidic epitopes also appeared to be slightly associated with high molecular mass (> 100 kDa) polydisperse ConA-reactive mannoproteins . For all the other Candida strains investigated, belonging to the species C . parapsilosis, C . krusei, C . glabrata and C . robusta (S . cerevisiae), beta-1,2-linked oligomannosidic epitopes were found to be present in association with medium molecular mass (18-100 kDa) and high molecular mass ConA-reactive mannoproteins, giving reproducible labelling profiles that varied between species.

Appl Microbiol Biotechnol, 1995 Oct, 43(5), 844 - 9
Stable multicopy integration of vector sequences in Hansenula polymorpha; Gatzke R et al.; Plasmids without an origin of replication, but bearing the URA3 gene of Saccharomyces cerevisiae as a selective marker for transformation, are shown to replicate autonomously in Hansenula polymorpha, indicating that parts of the S . cerevisiae URA3 gene can fulfil an autonomous replication and stabilization function in H . polymorpha . Such plasmids, replicated in low copy number in monomeric conformation, could be rescued in E . coli, and showed a low mitotic stability under selective and non-selective conditions . Selective propagation of such transformants, however, led to the integration of plasmid sequences into the H . polymorpha genome . The integration event usually occurred in high copy number (approx . 30-50) at a single non-homologous site of the genome . The plasmid sequences were found to be present in tandem array and stable under non-selective conditions . In contrast, the use of homologous URA3 gene under similar conditions led to low-copy-number transformants.

Mol Cell Biol, 1995 Oct, 15(10), 5757 - 61
Recruiting TATA-binding protein to a promoter: transcriptional activation without an upstream activator; Xiao H et al.; The binding of TATA-binding protein (TBP) to the TATA element is the first step in the initiation of RNA polymerase II transcription from many promoters in vitro . It has been proposed that upstream activator proteins stimulate transcription by recruiting TBP to the promoter, thus facilitating the assembly of a transcription complex . However, the role of activator proteins acting at this step to stimulate transcription in vivo remains largely speculative . To test whether recruitment of TBP to the promoter is sufficient for transcriptional activation in vivo, we constructed a hybrid protein containing TBP of the yeast Saccharomyces cerevisiae fused to the DNA-binding domain of GAL4 . Our results show that TBP recruited by the GAL4 DNA-binding domain to promoters bearing a GAL4-binding site can interact with the TATA element and direct high levels of transcription . This finding indicates that binding of TBP to promoters in S . cerevisiae is a major rate-limiting step accelerated by upstream activator proteins.

Mol Cell Biol, 1995 Oct, 15(10), 5470 - 81
Regulation of cation transport in Saccharomyces cerevisiae by the salt tolerance gene HAL3; Ferrando A et al.; Dynamic regulation of ion transport is essential for homeostasis as cells confront changes in their environment . The gene HAL3 encodes a novel component of this regulatory circuit in the yeast Saccharomyces cerevisiae . Overexpression of HAL3 improves growth of wild-type cells exposed to toxic concentrations of sodium and lithium and suppresses the salt sensitivity conferred by mutation of the calcium-dependent protein phosphatase calcineurin . Null mutants of HAL3 display salt sensitivity . The sequence of HAL3 gives little clue to its function . However, alterations in intracellular cation concentrations associated with changes in HAL3 expression suggest that HAL3 activity may directly increase cytoplasmic K+ and decrease Na+ and Li+ . Cation efflux in S . cerevisiae is mediated by the P-type ATPase encoded by the ENA1/PMR24 gene, a putative plasma membrane Na+ pump whose expression is salt induced . Acting in concert with calcineurin, HAL3 is necessary for full activation of ENA1 expression . This functional complementarity is also reflected in the participation of both proteins in recovery from alpha-factor-induced growth arrest . Recently, HAL3 was isolated as a gene (named SIS2) which when overexpressed partially relieves loss of transcription of G1 cyclins in mutants lacking the protein phosphatase Sit4p . Therefore, HAL3 influences cell cycle control and ion homeostasis, acting in parallel to the protein phosphatases Sit4p and calcineurin.

Mol Cell Biol, 1995 Oct, 15(10), 5312 - 21
Segregation of unreplicated chromosomes in Saccharomyces cerevisiae reveals a novel G1/M-phase checkpoint; Toyn JH et al.; Saccharomyces cerevisiae dbf4 and cdc7 cell cycle mutants block initiation of DNA synthesis (i.e., are iDS mutants) at 37 degrees C and arrest the cell cycle with a 1C DNA content . Surprisingly, certain dbf4 and cdc7 strains divide their chromatin at 37 degrees C . We found that the activation of the Cdc28 mitotic protein kinase and the Dbf2 kinase occurred with the correct relative timing with respect to each other and the observed division of the unreplicated chromatin . Furthermore, the division of unreplicated chromatin depended on a functional spindle . Therefore, the observed nuclear division resembled a normal mitosis, suggesting that S . cerevisiae commits to M phase in late G1 independently of S phase . Genetic analysis of dbf4 and cdc7 strains showed that the ability to restrain mitosis during a late G1 block depended on the genetic background of the strain concerned, since the dbf4 and cdc7 alleles examined showed the expected mitotic restraint in other backgrounds . This restraint was genetically dominant to lack of restraint, indicating that an active arrest mechanism, or checkpoint, was involved . However, none of the previously described mitotic checkpoint pathways were defective in the iDS strains that carry out mitosis without replicated DNA, therefore indicating that the checkpoint pathway that arrests mitosis in iDS mutants is novel . Thus, spontaneous strain differences have revealed that S . cerevisiae commits itself to mitosis in late G1 independently of entry into S phase and that a novel checkpoint mechanism can restrain mitosis if cells are blocked in late G1 . We refer to this as the G1/M-phase checkpoint since it acts in G1 to restrain mitosis.

J Bacteriol, 1995 Oct, 177(19), 5440 - 6
Overexpression of Mal61p in Saccharomyces cerevisiae and characterization of maltose transport in artificial membranes; van der Rest ME et al.; For maltose uptake in Saccharomyces cerevisiae, multiple kinetic forms of transport as well as inhibition of transport by high concentrations of maltose at the trans side of the plasma membrane have been described . Most of these studies were hampered by a lack of genetically well-defined mutants and/or the lack of an artificial membrane system to study translocation catalysis in vitro . A genetically well-defined S . cerevisiae strain lacking the various MAL loci was constructed by gene disruption . Expression of the maltose transport protein (Mal61p) was studied by using various plasmid vectors that differed in copy number and/or type of promoter . The expression levels were quantitated by immunoblotting with antibodies generated against the N-terminal half of Mal61p . The levels of expression as well as the initial uptake rates were increased 20-fold compared with those in a yeast strain carrying only one chromosomal MAL locus . Similar results were obtained when the transport activities were compared in hybrid membranes of the corresponding strains . To generate a proton motive force, isolated membranes were fused with liposomes containing cytochrome c oxidase as a proton pump . Fusion was achieved by a cycle of freeze-thawing, after which the hybrid membranes were passed through a filter with a defined pore size to obtain unilamellar membrane vesicles . Proton motive force-driven maltose uptake, maltose efflux down the concentration gradient, and equilibrium exchange of maltose in the hybrid membranes vesicles have been analyzed . The data indicate that maltose transport by the maltose transporter is kinetically monophasic and fully reversible under all conditions tested.

Biochim Biophys Acta, 1995 Sep 27, 1252(1), 23 - 7
Circular dichroism and Fourier transform infrared spectroscopic studies on the secondary structure of Saccharomyces cerevisiae and Escherichia coli phospho enolpyruvate carboxykinases; Encinas MV et al.; The secondary structure of Saccharomyces cerevisiae and Escherichia coli phospho enolpyruvate (PEP) carboxykinases was quantitatively examined using circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies . From CD analyses, values of 24% alpha-helix and 38% beta-sheet were obtained for the E . coli enzyme, while the corresponding values for the S . cerevisiae PEP carboxykinase were 20% and 36% . Analysis of the amide I' infrared band indicated 20% alpha-helix and 36% beta-sheet for the S . cerevisiae enzyme, while for the E . coli protein values of 40% beta-sheet and between 9 and 36% alpha-helix could be inferred . It is concluded that the bacterial enzyme has more secondary structure elements than the yeast protein . No alteration of the CD or FTIR spectra was detected upon substrate or metal ion binding to any enzyme.

Biochem Biophys Res Commun, 1995 Sep 25, 214(3), 1051 - 9
pH and the cAMP-dependent protein kinase mediate growth phase induction of the cytochrome c oxidase subunit VI gene, COX6, in Saccharomyces cerevisiae; Wright RM et al.; Respiratory adaptation in Saccharomyces cerevisiae is a complex genetic program that ensures ATP synthesis in a glucose-depleted environment . ATP is generated during respiration by the mitochondrial electron transport chain which is induced by respiratory adaptation . We have studied the terminal enzyme in mitochondrial electron transport, cytochrome c oxidase, from S . cerevisiae . Because subunits in this multisubunit enzyme are coordinately regulated, we have focused upon the well characterized subunit VI gene, COX6 . In yeast, COX6 transcription is regulated by several factors thought to mediate respiratory adaptation including growth phase induction, oxygen dependence, and glucose repression . In the present study, we found that in addition to these known regulators, COX6 expression also depends upon pH and the cAMP-dependent protein kinase which may comprise additional factors signaling respiratory adaptation.

EMBO J, 1995 Sep 15, 14(18), 4599 - 608
The location and structure of double-strand DNA breaks induced during yeast meiosis: evidence for a covalently linked DNA-protein intermediate; Liu J et al.; We have determined the precise location and structure of the double-strand DNA breaks (DSBs) formed during Saccharomyces cerevisiae meiosis . Breaks were examined at two recombination hot spots in both wild-type and rad50S mutant cells . At both loci, breaks occurred at multiple, irregularly spaced sites in a approximately 150 nucleotide interval contained within an area of nuclease-hypersensitive chromatin . No consensus sequence could be discerned at or around break sites . Patterns of cleavage observed on individual strands indicated that breaks initially form with a two nucleotide 5' overhang . Broken strands from rad50S mutant cells contained tightly bound protein at their 5' ends . We suggest that, in S.cerevisiae, meiotic recombination is initiated by a DSB-forming activity that creates a covalently linked protein-DNA intermediate.

Proc Natl Acad Sci U S A, 1995 Sep 12, 92(19), 8871 - 5
Identification of human cyclin-dependent kinase 8, a putative protein kinase partner for cyclin C; Tassan JP et al.; Metazoan cyclin C was originally isolated by virtue of its ability to rescue Saccharomyces cerevisiae cells deficient in G1 cyclin function . This suggested that cyclin C might play a role in cell cycle control, but progress toward understanding the function of this cyclin has been hampered by the lack of information on a potential kinase partner . Here we report the identification of a human protein kinase, K35 {cyclin-dependent kinase 8 (CDK8)}, that is likely to be a physiological partner of cyclin C . A specific interaction between K35 and cyclin C could be demonstrated after translation of CDKs and cyclins in vitro . Furthermore, cyclin C could be detected in K35 immunoprecipitates prepared from HeLa cells, indicating that the two proteins form a complex also in vivo . The K35-cyclin C complex is structurally related to SRB10-SRB11, a CDK-cyclin pair recently shown to be part of the RNA polymerase II holoenzyme of S . cerevisiae . Hence, we propose that human K35(CDK8)-cyclin C might be functionally associated with the mammalian transcription apparatus, perhaps involved in relaying growth-regulatory signals.

Cell, 1995 Sep 8, 82(5), 841 - 7
A checkpoint regulates the rate of progression through S phase in S . cerevisiae in response to DNA damage; Paulovich AG et al.; We demonstrate that in S . cerevisiae the rate of ongoing S phase is slowed when the DNA is subjected to alkylation . Slowing of replication is dependent on the MEC1 and RAD53 genes, indicating that lesions alone do not slow replication in vivo and that the slowing is an active process . While it has been shown that a MEC1- and RAD53-dependent checkpoint responds to blocked replication or DNA damage by inhibiting the onset of mitosis, we demonstrate that this checkpoint must also have an additional target within S phase that controls replication rate . MEC1 is a homolog of the human ATM gene, which is mutated in ataxia telangiectasia (AT) patients . Like mec1 yeast, AT cells are characterized by damage-resistant DNA synthesis, highlighting the congruence of the yeast and mammalian systems.

Cell, 1995 Sep 8, 82(5), 823 - 9
TEL1, a gene involved in controlling telomere length in S . cerevisiae, is homologous to the human ataxia telangiectasia gene; Greenwell PW et al.; Yeast chromosomes terminate in tracts of simple repetitive DNA (poly{G1-3T}) . Mutations in the gene TEL1 result in shortened telomeres . Sequence analysis of TEL1 indicates that it encodes a very large (322 kDa) protein with amino acid motifs found in phosphatidylinositol/protein kinases . The closest homolog to TEL1 is the human ataxia telangiectasia gene.

Cell, 1995 Sep 8, 82(5), 831 - 40
TEL1, an S . cerevisiae homolog of the human gene mutated in ataxia telangiectasia, is functionally related to the yeast checkpoint gene MEC1; Morrow DM et al.; Patients with the genetic disorder ataxia telangiectasia (AT) have mutations in the AT mutated (ATM) gene, which is homologous to TEL1 and the checkpoint gene MEC1 . A tel1 deletion mutant, unlike a mec1 deletion, is viable and does not exhibit increased sensitivity to DNA-damaging agents . However, increased dosage of TEL1 rescues sensitivity of a mec1 mutant, mec1-1, to DNA-damaging agents and rescues viability of a mec1 disruption . mec1-1 tel1 delta 1 double mutants are synergistically sensitive to DNA-damaging agents, including radiomimetic drugs . These data indicate that TEL1 and MEC1 are functionally related and that functions of the ATM gene are apparently divided between at least two S . cerevisiae homologs.

Curr Genet, 1995 Sep, 28(4), 367 - 73
Identification of the heterothallic mutation in HO-endonuclease of S . cerevisiae using HO/ho chimeric genes; Meiron H et al.; HO-endonuclease initiates a mating-type switch in the yeast S . cerevisiae by making a double-strand cleavage in the DNA of the mating-type gene, MAT . Heterothallic strains of yeast have a stable mating type and contain a recessive ho allele . Here we report the sequence of the ho allele; ho has four point mutations all of which encode for substitute amino acids . The fourth mutation is a leucine to histidine substitution within a presumptive zinc finger . Chimeric HO/ho genes were constructed in vivo by converting different parts of the sequence of the genomic ho allele to the HO sequence by gene conversion . HO activity was assessed by three bioassays: a mating-type switch, extinction of expression of an a-specific reporter gene, and the appearance of Canr Ade- papillae resulting from excision of an engineered Ty element containing the HO-endonuclease target site and a SUP4 degrees gene . We found that the replacement of the fourth point mutation in ho to the HO sequence restored HO activity to the chimeric endonuclease.

J Bacteriol, 1995 Sep, 177(17), 5009 - 15
Suppression of Escherichia coli alkB mutants by Saccharomyces cerevisiae genes; Wei YF et al.; The alkB gene is one of a group of alkylation-inducible genes in Escherichia coli, and its product protects cells from SN2-type alkylating agents such as methyl methanesulfonate (MMS) . However, the precise biochemical function of the AlkB protein remains unknown . Here, we describe the cloning, sequencing, and characterization of three Saccharomyces cerevisiae genes (YFW1, YFW12, and YFW16) that functionally complement E . coli alkB mutant cells . DNA sequence analysis showed that none of the three gene products have any amino acid sequence homology with the AlkB protein . The YFW1 and YFW12 proteins are highly serine and threonine rich, and YFW1 contains a stretch of 28 hydrophobic residues, indicating that it may be a membrane protein . The YFW16 gene turned out to be allelic with the S . cerevisiae STE11 gene . STE11 is a protein kinase known to be involved in pheromone signal transduction in S . cerevisiae; however, the kinase activity is not required for MMS resistance because mutant STE11 proteins lacking kinase activity could still complement E . coli alkB mutants . Despite the fact that YFW1, YFW12, and YFW16/STE11 each confer substantial MMS resistance upon E . coli alkB cells, S . cerevisiae null mutants for each gene were not MMS sensitive . Whether these three genes provide alkylation resistance in E . coli via an alkB-like mechanism remains to be determined, but protection appears to be specific for AlkB-deficient E . coli because none of the genes protect other alkylation-sensitive E . coli strains from killing by MMS.

J Biol Chem, 1995 Sep 1, 270(35), 20724 - 9
Cell cycle regulation of a novel DNA binding complex in Saccharomyces cerevisiae with E2F-like properties; Vemu S et al.; Using a biochemical approach, we have detected an activity in Saccharomyces cerevisiae extract that displays the same DNA binding specificity as the mammalian E2F transcription factor and interacts with TTTCGCGC promoter elements . Additional studies revealed that this factor, termed SCELA (S . cerevisiae E2F-like activity), also binds to the closely related SCB promoter sequences . SCB sites (consensus: TTTCGTG) are involved in the cell cycle regulation of several S . cerevisiae cyclin genes and have been shown to interact with the heterodimeric yeast Swi4-Swi6 complex . However, genetic studies clearly demonstrate that SCELA is not related to Swi4 or Swi6 . These experiments imply that SCB sites are able to interact with at least two activities: Swi4-Swi6 and SCELA . Because SCB sites are critical for the periodic activation of cell cycle genes, we asked whether SCELA is regulated during yeast cell cycle . Employing a temperature-sensitive strain, we were able to demonstrate that the DNA binding activity of SCELA oscillates during the cell cycle and reaches its maximum at the transition between the G1 and S phases . Preliminary studies suggest that this fluctuation is mediated by phosphorylation/dephosphorylation events . Further characterization of SCELA by UV cross-linking experiments indicate a molecular mass of 47 kDa for this activity . In addition, we present evidence strongly suggesting that SCELA is actually the DNA binding moiety of a large 300-kDa protein complex . Together, these studies firmly indicate that SCELA (as part of a larger complex) plays a critical role in cell cycle regulation of SCB-containing genes, such as CLN cyclins and HO endonuclease . This hypothesis is consistent with other studies that conclude that the SCB-mediated cell cycle oscillation of CLN cyclins and HO requires activities that are distinct from Swi4-Swi6 . Finally, it is worth mentioning that the similarities between SCELA and E2F, which is a crucial component in mammalian cell cycle regulation, extend well beyond the DNA binding specificity . In analogy to E2F, SCELA oscillates during the cell cycle, interacts with other cellular activities, and binds to promoter elements that are known mediators of cell cycle control . We will discuss possible functions for SCELA in yeast cell cycle regulation and its relationship to E2F.

Biochem J, 1995 Sep 1, 310 ( Pt 2), 605 - 14
Recombinant soluble human tissue factor secreted by Saccharomyces cerevisiae and refolded from Escherichia coli inclusion bodies: glycosylation of mutants, activity and physical characterization; Stone MJ et al.; Tissue factor (TF) is the cell-surface transmembrane receptor that initiates both the extrinsic and intrinsic blood coagulation cascades . The abilities of TF to associate with Factor VIIa and Factor X in a ternary complex and to enable proteolytic activation of Factor X by Factor VIIa reside in the extracellular domain of TF . We describe the expression of the surface domain of TF (truncated TF, tTF) in both Saccharomyces cerevisiae and Escherichia coli and the biochemical and physical characterization of the recombinant proteins . Wild-type tTF and several glycosylation-site mutants were secreted efficiently by S . cerevisiae under the control of the yeast prepro-alpha-signal sequence; the T13A,N137D double mutant was the most homogeneous variant expressed in milligram quantities . Wild-type tTF was expressed in a non-native state in E . coli inclusion bodies as a fusion protein with a poly(His) leader . The fusion protein could be fully renatured and the leader removed by proteolysis with thrombin; the correct molecular mass (24,729 Da) of the purified protein was confirmed by electrospray mass spectrometry . Recombinant tTFs from yeast, E . coli and Chinese hamster ovary cells were identical in their abilities to bind Factor VIIa, to enhance the catalytic activity of Factor VIIa and to enhance the proteolytic activation of Factor X by Factor VIIa . Furthermore, CD, fluorescence emission and NMR spectra of the yeast and E . coli proteins indicated that these proteins are essentially identical structurally.

Mol Cell Biol, 1995 Sep, 15(9), 4661 - 71
Characterization of the five replication factor C genes of Saccharomyces cerevisiae; Cullmann G et al.; Replication factor C (RFC) is a five-subunit DNA polymerase accessory protein that functions as a structure-specific, DNA-dependent ATPase . The ATPase function of RFC is activated by proliferating cell nuclear antigen . RFC was originally purified from human cells on the basis of its requirement for simian virus 40 DNA replication in vitro . A functionally homologous protein complex from Saccharomyces cerevisiae, called ScRFC, has been identified . Here we report the cloning, by either peptide sequencing or by sequence similarity to the human cDNAs, of the S . cerevisiae genes RFC1, RFC2, RFC3, RFC4, and RFC5 . The amino acid sequences are highly similar to the sequences of the homologous human RFC 140-, 37-, 36-, 40-, and 38-kDa subunits, respectively, and also show amino acid sequence similarity to functionally homologous proteins from Escherichia coli and the phage T4 replication apparatus . All five subunits show conserved regions characteristic of ATP/GTP-binding proteins and also have a significant degree of similarity among each other . We have identified eight segments of conserved amino acid sequences that define a family of related proteins . Despite their high degree of sequence similarity, all five RFC genes are essential for cell proliferation in S . cerevisiae . RFC1 is identical to CDC44, a gene identified as a cell division cycle gene encoding a protein involved in DNA metabolism . CDC44/RFC1 is known to interact genetically with the gene encoding proliferating cell nuclear antigen, confirming previous biochemical evidence of their functional interaction in DNA replication.

J Cell Biol, 1995 Sep, 130(6), 1373 - 85
Two microtubule-associated proteins required for anaphase spindle movement in Saccharomyces cerevisiae; Pellman D et al.; In many eucaryotic cells, the midzone of the mitotic spindle forms a distinct structure containing a specific set of proteins . We have isolated ASE1, a gene encoding a component of the Saccharomyces cerevisiae spindle midzone . Strains lacking both ASE1 and BIK1, which encodes an S . cerevisiae microtubule-associated protein, are inviable . The analysis of the phenotype of a bik1 ase1 conditional double mutant suggests that BIK1 and ASE1 are not required for the assembly of a bipolar spindle, but are essential for anaphase spindle elongation . The steady-state levels of Ase1p are regulated in a manner that is consistent with a function during anaphase: they are low in G1, accumulate to maximal levels after S phase and then drop as cells exit mitosis . Components of the spindle midzone may therefore be required in vivo for anaphase spindle movement . Additionally, anaphase spindle movement may depend on a dedicated set of genes whose expression is induced at G2/M.

Plant Mol Biol, 1995 Sep, 28(6), 997 - 1009
Expression of arginine decarboxylase is induced during early fruit development and in young tissues of Pisum sativum (L.); Perez-Amador MA et al.; A cDNA coding for arginine decarboxylase (ADC, EC 4.1.1.19) has been isolated from a cDNA library of parthenocarpic young fruits of Pisum sativum (L.) . The deduced aminoacid sequence is 74%, 46% and 35% identical to ADCs from tomato, oat and Escherichia coli, respectively . When the pea ADC cDNA was put under the control of the galactose inducible yeast promoter CYC1-GAL10 and introduced into Saccharomyces cerevisiae, it conferred galactose-regulated expression of the ADC activity . The ADC activity expressed in S . cerevisiae was inhibited 99% by alpha-DL-difluoromethylarginine (DFMA), a specific inhibitor of ADC activity . No activity was detected in the untransformed S . cerevisiae, nor when it was transformed with an antisense ADC construct . This provides direct evidence that the ADC cDNA from pea encoded a functional, specific ADC activity and that S . cerevisiae is able to process correctly the protein . In the pea plant, gene expression of the ADC is high in young developing tissues like shoot tips, young leaflets and flower buds . Fully expanded leaflets and roots have much lower, but still detectable, levels of the ADC transcript . In the ovary and fruit, they are developmentally regulated, showing high levels of expression during the early stages of fruit growth, which in pea is mainly due to cell expansion . The observed changes in the steady-state levels of ADC mRNA alone, however, cannot account for the differences in ADC activity suggesting that other regulatory mechanisms must be acting.

DNA Cell Biol, 1995 Sep, 14(9), 789 - 94
Characterization of rat neuronal nitric oxide synthase expressed in Saccharomyces cerevisiae; Black SM et al.; A cDNA encoding rat neuronal nitric oxide synthase (nNOS) was cloned into the yeast expression vector pMA56 to generate pA379 . Transformation of Saccharomyces cerevisiae strain BJ2168 with this plasmid resulted in the synthesis of nNOS at levels of 0.5-1.0 mg/liter . The protein is localized in the cytosol and is catalytically active as determined by the conversion of {3H}-L-arginine to {3H}-L-citrulline and NO . The enzyme was purified by calmodulin-Sepharose affinity chromatography and its catalytic activity was found to be both calcium and calmodulin dependent . Overexpression of nNOS in S . cerevisiae and purification of the recombinant protein will facilitate detailed characterization of this important enzyme.

Gene, 1995 Aug 30, 162(1), 111 - 5
Sequence of the met-10+ locus of Neurospora crassa: homology to a sequence of unknown function in Saccharomyces cerevisiae chromosome 8; Chow CM et al.; We have determined the sequence of the Neurospora crassa met-10+ gene and its flanking regions, and have isolated and analyzed cDNA clones for this region . We have identified two closely linked genes transcribed in the same orientation . The met-10+ gene is the downstream gene; an open reading frame (ORF) derived from five exons encodes a 475-amino-acid protein . The deduced protein lacks similarity to other characterized proteins . However, it exhibits a strong similarity to the product of an ORF of unknown function on Saccharomyces cerevisiae chromosome 8 . This sequence similarity suggests functional equivalence and should facilitate identification of the function of met-10+ using gene disruptions in S . cerevisiae.

Proc Natl Acad Sci U S A, 1995 Aug 29, 92(18), 8448 - 52
Isolation and characterization of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: identification of residues important for toxicity; Hur Y et al.; Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the 28S rRNA of eukaryotic ribosomes . PAP has potent antiviral activity against many plant and animal viruses, including human immunodeficiency virus . We describe here development of a positive selection system to isolate PAP mutants with reduced toxicity . In vitro translation in the presence or absence of microsomal membranes shows that PAP is synthesized as a precursor and undergoes at least two different proteolytic processing steps to generate mature PAP . The PAP cDNA was placed under control of the galactose-inducible GAL1 promoter and transformed into Saccharomyces cerevisiae . Induction of PAP expression was lethal to yeast . The PAP expression plasmid was mutagenized and plasmids encoding mutant PAP genes were identified by their failure to kill S . cerevisiae . A number of mutant alleles were sequenced . In one mutant, a point mutation at Glu-177 inactivated enzymatic function in vitro, suggesting that this glutamic acid residue is located at or near the catalytic site . Mutants with either point mutations near the N terminus or a nonsense mutation at residue 237 produced protein that was enzymatically active in vitro, suggesting that the toxicity of PAP is not due solely to enzymatic activity . Toxicity of PAP appears to be a multistep process that involves possibly different domains of the protein.

J Biol Chem, 1995 Aug 25, 270(34), 20090 - 7
Comparison of the reactivity of tetradecenoic acids, a triacsin, and unsaturated oximes with four purified Saccharomyces cerevisiae fatty acid activation proteins; Knoll LJ et al.; Saccharomyces cerevisiae contains at least five acyl-CoA synthetases (fatty acid activation proteins, or Faaps) . Four FAA genes have been recovered to date . Recent genetic studies indicate that Faa1p and Faa4p are involved in the activation of imported fatty acids, while Faa2p activates endogenous pools of fatty acids . We have now purified Faa4p from S . cerevisiae and compared its fatty acid substrate specificity in vitro with the specificities of purified Faa1p, Faa2p, and Faa3p . Among C8-C18 saturated fatty acids, Faa4p and Faa1p both prefer C14:0 . Surveys of C14 fatty acids with single cis-double bonds at C2-C12 indicated that Faa4p and Faa1p prefer Z9-tetradecenoic acid, although Faa4p's preference is much greater and also evident in C16 and C18 fatty acids . Faa4p's selectivity for fatty acids with a C9-C10 cis-double bond is a feature it shares with Faa3p and is notable since in yeast Ole1p, a microsomal cis-delta 9 desaturase, accounts for de novo production of monoenoic acyl-CoAs from saturated acyl-CoA substrates . Faa4p has no detectable acyl-CoA synthetase activity when incubated with tetradecenoic acids having a trans-double bond at C2-3, C4-5, C5-6, C6-7, C7-8, or C9-10 . Faa3p can only use E9-tetradecenoic acid as a substrate, while E4-, E6- and E9-tetradecenoic acids can be used by Faa1p and Faa2p . E2-tetradecenoic acid is an Faap inhibitor, with Faa2p exhibiting the greatest sensitivity (IC50 = 2.6 +/- 0.2 microM) . Triacsin C (1-hydroxy-3-(E,E,E,2',4',7'- undecatrienylidine)-1,2,3-triazene) has trans-double bonds at positions that correspond to those in E2-, E5-, and E7-tetradecenoic acids . This compound is a potent inhibitor of Faa2p (Ki = 15 +/- 1 nM; competitive with fatty acid), less potent against Faa4p (Ki = 2 microM), and not active against Faa1p or Faa3p (IC50 > 500 microM) . Analysis of an n-tetradecanal plus a series of oximes (tridecanal oxime, 1-azadeca-1,3,5-trienol, and 1-azaundeca-1,3,5-trienol) indicated that the combination of an azenol moiety (R-CH = N-OH) plus adjacent unsaturation are critical for triacsin C's selective inhibition of Faa2p . Triacsin C and oxime derivatives appear to be very useful for defining differences in molecular recognition among S . cerevisiae acyl-CoA synthetases . The > 25,000-fold range in the inhibitory effects of triacsin C on these four Faaps suggests that it may be possible to develop other selective inhibitors of eukaryotic acyl-CoA synthetases.

Biochem J, 1995 Aug 15, 310 ( Pt 1), 279 - 84
The interaction of coatomer with inositol polyphosphates is conserved in Saccharomyces cerevisiae; Ali N et al.; Coatomer is an oligomeric complex of coat proteins that regulates vesicular traffic through the Golgi complex and from the Golgi to the endoplasmic reticulum {Pelham (1994) Cell 79, 1125-1127} . We have investigated whether the binding of InsP6 to mammalian coatomer {Fleischer, Xie, Mayrleitner, Shears and Fleischer (1994) J . Biol . Chem . 269, 17826-17832} is conserved in the genetically amenable model Saccharomyces cerevisiae . We have isolated coatomer from S . cerevisiae and found it to bind InsP6 at two apparent classes of binding sites (KD1 = 0.8 +/- 0.2 nM; KD2 = 361 +/- 102 nM) . Ligand specificity was studied by displacing 4.5 nM {3H}InsP6 from coatomer with various Ins derivatives . The following IC50 values (nM) were obtained: myo-InsP6 = 6; bis(diphospho)inositol tetrakisphosphate = 6; diphosphoinositol pentakisphosphate = 6; scyllo-InsP6 = 12; Ins(1,3,4,5,6)P5 = 13; Ins(1,2,4,5,6)P5 = 22; Ins(1,3,4,5)P4 = 22; 1-O-(1,2-di-O-octanoyl-sn-glycero-3-phospho)-D-Ins(3,4,5)P3 = 290 . Less than 10% of the 3H label was displaced by 1 microM of either Ins(1,4,5)P3 or inositol hexakis-sulphate . A cell-free lysate of S . cerevisiae synthesized diphosphoinositol polyphosphates (PP-InsPn) from InsP6, but our binding data, plus measurements of the relative levels of inositol polyphosphates in intact yeast {Hawkins, Stephens and Piggott (1993) J . Biol . Chem . 268, 3374-3383}, indicate that InsP6 is the major physiologically relevant ligand . Thus a reconstituted vesicle trafficking system using coatomer and other functionally related components isolated from yeast should be a useful model for elucidating the functional significance of the binding of InsP6 by coatomer.

FEBS Lett, 1995 Aug 14, 370(1-2), 41 - 5
Glycoprotein biosynthesis in Saccharomyces cerevisiae: ngd29, an N-glycosylation mutant allelic to och1 having a defect in the initiation of outer chain formation; Lehle L et al.; Outer chain glycosylation in Saccharomyces cerevisiae leads to heterogeneous and immunogenic asparagine-linked saccharide chains containing more than 50 mannose residues on secreted glycoproteins . Using a {3H}mannose suicide selection procedure a collection of N-glycosylation defective mutants (designated ngd) was isolated . One mutant, ngd29, was found to have a defect in the initiation of the outer chain and displayed a temperature growth sensitivity at 37 degrees C allowing the isolation of the corresponding gene by complementation . Cloning, sequencing and disruption of NGD29 showed that it is a non lethal gene and identical to OCH1 . It complemented both the glycosylation and growth defect . Membranes isolated from an ngd29 disruptant or an ngd29mnn1 double mutant were no longer able, in contrast to membranes from wild type cells, to transfer mannose from GDPmannose to Man8GlcNAc2, the in vivo acceptor for building up the outer chain . Heterologous expression of glucose oxidase from Aspergillus niger in an ngd29mnn1 double mutant produced a secreted uniform glycoprotein with exclusively Man8GlcNAc2 structure that in wild type yeast is heavily hyperglycosylated . The data indicate that this mutant strain is a suitable host for the expression of recombinant glycoproteins from different origin in S . cerevisiae to obtain mammalian oligomannosidic type N-linked carbohydrate chains.

Nucleic Acids Res, 1995 Aug 11, 23(15), 2831 - 6
Identity elements of tRNA(Thr) towards Saccharomyces cerevisiae threonyl-tRNA synthetase; Nameki N; Identity elements of tRNA(Thr) towards Saccharomyces cerevisiae threonyl-tRNA synthetase were examined using in vitro transcripts . By mutation studies, a marked decrease in aminoacylation with threonine showed that the first base pair in the acceptor stem and the second and third positions of the anticodon are major identity elements of tRNA(Thr), which are essentially the same as those of Escherichia coli tRNA(Thr) . Base substitution of the discriminator base, A73, by G73 or C73 impaired the threonine accepting activity, but not that by U73, suggesting that this position contributes to discrimination from other tRNAs possessing G73 or C73 . No effects on aminoacylation were observed with substitutions at the second base pair in the acceptor stem . These are in contrast to E.coli tRNA(Thr) where the second base pair is required for the specific aminoacylation, with the discriminator base playing no roles . Of several mutations at the third base pair in the acceptor stem, only the G3-U70 mutation impaired the activity, suggesting that the G3-U70 wobble pair, the identity determinant of tRNAAla, acts as a negative element for threonyl-tRNA synthetase . These findings indicate that while the first base pair in the acceptor stem and the anticodon nucleotides have been retained as major recognition sites between S . cerevisiae and E.coli tRNA(Thr), the mechanism by which the synthetase recognizes the vicinity of the top of the acceptor stem seems to have diverged with the species.

Cell, 1995 Aug 11, 82(3), 495 - 506
Identification of a novel nuclear pore-associated protein as a functional target of the HIV-1 Rev protein in yeast; Stutz F et al.; The HIV-1 Rev protein increases the cytoplasmic levels of unspliced and singly spliced target transcripts in metazoan systems . Based on experiments that indicate a similar function of Rev in the yeast S . cerevisiae, we have identified a yeast protein that interacts with the effector domain of Rev . The protein, Rip1p, is a novel small nucleoporin-like protein, some of which is associated with nuclear pores . Its closest known yeast relative is a nuclear pore component also implicated in mRNA transport from nucleus to cytoplasm . Analysis of strains that overexpress Rip1p or that are deleted for the RIP1 gene show that Rip1p is important for the effect of Rev on gene expression, indicating that the physical interaction is of functional significance in vivo . The results suggest that Rev directly promotes the cytoplasmic transport of suitable transcripts by targeting them to the nuclear pore.

Cell, 1995 Aug 11, 82(3), 425 - 34
Rbl2p, a yeast protein that binds to beta-tubulin and participates in microtubule function in vivo; Archer JE et al.; Genetic configurations resulting in high ratios of beta-tubulin to alpha-tubulin are toxic in S . cerevisiae, causing microtubule disassembly and cell death . We identified three non-tubulin yeast genes that, when overexpressed, rescue cells from excess beta-tubulin . One, RBL2, rescues beta-tubulin lethality as efficiently as does alpha-tubulin . Rbl2p binds to beta-tubulin in vivo . Deficiencies or excesses of either Rbl2p or alpha-tubulin affect microtubule-dependent functions in a parallel fashion . Rbl2p has functional homology with murine cofactor A, a protein important for in vitro assays of beta-tubulin folding . The results suggest that Rbl2p participates in microtubule morphogenesis but not in the assembled polymer.

J Biol Chem, 1995 Aug 4, 270(31), 18491 - 9
Cloning and characterization of a Saccharomyces cerevisiae gene encoding the low molecular weight protein-tyrosine phosphatase; Ostanin K et al.; The low molecular weight protein-tyrosine phosphatase (low M(r) PTPase) is an 18-kDa cytoplasmic enzyme of unknown function that has been previously found in several vertebrates . Using an oligonucleotide probe derived from the active site sequence of the mammalian low M(r) PTPases, a Saccharomyces cerevisiae gene that encodes a homolog of this enzyme was cloned by low stringency hybridization . This gene, LTP1, together with a neighboring gene, TKL1, is shown to be located on the right arm of chromosome XVI . The deduced amino acid sequence of its 161-amino acid residue product shows a 39% average identity with that of the mammalian enzymes . The yeast Ltp1 protein was expressed in Escherichia coli, purified to homogeneity, and shown to possess PTPase activity . The recombinant Ltp1 efficiently hydrolyzes phosphotyrosine and a phosphotyrosine-containing peptide, Tyr531-fyn, but it shows low activity toward phosphoserine and phosphothreonine . The catalytic activity of Ltp1 toward a number of substrates was approximately 30-fold lower than the corresponding values measured for the bovine low M(r) PTPase . However, the yeast enzyme was markedly activated by adenine and some purine nucleosides and nucleotides, including cAMP and cGMP . In the case of adenine, the activity of Ltp1 was increased by approximately 30-fold . The high degree of evolutionary conservation of the low M(r) PTPases implies a significant role for this enzyme . However, neither the disruption of the LTP1 gene nor an approximately 10-fold overexpression of its product in S . cerevisiae caused any apparent phenotypic changes under the conditions tested . No proteins related to Ltp1 could be detected in extracts of the ltp1 null mutant, either by immunoblotting or by gel-filtration analysis accompanied by extended kinetic assays, consistent with the conclusion that LTP1 is the only low M(r) PTPase-encoding gene in S . cerevisiae.

J Biol Chem, 1995 Aug 4, 270(31), 18408 - 12
Identification of YHR068w in Saccharomyces cerevisiae chromosome VIII as a gene for deoxyhypusine synthase . Expression and characterization of the enzyme; Kang KR et al.; Deoxyhypusine synthase catalyzes the formation of deoxyhypusine, the first step in hypusine biosynthesis . Amino acid sequences of five tryptic peptides from rat deoxyhypusine synthase were found to match partially the deduced amino acid sequence of the open reading frame of gene YHR068w of Saccharomyces cerevisiae chromosome VIII (AC:U00061) . In order to determine whether the product of this gene corresponds to yeast deoxyhypusine synthase,a 1.17-kilobase pair cDNA with an identical nucleotide sequence to that of the YHR068w coding region was obtained from S . cerevisiae cDNA by polymerase chain reaction and was expressed in Escherichia coli B strain BL21 (DE3) . The recombinant protein was found mostly in the E . coli cytosol fraction and comprised approximately 20% of the total soluble protein . The purified form of the expressed protein effectively catalyzed the formation of deoxyhypusine in yeast eIF-5A precursors as well as in human precursor and in those from Chinese hamster ovary cells . The molecular mass of the enzyme was estimated to be 172,000 +/- 4,300 Da by equilibrium centrifugation . The mass of its polypeptide subunit was determined to be approximately 43,000 Da, in close agreement with that calculated for the coding region of the YHRO68w gene . These findings show that this gene is a coding sequence for yeast deoxyhypusine synthase and that the product of this gene exists in a tetrameric form.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1995 Aug, 17(4), 274 - 80
{Interleukin-2 gene cloning and expression in S . cerevisiae}; Zhang X et al.; Mature human Interleukin-2 gene was amplified from IL-2 cDNA by PCR methods . The PCR product was cloned into pUC12 plasmid at Sma I site, then precisely engineered into an intermidiate vector pSK43SB which were digested with Hind III, mung bean nuclease, and Sal I . Added BamH I linker to Cla I site of pSK43SB-IL2 to produce one more BamH I site, then digested pSK43DB-IL2 with restriction enzyme BamH I, the BamH I fragment with an alpha-factor leading sequence, IL2 gene and cycl terminater were produced . It was then cloned into a typical high efficiency episomal expression vector YEpHc8 . Human IL-2 protein was purified from supernatant secreted by yeast transformants of YEpHc8-IL2 leading by alpha-factor promoter with IL-2 right translational reading frame and accurate cleavage site . High level expression of IL-2 with highly activity has been purified . A sugar specific silver staining method and 3H sugar labelling experiment has shown the detection of carbohydrate component.

Yeast, 1995 Aug, 11(10), 967 - 74
The sequence of a 44 420 bp fragment located on the left arm of chromosome XIV from Saccharomyces cerevisiae; Bergez P et al.; We have determined the complete nucleotide sequence of a 44 420 bp DNA fragment from chromosome XIV of Saccharomyces cerevisiae . The sequence data revealed 23 open reading frames (ORFs) larger than 300 bp, covering 73.5% of the sequence . The ORFs N2418, N2428, N2441, N2474 and N2480 correspond to previously sequenced S . cerevisiae genes coding respectively for the mitochondrial import protein Mas5, the nucleolar protein Nop2, the outer mitochondrial membrane porin Por1, the cytochrome c oxidase polypeptide VA precursor CoxA and the yeast protein tyrosine phosphatase Msg5 . Translation products of three other ORFs N2406, N2411 and N2430 exhibit similarity to previously known S . cerevisiae proteins: the ribosomal protein YL9A, the protein Nca3 involved in the mitochondrial expression of subunits 6 and 8 of the ATP synthase and actin; in addition N2505 presents strong similarity to an ORF of chromosome IX . The predicted protein products of ORFs N2417 and N2403 present similarities with domains from proteins of other organisms: the Candida maltosa cycloheximide-resistance protein, the human interleukin enhancer-binding factor (ILF-2) . The 12 remaining ORFs show no significant similarity to known proteins . In addition, we have detected a DNA region very similar to the yeast transposon Ty 1-15 of which insertion has disrupted a tRNA(Asp) gene.

Yeast, 1995 Aug, 11(10), 953 - 9
The effect of ethanol and specific growth rate on the lipid content and composition of Saccharomyces cerevisiae grown anaerobically in a chemostat; Arneborg N et al.; The effects of produced ethanol and specific growth rate on the lipid content and composition of Saccharomyces cerevisiae CBS 2806 were studied using anaerobic chemostat cultures . The cells adapted to increased concentrations of produced ethanol by increasing the proportion of ergosterol at the expense of lanosterol, by increasing the proportion of phosphatidylinositol at the expense of phosphatidylcholine, and by increasing the amount of C18:0 fatty acids in total phospholipids at the expense of C16:0 fatty acids . The produced ethanol had no effect on the phospholipid content nor on the proportion of unsaturated fatty acids in the phospholipids . The specific growth rate had no effect on the phospholipid content, the sterol composition, the phospholipid composition, the fatty acid composition of total phospholipids, or on the proportion of unsaturated fatty acids in the phospholipids of S . cerevisiae . It was not possible to separate the effects of produced ethanol and growth rate on the ergosterol content of the chemostat-grown S . cerevisiae cells.

Yeast, 1995 Aug, 11(10), 929 - 44
Scp160p, a new yeast protein associated with the nuclear membrane and the endoplasmic reticulum, is necessary for maintenance of exact ploidy; Wintersberger U et al.; We have cloned a new gene, SCP160, from Saccharomyces cerevisiae, the deduced amino acid sequence of which does not exhibit overall similarity to any known yeast protein . A weak resemblance between the C-terminal part of the Scp160 protein and regulatory subunits of cAMP-dependent protein kinases from eukaryotes as well as the pstB protein of Escherichia coli was observed . The SCP160 gene resides on the left arm of chromosome X and codes for a polypeptide of molecular weight around 160 kDa . By immunofluorescence microscopy the Scp160 protein appears to be localized to the nuclear envelope and to the endoplasmic reticulum (ER) . However, no signal sequence or membrane-spanning region exists, suggesting that the Scp160 protein is attached to the cytoplasmic surface of the ER-nuclear envelope membranes . Disruption of the SCP160 gene is not lethal but results in cells of decreased viability, abnormal morphology and increased DNA content . This phenotype is not reversible by transformation with a plasmid carrying the wild-type gene . Crosses of SCP160 deletion mutant strains among each other or with unrelated strains lead to irregular segregation of genetic markers . Taken together the data suggest that the Scp160 protein is required during cell division for faithful partitioning of the ER-nuclear envelope membranes which in S . cerevisiae enclose the duplicated chromosomes.

Curr Genet, 1995 Aug, 28(3), 217 - 24
The S . cerevisiae nuclear gene SUV3 encoding a putative RNA helicase is necessary for the stability of mitochondrial transcripts containing multiple introns; Golik P et al.; The product of the nuclear gene SUV3 is implicated in a variety of post-transcriptional processes in yeast mitochondria . We have analysed the effect of SUV3 gene-disruption on the expression of intron-containing alleles of the mitochondrial cytb and cox1 genes . We have constructed several strains with mitochondrial genomes containing different combinations of cytb and cox1 introns, and associated these genomes with the disruption of SUV3 . The resulting strains were tested for their respiratory competence and spectral cytochrome content . All the strains containing only two or three introns showed normal expression of cytb and cox1, whereas the strains containing more introns were unable to express the appropriate gene . The analysis of mitochondrial RNAs by Northern hybridisation showed that the loss of respiratory competence in the strains containing more introns is due to the decrease of mRNA level with no over-accumulation of high-molecular-weight precursors . However, the transcription of the genes was not affected . These results led us to the notion that SUV3 is required for the stability of intron-containing cytb and cox1 transcripts in a cumulative way, not dependent on any particular intron.

Trends Biochem Sci, 1995 Aug, 20(8), 319 - 23
Yeast DNA polymerases and their role at the replication fork; Sugino A; The DNA polymerases of the yeast Saccharomyces cerevisiae serve as a model system for the study of the replication fork during DNA replication . To date, six S . cerevisiae DNA polymerases have been at least partially characterized (compared with four in mammals so far), with further candidates being identified as open reading frames in the yeast genome sequencing project . Here, we review the current state of knowledge of the yeast polymerases, and discuss, where possible, their biological role during DNA replication.

Bioessays, 1995 Aug, 17(8), 669 - 72
Is there a unique form of chromatin at the Saccharomyces cerevisiae centromeres?
Basrai MA, Hieter P.
Chromosome transmission in S . cerevisiae requires the activities of many structural and regulatory proteins required for the replication, repair, recombination and segregation of chromosomal DNA, and co-ordination of the chromosome cycle with progression through cell cycle . An important structural domain on each chromosome is the kinetochore (centromere DNA and associated proteins), which provides the site of attachment of chromosomes to the spindle microtubules . Stoler et al . have recently reported the cloning of an essential gene CSE4, mutations in which cause chromosome nondisjunction of a marked chromosome bearing a centromere DNA mutation . The cse4-1 mutation causes cells to arrest in the G2/M phase of the cell cycle with a 2N DNA content in a RAD9 checkpoint-independent manner . The carboxyl terminus of Cse4p and the human centromere-localized protein CENP-A have a high degree of homology to the C-terminal domain of histone H3 . Since both CENP-A and Cse4p also have biochemical properties similar to histones H3 and H4, it is tempting to speculate that these histone H3-like proteins are components of specialized nucleosomes, a class of which may be unique to the centromeres.

FEMS Microbiol Lett, 1995 Aug 1, 130(2-3), 245 - 51
Distribution of individual cytoplasmic pH values in a population of the yeast Saccharomyces cerevisiae; Cimprich P et al.; Fluorescence ratio imaging microscopy using pH-sensitive fluorescent dyes makes it possible to evaluate statistical distribution of intracellular pH in a population of the yeast S . cerevisiae examined in a thin layer of suspension in a Petri dish . The distribution appears to fit a Gaussian curve with a half-width around the 0.4 pH unit . The curve became slightly narrower after resuspension in a strong buffer; the mean values shifted with the pH of the buffer . The shape of the distribution curves of both resting and growing cells in various phases of growth does not change significantly . Likewise, addition of 1% of glucose, 50 microM suloctidil or 100 microM diethylstilbestrol brings about no alteration . The only value which clearly changes is the average cytoplasmic pH.

FEMS Microbiol Lett, 1995 Aug 1, 130(2-3), 221 - 9
The gene encoding the biotin-apoprotein ligase of Saccharomyces cerevisiae; Cronan JE Jr et al.; We report the isolation, genomic mapping, and DNA sequence of the BPL1 gene encoding the biotin-apoprotein ligase of Saccharomyces cerevisiae . The gene was isolated by complementation of an Escherichia coli birA (biotin-apoprotein ligase) mutant indicating that the expressed yeast protein modified the essential biotinated protein of the bacterial host . The BPL1 gene encodes a protein of 690 residues (M(r) 76.4 kDa) with strong sequence similarities to the E . coli and human biotin-apoprotein ligases . BPL1 was mapped to chromosome IV, is allelic to the previously described ACC2 gene, and encodes the major (if not the only) biotin-apoprotein ligase activity of S . cerevisiae.

Eur J Biochem, 1995 Aug 1, 231(3), 845 - 54
Characterization and gene cloning of 1,3-beta-D-glucan synthase from Saccharomyces cerevisiae; Inoue SB et al.; 1,3-beta-D-Glucan synthase of Saccharomyces cerevisiae was solubilized and purified up to 700-fold by product entrapment . The specific activity of the partially purified enzyme was around 4 mumol glucose incorporated.min-1.mg protein-1 . In SDS/PAGE, enrichment of a 200-kDa protein was clearly observed in parallel with the increase in specific activity . mAbs that could immunoprecipitate the 1,3-beta-D-glucan synthase activity were isolated, and some of them also recognized this 200-kDa protein in the Western blot . Internal amino acid sequences of this 200-kDa protein were determined after lysyl endopeptidase digestion . With the information of these amino acid sequences, we cloned two genes, GSC1 and GSC2 (glucan synthase of S . cerevisiae 1 and 2), which are very similar to each other (88% at the amino acid level); hydropathy profiles of both proteins suggest that these genes encode integral membrane proteins which can be assumed to have approximately 16 transmembrane domains . Disruption of each gene was not lethal, but disruption of both genes was lethal . The 1,3-beta-D-glucan synthase activities of membrane and partially purified enzyme of gsc1::URA3 cells were significantly lower than those of the wild-type and gsc2::LEU2 cells.

J Cell Biol, 1995 Aug, 130(4), 835 - 45
A truncated form of the Pho80 cyclin redirects the Pho85 kinase to disrupt vacuole inheritance in S . cerevisiae; Nicolson TA et al.; Partitioning of the vacuole during cell division in Saccharomyces cerevisiae begins during early S phase and ends in late G2 phase before the yeast nucleus migrates into the bud neck . We have isolated and characterized a new mutant, vac5-1, which is defective in vacuole segregation . Cells with the vac5-1 mutation can form large buds without vacuoles . The VAC5 gene was cloned and is identical to PHO80 . PHO80 encodes a cyclin which acts in a complex with a cdc-like kinase, PHO85, as a negative regulator of two transcription factors (PHO2 and PHO4) that govern the expression of metabolic phosphatases . The vacuole inheritance defect in vac5-1 cells is dependent on the presence of the Pho85 kinase and its targets Pho4p and Pho2p . As with other alleles of PHO80, phosphatase levels are elevated in vac5-1 mutants . A suppressor, the COOH-terminal half of the Gal11 transcription factor, rescues the vac5-1 phenotype of defective vacuole inheritance without altering the vac5-1 phenotype of elevated phosphatase levels . In addition, neither maximal nor minimal levels of expression of the inducible "PHO" system phosphatases causes a vacuole inheritance defect . Though vac5-1 is recessive, pho80 delta or pho85 delta strains do not show a defect in vacuole inheritance, suggesting that vac5-1 is not a complete loss-of-function allele . Sequence analysis shows that the vac5-1 allele encodes a truncated form of the Pho80 cyclin and overexpression of vac5-1 in pho80 delta cells causes a vacuole inheritance defect . We conclude that the vac5-1 allele directs the Pho85 kinase to regulate, via transcription factors Pho4 and Pho2, genes that affect vacuole inheritance but which are not known to be under normal PHO pathway control.

Proc Natl Acad Sci U S A, 1995 Aug 1, 92(16), 7525 - 9
A family of Ran binding proteins that includes nucleoporins; Dingwall C et al.; Ran, a small nuclear GTP binding protein, is essential for the translocation of nuclear proteins through the nuclear pore complex . We show that several proteins, including the Saccharomyces cerevisiae Nup2p and Caenorhabditis elegans F59A2.1 nucleoporins, contain domains similar to the previously characterized murine Ran binding protein (RBP, termed RBP1) . To test the significance of this similarity, we have used the corresponding domains of Nup2p and a putative S . cerevisiae RBP in Ran binding assays and the yeast two-hybrid system . Both proteins bind S . cerevisiae Ran, but only the putative S . cerevisiae RBP binds human Ran . Two-hybrid analysis revealed Ran-Ran interactions and that yeast and human Rans can interact . These data identify Nup2p as a target for Ran in the nuclear pore complex, suggesting a direct role for it in nuclear-cytoplasmic transport . We discuss the possibility that proteins harboring Ran binding domains link the Ran GTPase cycle to specific functions in the nucleus.

Mol Cell Biol, 1995 Aug, 15(8), 4578 - 84
Identification and characterization of Ral-binding protein 1, a potential downstream target of Ral GTPases; Cantor SB et al.; Ral proteins constitute a distinct family of Ras-related GTPases . Although similar to Ras in amino acid sequence, Ral proteins are activated by a unique nucleotide exchange factor and inactivated by a distinct GTPase-activating protein . Unlike Ras, they fail to promote transformed foci when activated versions are expressed in cells . To identify downstream targets that might mediate a Ral-specific function, we used a Saccharomyces cerevisiae-based interaction assay to clone a novel cDNA that encodes a Ral-binding protein (RalBP1) . RalBP1 binds specifically to the active GTP-bound form of RalA and not to a mutant Ral with a point mutation in its putative effector domain . In addition to a Ral-binding domain, RalBP1 also contains a Rho-GTPase-activating protein domain that interacts preferentially with Rho family member CDC42 . Since CDC42 has been implicated in bud site selection in S . cerevisiae and filopodium formation in mammalian cells, Ral may function to modulate the actin cytoskeleton through its interactions with RalBP1.

J Cell Biol, 1995 Aug, 130(3), 661 - 73
Members of the NAP/SET family of proteins interact specifically with B-type cyclins; Kellogg DR et al.; Cyclin-dependent kinase complexes that contain the same catalytic subunit are able to induce different events at different times during the cell cycle, but the mechanisms by which they do so remain largely unknown . To address this problem, we have used affinity chromatography to identify proteins that bind specifically to mitotic cyclins, with the goal of finding proteins that interact with mitotic cyclins to carry out the events of mitosis . This approach has led to the identification of a 60-kD protein called NAP1 that interacts specifically with members of the cyclin B family . This interaction has been highly conserved during evolution: NAP1 in the Xenopus embryo interacts with cyclins B1 and B2, but not with cyclin A, and the S . cerevisiae homolog of NAP1 interacts with Clb2 but not with Clb3 . Genetic experiments in budding yeast indicate that NAP1 plays an important role in the function of Clb2, while biochemical experiments demonstrate that purified NAP1 can be phosphorylated by cyclin B/p34cdc2 kinase complexes, but not by cyclin A/p34cdc2 kinase complexes . These results suggest that NAP1 is a protein involved in the specific functions of cyclin B/p34cdc2 kinase complexes . In addition to NAP1, we found a 43-kD protein in Xenopus that is homologous to NAP1 and also interacts specifically with B-type cyclins . This protein is the Xenopus homolog of the human SET protein, which was previously identified as part of a putative oncogenic fusion protein (Von Lindern et al., 1992).

Int J Biochem Cell Biol, 1995 Aug, 27(8), 767 - 73
Quantitation of protein phosphatase 1 and 2A in extracts of the budding yeast and fission yeast; Farkas I et al.; Serine/threonine protein phosphatases are also involved in the control of cell division . The aim of the present study was to compare the activity of protein phosphatase 1 (PP1) and 2A (PP2A) in cell extracts of the budding and fission yeast, made at different phases of growth . The activities of PP1 and PP2A toward phosphorylase were similar in extracts of S . cerevisiae . In S . pombe extracts, PP1 was responsible for more than 80% of the phosphorylase phosphatase activity . Ammonium sulfate-ethanol treatment increased the specific activity of the phosphatases and the percentage of PP2A in S . cerevisiae extracts . No increase in the proportion of PP2A was observed upon the same treatment of S . pombe extracts . The above results were confirmed by fractionation of PP1 and PP2A activities on a heparin-Sepharose column . The proportion of PP1 and PP2A activities did not change significantly during exponential cell growth but cells from stationary phase exhibited lower phosphatase activities . These results may indicate a lower level of expression of the PP2A genes in S . pombe and/or differences in the structure of the holoenzymes or their regulators in the two genera.

Biochem Mol Med, 1995 Aug, 55(2), 122 - 30
Antigenic and functional conservation of an integrin I-domain in Saccharomyces cerevisiae; Hostetter MK et al.; We sought evidence for precursors of the leukocyte integrin subunits alpha M and alpha X among unicellular eukaryotes such as Saccharomyces cerevisiae . Chromatography of cytosolic extracts of Saccharomyces cerevisiae on an affinity matrix coupled to BU-15, a monoclonal antibody that recognizes alpha X, revealed a band of M(r) > 205 kDa under nonreducing conditions . Screening a lambda gt11 library of S . cerevisiae DNA with BU-15 (anti-alpha X) and anti-Mo1 (anti-alpha M) led to the isolation of a 3.7-kb EcoRI fragment containing the 3' end of an open reading frame sufficient to encode a polypeptide in excess of 118 kDa . On the basis of Southern blotting at high stringency, this gene was present in S . cerevisiae, but not in other yeast species such as Candida glabrata . Analysis of the derived amino acid sequence demonstrated > 98% identity with the S . cerevisiae protein Uso1p, a myosin-like polypeptide found exclusively in the cytosol . The C-terminal 1016 aa, expressed from the 3.7-kb EcoRI fragment in Escherichia coli as a beta-galactosidase fusion protein, bound iC3b, a ligand for the I-domain in alpha M and alpha X, and were recognized by Mn41, a monoclonal antibody specific for the alpha M I-domain . Antigenic and functional conservation of an I-domain in S . cerevisiae suggests that this domain may be a prototype for integrin-like proteins in other primitive eukaryotes.

Genetics, 1995 Aug, 140(4), 1259 - 75
Mutational analysis of morphologic differentiation in Saccharomyces cerevisiae; Blacketer MJ et al.; A genetic analysis was undertaken to investigate the mechanisms controlling cellular morphogenesis in Saccharomyces cerevisiae . Sixty mutant strains exhibiting abnormally elongated cell morphology were isolated . The cell elongation phenotype in at least 26 of the strains resulted from a single recessive mutation . These mutations, designated generically elm (elongated morphology), defined 14 genes; two of these corresponded to the previously described genes GRR1 and CDC12 . Genetic interactions between mutant alleles suggest that several ELM genes play roles in the same physiological process . The cell and colony morphology and growth properties of many elm mutant strains are similar to those of wild-type yeast strains after differentiation in response to nitrogen limitation into the pseudohyphal form . Each elm mutation resulted in multiple characteristics of pseudohyphal cells, including elongated cell shape, delay in cell separation, simultaneous budding of mother and daughter cells, a unipolar budding pattern, and/or the ability to grow invasively beneath the agar surface . Mutations in 11 of the 14 ELM gene loci potentiated pseudohyphal differentiation in nitrogen-limited medium . Thus, a subset of the ELM genes are likely to affect control or execution of a defined morphologic differentiation pathway in S . cerevisiae.

J Biol Chem, 1995 Jul 28, 270(30), 18099 - 109
An essential yeast gene encoding a homolog of ubiquitin-activating enzyme; Dohmen RJ et al.; Ubiquitin (Ub) activation by the Ub-activating (E1) enzyme is the initial and essential step common to all of the known processes that involve post-translational conjugation of Ub to itself or other proteins . The "activated" Ub, linked via a thioester bond to a specific cysteine residue in one of several Ub-conjugating (E2) enzymes, which catalyze the formation of isopeptide bonds between the C-terminal glycine of Ub and lysine residues of acceptor proteins . In the yeast Saccharomyces cerevisiae, a 114-kDa E1 enzyme is encoded by an essential gene termed UBA1 (McGrath, J.P., Jentsch, S., and Varshavsky, A . (1991) EMBO J . 10, 227-236) . We describe the isolation and analysis of another essential gene, termed UBA2, that encodes a 71-kDa protein with extensive sequence similarities to both the UBA1-encoded yeast E1 and E1 enzymes of other organisms . The regions of similarities between Uba1p and Uba2p encompass a putative ATP-binding site as well as a sequence that is highly conserved between the known E1 enzymes and contains the active-site cysteine of E1 . This cysteine is shown to be required for an essential function of Uba2p, suggesting that Uba2p-catalyzed reactions involved a transient thioester bond between Uba2p and either Ub or another protein . Uba2p is located largely in the nucleus . The putative nuclear localization signal of Uba2p is near its C terminus . The Uba1p (E1 enzyme) and Uba2p cannot complement each others essential functions even if their subcellular localization is altered by mutagenesis . Uba2p appears to interact with itself and several other S . cerevisiae proteins with apparent molecular masses of 52, 63, 87, and 120 kDa . Uba2p is multiubiquitinated in vivo, suggesting that at least a fraction of Uba2p is metabolically unstable . Uba2p is likely to be a component of the Ub system that functions as either an E2 or E1/E2 enzyme.

J Biol Chem, 1995 Jul 28, 270(30), 17660 - 3
Lethality in yeast of trichothiodystrophy (TTD) mutations in the human xeroderma pigmentosum group D gene . Implications for transcriptional defect in TTD; Guzder SN et al.; Mutations in the human XPD gene result in a defect in nucleotide excision repair of ultraviolet damaged DNA and cause the cancer-prone syndrome xeroderma pigmentosum (XP) . Besides XP, mutations in XPD can cause another seemingly unrelated syndrome, trichothiodystrophy (TTD), characterized by sulfur-deficient brittle hair, ichthyosis, and physical and mental retardation . To ascertain the underlying defect responsible for TTD, we have expressed the TTD mutant proteins in the yeast Saccharomyces cerevisiae and determined if these mutations can rescue the inviability of a rad3 null mutation . RAD3, the S . cerevisiae counterpart of XPD, is required for nucleotide excision repair and also has an essential role in RNA polymerase II transcription . Expression of the wild type XPD protein or the XPD Arg-48 protein carrying a mutation in the DNA helicase domain restores viability to the rad3 null mutation . Interestingly, the XPD variants containing TTD mutations fail to complement the lethality of the rad3 null mutation, strongly suggesting that TTD mutations impair the ability of XPD protein to function normally in RNA polymerase II transcription . From our studies, we conclude that XPD DNA helicase activity is not essential for transcription and infer that TTD mutations in XPD result in a defect in transcription.

Nucleic Acids Res, 1995 Jul 25, 23(14), 2669 - 76
U24, a novel intron-encoded small nucleolar RNA with two 12 nt long, phylogenetically conserved complementarities to 28S rRNA; Qu LH et al.; Following computer searches of sequence banks, we have positively identified a novel intronic snoRNA, U24, encoded in the ribosomal protein L7a gene in humans and chicken . Like previously reported intronic snoRNAs, U24 is devoid of a 5'-trimethyl-cap . U24 is immunoprecipitated by an antifibrillarin antibody and displays an exclusively nucleolar localization by fluorescence microscopy after in situ hybridization with antisense oligonucleotides . In vertebrates, U24 is a 76 nt long conserved RNA which is metabolically stable, present at approximately 14,000 molecules per human HeLa cell . U24 exhibits a 5'-3' terminal stem-box C-box D structure, typical for several snoRNAs, and contains two 12 nt long conserved sequences complementary to 28S rRNA . It is, therefore, strikingly related to U14, U20 and U21 snoRNAs which also possess long sequences complementary to conserved sequences of mature 18S or 28S rRNAs . In 28S rRNA the two tracts complementary to U24 are adjacent to each other, they involve several methylated nucleotides and are surprisingly close, within the rRNA secondary structure, to complementarities to snoRNAs U18 and U21 . Identification of the yeast Saccharomyces cerevisiae U24 gene directly confirms the outstanding conservation of the complementarity to 28S rRNA during evolution, suggesting a key role of U24 pairing to pre-rRNA during ribosome biogenesis, possible in the control of pre-rRNA folding . Yeast S.cerevisiae U24 is also intron-encoded but not in the same host-gene as in humans or chicken.

Biochim Biophys Acta, 1995 Jul 25, 1263(1), 45 - 52
Heterologous expression of the highly conserved acidic ribosomal phosphoproteins from Dictyostelium (changed from Dictyosteliumm) discoideum in Saccharomyces cerevisiae; Bermejo B et al.; The genes encoding the acidic ribosomal phosphoproteins DdP1 and DdP2 from Dictyostelium discoideum have been cloned into yeast plasmid vectors under the control of the inducible GAL1 promoter . These constructions have been used to transform S . cerevisiae strains D45 and D67 lacking the equivalent ribosomal components . The D . discoideum genes are properly transcribed when cells are grown in the presence of the inducer galactose and the mRNAs incorporated into polysomes . However, the heterologous ribosomal proteins are not able to rescue the growth deficiency in S . cerevisiae caused by the absence of their own ribosomal proteins . When the heterologous proteins are analyzed using specific antibodies, only protein DdP1 is found in the ribosomes of the transformed S . cerevisiae D67 strain . No other heterologous protein is found in any other transformed strain, suggesting that the heterologous acidic ribosomal components are rapidly degraded when they are not bound to the ribosomes . The results indicate that D . discoideum DdP1 protein is able to interact with the yeast ribosome, though the interaction is functionally inefficient . Protein DdP2, in spite of having a higher sequence similarity to its yeast counterparts, is completely inactive in S . cerevisiae . Since the P proteins from both organisms have extensive amino acid sequence similarity ranging from 60% to 70%, these results warns about establishing a direct relationship between the extent of amino acid sequence similarity and the capacity of heterologous proteins to be functional in host species . Moreover, our data suggest that evolution affected the interaction of the acidic proteins with the ribosome rather than the structural features responsible for their primary functions.

FEBS Lett, 1995 Jul 24, 368(3), 435 - 40
Conserved ATPase and luciferase refolding activities between bacteria and yeast Hsp70 chaperones and modulators; Levy EJ et al.; We have reconstituted an ATP-dependent protein folding machinery using purified yeast cytosolic proteins . The S . cerevisiae Hsp70 Ssa1p and the DnaJ homolog Ydj1p refolded denatured firefly luciferase . In E . coli, efficient refolding of luciferase requires the Hsp70 DnaK and two modulators, DnaJ and GrpE, that synergistically stimulate its ATPase activity . Exchanging DnaJ homologs between the S . cerevisiae and E . coli systems revealed that their ability to stimulate Hsp70 ATPase activity was conserved . In contrast, GrpE further stimulated only DnaK's ATPase activity . Efficient refolding of luciferase by Ssa1p and DnaJ, but not by DnaK and Ydj1p, suggests that a compatible Hsp70/DnaJ homolog pair can act as a protein folding machinery.

Science, 1995 Jul 21, 269(5222), 396 - 400
Telomerase in yeast; Cohn M et al.; The ribonucleoprotein enzyme telomerase synthesizes telomeric DNA by copying an internal RNA template sequence . The telomerase activities of the yeasts Saccharomyces castellii and Saccharomyces cerevisiae--with regular and irregular telomeric sequences, respectively--have now been identified and characterized . The S . cerevisiae activity required the telomerase RNA gene TLC1 but not the EST1 gene, both of which are required for normal telomere maintenance in vivo . This activity exhibited low processivity and produced no regularly repeated products . An inherently high stalling frequency of the S . cerevisiae telomerase may account for its in vitro properties and for the irregular telomeric sequences of this yeast.

FEBS Lett, 1995 Jul 10, 368(1), 73 - 6
Oxidative stress response in yeast: effect of glutathione on adaptation to hydrogen peroxide stress in Saccharomyces cerevisiae; Izawa S et al.; Role of intracellular glutathione in the response of Saccharomyces cerevisiae to H2O2 was investigated . Depletion of cellular glutathione or inhibition of gamma-glutamylcysteine synthetase (GSH-I) enhanced the sensitivity to H2O2 and suppressed the adaptation to H2O2 . A mutant deficient in GSH-I also showed the hypersensitivity and could not adapt to H2O2 . Incubation of the cell with amino acids constituting glutathione (L-Glu, L-Cys, Gly) increased the intracellular glutathione content, and subsequently the cell acquired resistance against H2O2 . These results strongly suggest that intracellular glutathione plays an important role in the adaptive response in S . cerevisiae to oxidative damage.

EMBO J, 1995 Jul 3, 14(13), 3021 - 7
A potassium transporter of the yeast Schwanniomyces occidentalis homologous to the Kup system of Escherichia coli has a high concentrative capacity; Banuelos MA et al.; The yeast Schwanniomyces occidentalis has a high-affinity K+ uptake system with a high concentrative capacity, which is able to deplete the external K+ to < 0.03 microM . We have cloned the gene HAK1 of S.occidentalis which complements defective K+ uptake by trk1 and trk1 trk2 mutants of Saccharomyces cerevisiae . When HAK1 was expressed in a trk1 trk2 S.cerevisiae mutant, transport affinities for K+ and other alkali cations resembled those of S.occidentalis . The predicted amino acid sequence of the HAK1 protein shows significant homology with the hydrophobic region of the Kup transporter of Escherichia coli . In S.occidentalis HAK1 expresses in K(+)-limiting conditions . Our data indicate that in K(+)-starved cells the system encoded by HAK1 is the major K+ transporter of S.occidentalis.

Biochem Mol Biol Int, 1995 Jul, 36(4), 883 - 8
Pyrimidine moiety of thiamin is biosynthesized from pyridoxine and histidine in Saccharomyces cerevisiae; Tazuya K et al.; The isotopes of {6-13C}- and {5'-2H2}pyridoxine were incorporated efficiently into the pyrimidine moiety of thiamin in S . cerevisiae . The mass fragmentation pattern showed that the C-6 and H-5' atoms of pyridoxine were incorporated into the C-6, and H-5' atoms of the pyrimidine, respectively . These findings, along with out previous results, show that the C-2', C-2, N-1, C-6, C-5, and C-5' unit of the pyrimidine are derived from the C-2', C-2, N-1, C-6, C-5 and C-5' unit of pyridoxine, and that pyrimidine is biosynthesized from pyridoxine and histidine.

Biochem Mol Biol Int, 1995 Jul, 36(4), 781 - 91
Molecular cloning and expression in S . cerevisiae of two exochitinases from Trichoderma harzianum; Draborg H et al.; The synthetic exochitinase substrate 4-methylumbelliferyl N-acetylglucosamine was used to identify seven full-length exochitinase-encoding cDNAs from a Trichoderma harzianum cDNA library by expression in yeast . The cDNA clones represented transcripts of two exochitinase genes, designated as exc1 and exc2, which cross-hybridized under moderate stringency conditions in genomic Southern blots . The exc1 cDNA encodes a 578 amino acid polypeptide showing 72% similarity to the exc2-encoded 602-residue polypeptide . The deduced exochitinase amino acid sequences were found to be homologous with mammalian and fungal hexosaminidases as well as a bacterial chitobiosidase . The substrate specificity of the recombinant enzymes expressed in S . cerevisiae indicates that the enzymes are N-acetylglucosaminidases releasing single N-acetylglucosamine residues from the non-reducing end of the chitin substrate.

Mol Cell Biol, 1995 Jul, 15(7), 3487 - 95
Identification of a mouse protein whose homolog in Saccharomyces cerevisiae is a component of the CCR4 transcriptional regulatory complex; Draper MP et al.; The CCR4 protein from Saccharomyces cerevisiae is a component of a multisubunit complex that is required for the regulation of a number of genes in yeast cells . We report here the identification of a mouse protein (mCAF1 {mouse CCR4-associated factor 1}) which is capable of interacting with and binding to the yeast CCR4 protein . The mCAF1 protein was shown to have significant similarity to proteins from humans, Caenorhabditis elegans, Arabidopsis thaliana, and S . cerevisiae . The yeast gene (yCAF1) had been previously cloned as the POP2 gene, which is required for expression of several genes . Both yCAF1 (POP2) and the C . elegans homolog of CAF1 were shown to genetically interact with CCR4 in vivo, and yCAF1 (POP2) physically associated with CCR4 . Disruption of the CAF1 (POP2) gene in yeast cells gave phenotypes and defects in transcription similar to those observed with disruptions of CCR4, including the ability to suppress spt10-enhanced ADH2 expression . In addition, yCAF1 (POP2) when fused to LexA was capable of activating transcription . mCAF1 could also activate transcription when fused to LexA and could functionally substitute for yCAF1 in allowing ADH2 expression in an spt10 mutant background . These data imply that CAF1 is a component of the CCR4 protein complex and that this complex has retained evolutionarily conserved functions important to eukaryotic transcription.

Genetics, 1995 Jul, 140(3), 957 - 63
Specialization of B-type cyclins for mitosis or meiosis in S . cerevisiae; Dahmann C et al.; The CLB1, CLB2, and CLB3 genes encode B-type cyclins important for mitosis in Saccharomyces cerevisiae, while a fourth B-type cyclin gene, CLB4, has no clear role . The effects of homozygous clb mutations on meiosis were examined . Mutants homozygous for clb1 clb3, or for clb1 clb4, gave high levels of sporulation, but produced mainly two-spored asci instead of four-spored asci . The cells had completed meiosis I but not meiosis II, producing viable diploid ascospores . CLB1 and CLB4 seem to be much more important for meiosis than for mitosis and may play some special role in meiosis II . In contrast, CLB2 is important for mitosis but not meiosis . The level of Cdc28-Clb activity may be important in determining whether meiosis II will occur.

Bioessays, 1995 Jul, 17(7), 639 - 50
A 200-amino acid ATPase module in search of a basic function; Confalonieri F et al.; A fast growing family of ATPases has recently been highlighted . It was named the AAA family, for ATPases Associated to a variety of cellular Activities . The key feature of the family is a highly conserved module of 230 amino acids present in one or two copies in each protein . Despite extensive sequence conservation, the members of the family fulfil a large diversity of cellular functions: cell cycle regulation, gene expression in yeast and HIV, vesicle-mediated transport, peroxisome assembly, 26S protease function etc . In addition, several members of this family can be found in the same organism (up to 17 in S . cerevisiae) . The contrast between functional diversity and structural conservation of the module, from archaebacteria to mammals, suggests that it plays an essential, but as yet unknown, role at key points of the cellular machinery . Two (non-exclusive) such possibilities are: (1) ATP-dependent proteasome function and (2) ATP-dependent anchorage of proteins . Finally, the basic biochemical activity of the AAA module is still a matter of speculation, and we propose that it acts as an ATP-dependent protein clamp.

DNA Cell Biol, 1995 Jul, 14(7), 619 - 28
In vivo reconstitution of highly active Candida maltosa cytochrome P450 monooxygenase systems in inducible membranes of Saccharomyces cerevisiae; Zimmer T et al.; To establish a system for functional characterization of individual Candida maltosa cytochrome P450 monooxygenases, the NADPH-cytochrome P450 reductase from this yeast species was co-expressed in Saccharomyces cerevisiae with each of the following cytochrome P450 forms; P450Cm1 (CYP52 A3), P450Cm2 (CYP52 A4), and P450AlK2A (CYP52 A5) . For this purpose, a multicopy plasmid was constructed that contained two independent expression units controlled by the galactose-inducible GAL10 promoter . As shown by spectral and immunological methods, large amounts of the desired monooxygenase components could be simultaneously produced in the respective S . cerevisiae transformants . It was important, however, to adjust semi-anaerobic cultivation conditions during induction by galactose to minimize a mutual impairment of cytochrome P450 and NADPH-cytochrome P450 reductase formation . Compared to the specific cellular content of the host-own enzyme, a 75- to 100-fold overproduction of the reductase component was obtained resulting in P450/reductase molar ratios of about 1:3 in the microsomal fractions prepared from the co-expression strains . At the same time, the rates of cytochrome P450-dependent lauric acid hydroxylation increased more than 10-fold, showing a proper reconstitution of the C . maltosa monooxygenase systems in S . cerevisiae . Using intact cells, an efficient biotransformation of lauric acid to omega-hydroxylauric acid and dodecanedioic acid was found . S . cerevisiae cells coexpressing cytochrome P450 and NADPH-cytochrome P450 reductase were characterized by a marked proliferation of the endoplasmic reticulum . Immunoelectron microscopy revealed a colocalization of the monooxygenase components produced to these newly formed membrane structures.

Cell Growth Differ, 1995 Jul, 6(7), 789 - 98
Genetic analysis of growth inhibition by GAL4-L kappa B-alpha in Saccharomyces cerevisiae; Morin PJ et al.; I kappa B proteins bind to and regulate Rel/NF- kappa B transcription factors . We showed previously that a fusion protein (GAL4-p40) containing the DNA-binding domain of GAL4 and sequences of chicken l kappa B-alpha (p40) inhibits growth in the yeast Saccharomyces cerevisiae . We now show that p40 must be bound to DNA to inhibit yeast growth, p40 proteins, bound to DNA either as GAL4 or LEXA fusion proteins, inhibit yeast growth . In contrast, p40 proteins that cannot bind to DNA, such as full-length p40, a GAL4-l kappa B fusion protein containing a mutant GAL4 DNA-binding domain, and a fusion protein (GAD-p40) containing the transcriptional activation domain of GAL4 fused to p40, each failed to inhibit cell growth . As with GAL4-VP16, GAL4-p40 needs a functional cellular ADA2 gene to exert its growth-inhibitory effect in S . cerevisiae . Using a high copy suppression strategy, we have isolated three S . cerevisiae genes that restore normal growth to yeast expressing GAL4-p40 or LEXA-p40 . We have termed these rescuing genes collectively as SIK genes, for "Suppressors of 1 kappa B." Expression of the SIK genes specifically suppresses the growth-inhibitory activity of GAL4-p40 and LEXA-p40 because SIK gene expression cannot block GAL4-VP16-mediated growth inhibition in S . cerevisiae . SIK1 encodes a novel protein that contains a COOH-terminal repeat that has been found in many microtubule-binding proteins . SIK2 encodes NH2-terminal acetyltransferase, and SIK3 encodes the yeast ribosomal S4 protein . None of the SIK proteins binds directly to p40 sequences in vitro, suggesting that the SIK proteins are likely to act downstream of the direct point of growth inhibition by GAL4-p40 . Our results may be useful for devising strategies for identifying vertebrate inhibitors of l kappa B proteins and of other proteins that inhibit growth in S . cerevisiae.

Yeast, 1995 Jun 30, 11(8), 783 - 7
The S1, S2 and SGA1 ancestral genes for the STA glucoamylase genes all map to chromosome IX in Saccharomyces cerevisiae; Lambrechts MG et al.; The polymorphic extracellular glucoamylase-encoding genes STA1 (chr . IV), STA2 (chr . II) and STA3 (chr . XIV), from Saccharomyces cerevisiae var . diastaticus probably evolved by genomic rearrangement of DNA regions (S1, S2 and SGA1) present in S . cerevisiae, and subsequent translocation to unlinked regions of chromosomal regions . S1, encoding a homologue to the threonine/serine-rich domain of STA glucoamylases (GAI-III), mapped to the right arm of chromosome IX . S2, encoding the hydrophobic leader peptide of GAI-III), was also mapped on the right arm of chromosome IX, next to S1, close to DAL81 . The SGA1 sporulation-specific, intracellular glucoamylase-encoding gene is located on the left arm of chromosome IX, 32 kb proximal of HIS5.

Yeast, 1995 Jun 30, 11(8), 767 - 74
The DNA sequence of a 7941 bp fragment of the left arm of chromosome VII of Saccharomyces cerevisiae contains four open reading frames including the multicopy suppressor gene of the pop2 mutation and a putative serine/threonine protein kinase gene; Coglievina M et al.; We report the sequence of a 7941 bp DNA fragment from the left arm of chromosome VII of Saccharomyces cerevisiae which contains four open reading frames (ORFs) of greater than 100 amino acid residues . ORF biC834 shows 100% bp identity with the recently identified multicopy suppressor gene of the pop2 mutation (MPT5); its deduced protein product carries an eight-repeat domain region, homologous to that found in the hypothetical regulatory YGL023 protein of S . cerevisiae and the Pumilio protein of Drosophila . ORF biE560 protein exhibits patterns typical of serine/threonine protein kinases, with which it shares high degrees of homology.

Yeast, 1995 Jun 30, 11(8), 747 - 59
A regulated MET3-GLC7 gene fusion provides evidence of a mitotic role for Saccharomyces cerevisiae protein phosphatase 1; Black S et al.; Saccharomyces cerevisiae possesses a single essential gene (GLC7) encoding protein phosphatase 1 (PP1) . Elevated expression of this gene from the GAL1 promoter is highly detrimental to the cell, causing a growth defect and aberrant bud morphology, which leads to cells exhibiting long, extended buds . By comparison, expression of GLC7 from the weaker MET3 promoter was without significant effect on either growth or morphology . However, repression of GLC7 expression from the MET3 promoter in cells where the MET3-GLC7 fusion was the sole source of PP1 resulted in a mitotic delay . Such cultures showed a massive decrease in the rate of proliferation in conjunction with a significant increase in the proportion of large, budded cells . 4'6-diamidino-2-phenylindole dihydrochloride (DAPI) staining and anti-tubulin immunofluorescence analysis of these cells revealed that many were blocked in mitosis, with a short spindle and DAPI-stained material stretched between the mother and daughter cell within the bud neck . These results support a role for PP1 in the completion of mitosis in S . cerevisiae.

J Mol Biol, 1995 Jun 30, 250(1), 24 - 36
Evolutionarily conserved structural elements are critical for processing of Internal Transcribed Spacer 2 from Saccharomyces cerevisiae precursor ribosomal RNA; van Nues RW et al.; Structural features of Internal Transcribed Spacer 2 (ITS2) important for the correct and efficient removal of this spacer from Saccharomyces cerevisiae pre-rRNA were identified by in vivo mutational analysis based upon phylogenetic comparison with its counterparts from four different yeast species . Compatibility between ITS2 structure and the S . cerevisiae processing machinery was found to have been maintained over only a short evolutionary distance, in contrast to the situation for ITS1 . Nevertheless, cis-acting elements required for correct and efficient processing are confined predominantly to those regions of the spacer that show the highest degree of evolutionary conservation . Mutation or deletion of each of these regions severely reduced production of mature 26 S, but not 17 S rRNA, mainly by impeding processing of the 29 SB precursor . In some cases, however, conversion of 29SA into 29 SB pre-rRNA also appeared to be affected . Deletion of non-conserved segments, on the other hand, caused little or no disturbance in processing . Surprisingly, some combinations of such individually neutral deletions had a severe negative effect on the removal of ITS2, suggesting a requirement for a higher-order structure of ITS2 . Finally, even structural alterations of ITS2 that did not noticeably affect processing, significantly reduced the growth rate of cells that exclusively express the mutant rDNA units . We take this as further evidence for a direct role of ITS2 in the formation of fully functional 60 S ribosomal subunits.

Cell, 1995 Jun 30, 81(7), 1127 - 35
An in vitro assay for Saccharomyces telomerase requires EST1; Lin JJ et al.; Telomerase activity was demonstrated in cell-free extracts from S . cerevisiae through the use of a PCR-based assay . As expected, this activity was eliminated by RNase or phenol treatment of the extract and was dependent on dGTP and dTTP . Telomerase was not detected in extracts prepared from cells grown for approximately 30 or more cell divisions in the absence of the EST1 product, Est1p . TLC1 RNA, which determines the sequence of telomeric DNA in vivo, was present in normal amounts in est1 delta cells . Moreover, TLC1 RNA specifically precipitated with epitope-tagged Est1p . These data indicate that Est1p is either a subunit of yeast telomerase or an accessory protein associated with telomerase that is essential in vitro for its activity.

Cell, 1995 Jun 30, 81(7), 1075 - 84
SRP samples nascent chains for the presence of signal sequences by interacting with ribosomes at a discrete step during translation elongation; Ogg SC et al.; The signal recognition particle (SRP) binds to ribosomes that synthesize nascent chains bearing signal sequences and catalyzes their targeting to the endoplasmic reticulum membrane . In S . cerevisiae, a temperature-sensitive mutation in the SEC65 gene, encoding an SRP subunit, results in lowered levels of SRP . Growth and protein translocation defects induced by this mutation can be suppressed specifically by sublethal doses of cycloheximide but not anisomycin, each inhibitors of different steps of translation elongation . Cycloheximide also suppresses protein translocation defects caused by depletion of a different SRP subunit . We propose that reduced elongation rates in the presence of cycloheximide allow otherwise insufficient SRP to interact efficiently with ribosomes . These results are consistent with a sampling model in which SRP cycles on and off of translating ribosomes at specific steps during the elongation cycle to inspect all nascent chains for the presence of signal sequences.

Mol Gen Genet, 1995 Jun 25, 247(6), 709 - 15
Isolation of a cDNA from Saccharomyces cerevisiae that encodes a high affinity sulphate transporter at the plasma membrane; Smith FW et al.; Resistance to selenate and chromate, toxic analogues of sulphate, was used to isolate a mutant of Saccharomyces cerevisiae deficient in the capacity to transport sulphate into the cells . A clone which complements this mutation was isolated from a cDNA library prepared from S . cerevisiae poly(A)+ RNA . This clone contains an insert which is 2775 bp in length and has a single open reading frame that encodes a 859 amino acid polypeptide with a molecular mass of 96 kDa . Sequence motifs within the deduced amino acid sequence of this cDNA (SUL1) show homology with conserved areas of sulphate transport proteins from other organisms . Sequence analysis predicts the position of 12 putative membrane spanning domains in SUL1 . When the cDNA for SUL1 was expressed in S . cerevisiae, a high affinity sulphate uptake activity (Km = 7.5 +/- 0.6 microM for SO2-4) was observed . A genomic mutant of S . cerevisiae in which 1096 bp were deleted from the SUL1 coding region was constructed . This mutant was unable to grow on media containing less than 5 mM sulphate unless complemented with a plasmid containing the SUL1 cDNA . We conclude that the SUL1 cDNA encodes a S . cerevisiae high affinity sulphate transporter that is responsible for the transfer of sulphate across the plasma membrane from the external medium.

Cell, 1995 Jun 16, 81(6), 947 - 55
A role for recombination junctions in the segregation of mitochondrial DNA in yeast; Lockshon D et al.; In S . cerevisiae, mitochondrial DNA (mtDNA) molecules, in spite of their high copy number, segregate as if there were a small number of heritable units . The rapid segregation of mitochondrial genomes can be analyzed using mtDNA deletion variants . These small, amplified genomes segregate preferentially from mixed zygotes relative to wild-type mtDNA . This segregation advantage is abolished by mutations in a gene, MGT1, that encodes a recombination junction-resolving enzyme . We show here that resolvase deficiency causes a larger proportion of molecules to be linked together by recombination junctions, resulting in the aggregation of mtDNA into a small number of cytological structures . This change in mtDNA structure can account for the increased mitotic loss of mtDNA and the altered pattern of mtDNA segregation from zygotes . We propose that the level of unresolved recombination junctions influences the number of heritable units of mtDNA.

Mol Gen Genet, 1995 Jun 10, 247(5), 639 - 45
Molecular characterization and heterologous expression of an endo-beta-1,6-glucanase gene from the mycoparasitic fungus Trichoderma harzianum; Lora JM et al.; Hydrolytic enzymes from the filamentous fungus Trichoderma harzianum have been described as critical elements of the mycoparasitic action of Trichoderma against fungal plant pathogens . In this report we describe the first genomic and cDNA clones encoding a beta-1,6-endoglucanase gene . The deduced protein sequence has limited homology with other beta-glucanases . Northern experiments show a marked repression of mRNA accumulation by glucose . The protein has been successfully produced in Saccharomyces cerevisiae upon construction of a transcriptional fusion of the cDNA with a yeast promoter . This S . cerevisiae recombinant strain shows a strong lytic action on agar plates containing beta-1,6-glucan.

FEBS Lett, 1995 Jun 5, 366(1), 43 - 5
Human prothymosin alpha inhibits division of yeast Saccharomyces cerevisiae cells, while its mutant lacking nuclear localization signal does not; Pavlov N et al.; Effect of human prothymosin alpha and its mutant over-produced in S . cerevisiae on yeast cell division was studied . Wild-type prothymosin alpha appeared to block division of yeast cells . Its inhibitory action could be abolished by deletion of the last nine carboxy-terminal amino acids of prothymosin alpha containing nuclear localization signal, thus pointing to the nucleus as a compartment, where prothymosin alpha performs its action.

Cell, 1995 Jun 2, 81(5), 667 - 76
ORC and Cdc6p interact and determine the frequency of initiation of DNA replication in the genome; Liang C et al.; The origin recognition complex (ORC) binds replicators in the yeast S . cerevisiae in a manner consistent with it being an initiator protein for DNA replication . Two-dimensional (2D) gel techniques were used to examine directly initiation of chromosomal DNA replication in temperature-sensitive orc mutants . Unlike in wild-type cells, in orc2-1 and orc5-1 mutant cells, only a subset of replicators formed active origins of DNA replication at the permissive temperature . At the restrictive temperature, the number of active replicators was diminished further . Using a genetic screen, CDC6 was identified as a multicopy suppressor of orc5-1 . 2D gel and biochemical analyses demonstrated that Cdc6p interacted functionally and physically with ORC . We suggest that ORC and Cdc6p form a prereplication complex at individual replicators and therefore cooperate to determine the frequency of initiation of DNA replication in the genome.

J Biol Chem, 1995 Jun 2, 270(22), 13503 - 11
EEA1, an early endosome-associated protein . EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine "fingers" and contains a calmodulin-binding IQ motif; Mu FT et al.; Early endosomes are cellular compartments receiving endocytosed material and sorting them for vesicular transport to late endosomes and lysosomes or for recycling to the plasma membrane . We have cloned a human cDNA encoding an evolutionarily conserved 180-kDa protein on early endosomes named EEA1 (Early Endosome Antigen1) . EEA1 is associated with early endosomes since it co-localizes by immunofluorescence with the transferrin receptor and with Rab5 but not with Rab7 . Immunoelectron microscopy shows that it is associated with tubulovesicular early endosomes containing internalized bovine serum albumin-gold . EEA1 is a hydrophilic peripheral membrane protein present in cytosol and membrane fractions . It partitions in the aqueous phase after Triton X-114 solubilization and is extracted from membranes by 0.3 M NaCl . It is a predominantly alpha-helical protein sharing 17-20% sequence identity with the myosins and contains a calmodulin-binding IQ motif . It is flanked by metal-binding, cysteine "finger" motifs . The COOH-terminal fingers, Cys-X2-Cys-X12-Cys-X2-Cys and Cys-X2-Cys-X16-Cys-X2-Cys, are present within a region that is strikingly homologous with Saccharomyces cerevisiae FAB1 protein required for endocytosis and with Caenorhabditis elegans ZK632 . These fingers also show limited conservation with S . cerevisiae VAC1, Vps11, and Vps18p proteins implicated in vacuolar transport . We propose that EEA1 is required for vesicular transport of proteins through early endosomes and that its finger motifs are required for this activity.

Curr Genet, 1995 Jun, 28(1), 39 - 53
The complete mitochondrial DNA sequence of Hansenula wingei reveals new characteristics of yeast mitochondria; Sekito T et al.; The complete 27,694-bp mitochondrial (mt) DNA sequence of Hansenula wingei, which is a typical budding yeast and contains circular mitochondrial DNA, has been determined . The mt sequence contains genes encoding large and small ribosomal RNAs, 25 tRNAs, three subunits of cytochrome c oxidase (subunits 1, 2 and 3), three subunits of ATPase (subunits 6, 8 and 9), apocytochrome b, seven subunits of NADH dehydrogenase (subunits 1, 2, 3, 4, 4L, 5 and 6), and a ribosomal protein, VAR1 . The VAR1 gene is considered to be a typical yeast type . This is consistent with data on DNA and the deduced amino-acid sequence homology comparisons of genes ubiquitous in yeast and fungi . However, we have identified seven genes encoding NADH dehydrogenase subunits, which are not found in other yeast mitochondrial genomes, thus placing the H . wingei mitochondrial genome in a unique position . In addition the H . wingei mitochondrial genome also encodes one tRNA pseudogene and one short unidentified ORF . The genome is compact with only two introns both of which contain an ORF . One intron lies in the large rRNA gene while the other is situated in the cytochrome c oxidase subunit-1 gene . The conserved nonanucleotide motif (A/T)TATAAG (T/A)(A/T), which is a transcription start signal in Saccharomyces cerevisiae mitochondria, has also been found in the H . wingei mitochondrial genome . The codon assignments for ATA and CTN in H . wingei mitochondria are different from those in S . cerevisiae mitochondria . These results indicate a unique and novel structure for the H . wingei mitochondrial genome in terms of characteristics which are typical for both yeast and for filamentous fungi . This is the first complete mt DNA sequence report in yeast.

J Cell Biol, 1995 Jun, 129(6), 1617 - 28
Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function; Golsteyn RM et al.; Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells . Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes . In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions . Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S . cerevisiae Cdc5p . With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity . We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis . We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase . Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase . These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression . Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation.

J Bacteriol, 1995 Jun, 177(12), 3379 - 85
Production and reutilization of an extracellular phosphatidylinositol catabolite, glycerophosphoinositol, by Saccharomyces cerevisiae; Patton JL et al.; Phosphatidylinositol catabolism in Saccharomyces cerevisiae is known to result in the formation of extracellular glycerophosphoinositol (GroPIns) . We now report that S . cerevisiae not only produces but also reutilizes extracellular GroPIns and that these processes are regulated in response to inositol availability . A wild-type strain uniformly prelabeled with {3H} inositol displayed dramatically higher extracellular GroPIns levels when cultured in medium containing inositol than when cultured in medium lacking inositol . This difference in extracellular accumulation of GroPIns in response to inositol availability was shown to be a result of both regulated production and regulated reutilization . In a strain in which a negative regulator of phospholipid and inositol biosynthesis had been deleted (an opi1 mutant), this pattern of extracellular GroPIns accumulation in response to inositol availability was altered . An inositol permease mutant (itr1 itr2), which is unable to transport free inositol, was able to incorporate label from exogenous glycerophospho {3H}inositol, indicating that the inositol label did not enter the cell solely via the transporters encoded by itr1 and itr2 . Kinetic studies of a wild-type strain and an itr1 itr2 mutant strain revealed that at least two mechanisms exist for the utilization of exogenous GroPIns: an inositol transporter-dependent mechanism and an inositol transporter-independent mechanism . The inositol transporter-independent pathway of exogenous GroPIns utilization displayed saturation kinetics and was energy dependent . Labeling studies employing {14C}glycerophospho{3H} inositol indicated that, while GroPIns enters the cell intact, the inositol moiety but not the glycerol moiety is incorporated into lipids.

Mol Cell Biol, 1995 Jun, 15(6), 2983 - 92
KIN28 encodes a C-terminal domain kinase that controls mRNA transcription in Saccharomyces cerevisiae but lacks cyclin-dependent kinase-activating kinase (CAK) activity; Cismowski MJ et al.; The Saccharomyces cerevisiae gene KIN28 is a member of the cyclin-dependent kinase (CDK) family . The Kin28 protein shares extensive sequence identity with the vertebrate CDK-activating kinase MO15 (Cdk7), which phosphorylates CDKs in vitro on a critical threonine residue . Kin28 and MO15 have recently been found to copurify with the transcription factor IIH (TFIIH) holoenzyme of yeast and human cells, respectively . Although TFIIH is capable of phosphorylating the C-terminal domain (CTD) of RNA polymerase II, it has been unclear whether Kin28 is the physiologically relevant CTD kinase or what role CTD phosphorylation plays in transcription . In this study, we used a thermosensitive allele of KIN28 and a hemagglutinin epitope-tagged Kin28 protein to investigate Kin28 function in transcription and in the cell cycle . We show that Kin28 acts as a positive regulator of mRNA transcription in vivo and possesses CTD kinase activity in vitro . However, Kin28 neither regulates the phosphorylation state of the yeast cell cycle CDK, Cdc28, nor possesses CDK-activating kinase activity in vitro . We conclude that Kin28 is a strong candidate for the physiological CTD kinase of S . cerevisiae and that Kin28 function is required for mRNA transcription.

Cell Biochem Funct, 1995 Jun, 13(2), 91 - 8
Assessment of growth inhibition by aldehydic lipid peroxidation products and related aldehydes by Saccharomyces cerevisiae; Wonisch W et al.; The effect of pretreatment with aldehydes on the subsequent colony forming efficiency (CFE) of Saccharomyces cerevisiae was investigated . All 21 aldehydes tested inhibited CFE in a dose-dependent manner . The effective doses, however, differed markedly from 300 mM to 0.07 mM depending on the functional groups and chain length of the aldehydes . Amongst the nine representatives of n-alkanals, formaldehyde was the most potent inhibitor, reducing CFE to 50 per cent at a dose of 0.3 mM (IC50) . In the series of 2-trans-alkenals, acrolein was most effective with an IC50 of 0.08 mM and amongst the 4-hydroxy 2-trans-alkenals, 4-hydroxynonenal was most effective with IC50 of 0.07 mM . In general, effectiveness decreased in the order: 4-hydroxyalkenals > 2-alkenals >> n-alkenals . It is proposed that S . cerevisiae is a promising target cell to elucidate further the molecular mechanisms by which aldehydes, particularly the lipid peroxidation product 4-hydroxynonenal, inhibits cell proliferation.

J Ind Microbiol, 1995 Jun, 14(6), 440 - 50
Chemical and cytological changes during the autolysis of yeasts; Hernawan T et al.; Cell suspensions of Sacharomyces cerevisiae, Kloeckera apiculata and Candida stellata were autolyzed in phosphate buffer, pH 4.5, for up to 10 days . Cell dry weights decreased by 25-35% after 10 days . Based on initial cell dry weight, the soluble autolysate consisted of: carbohydrate (principally polysaccharide) 3-7%; organic acids 3-6%; protein 12-13%; free amino acids 8-12%; nucleic acid products 3-5%; and lipids 1-2% . The main organic acids in autolysates were propionic, succinic and acetic and the main amino acids were phenylalanine, glutamic acid, leucine, alanine and arginine . Approximately 85-90% of cellular RNA and 25-40% of cellular DNA were degraded during autolysis . Both neutral lipid and phospholipid components were degraded, with neural lipids but not phospholipids being found in autolysates . Scanning and transmission electron micrographs showed retention of cell wall structure and shape during autolysis, but there was extensive intracellular disorganization within S . cerevisiae and C . stellata . There were differences in the autolytic behavior of K . apiculata compared with S . cerevisiae and C . stellata.

Proc Natl Acad Sci U S A, 1995 May 23, 92(11), 4907 - 11
Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes; Singh KK et al.; Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism . Induction of the S . cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter . Sequences similar to the MAG URS elements are present in at least 11 other S . cerevisiae DNA repair and metabolism genes . Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination . We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa . Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes . These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes.

Biochemistry, 1995 May 16, 34(19), 6382 - 8
Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: revised amino acid sequence, site-directed mutagenesis, and microenvironment characteristics of cysteines 365 and 458; Krautwurst H et al.; Two cysteine residues in phosphoenolpyruvate (PEP) carboxykinase from Saccharomyces cerevisiae {ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49} the modification of which leads to enzyme inactivation have been subjected to site-directed mutagenesis . PEP carboxykinase is inactivated by alkylation of Cys365 or Cys458; however, mutation of either or both of these residues to serine has little effect on the enzymatic activity . These results eliminate any possible catalytic function for these cysteinyl residues . In the course of this work, discrepancies in the published nucleotide sequence of the S . cerevisiae PEP carboxykinase gene were detected that alter the deduced amino acid sequence . Several of these discrepancies were verified through the sequencing of proteolytic peptides . Our results indicate that the protein corresponds to a 549 amino acid polypeptide and that the positions previously assigned to Cys364 and Cys457 correspond to Cys365 and Cys458 . The individual reactivities and the microenvironment characteristics around these sulfhydryl groups were investigated by their selective modification with the fluorescent reagent N-(1-pyrenyl)maleimide (PyM) . Our findings indicate that Cys458 is 7-fold more reactive toward the sulfhydryl-directed probe than Cys365, while quenching experiments of PyM-labeled mutant enzymes suggest that the former residue is located in a region more accessible to water than the latter.

Nucleic Acids Res, 1995 May 11, 23(9), 1557 - 60
Versatile vectors to study recoding: conservation of rules between yeast and mammalian cells; Stahl G et al.; In many viruses and transposons, expression of some genes requires alternative reading of the genetic code, also called recoding . Such events depend on specific mRNA sequences and can lead to read through of an in-frame stop codon or to +1 or -1 frameshifting . Here, we addressed the issue of conservation of recoding rules between the yeast Saccharomyces cerevisiae and mammalian cells by establishing a versatile vector that can be used to study recoding in both species . We first assessed this vector by analysing the site of +1 frameshift of the Ty1 transposon . Two sequences from higher organisms were then tested in both yeast and mammalian cells: the gag-pol junction of human immunodeficiency virus type 1 (HIV-1) (a site of -1 frameshift), and the stop codon region of the replicase cistron from the tobacco mosaic virus (a site of UAG read through) . We show that both sequences direct a high level of recoding in yeast . Furthermore, different mutations of the target sequences have similar effects on recoding in yeast and in mouse cells . Most notably, a strong decrease of frameshifting was observed in the absence of the HIV-1 stem-loop stimulatory signal . Taken together, these data suggest that mechanisms of some recoding events are conserved between lower and higher eukaryotes, thus allowing the use of S . cerevisiae as a model system to study recoding on target sequences from higher organisms.

Mol Gen Genet, 1995 May 10, 247(3), 379 - 86
Incorporation of the yeast mitochondrial ribosomal protein Mrp2 into ribosomal subunits requires the mitochondrially encoded Var1 protein; Davis SC et al.; Mrp2 is a protein component of the small subunit of mitochondrial ribosomes in the yeast Saccharomyces cerevisiae . We have examined the expression of Mrp2 in yeast mutants lacking mitochondrial DNA and found that the steady-state level of Mrp2 is dramatically decreased relative to wild type . These data suggest that the accumulation of Mrp2 depends on the expression of one or more mitochondrial gene products . The mitochondrial genome of S . cerevisiae encodes two components of the small ribosomal subunit, 15S rRNA and the Var1 protein, both of which are necessary for the formation of mature 37S subunits . Several studies have shown that in the absence of Var1 incomplete subunits accumulate, which lack a limited number of ribosomal proteins . Here, we show that Mrp2 is one of the proteins absent from subunits lacking Var1, indicating that Var1 plays an important role in the incorporation of Mrp2 into mitochondrial ribosomal subunits.

Proc Natl Acad Sci U S A, 1995 May 9, 92(10), 4219 - 23
Escherichia coli iron superoxide dismutase targeted to the mitochondria of yeast cells protects the cells against oxidative stress; Balzan R et al.; A gene encoding a fusion protein consisting of Escherichia coli iron superoxide dismutase (FeSOD) with the mitochondrial targeting presequence of yeast manganese superoxide dismutase (MnSOD) was cloned and expressed in E . coli and in Saccharomyces cerevisiae DL1Mn- yeast cells deficient in MnSOD . In the yeast cells the fusion protein was imported into the mitochondrial matrix . However, the presequence was not cleaved . In a control set of experiments, the E . coli FeSOD gene without the yeast MnSOD leader sequence was also cloned and expressed in S . cerevisiae DL1Mn- cells . In this case the FeSOD was located in the cytosol and was not imported into the mitochondrial matrix . E . coli FeSOD, with and without the yeast MnSOD presequence, proved to be active in yeast, but, whereas the FeSOD targeted to the mitochondria of yeast cells deficient in MnSOD protected the cells from the toxic effects of oxidative stress, FeSOD without the yeast MnSOD presequence did not protect the yeast cells deficient in MnSOD against oxidative stress.

Biochem Biophys Res Commun, 1995 May 5, 210(1), 126 - 32
Novel system for monitoring autophagy in the yeast Saccharomyces cerevisiae; Noda T et al.; The yeast S . cerevisiae imports cytosolic components into the vacuole non-selectively by autophagy and degrades them by vacuolar hydrolases under nutrient starvation conditions . We developed a novel system for monitoring autophagy by constructing cells in which modified vacuolar alkaline phosphatase is expressed as an inactive precursor form in the cytosol . Under starvation conditions, the processing of the precursor to the mature form and phosphatase activity appeared gradually, and the mature form was located in the vacuole . Disruption of APG1, an essential gene for autophagy, resulted in no processing or phosphatase activity . These results indicate that the precursor form in the cytosol is transferred to the vacuole by autophagy and converted to the active form by vacuolar proteinases . Thus, autophagy could be determined easily and accurately by measuring the phosphatase activity.

Microbiology, 1995 May, 141 ( Pt 5), 1101 - 8
Nitrogen-regulated transcription and enzyme activities in continuous cultures of Saccharomyces cerevisiae; ter Schure EG et al.; Variations in the transcription of nitrogen-regulated genes and in the activities of nitrogen-regulated enzymes of the yeast Saccharomyces cerevisiae were studied by changing the carbon and nitrogen fluxes . S . cerevisiae was grown in continuous culture at various dilution rates (D) under nitrogen limitation with NH4Cl as sole nitrogen source . With an increase in D from 0.05 to 0.29 h-1, both the glucose and the ammonia flux increased sixfold . The activities of the two ammonia-incorporating enzymes, NADPH-dependent glutamate dehydrogenase (NADPH-GDH) and glutamine synthetase (GS), encoded by GDH1 and GLN1, respectively, increased with increasing D, while the activity of the glutamate-degrading enzyme, NAD-dependent glutamate dehydrogenase (NAD-GDH), decreased . Surprisingly, no changes were observed in the transcription of GDH1 and GLN1; however increased D was accompanied by an increase in GAP1 transcription . At the metabolite level, the increase in the glucose and nitrogen flux did not result in changes in the intracellular 2-oxoglutarate, glutamate or glutamine concentrations . It is shown that growth on ammonia alone is not sufficient to cause repression of GAP1 and GLN1 transcription and that the regulation of GAP1 transcription and both NADPH-GDH and GS activity is not an on/off switch, but is gradually modulated in correlation with the ammonia concentration.

Biochem J, 1995 May 1, 307 ( Pt 3), 657 - 61
The mechanism for the ATP-induced uncoupling of respiration in mitochondria of the yeast Saccharomyces cerevisiae; Prieto S et al.; We have recently reported that ATP induces an uncoupling pathway in Saccharomyces cerevisiae mitochondria {Prieto, Bouillaud, Ricquier and Rial (1992) Eur . J . Biochem . 208, 487-491} . The presence of this pathway would explain the reported low efficiency of oxidative phosphorylation in S . cerevisiae, and may represent one of the postulated energy-dissipating mechanisms present in these yeasts . In this paper we demonstrate that ATP exerts its action in two steps: first, at low ATP/Pi ratios, it increases the respiratory-chain activity, probably by altering the kinetic properties of cytochrome c oxidase . Second, at higher ATP/Pi ratios, an increase in membrane permeability leads to a collapse in membrane potential . The ATP effect on cytochrome c oxidase corroborates a recent report showing that ATP interacts specifically with yeast cytochrome oxidase, stimulating its activity {Taanman and Capaldi (1993) J . Biol . Chem . 268, 18754-18761}.

Mol Cell Biol, 1995 May, 15(5), 2728 - 36
Regulation and intracellular localization of Saccharomyces cerevisiae strand exchange protein 1 (Sep1/Xrn1/Kem1), a multifunctional exonuclease; Heyer WD et al.; The Saccharomyces cerevisiae strand exchange protein 1 (Sep1; also referred to as Xrn1, Kem1, Rar5, or Stp beta) catalyzes the formation of hybrid DNA from model substrates in vitro . The protein is also a 5'-to-3' exonuclease active on DNA and RNA . Multiple roles for the in vivo function of Sep1, ranging from DNA recombination and cytoskeleton to RNA turnover, have been proposed . We show that Sep1 is an abundant protein in vegetative S . cerevisiae cells, present at about 80,000 molecules per diploid cell . Protein levels were not changed during the cell cycle or in response to DNA-damaging agents but increased twofold during meiosis . Cell fractionation and indirect immunofluorescence studies indicated that > 90% of Sep1 was cytoplasmic in vegetative cells, and indirect immunofluorescence indicated a cytoplasmic localization in meiotic cells as well . The localization supports the proposal that Sep1 has a role in cytoplasmic RNA metabolism . Anti-Sep1 monoclonal antibodies detected cross-reacting antigens in the fission yeast Schizosccharomyces pombe, in Drosophila melanogaster embryos, in Xenopus laevis, and in a mouse pre-B-cell line.

Hum Mol Genet, 1995 May, 4(5), 791 - 9
A human homolog of the S . cerevisiae HIR1 and HIR2 transcriptional repressors cloned from the DiGeorge syndrome critical region; Lamour V et al.; The DiGeorge syndrome (DGS) is a developmental disorder affecting derivatives of the third and fourth pharyngeal pouches . DGS patients present an interstitial deletion in one of their two chromosomes 22 . Cosmid DAC30 was mapped to the DGS smallest critical region . Iterative cDNA library screening initiated with a DAC30 gene fragment candidate yielded a cDNA contig whose assembled nucleotide sequence is consistent with the widely transcribed, 4.2-4.4 kb long, messengers detected by northern analysis . The deduced protein sequence, 1017 amino acids in length, entirely encompasses the 766 amino acids previously designated as TUPLE1 . The completed protein has been renamed HIRA because it contains various features matching those found in HIR1 and HIR2, two repressors of histone gene transcription characterized in the yeast Saccharomyces cerevisiae . Strikingly alike in their N-terminal third, HIRA and HIR1 contain seven copies of the WD repeat, a motif implicated in protein-protein interactions, suggesting that they might define a new subfamily of functionally homologous proteins . The remainder of the human polypeptide highly resembles a corresponding fragment in HIR2 . We propose that HIRA, alone, could have a part in mechanisms of transcriptional regulation similar to that played by HIR1 and HIR2 together . The presence of a single copy of the HIRA gene in DGS patients possibly accounts for some of the abnormalities associated with this syndrome.

Appl Microbiol Biotechnol, 1995 May-Jun, 43(2), 321 - 4
Influence of pH, malic acid and glucose concentrations on malic acid consumption by Saccharomyces cerevisiae; Delcourt F et al.; Malic acid consumption by Saccharomyces cerevisiae was studied in a synthetic medium . The extent of malic acid degradation is affected by its initial concentration, the extent and the rate of deacidification increased with initial malate concentration up to 10 milligrams . For malic acid consumption, an optimal pH range of 3-3.5 was found, confirming that non-dissociated organic acids enter S . cerevisiae cells by simple diffusion . A full factorial design has been employed to describe a statistical model of the effect of sugar and malic acid on the quantity of malate degraded (milligrams) by a given amount of biomass (milligrams) . The results indicated that the initial malic acid concentration is very important for the ratio of malate consumption to quantity of biomass . The yeast was found to be most efficient at higher levels of malate.

J Ind Microbiol, 1995 May, 14(5), 355 - 64
The Thom Award address . Industrial mycology and the new genetics; Lemke PA; The genetic investigation of fungi has been extended substantially by DNA-mediated transformation, providing a supplement to more conventional genetic approaches based upon sexual and parasexual processes . Initial transformation studies with the yeast Saccharomyces cerevisiae provided the model for transformation systems in other fungi with regard to methodology, vector construction and selection strategies . There are, however, certain differences between S . cerevisiae and filamentous fungi with regard to type of genomic insertion and the availability of shuttle vectors . Single-site linked insertions are common in yeast due to the high level of homology required for recombination between vectored and genomic sequences, whereas mycelial fungi often show a high frequency of heterologous and unlinked insertions, often in the form of random and multiple-site integrations . While extrachromosomally-maintained or replicative vectors are readily available for use with yeasts, such vectors have been difficult to construct for use with filamentous fungi . The development of vectors for replicative transformation with these fungi awaits further study . It is proposed that replicative vectors may be inherently less efficient for use with mycelial fungi relative to yeasts, since the mycelium, as an extended and semicontinuous network of cells, may delimit an adequate diffusion of the vector carrying the selectable gene, thus leading to a high frequency of abortive or unstable transformants.

Curr Genet, 1995 May, 27(6), 517 - 23
Use of the polymerase chain reaction to identify coding sequences for chitin synthase isozymes in Phialophora verrucosa; Peng M et al.; Based on conserved amino-acid regions predicted for the chitin synthases (Chs) of Saccharomyces cerevisiae, two different primer sets were synthesized and used in polymerase chain reactions (PCRs) to amplify 614-bp and 366-bp sequences from genomic DNA of the zoopathogenic fungus Phialophora verrucosa . DNA-sequencing and Southern-blotting analyses of the 614-bp DNA amplification products suggested that portions of two distinct P . verrucosa chitin synthase genes (PvCHS1, PvCHS2), coding for two different zymogenic-type PvChs isozymes, had been identified . The deduced amino-acid sequence of each fell into different Chs classes, namely class I and class II . In addition, the 366-bp DNA segment was shown to code for a conserved region having homology with the CSD2/CAL1 gene of S . cerevisiae, which encodes a nonzymogenic-type enzyme, Chs3, in that fungus . The amino-acid sequence derived from PvCHS3 exhibits 88.2% similarity and 78.4% identity to the same amino-acid region of the S . cerevisiae enzyme . These results provide a critical first step toward investigating the molecular and pathogenic importance of CHS gene regulation in this fungus and for exploring steps leading to Chs function as potential targets for the design of new therapeutic agents.

Curr Genet, 1995 May, 27(6), 493 - 5
Gene PSO5 of Saccharomyces cerevisiae, involved in repair of oxidative DNA damage, is allelic to RAD16; Paesi-Toresan SO et al.; The pos5-1 mutation renders Saccharomyces cerevisiae cells sensitive to DNA-damaging agents . We have isolated plasmids from a S . cerevisiae genomic library capable of restoring wild-type levels of 254-nm ultraviolet light sensitivity of the pso5-1 mutant . DNA sequence analysis revealed that the complementing activity resides in RAD16, a gene involved in excision repair . Tetrad analysis showed that PSO5, like RAD16, is tightly linked to LYS2 on chromosome II . Moreover, allelism between the pso5-1 and rad16 mutants was demonstrated by the comparison of mutagen sensitivity phenotypes, complementation tests, and by meiotic analysis . The cloned RAD16 gene was capable of restoring wild-type resistance of the pso5-1 mutant to H2O2 and photoactivated 3-carbethoxypsoralen, both treatments generating oxidative stress-related DNA damage . This indicates that RAD16/PSO5 might also participate in the repair of oxidative base damage.

Yeast, 1995 Apr 30, 11(5), 447 - 53
Saccharomyces cerevisiae mRNA 3' end forming signals are also involved in transcription termination; Russo P; Previously, a 38-base-pair (bp) region in the 3' untranslated portion of the Saccharomyces cerevisiae iso-1-cytochrome c gene, was shown to be required for both normal CYC1 mRNA 3' end formation (Zaret and Sherman, 1982), and efficient transcription termination (Russo and Sherman, 1989) . In another study, specific sequences such as TATATA, TACATA, and TAGTAGTA were shown to be involved in mRNA 3' end formation in S . cerevisiae (Russo et al., 1991) . In this report, an in vivo plasmid stability assay has been utilized to show that these and related sequences are also involved in transcription termination, at varying efficiencies, and in an orientation-dependent manner . For example: the sequence TATATA appeared to terminate transcription almost as efficiently as the original wild type 38-bp region; whereas, the sequences TAGATATATGTAA and TACATA were less efficient, and TTTTTTTATA had little, if any, transcription termination function . In contrast, none of these sequences appeared to terminate transcription in the reverse orientation . Therefore, it appears that certain sequence signals capable of promoting mRNA 3' end formation in yeast, are also directly involved in transcription termination.

Yeast, 1995 Apr 30, 11(5), 425 - 33
Cloning and sequencing of the URA5 gene from the yeast Yarrowia lipolytica; Sanchez M et al.; The URA5 gene of Yarrowia lipolytica encoding the orotate phosphoribosyl transferase (OPRTase, EC2.4.2.10) was isolated by target integration in a mutant strain originally named ura2.21 . The nucleotide sequence of the gene predicts a protein with high similarities with the OPRTases from Saccharomyces cerevisiae, Podospora anserina and Escherichia coli and to a lesser extent with that of Dictyostelium discoideum . The transcription start point has been mapped by primer extension analysis and indicates the existence of a long leader sequence in the corresponding mRNA . Northern-blot hybridization revealed the URA5 transcript to be approximately 0.94 kb . Deletion of the URA5 gene in Y . lipolytica produced a leaky phenotype similar to the one described for the ura5 mutation in S . cerevisiae . The URA5 gene of Y . lipolytica was able to complement functionally the ura5 mutation of S . cerevisiae.

Yeast, 1995 Apr 30, 11(5), 407 - 18
Regulation of carbon metabolism in chemostat cultures of Saccharomyces cerevisiae grown on mixtures of glucose and ethanol; de Jong-Gubbels P et al.; Growth efficiency and regulation of key enzyme activities were studied in carbon- and energy-limited chemostat cultures of Saccharomyces cerevisiae grown on mixtures of glucose and ethanol at a fixed dilution rate . Biomass yields on substrate carbon and oxygen could be adequately described as the net result of growth on the single substrates . Activities of isocitrate lyase and malate synthase were not detected in cell-free extracts of glucose-limited cultures . However, both enzymes were present when the ethanol fraction in the reservoir medium exceeded the theoretical minimum above which the glyoxylate cycle is required for anabolic reactions . Fructose-1,6-bisphosphatase activity was only detectable at high ethanol fractions in the feed, when activity of this enzyme was required for synthesis of hexose phosphates . Phospho-enol-pyruvate-carboxykinase activity was not detectable in extracts from glucose-grown cultures and increased with the ethanol fraction in the feed . It is concluded that, during carbon-limited growth of S . cerevisiae on mixtures of glucose and ethanol, biosynthetic intermediates with three or more carbon atoms are preferentially synthesized from glucose . Synthesis of the key enzymes of gluconeogenesis and the glyoxylate cycle is adapted to the cells' requirement for these intermediates . The gluconeogenic enzymes and their physiological antagonists (pyruvate kinase, pyruvate carboxylase and phosphofructokinase) were expressed simultaneously at high ethanol fractions in the feed . If futile cycling is prevented under these conditions, this is not primarily achieved by tight control of enzyme synthesis.

Nucleic Acids Res, 1995 Apr 25, 23(8), 1350 - 3
Correlation of GC content with replication timing and repair mechanisms in weakly expressed E.coli genes; Deschavanne P et al.; Regional variations of DNA GC content are observed in species as different as S.cerevisiae and humans . In vertebrates and yeast they are correlated with replication timing; late replicating chromosomal regions are more AT-rich than early replicating regions . We show here that gene composition in E.coli also has long range variations which are similarly correlated with replication timing . We suggest that the enrichment in AT base pairs in late replicating DNA reflects differences in DNA repair modes . These sequences, which are in single copy for a greater part of the cell cycle than origin-linked genes, have less opportunity to engage in repair via homologous recombination and therefore may resort more often to translesion synthesis involving the misincorporation of adenine opposite modified nucleotides.

Nucleic Acids Res, 1995 Apr 25, 23(8), 1307 - 10
Cloning, sequencing and bacterial expression of human glycine tRNA synthetase; Williams J et al.; The human glycine tRNA synthetase gene (GlyRS) has been cloned and sequenced . The 2462 bp cDNA for this gene contains a large open reading frame (ORF) encoding 685 amino acids with predicted M(r) = 77,507 Da . The protein sequence has approximately 60% identity with B . mori GlyRS and 45% identity with S . cerevisiae GlyRS and contains motifs 2 and 3 characteristic of Class II tRNA synthetases . A second ORF encoding 47 amino acids is found upstream of the large ORF . Translation of this ORF may precede the expression of GlyRS as a possible regulatory mechanism . The enzyme was expressed in E . coli as a fusion protein with a 13 kDa biotinylated tag with an apparent M(r) = 90 kDa . The fusion protein was immunoprecipitated from crude bacterial extract with human EJ serum, which contains autoantibodies directed against GlyRS, and with rabbit polyclonal serum raised against a synthetic peptide derived from the predicted amino acid sequence of human GlyRS . Bacterial extract containing the fusion protein catalyses the aminoacylation of bovine tRNA with {14C}-gly at 10-fold increased level above normal bacterial extract and confirms that the cDNA encodes human GlyRS.

Cell, 1995 Apr 21, 81(2), 261 - 8
CDC27Hs colocalizes with CDC16Hs to the centrosome and mitotic spindle and is essential for the metaphase to anaphase transition; Tugendreich S et al.; We have isolated cDNAs and raised antibodies corresponding to the human homologs of the S . cerevisiae CDC27 and CDC16 proteins, which are tetratrico peptide repeat (TPR)-containing proteins essential for mitosis in budding yeast . We find that the CDC27Hs and CDC16Hs proteins colocalize to the centrosome at all stages of the mammalian cell cycle, and to the mitotic spindle . Injection of affinity-purified anti-CDC27Hs antibodies into logarithmically growing HeLa cells causes a highly reproducible cell cycle arrest in metaphase with apparently normal spindle structure . We conclude that CDC27 and CDC16 are evolutionarily conserved components of the centrosome and mitotic spindle that control the onset of postmetaphase events during mitosis.

Cell, 1995 Apr 21, 81(2), 253 - 60
Sgs1: a eukaryotic homolog of E . coli RecQ that interacts with topoisomerase II in vivo and is required for faithful chromosome segregation; Watt PM et al.; Topoisomerase II (topo II) catalyzes the decatenation of interlinked DNA molecules and is essential for chromosome segregation . To test the hypothesis that the noncatalytic C-terminal domain of topo II is necessary for mediating interactions with other proteins required for chromosome segregation, we used a two-hybrid cloning strategy to identify proteins that interact with S . cerevisiae topo II in vivo . One protein identified (Sgs1p) is structurally related to E . coli RecQ protein and contains helicase signature motifs . Strains lacking Sgs1p exhibit elevated levels of chromosome misseggregation during both mitotic and meiotic division . We propose a model to account for the interaction of a topoisomerase and a helicase in the faithful segregation of newly replicated eukaryotic chromosomes.

Yeast, 1995 Apr 15, 11(4), 371 - 82
Sequence analysis of the right end of chromosome XV in Saccharomyces cerevisiae: an insight into the structural and functional significance of sub-telomeric repeat sequences; Pryde FE et al.; Approximately 3.9 kb of DNA, centromere proximal to the previously sequenced Y' element at the right end of chromosome XV in Saccharomyces cerevisiae strain YP1, has been sequenced . A number of the known sub-telomeric repeat sequences were identified, including Y', core X and STRs A, B . C and D . Several of these repeat elements contain potentially functional sequences . In addition, two other members of repeated gene families were identified . The first of these shows 61% and 60% DNA sequence identity to Enolases 1 and 2 respectively . The Enolase-like sequence appears to be species specific, with three copies being found in all strains of S . cerevisiae studied . The location of the three copies is the same for all strains . The second repeated sequence has homology with known open reading frames on chromosomes III, V and XI . There are five or six copies of this sequence in all S . cerevisiae and S . paradoxus strains studied and three in S . bayanus strains . The analysis of this region and comparison to sub-telomeric regions on other chromosomes gives some indication as to the potential functional and structural significance of sub-telomeric repeat sequences . In addition, these findings are consistent with the idea that sub-telomeric regions may be targets for unusual recombination events.

Biochim Biophys Acta, 1995 Apr 6, 1266(1), 45 - 9
Cytokinins affect spore formation but not cell division in the yeast Saccharomyces cerevisiae; Laten HM; Cytokinins are N6-substituted adenine derivatives that function as essential growth hormones in higher plants . In experimental systems, cytokinins can influence cell growth and differentiation among both plant and non-plant tissues . The single-celled yeast, Saccharomyces cerevisiae, has served as an effective and useful model system for the study of a wide range of cellular phenomena generally associated with higher eukaryotes, including mammals . In an attempt to assess the efficacy of its use to dissect the molecular basis for plant hormone action, the effects of cytokinins on S . cerevisiae with respect to cell division rates and sporulation efficiencies were monitored . While none of the cytokinins tested influenced mitotic generation times, micromolar concentrations of kinetin enhanced the formation of yeast haploid ascospores and even lower concentrations of isopentenyladenine inhibited ascus formation.

Biochim Biophys Acta, 1995 Apr 5, 1248(1), 75 - 9
Affinity-purification and identification of GrpE homologues from mammalian mitochondria; Naylor DJ et al.; We used affinity chromatography on DnaK columns to identify a mitochondrial GrpE homologue from bovine, porcine and rat liver mitochondria . The 24 kDa GrpE homologue bound specifically to the DnaK column and was not eluted with 1 M KCl but readily with 5 mM ATP . Sequence analysis of the bovine homologue (85 residues) revealed 42% positional identity to mitochondrial GrpEp from S . cerevisiae and about 30% identity to the bacterial counterparts . Thus, GrpE homologues from higher and lower eukaryotes are highly conserved.

EMBO J, 1995 Apr 3, 14(7), 1453 - 67
The sequence and binding specificity of UaY, the specific regulator of the purine utilization pathway in Aspergillus nidulans, suggest an evolutionary relationship with the PPR1 protein of Saccharomyces cerevisiae; Suarez T et al.; The uaY gene codes for a transcriptional activator mediating the induction of a number of unlinked genes involved in purine utilization in Aspergillus nidulans . Here we present the complete genomic and cDNA nucleotide sequence of this gene . The gene contains two introns . The derived polypeptide of 1060 residues contains a typical zinc binuclear cluster domain and shows a number of similarities with the PPR1 regulatory gene of Saccharomyces cerevisiae . These similarities are most striking in the putative linker and dimerization regions following the zinc cluster . Gel-shift and DNase I footprinting experiments have been carried out for three genes subject to UaY-mediated induction . The binding sequence is 5'-TCGG-6X-CCGA, which is identical to the proposed PPR1 binding sites . Nevertheless, the identity of the base immediately 3' of the 5'-TCGG sequence clearly affects the affinity of the site . The site upstream of the uapA gene has been shown to be active in vivo . Binding to this site has been analysed by a number of interference techniques . There is an interesting chemical similarity between the co-inducer of the purine utilization pathway (uric acid) and that of the genes of the pyrimidine biosynthetic pathway (dihydroorotic acid) and we show that dihydroorotic acid can act as a poor inducer of at least one activity under UaY control . These striking similarities, together with the unique pattern of regulation of pyrimidine biosynthesis in S . cerevisiae, suggest that PPR1 evolved through recruitment into the pyrimidine biosynthetic pathway of an ancestral gene related to uaY.

J Bacteriol, 1995 Apr, 177(7), 1817 - 23
Sterol uptake induced by an impairment of pyridoxal phosphate synthesis in Saccharomyces cerevisiae: cloning and sequencing of the PDX3 gene encoding pyridoxine (pyridoxamine) phosphate oxidase; Loubbardi A et al.; Exogenous sterols do not permeate wild-type Saccharomyces cerevisiae in aerobic conditions . However, mutant strain FKerg7, affected in lanosterol synthase, is a sterol auxotroph which is able to grow aerobically in the presence of ergosterol . Viability of this strain depends on the presence of an additional mutation, aux30, that leads to sterol permeability . Cells bearing the aux30 mutation fail to grow in standard yeast nitrogen base medium containing pyridoxine but grow normally if pyridoxine is replaced by either pyridoxal or pyridoxamine . These mutants are characterized by a lack in pyridoxine (pyridoxamine) phosphate oxidase {P(N/M)P oxidase} (EC 1.4.3.5) activity . The pleiotropic phenotype induced by the aux30 mutation includes a strong perturbation in amino acid biosynthesis . Strains bearing the aux30 mutation also display atypic fatty acid, sterol, and cytochrome patterns . Transformation of an aux30 strain with a replicative vector carrying the wild-type PDX3 gene encoding P(N/M)P oxidase restored wild-type fatty acid, sterol, and cytochrome patterns and suppressed exogenous sterol accumulation . It is proposed that sterol permeation of aux30 strains in mainly the consequence of their leaky Hem- character . The amino acid sequence of S . cerevisiae P(N/M)P oxidase inferred from the nucleotide sequence of PDX3 shows a high percentage of homology with the corresponding enzymes from Escherichia coli and Myxococcus xanthus . Several putative Gcn4p binding sequences are present in the PDX3 promoter region, leading to the assumption that transcription of this gene is under the general control of nitrogen metabolism.

Trends Biochem Sci, 1995 Apr, 20(4), 143 - 6
The SWI-SNF complex: a chromatin remodeling machine?
Peterson CL, Tamkun JW.
The SWI-SNF complex plays a key role in the regulation of eukaryotic gene expression . Genetic studies in the yeast Saccharomyces cerevisiae suggest that one role for the complex is to antagonize chromatin-mediated repression of transcription . Recent biochemical studies indicate that S . cerevisiae and putative human SWI-SNF complexes use the energy of ATP hydrolysis to disrupt nucleosome structure.

Mol Microbiol, 1995 Apr, 16(1), 157 - 67
Identification of novel HXT genes in Saccharomyces cerevisiae reveals the impact of individual hexose transporters on glycolytic flux; Reifenberger E et al.; In Saccharomyces cerevisiae, hexose uptake is mediated by HXT proteins which belong to a superfamily of monosaccharide facilitators . We have identified three more genes that encode hexose transporters (HXT5, 6, 7) . Genes HXT6 and HXT7 are almost identical and located in tandem 3' adjacent to HXT3 on chromosome IV . We have constructed a set of congenic strains expressing none or any one of the seven known HXT genes and followed growth and flux rates for glucose utilization . The hxt null strain does not grow on glucose, fructose or mannose, and both glucose uptake and flux rate were below the detection level . Expression of either HXT1, 2, 3, 4, 6 or 7 is basically sufficient for aerobic growth on these sugars . In most of the constructs, glucose was the preferred substrate compared to fructose or mannose . There is a considerable variation in flux and growth rates with 1% glucose, dependent on the expression of the individual HXT genes . Expression of either HXT2, 6 or 7 in the null background is sufficient for growth on 0.1% glucose, while growth of strains with only HXT1, 3 or 4 requires higher (> or = 1%) glucose concentrations . These results demonstrate that individual HXT proteins can function independently as hexose transporters, and that most of the metabolically relevant HXT transporters from S . cerevisiae have been identified.

Curr Genet, 1995 Apr, 27(5), 427 - 34
Mutants of Saccharomyces cerevisiae sensitive to oxidative and osmotic stress; Krems B et al.; Although oxidative stress is involved in many human diseases, little is known of its molecular basis in eukaryotes . In a genetic approach, S . cerevisiae was used to identify elements involved in oxidative stress . By using hydrogen peroxide as an agent for oxidative stress, 34 mutants were identified . All mutants were recessive and fell into 16 complementation groups (pos1 to pos16 for peroxide sensitivity) . They corresponded to single mutations as shown by a 2:2 segregation pattern . Enzymes reportedly involved in oxidative stress, such as glucose-6-phosphate dehydrogenase, glutathione reductase, superoxide dismutase, as well as glutathione concentrations, were investigated in wild-type and mutant-cells . One complementation group lacked glucose-6-phosphate dehydrogenase and was shown to be allelic to the glucose-6-phosphate dehydrogenase structural gene ZWF1/MET19 . In other mutants all enzymes supposedly involved in oxidative-stress resistance were still present . However, several mutants showed strongly elevated levels of glutathione reductase, gluconate-6-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase . One complementation group, pos9, was highly sensitive to oxidative stress and revealed the same growth phenotype as the previously described yap1/par1 mutant coding for the yeast homologue of mammalian transcriptional activator protein, c-Jun, of the proto-oncogenic AP-1 complex . However, unlike par1 mutants, which showed diminished activities of oxidative-stress enzymes and glutathion level, the pos9 mutants did not reveal any such changes . In contrast to other recombinants between pos mutations and par1, the sensitivity did not further increase in par1 pos9 recombinants, which may indicate that both mutations belong to the same regulating circuit.(ABSTRACT TRUNCATED AT 250 WORDS)

J Cell Biol, 1995 Apr, 129(1), 65 - 80
PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intra-peroxisomal protein that is a member of the WD repeat family and is essential for the import of thiolase into peroxisomes; Zhang JW et al.; We have previously described mutant S . cerevisiae that are defective in peroxisome biogenesis (peb mutants) (Zhang, J . W., Y . Han, and P . B . Lazarow . 1993 . J . Cell Biol . 123:1133-1147.) . In some mutants, peroxisomes are undetectable . Other mutants contain normal-looking peroxisomes but fail to package subsets of peroxisomal proteins into the organelle (Zhang, J . W., C . Luckey, and P . B . Lazarow . 1993 . Mol . Biol . Cell . 4:1351-1359.) . In peb1 (pas7) cells, for example, the peroxisomes contain proteins that are targeted by COOH-terminal tripeptides and contain acyl-CoA oxidase (which is probably targeted by internal oligopeptides), but fail to import thiolase (which is targeted by an NH(2)-terminal 16-amino acid sequence) . These and other data suggest that there are three branches in the pathway for the import of proteins into peroxisomes, each of which contains a receptor for one type of peroxisomal topogenic information . Here, we report the cloning and characterization of the PEB1 gene, that encodes a 42,320-Da hydrophilic protein with no predicted transmembrane segment . The protein contains six WD repeats, a motif which has been found in 27 proteins involved in diverse cellular functions . The PEB1 gene product was tagged with the hemagglutinin epitope and found to rescue thiolase import in the peb1 null mutant . The epitope-tagged protein was shown to be inside of peroxisomes by immunofluorescence, digitonin permeabilization, equilibrium density centrifugation, immunoelectron microscopy, and proteinase K protection studies . The PEB1 gene product does not cleave the thiolase-targeting sequence . It may function to draw thiolase into peroxisomes.

Eur J Biochem, 1995 Mar 15, 228(3), 727 - 31
A novel approach for investigating reaction mechanisms in cells . Mechanism of deoxy-trehalose synthesis in Saccharomyces cerevisiae studied by 1H-NMR spectroscopy; Tran-Dinh S et al.; A new approach is proposed for investigating the mechanism of metabolite synthesis in cells . This method, based on the competition between various substrates, allows the flux along a pathway, which is normally independent of the concentration of the corresponding precursor in the external medium, to be divided into partial fluxes . In particular, the mechanism deoxy-trehalose synthesis in glucose-grown repressed Saccharomyces cerevisiae was studied, by 1H-NMR spectroscopy, using the competition between 2-deoxy-D-glucose (dGlc) and 2-fluoro-deoxy-D-glucose (FdGlc) with respect to hexokinase . S . cerevisiae cells, suspended in a standard pyrophosphate medium containing about 5 x 10(7) cells/ml, were incubated with 30 mM glucose and various concentrations of dGlc and FdGlc . Apart from dGlc6P and FdGlc6P, trehalose and the dissacharides relative to dGlc, i.e . dideoxy-trehalose (dGlc-dGlc) and deoxytrehalose (dGlc-Glc), are observed while their analogues relative to FdGlc (FdGlc-FdGlc, FdGlc-Glc) are surprisingly absent . For the same external concentration of dGlc and FdGlc, the internal concentration of FdGlc6P is about three times larger than that of dGlc6P . The ratio of the FdGlc6P and dGlc6P concentrations is independent of the incubation times and proportional to the FdGlc and dGlc concentrations in the suspension . The dGlc6P concentration can thus be reduced at will by increasing the {FdGlc}/{dGlc} ratio . Under these conditions, the dGlc-Glc concentration was found to vary linearly with that of dGlc6P . The present data clearly show that deoxy-trehalose is not synthesized from UDP-dGlc and Glc6P but from UDP-Glc and dGlc6P . This conclusion was also confirmed by an experiment in which S . cerevisiae cells were previously charged with dGlc6P and then incubated with glucose.

EMBO J, 1995 Mar 15, 14(6), 1057 - 66
A role of Sep1 (= Kem1, Xrn1) as a microtubule-associated protein in Saccharomyces cerevisiae; Interthal H et al.; Saccharomyces cerevisiae cells lacking the SEP1 (also known as XRN1, KEM1, DST2, RAR5) gene function exhibit a number of phenotypes in cellular processes related to microtubule function . Mutant cells show increased sensitivity to the microtubule-destabilizing drug benomyl, increased chromosome loss, a karyogamy defect, impaired spindle pole body separation, and defective nuclear migration towards the bud neck . Analysis of the arrest morphology and of the survival during arrest strongly suggests a structural defect accounting for the benomyl hypersensitivity, rather than a regulatory defect in a checkpoint . Biochemical analysis of the purified Sep1 protein demonstrates its ability to promote the polymerization of procine brain and authentic S.cerevisiae tubulin into flexible microtubules in vitro . Furthermore, Sep1 co-sediments with these microtubules in sucrose cushion centrifugation . Genetic analysis of double mutant strains containing a mutation in SEP1 and in one of the genes coding for alpha- or beta-tubulin further suggests interaction between Sep1 and microtubules . Taken together these three lines of evidence constitute compelling evidence for a role of Sep1 as an accessory protein in microtubule function in the yeast S.cerevisiae.

J Biol Chem, 1995 Mar 10, 270(10), 5483 - 9
Golgi localization in yeast is mediated by the membrane anchor region of rat liver sialyltransferase; Schwientek T et al.; To investigate the function of the membrane anchor region of a mammalian glycosyltransferase in yeast we constructed a fusion gene that encodes the 34 amino-terminal residues of rat liver beta-galactoside alpha-2,6-sialyl-transferase (EC 2.4.99.1) (ST) fused to the mature form of yeast invertase . Transformants of Saccharomyces cerevisiae expressing the fusion gene produced an intracellular heterogeneously N-glycosylated fusion protein of intermediate molecular weight between the core and fully extended N-glycosylated form of invertase, suggesting a post-endoplasmic reticulum (ER) localization . In two types of cell fractionation using sucrose density gradients the ST-invertase fusion protein cofractionated with Golgi marker proteins, whereas a minor fraction (about 30%) comigrated with a vacuolar marker; ST-invertase was not detected in other cell fractions including the ER and the plasma membrane . Consistent with Golgi localization, about 70% of the total amount of the ST-invertase fusion was immunoprecipitated with an antibody directed against alpha-1,6-mannose linkages . The results demonstrate that the membrane anchor region of a mammalian type II glycosyltransferase is able to target a protein to the secretory pathway and to a Golgi compartment of the yeast S . cerevisiae, indicating conservation of targeting mechanisms between higher and lower eukaryotes . Since typical yeast Golgi localization signals are missing in the ST-membrane anchor region the results also suggest that yeast as mammalian cells utilize diverse mechanisms to direct proteins to the Golgi.

J Biol Chem, 1995 Mar 3, 270(9), 4503 - 8
DNA structural elements required for FEN-1 binding; Harrington JJ et al.; In eukaryotic cells, a 5'-flap DNA endonuclease and a double-stranded DNA 5'-exonuclease activity reside within a 42-kDa enzyme called FEN-1 (flap endonuclease-1 and 5(five)'-exonuclease) . This endo/exonuclease has been shown to be highly homologous to human XP-G, Saccharomyces cerevisiae RAD2, and S . cerevisiae YKL510 . Like FEN-1, these related structure-specific nucleases recognize and cleave a branched DNA structure called a DNA flap and its derivative, called a pseudo Y-structure . To dissect the important structural components of the DNA flap structure, we have developed a mobility shift assay . We find that the Fadj strand (located adjacent to the displaced flap strand) is necessary for efficient binding and cleavage of flap structures by FEN-1 . When this strand is absent or when it is present, but recessed from the elbow of the flap strand, binding efficiency drops . Further investigation of the role of the Fadj strand using double flap structures reveals that the Fadj strand is necessary to provide a double-stranded template upon which FEN-1 can bind near the elbow of the flap strand . These results provide a basis for understanding how this structure-specific nuclease recognizes a variety of DNA substrates.

Mol Cell Biol, 1995 Mar, 15(3), 1632 - 41
A mutation in the gene encoding the Saccharomyces cerevisiae single-stranded DNA-binding protein Rfa1 stimulates a RAD52-independent pathway for direct-repeat recombination; Smith J et al.; In the yeast Saccharomyces cerevisiae, recombination between direct repeats is synergistically reduced in rad1 rad52 double mutants, suggesting that the two genes define alternate recombination pathways . Using a classical genetic approach, we searched for suppressors of the recombination defect in the double mutant . One mutation that restores wild-type levels of recombination was isolated . Cloning by complementation and subsequent physical and genetic analysis revealed that it maps to RAF1 . This locus encodes the large subunit of the single-stranded DNA-binding protein complex, RP-A, which is conserved from S . cerevisiae to humans . The rfa1 mutation on its own causes a 15-fold increase in direct-repeat recombination . However, unlike most other hyperrecombination mutations, the elevated levels in rfa1 mutants occur independently of RAD52 function . Additionally, rfa1 mutant strains grow slowly, are UV sensitive, and exhibit decreased levels of heteroallelic recombination . DNA sequence analysis of rfa1 revealed a missense mutation that alters a conserved residue of the protein (aspartic acid 228 to tyrosine {D228Y}) . Biochemical analysis suggests that this defect results in decreased levels of RP-A in mutant strains . Overexpression of the mutant subunit completely suppresses the UV sensitivity and partially suppresses the recombination phenotype . We propose that the defective complex fails to interact properly with components of the repair, replication, and recombination machinery . Further, this may permit the bypass of the recombination defect of rad1 rad52 mutants by activating an alternative single-stranded DNA degradation pathway.

Mol Cell Biol, 1995 Mar, 15(3), 1499 - 512
Genetic dissection of thyroid hormone receptor beta: identification of mutations that separate hormone binding and transcriptional activation; Uppaluri R et al.; The thyroid hormone receptors (TR) are members of the nuclear receptor family of ligand-mediated transcription factors . The large region of TR that lies C-terminal to its DNA-binding domain subserves functions of ligand binding, dimerization, and transactivation . Little is known regarding the structural or functional determinants of these processes . We have utilized genetic screening in the yeast Saccharomyces cerevisiae to identify residues involved in these functions . Random mutations of the rat TR beta 1 isoform between amino acid residues 179 and 456 were screened, and mutants with reduced hormone-dependent activation of reporter gene activity were isolated . In this paper we describe the characterization of a class of mutants that exhibit a dissociation between hormone binding and transcriptional activation . These mutants retained hormone binding (> 15% of the wild-type level) yet failed to transactivate a reporter gene . A number of these mutations occurred within the D region, which links the DNA-binding and ligand-binding domains of the receptor . One subset of these mutations abrogated DNA binding, supporting a role of the D region in this process . The remainder retain DNA binding and thus highlight residues critical for receptor activation . In addition, an unexpected group of "superactivator" mutations that led to enhanced hormone-dependent activation in S . cerevisiae were found . These mutations localized to the carboxy-terminal portion of the receptor in a region which contains elements conserved across the superfamily of nuclear receptors . The hormone-dependent phenotype of these superactivator mutations suggests an important role of this segment in ligand-mediated transcriptional activation.

Genetics, 1995 Mar, 139(3), 1175 - 88
Mismatch correction acts as a barrier to homeologous recombination in Saccharomyces cerevisiae; Selva EM et al.; A homeologous mitotic recombination assay was used to test the role of Saccharomyces cerevisiae mismatch repair genes PMS1, MSH2 and MSH3 on recombination fidelity . A homeologous gene pair consisting of S . cerevisiae SPT15 and its S . pombe homolog were present as a direct repeat on chromosome V, with the exogenous S . pombe sequences inserted either upstream or downstream of the endogenous S . cerevisiae gene . Each gene carried a different inactivating mutation, rendering the starting strain Spt15- . Recombinants that regenerated SPT15 function were scored after nonselective growth of the cells . In strains wild type for mismatch repair, homeologous recombination was depressed 150- to 180-fold relative to homologous controls, indicating that recombination between diverged sequences is inhibited . In one orientation of the homeologous gene pair, msh2 or msh3 mutations resulted in 17- and 9.6-fold elevations in recombination and the msh2 msh3 double mutant exhibited an 43-fold increase, implying that each MSH gene can function independently in trans to prevent homeologous recombination . Homologous recombination was not significantly affected by the msh mutations . In the other orientation, only msh2 strains were elevated (12-fold) for homeologous recombination . A mutation in MSH3 did not affect the rate of recombination in this orientation . Surprisingly, a pms1 deletion mutant did not exhibit elevated homeologous recombination.

Biosci Biotechnol Biochem, 1995 Mar, 59(3), 482 - 6
The physiological roles of membrane ergosterol as revealed by the phenotypes of syr1/erg3 null mutant of Saccharomyces cerevisiae; Hemmi K et al.; Ergosterol is a major sterol component of fungal plasma membranes . The effects of disrupting the Saccharomyces cerevisiae SYR1/ERG3 gene, which encodes sterol C-5 desaturase, an enzyme of ergosterol biosynthesis pathway, were markedly different for different S . cerevisiae strains and growth temperatures . The null mutation of SYR1 (delta syr1) in strain RAY-3A had only a slight effect on the growth rate at 28 degrees C . However, at this temperature, the same mutation caused poor growth in strain KA-311A and no growth in strain W303-1A . The delta syr1 disruptant of these strains were able to grow at 37 degrees C, as well as their parental strains . Moreover, the growth of the delta syr1 disruptant of W303-1A and KA-311A strains were severely inhibited at 16 degrees C . These results indicated that ergosterol is essential for growth at low temperatures, and the effects of the gene disruption are variable by the genetic background . The growth defect at low temperatures appeared to be due to the defect of tryptophan uptake in the delta syr1 mutants . The delta syr1 mutants were sensitive to a wide variety of drugs, chemicals, and ions, suggesting that yeast ergosterol is important as permeability barrier against various chemical stresses.

Biosci Biotechnol Biochem, 1995 Mar, 59(3), 382 - 7
High level secretion by Saccharomyces cerevisiae of human apolipoprotein E as a fusion to Rhizomucor rennin; Nomura N et al.; As the first step for production of human apolipoprotein E (hApoE) in Saccharomyces cerevisiae, the hApoE cDNA was cloned in Escherichia coli, on the basis of the nucleotide sequence reported previously . When the hApoE cDNA including its pre-sequence-encoding region was expressed under the control of the GAL7 promoter, no protein immunoreactive with anti-hApoE antibody was detected either in the culture medium or inside the cells . For efficient production and secretion of hApoE in S . cerevisiae, the mature hApoE-encoding region was fused to the prepro-sequence region of Rhizomucor rennin (MPR) and to the whole MPR gene including its prepro- and mature-MPR regions . When the fusion gene consisting of the prepro-sequence-encoding region and hApoE regions was expressed in S . cerevisiae, no protein reactive with the anti-hApoE antibody was detected in any fraction of the yeast cells, probably due to rapid degradation of the hApoE protein by yeast proteases . On the other hand, when hApoE was expressed as a fusion to the whole MPR protein, a considerable amount of the fused protein was secreted into the medium . The prepro-sequence of MPR was correctly processed from the fused protein in the medium by autocatalytic activity of MPR and by a protease(s) of the host cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Microbiol Biotechnol, 1995 Mar, 42(6), 865 - 70
Secretion by Saccharomyces cerevisiae of rat apolipoprotein E as a fusion to Mucor rennin; Nomura N et al.; As the first step for production of rat apolipoprotein E (rApoE) in Saccharomyces cerevisiae, the rApoE cDNA was cloned and its nucleotide sequence was determined . When the intact rApoE gene including the presequence-encoding region was expressed under the control of the yeast GAL7 promoter, no protein immunoreactive with anti-rApoE antibody was detected either in the culture medium or inside the cells . For the purpose of the extracellular production of rApoE, three fusion genes were constructed in which the mature rApoE-encoding sequence was connected after the pre, prepro, and whole regions of the gene encoding a fungal aspartic proteinase, Mucor pusillus rennin (MPP), since MPP is efficiently secreted from recombinant S . cerevisiae containing the MPP gene . When these three fusion genes were expressed under the control of the GAL7 promoter, only one, encoding the mature rApoE connected to the whole MPP sequence, directed efficient secretion of the fused protein . The maximum yield of the fused protein secreted into the medium reached 11.8 mg/l and the calculated rApoE part was 5.3 mg in the fused protein . The excreted fusion protein was glycosylated at the original two sites in the MPP part . The fused protein was gradually degraded in the medium probably by proteases of the host cell, because no such degradation occurred in a yeast pep4mutant strain.

J Eukaryot Microbiol, 1995 Mar-Apr, 42(2), 142 - 9
Cloning and identification of Arp1, an actin-related protein from Pneumocystis carinii; Christopher LJ et al.; The complete Pneumocystis carinii Arp1 gene has been sequenced from two cDNA clones . The gene encodes a protein 385 bp in length with an estimated size of 45,000 kD . The A + T% for the Arp1 gene and a 900-bp sequence upstream of the gene were 63.7% and 70.3%, respectively . These values are consistent with A + T codon preference displayed by P . carinii and are similar to values reported for other P . carinii genes . The predicted amino acid sequence of the P . carinii Arp1 protein had a similarity of 87.6% with Neurospora crassa Arp1, 82.1% similarity with vertebrate centractin, and 71.2% similarity with the Saccharomyces cerevisiae Act5p . Expression of Arp1 mRNA in P . carinii was detectable via synthesis of cDNA and subsequent PCR amplification . Affinity purified antibodies against S . cerevisiae Act5p, and canine centractin recognized both the recombinantly expressed protein and a 45,000 kD protein in P . carinii nuclear extracts . The Arp1 gene is the second member of the actin multigene family that has been identified in P . carinii.

Curr Genet, 1995 Mar, 27(4), 330 - 8
Molecular and biochemical characterization of the hexokinase from the starch-utilizing yeast Schwanniomyces occidentalis; Rose M; Hexose-phosphorylating enzymes from the starch-utilizing yeast Schwanniomyces occidentalis were purified and two isoenzymes separated . The substrate pattern characterized one of these as a hexokinase phosphorylating glucose and fructose and the other as a glucokinase unable to phosphorylate fructose . The purified Schw . occidentalis hexokinase had a KM value of 0.98 mM for glucose and 9.3 mM for fructose . The hexokinase gene was cloned by cross hybridization with a probe from the Saccharomyces cerevisiae HXK2 gene . Deletion of Schw . occidentalis hexokinase by gene replacement yielded a mutant unable to grow on fructose as sole carbon source, but still growing on glucose . Deletion mutants of Schw . occidentalis hexokinase prevented glucose repression of invertase and maltase . Growth deficiencies and the defect of glucose repression of a S . cerevisiae hexokinase null mutant could be restored by heterologous expression of the Schw . occidentalis hexokinase . Moreover, the results clearly showed the existence of a separate glucokinase in Schw . occidentalis.

Protein Eng, 1995 Mar, 8(3), 261 - 73
Engineering ribonuclease A: production, purification and characterization of wild-type enzyme and mutants at Gln11; delCardayre SB et al.; Bovine pancreatic ribonuclease A (RNase A) has been the object of much landmark work in biological chemistry . Yet the application of the techniques of protein engineering to RNase A has been limited by problems inherent in the isolation and heterologous expression of its gene . A cDNA library was prepared from cow pancreas, and from this library the cDNA that codes for RNase A was isolated . This cDNA was inserted into expression plasmids that then directed the production of RNase A in Saccharomyces cerevisiae (fused to a modified alpha-factor leader sequence) or Escherichia coli (fused to the pelB signal sequence) . RNase A secreted into the medium by S.cerevisiae was an active but highly glycosylated enzyme that was recoverable at 1 mg/l of culture . RNase A produced by E.coli was in an insoluble fraction of the cell lysate . Oxidation of the reduced and denatured protein produced active enzyme which was isolated at 50 mg/l of culture . The bacterial expression system is ideal for the large-scale production of mutants of RNase A . This system was used to substitute alanine, asparagine or histidine for Gln11, a conserved residue that donates a hydrogen bond to the reactive phosphoryl group of bound substrate . Analysis of the binding and turnover of natural and synthetic substrates by the wild-type and mutant enzymes shows that the primary role of Gln11 is to prevent the non-productive binding of substrate.

Gene, 1995 Feb 27, 154(1), 99 - 103
Expression of a synthetic gene encoding P2 ribonuclease from the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus in mesophylic hosts; Fusi P et al.; This work reports the molecular cloning and expression of a synthetic gene encoding P2, a 7-kDa ribonuclease (RNase) previously isolated in our laboratory from the archaebacterium Sulfolobus solfataricus {Fusi et al., Eur . J . Biochem . 211 (1993) 305-310} . The P2-encoding synthetic gene was expressed in E . coli and in Saccharomyces cerevisiae . The recombinant (re-) protein was produced to approx . 1.5% of the total protein content in S . cerevisiae using the galactose-inducible GAL1 promoter and to 3% (tac/lac tandem promoters) or 6.5% (T7 promoter) in E . coli as judged by immunological and biochemical criteria . E . coli-produced P2 was purified to electrophoretic homogeneity through a one-step procedure, i.e., DEAE-Sephacel chromatography at pH 9.3 . S . cerevisiae-produced P2 additionally required filtration through a Centricon-10 microconcentrator to obtain the same purity . The re-P2 was found to be indistinguishable from the Su . solfataricus enzyme on the basis of heat stability, pH optimum and RNA digestion pattern . Furthermore, monodimensional nuclear magnetic resonance showed that the E . coli- and Su . solfataricus-produced enzymes were structurally identical, the only exceptions being that Lys4 and Lys6 were not methylated in the re-enzyme, thus showing that lysine methylation does not play a role in P2 thermostabilization.

Nucleic Acids Res, 1995 Feb 25, 23(4), 558 - 64
Two mutually exclusive regulatory systems inhibit UASGATA, a cluster of 5'-GAT(A/T)A-3' upstream from the UGA4 gene of Saccharomyces cerevisiae; Andre B et al.; The S . cerevisiae Uga43(Dal80) protein down-regulates the expression of multiple nitrogen pathway genes . It contains a zinc-finger motif similar to the DNA-binding domain of the vertebrate GATA family of transcription factors; this domain is known to direct binding to 5'-GATA-3' core sequences . The inducible UGA4 gene, which encodes the specific gamma-aminobutyrate permease, undergoes strong repression by Uga43p . This study shows that the 5' region of UGA4 contains a UAS element made of four directly repeated 5'-CGAT(A/T) AG-3' sequences . This element, called UASGATA, can potentially confer to the UGA4 gene high-level expression in the absence of inducer, but this potential activity is inhibited by two distinct repression systems . One system is Uga43p-dependent; it operates in cells grown on a poor nitrogen source . The other is the nitrogen repression system, which relies on Ure2p and glutamine and operates when a good nitrogen source is present . Nitrogen repression also blocks the synthesis of Uga43p, making the two repression systems mutually exclusive . Previous studies have shown that expression supported by 5'-GATA-3'-containing UAS elements requires Gln3p, another global nitrogen regulatory factor containing a GATA zinc-finger domain . Although Gln3p contributes to UASGATA activity, evidence suggests that a second factor can potentially direct expression through UASGATA . Expression conferred by this putative factor is subject to both Uga43p- and Ure2p-mediated repression . The role of UASGATA in the expression of the UGA4 gene is discussed in relation to its sensitivity to the two distinct repression systems.

Nucleic Acids Res, 1995 Feb 25, 23(4), 550 - 7
Cis- and trans-acting elements determining induction of the genes of the gamma-aminobutyrate (GABA) utilization pathway in Saccharomyces cerevisiae; Talibi D et al.; In S . cerevisiae, gamma-aminobutyrate (GABA) induces transcription of the UGA genes required for its utilization as a nitrogen source . Analysis of the 5' region of the UGA1 and UGA4 genes led to the identification of a conserved GC-rich sequence (UASGABA) essential to induction by gamma-aminobutyrate . Alone, this UASGABA element also supported some levels of reporter gene transcription in the presence of gamma-aminobutyrate . To be effective, UASGABA requires two positive-acting proteins that both contain a Cys6-Zn2 type zinc-finger motif, namely pathway-specific Uga3p and pleiotropic Uga35p(Dal81p/DurLp) . Further analysis of the UGA4 gene revealed that Gln3p, a global nitrogen regulatory protein containing a GATA zinc-finger domain, is required in order to reach high levels of gamma-aminobutyrate-induced transcription . The Gln3p factor exerts its function mainly through a cluster of 5'-GAT(A/T)A-3'(UASGATA) situated just upstream from UASGABA . The role of Gln3p is less predominant in UGA1 than in UGA4 gene expression . We propose that tight coupling between the UASGABA and UASGATA elements enables the cell to integrate, according to its nitrogen status, the induced expression levels of UGA4.

Cell, 1995 Feb 24, 80(4), 583 - 92
Histone H3 and H4 N-termini interact with SIR3 and SIR4 proteins: a molecular model for the formation of heterochromatin in yeast; Hecht A et al.; The silent mating loci and chromosomal regions adjacent to telomeres of S . cerevisiae have features similar to heterochromatin of more complex eukaryotes . Transcriptional repression at these sites depends on the silent information regulators SIR3 and SIR4 as well as histones H3 and H4 . We show here that the SIR3 and SIR4 proteins interact with specific silencing domains of the H3 and H4 N-termini in vitro . Certain mutations in these factors, which affect their silencing functions in vivo, also disrupt their interactions in vitro . Immunofluorescence studies with antibodies against RAP1 and SIR3 demonstrate that the H3 and H4 N-termini are required for the association of SIR3 with telomeric chromatin and the perinuclear positioning of yeast telomeres . Based on these interactions, we propose a model for heterochromatin-mediated transcriptional silencing in yeast, which may serve as a paradigm for other eukaryotic organisms as well.

Biochem Biophys Res Commun, 1995 Feb 15, 207(2), 559 - 64
Novel antagonist to agonist switch in chimeric G protein-coupled alpha-factor peptide receptors; Sen M et al.; The alpha-factor analog desTrp1,Ala3 alpha-factor (dTA-alpha-factor) acts as an antagonist for both the S . cerevisiae alpha-factor receptor (c-alpha-FR) and the S . kluyveri alpha-factor receptor (k-alpha FR) . Chimeric alpha-factor receptors in which a portion (residues 250-303) of the seven hydrophobic segments were derived from the k-alpha FR and the rest from c-alpha FR conferred potent activation of cellular responses by dTA-alpha-factor including induction of FUS1 expression without significantly affecting binding . Chimeric receptors with all hydrophobic segments derived from one receptor did not express sensitivity . We propose that dTA-alpha-factor represents an antagonist that that has an activation energy barrier to activating c-alpha FR and k-alpha FR but which retains a latent potential to act as an agonist.

Cell, 1995 Feb 10, 80(3), 485 - 96
Mutation in the silencing gene SIR4 can delay aging in S . cerevisiae; Kennedy BK et al.; Aging in S . cerevisiae is exemplified by the fixed number of cell divisions that mother cells undergo (termed their life span) . We have exploited a correlation between life span and stress resistance to identify mutations in four genes that extend life span . One of these, SIR4, encodes a component of the silencing apparatus at HM loci and telomeres . The sir4-42 mutation extends life span by more than 30% and is semidominant . Our findings suggest that sir4-42 extends life span by preventing recruitment of the SIR proteins to HM loci and telomeres, thereby increasing their concentration at other chromosomal regions . Maintaining silencing at these other regions may be critical in preventing aging . Consistent with this view, expression of only the carboxyl terminus of SIR4 interferes with silencing at HM loci and telomeres, which also extends life span . Possible links among silencing, telomere maintenance, and aging in other organisms are discussed.

J Biol Chem, 1995 Feb 10, 270(6), 2770 - 5
Protein O-glycosylation in yeast . The PMT2 gene specifies a second protein O-mannosyltransferase that functions in addition to the PMT1-encoded activity; Lussier M et al.; The PMT2 gene from Saccharomyces cerevisiae was identified as FUN25, a transcribed open reading frame on the left arm of chromosome I (Ouellette, B . F . F., Clark, M . W . C., Keng, T., Storms, R . G., Zhong, W., Zeng, B., Fortin, N., Delaney, S., Barton, A., Kaback, D.B., and Bussey, H . (1993) Genome 36, 32-42) . The product encoded by the PMT2 gene shows significant similarity with the dolichyl phosphate-D-mannose:protein O-D-mannosyltransferase, Pmt1p (EC 2.4.1.109), which is required for initiating the assembly of O-linked oligosaccharides in S . cerevisiae (Strahl-Bolsinger, S., Immervoll, T., Deutzmann, R., and Tanner, W . (1993) Proc . Natl . Acad . Sci . U.S.A . 90, 8164-8168) . The PMT2 gene encodes a new protein O-D-mannosyltransferase . Yeast cells carrying a PMT2 disruption show a diminished in vitro and in vivo O-mannosylation activity and resemble mutants with a nonfunctional PMT1 gene . Strains bearing a pmt1 pmt2 double disruption show a severe growth defect but retain residual O-mannosylation activity indicating the presence of at least one more protein-O-mannosyltransferase.

Mol Gen Genet, 1995 Feb 6, 246(3), 353 - 9
A multigene family related to chitin synthase genes of yeast in the opportunistic pathogen Aspergillus fumigatus; Mellado E et al.; Two approaches were used to isolate fragments of chitin synthase genes from the opportunistic human pathogen Aspergillus fumigatus . Firstly, regions of amino acid conservation in chitin synthases of Saccharomyces cerevisiae were used to design degenerate primers for amplification of portions of related genes, and secondly, a segment of the S . cerevisiae CSD2 gene was used to screen an A . fumigatus lambda genomic DNA library . the polymerase chain reaction (PCR)-based approach led to the identification of five different genes, designated chsA, chsB, chsC, chsD and chsE . chsA, chsB, and chsC fall into Classes I, II and III of the 'zymogen type' chitin synthases, respectively . The chsD fragment has approximately 35% amino acid sequence identity to both the zymogen type genes and the non-zymogen type CSD2 gene . chsF appears to be a homologue of CSD2, being 80% identical to CSD2 over 100 amino acids . An unexpected finding was the isolation by heterologous hybridization of another gene (chsE), which also has strong sequence similarity (54% identity at the amino acid level over the same region as chsF) to CSD2 . Reverse transcriptase-PCR was used to show that each gene is expressed during hyphal growth in submerged cultures.

J Biol Chem, 1995 Feb 3, 270(5), 1983 - 91
A new old yellow enzyme of Saccharomyces cerevisiae; Niino YS et al.; In 1993, the first gene of Old Yellow Enzyme (OYE) of Saccharomyces cerevisiae was cloned (Stott, K., Saito, K., Thiele, D . J., and Massey, V . (1993) J . Biol . Chem . 268, 6097-6106) and named OYE2 to distinguish it from the first OYE gene cloned from Saccharomyces carlsbergenesis (Saito, K., Thiele, D . J., Davio, M., Lockridge, O., and Massey, V . (1991) J . Biol . Chem . 266, 20720-20724) . The analysis of an OYE2 deletion mutant suggested that S . cerevisiae had at least two OYE genes . In the present study, we cloned a new OYE species named OYE3 and analyzed the OYE3 protein expressed in Escherichia coli . OYE3 consists of 400 amino acid residues and its molecular mass calculated by electrospray mass spectrometry is 44,788 daltons, in good agreement with the value of 44,920 daltons predicted from the amino acid sequence derived from the DNA sequence . In the downstream region of the OYE3 gene, the cytochrome oxidase (COX10) gene exists with a 426-base pair intermediate sequence . Some of the physicochemical and kinetic properties of OYE2 and OYE3 have been determined . Although the two enzymes are clearly closely related, they show differences in ligand binding properties and in their catalytic activities with oxygen and cyclohexen-2-one as acceptors.

J Biol Chem, 1995 Feb 3, 270(5), 1975 - 8
Ran-binding protein-1 is an essential component of the Ran/RCC1 molecular switch system in budding yeast; Ouspenski II et al.; We have performed a screen for genes that affect chromosome stability when overexpressed in the budding yeast Saccharomyces cerevisiae . Two of the genes recovered in the screen, CST17 and CST20, share a number of phenotypic properties, suggesting their involvement in the same cellular process . DNA sequence analysis of these genes revealed that they encode components of the Ran/RCC1 molecular switch system: CST17 is Ran itself (Ras-like nuclear protein) and CST20 is a novel yeast protein with a high degree of similarity to mammalian RanBP1, which is known to interact with Ran-GTP in vitro . We demonstrate that the CST20 protein can interact with Ran-GTP in vitro under similar conditions, indicating that it is the functional yeast homolog of mammalian RanBP1 . The results of immunoprecipitation experiments show that the two yeast proteins form a complex in vivo . Deletion of the gene encoding RanBP1 revealed that it is essential for viability, as are Ran and RCC1 . Similar phenotypic consequences of overproduction of either Ran or RanBP1 indicate that the latter protein is a functional component of the Ran/RCC1 molecular switch system, which is implicated in the control of a number of nuclear functions . Our finding that overproduction of two components of this system results in mitotic chromosome nondisjunction and sensitivity to an anti-microtubule drug benomyl suggest their involvement in mitosis as well . Thus RanBP1 is a functional component of a highly conserved molecular system that affects diverse cellular processes . The availability of this gene in S . cerevisiae provides a genetic system for the analysis of RanBP1 function in vivo.

Arch Biochem Biophys, 1995 Feb 1, 316(2), 773 - 9
Mannosylphosphoryldolichol-mediated O-mannosylation of yeast glycoproteins: stereospecificity and recognition of the alpha-isoprene unit by a purified mannosyltransferase; Dotson SB et al.; Mannosylphosphoryldolichol (Man-P-Dol):protein O-mannosyltransferase (PMT1) was solubilized by extracting a crude microsomal fraction from Saccharomyces cerevisiae with 1.2% Chaps-0.5% desoxycholate and purified 120-fold by standard chromatographic procedures . These very stable, partially purified preparations of PMT1 catalyzed the transfer of mannosyl units from exogenous Man-P-Dol to serine/threonine residues in the synthetic peptide acceptor, Tyr-Asn-Pro-Thr-Ser-Val-NH2, forming O-mannosidic linkages of the alpha-configuration . The specificity of yeast PMT1 was defined with respect to the recognition of the saturated alpha-isoprene unit, the chain length of the dolichyl moiety, and the anomeric configuration of the mannosyl-phosphoryl linkage of the lipophilic mannosyl donor . When Man-P-Dol95 and mannosylphosphorylpolyprenol (Man-P-Poly95), which contains a fully unsaturated polyprenyl chain, were compared as substrates, the initial rate for peptide mannosylation was dramatically higher with Man-P-Dol95 relative to Man-P-Poly95 . The chain length of the dolichyl moiety also influenced the mannolipid-enzyme interaction as the partially purified PMT1 had a higher affinity for Man-P-Dol95 than for Man-P-Dol55 . When beta-Man-P-Dol95 was compared with chemically synthesized alpha-Man-P-Dol95 as a mannosyl donor, a strict stereo-specificity was observed for the presence of a beta-mannosyl-phosphoryl linkage . In summary, a procedure for isolating a stable, partially purified preparation of PMT1 from S . cerevisiae is described . Enzymological studies with these preparations of PMT1 provide the first evidence that the recognition of the lipophilic mannosyl donor is stereospecific . These results also demonstrate that maximal O-mannosylation of serine/threonine residues in yeast glycoproteins catalyzed by the partially purified preparation of PMT1 requires the presence of a saturated alpha-isoprene unit in the dolichyl moiety of Man-P-Dol.

J Cell Biol, 1995 Feb, 128(4), 617 - 24
Saccharomyces cerevisiae kinesin- and dynein-related proteins required for anaphase chromosome segregation; Saunders WS et al.; The Saccharomyces cerevisiae kinesin-related gene products Cin8p and Kip1p function to assemble the bipolar mitotic spindle . The cytoplasmic dynein heavy chain homologue Dyn1p (also known as Dhc1p) participates in proper cellular positioning of the spindle . In this study, the roles of these motor proteins in anaphase chromosome segregation were examined . While no single motor was essential, loss of function of all three completely halted anaphase chromatin separation . As combined motor activity was diminished by mutation, both the velocity and extent of chromatin movement were reduced, suggesting a direct role for all three motors in generating a chromosome-separating force . Redundancy for function between different types of microtubule-based motor proteins was also indicated by the observation that cin8 dyn1 double-deletion mutants are inviable . Our findings indicate that the bulk of anaphase chromosome segregation in S . cerevisiae is accomplished by the combined actions of these three motors.

J Bacteriol, 1995 Feb, 177(4), 898 - 906
The gene for a major exopolyphosphatase of Saccharomyces cerevisiae; Wurst H et al.; The gene encoding a major exopolyphosphatase (scPPX1) in Saccharomyces cerevisiae (H . Wurst and A . Kornberg, J . Biol . Chem . 269:10996-11001, 1994) has been isolated from a genomic library . The gene, located at 57 kbp from the end of the right arm of chromosome VIII, encodes a protein of 396 amino acids . Overexpression in Escherichia coli allowed the ready purification of a recombinant form of the enzyme . Disruption of the gene did not affect the growth rate of S . cerevisiae . Lysates from the mutants displayed considerably lower exopolyphosphatase activity than the wild type . The enzyme is located in the cytosol, whereas the vast accumulation of polyphosphate (polyP) of the yeast is in the vacuole . Disruption of PPX1 in strains with and without deficiencies in vacuolar proteases allowed the identification of exopolyphosphatase activity in the vacuole . This residual activity was strongly reduced in the absence of vacuolar proteases, indicating a dependence on proteolytic activation . A 50-fold-lower protease-independent activity could be distinguished from this protease-dependent activity by different patterns of expression during growth and activation by arginine . With regard to the levels of polyP in various mutants, those deficient in vacuolar ATPase retain less than 1% of the cellular polyP, a loss that is not offset by additional mutations that eliminate the cytosolic exopolyphosphatase and the vacuolar polyphosphatases dependent on vacuolar protease processing.

J Cell Biol, 1995 Feb, 128(3), 383 - 92
Tropomyosin is essential in yeast, yet the TPM1 and TPM2 products perform distinct functions; Drees B et al.; Sequence analysis of chromosome IX of Saccharomyces cerevisiae revealed an open reading frame of 166 residues, designated TPM2, having 64.5% sequence identity to TPM1, that encodes the major form of tropomyosin in yeast . Purification and characterization of Tpm2p revealed a protein with the characteristics of a bona fide tropomyosin; it is present in vivo at about one sixth the abundance of Tpm1p . Biochemical and sequence analysis indicates that Tpm2p spans four actin monomers along a filament, whereas Tpmlp spans five . Despite its shorter length, Tpm2p can compete with Tpm1p for binding to F-actin . Over-expression of Tpm2p in vivo alters the axial budding of haploids to a bipolar pattern, and this can be partially suppressed by co-over-expression of Tpm1p . This suggests distinct functions for the two tropomyosins, and indicates that the ratio between them is important for correct morphogenesis . Loss of Tpm2p has no detectable phenotype in otherwise wild type cells, but is lethal in combination with tpm1 delta . Over-expression of Tpm2p does not suppress the growth or cell surface targeting defects associated with tpm1 delta, so the two tropomyosins must perform an essential function, yet are not functionally interchangeable . S . cerevisiae therefore provides a simple system for the study of two tropomyosins having distinct yet overlapping functions.

Mol Cell Biol, 1995 Feb, 15(2), 983 - 8
The glutamine-rich activation domains of human Sp1 do not stimulate transcription in Saccharomyces cerevisiae; Ponticelli AS et al.; Eukaryotic transcriptional activators have been classified on the basis of the characteristics of their activation domains . Acidic activation domains, such as those in the yeast GAL4 or GNC4 proteins and the herpes simplex virus activator VP16, stimulate RNA polymerase II transcription when introduced into a variety of eukaryotic cells . This species interchangeability demonstrates that the mechanism by which acidic activation domains function is highly conserved in the eukaryotic kingdom . To determine whether such a conservation of function exists for a different class of activation domain, we have tested whether the glutamine-rich activation domains of the human transcriptional activator Sp1 function in the yeast Saccharomyces cerevisiae . We report here that the glutamine-rich domains of Sp1 do not stimulate transcription in S . cerevisiae, even when accompanied by human TATA-box binding protein (TBP) or human-yeast TATA-box binding protein hybrids . Thus, in contrast to the case for acidic activation domains, the mechanism by which glutamine-rich domains stimulate transcription is not conserved between S . cerevisiae and humans.

Infect Immun, 1995 Feb, 63(2), 478 - 85
Pathogenicity of Saccharomyces cerevisiae in complement factor five-deficient mice; Byron JK et al.; We have previously determined the relative virulence of isolates of Saccharomyces cerevisiae on the basis of differences in proliferation and resistance to clearance in CD-1 mice . These infections were not fatal . To further characterize S . cerevisiae pathogenesis, we studied a virulent clinical isolate, YJM128, and an avirulent nonclinical isolate, Y55, in C5-deficient mice . DBA/2N mice were infected intravenously with YJM128 or Y55, and temporal burdens of yeast cells in various organs were determined . After infection with 10(7) CFU, Y55 increased by 13-fold and YJM128 increased by 20-fold in the brain from day 0 to 3 . In addition, YJM128 increased by 4-fold in the kidneys, whereas Y55 decreased by 16-fold . Both isolates declined in number in other organs . In all studies, 90% of mice infected with 10(7) CFU of YJM128 died between days 2 and 7, whereas no mice infected with equivalent numbers of Y55 died . No mice died after infection with 10(6) CFU of Y55 or YJM128 . The importance of C5 was confirmed by studies using B10.D2/oSnJ (C5-) mice and their congenic C5+ counterparts . Again, the C5- mice were most susceptible to infection with S . cerevisiae, with 63% infected with YJM128 dying by day 7; no C5+ mice died . No Y55-infected mice died, and mean burdens in the brain at day 14 were sevenfold lower in C5+ mice than in C5- mice . Seven of 10 other S . cerevisiae isolates were also more virulent in DBA/2N than CD-1 mice, causing > or = 40% mortality . These data indicate that C5 is a critical factor in host resistance against S . cerevisiae infections and further confirm the pathogenic potential of some isolates of S . cerevisiae.

Int J Biochem Cell Biol, 1995 Feb, 27(2), 169 - 73
Evidence that 4-aminobutyric acid and 5-aminolevulinic acid share a common transport system into Saccharomyces cerevisiae; Bermudez Moretti M et al.; It has been previously reported that 5-aminolevulinic acid (ALA) and 4-aminobutyric acid (GABA) share a common permease in Saccharomyces cerevisiae (Bermudez Moretti et al., 1993) . The aim of the present work was to determine the relationship between the transport of these compounds in isolated cells . Assessment of amino acid incorporation was performed in S . cerevisiae using 14C-ALA or 3H-GABA . Initial rates of ALA incorporation in cells grown in the presence of 5 mM ALA and 5 mM GABA, were three to four times lower than in cells grown without supplements . Kinetic studies indicate that GABA competitively inhibits ALA transport . During the growth phase GABA uptake was also inhibited by 74% and 60% in the presence of ALA and GABA, respectively . These findings indicate that in S . cerevisiae the structurally related compounds, ALA and GABA, may be incorporated into the cells by a common carrier protein . Should this occur in other lukaryotic cells it may explain the neurotoxic effect attributed to ALA in the pathogenesis of acute porphyrias.

Bioessays, 1995 Feb, 17(2), 147 - 59
Genetic control of intrachromosomal recombination; Klein HL; Intrachromosomal recombination between direct repeats can occur either as gene conversion events, which maintain exactly the number of repeat units, or as deletions, which reduce the number of repeat units . Gene conversions are classical recombination events that utilize the standard chromosome recombination machinery . Spontaneous deletions between direct repeats are generally recA-independent in E . coli and RAD52-independent in S . cerevisiae . This independence from the major recombination genes does not mean that deletions form through a nonrecombinational process . Deletions have been suggested to result from sister chromatid exchange at the replication fork in a recA-independent process . The same type of exchange is proposed to be RAD52-independent in Saccharomyces cerevisiae . RAD52-dependent events encompass all events that involve the initial steps of a recombination reaction, which include strand invasion to form a heteroduplex intermediate.

Curr Genet, 1995 Feb, 27(3), 217 - 28
Determination of chromosome copy numbers in Saccharomyces cerevisiae strains via integrative probe and blot hybridization techniques; Hadfield C et al.; Methods have been devised for analyzing chromosome copy numbers in S . cerevisiae strains that may be polyploid or aneuploid, as is apparent in the case of many industrial strains . The initial step involved transformation of a strain with an integrative "ploidy probe" transplacement fragment that enabled the copy number of the targeted chromosomal locus to be determined via genomic Southern blotting and quantitative probe hybridization . Dual probe co-hybridization to Southern genomic DNA blots was used to extend such locus copy number determinations to other loci within the same chromosome, thereby screening for internal consistency along the length of the chromosome . This approach was also used to extend the analysis to other chromosomes in the genome . The method was established and verified with euploid series laboratory strains and then used to examine chromosome copy numbers in three industrial strains . One brewing strain apparently contained three copies of the chromosomes tested, whilst another brewing and a baking strain showed evidence of aneuploidy.

Curr Genet, 1995 Feb, 27(3), 207 - 12
The PSO4 gene of S . cerevisiae is important for sporulation and the meiotic DNA repair of photoactivated psoralen lesions; da Silva KV et al.; We have evaluated the effect of the Saccharomyces cerevisiae pso4-1 mutation in sporulation and DNA repair during meiosis . We have found that pso4-1 cells were arrested in an early step of meiosis, before premeiotic DNA synthesis, and hence did not produce spores . These results suggest that the PSO4 gene may act at the start point of the cell cycle, as do some SPO and CDC genes . The pso4-1 mutant cells are specifically sensitive to 8-MOP- and 3-CPs-photoinduced lesions, and are found to be severely affected in meiotic recombination as well as impaired in the mutagenic response, as previously described for mitosis . This means that the PSO4 gene is important for the repair 8-MOP-photoinduced lesions, mainly double-strand breaks, and the processing of these lesions into recombinogenic intermediates.

Curr Genet, 1995 Feb, 27(3), 201 - 6
Abnormal growth induced by expression of HBsAg in the secretion pathway of S . cerevisiae pep4 mutants; Chen DC et al.; The toxicity of HBsAg in the secretion pathway of pep4 strains can be progressively reduced in modified SD media containing lower concentrations of ammonium sulphate . A procedure, combining a reduction of ammonium sulphate concentration in SD media with the disruption of the PEP4 gene of the host strain, was developed to enrich transformants which are not inhibited by HBsAg expressed in the secretion pathway . Abnormal growth of these non-inhibited transformants is characterized by the enlargement of cell morphology, a transition to pseudohyphal-like growth in nitrogen-starved media, an increase in HBsAg particle production, and the enhancement of growth rate in liquid media . This suggests a new approach to overcoming the toxicity of heterologous protein in the yeast secretion pathway.

Mutat Res, 1995 Feb, 326(2), 211 - 8
Genetic control of RBE of alpha-particles for yeast cells irradiated in stationary and exponential phase of growth; Petin DV et al.; Survival curves of S . cerevisiae wild type and rad 50, 51, 52 and 54 mutants in haploid and diploid strains were measured after gamma-ray and alpha-particle irradiation in stationary and exponential phase of growth . The values of RBE of high-LET radiation, defined as the ratio of the mean lethal doses after sparsely and densely ionizing radiations, were determined . A correlation between the RBE of alpha-particles and cell repair capacity was supported for stationary phase cultures . For the first time, it was shown for all strains studied that at exponential phase of growth the RBE of alpha-particle-induced survival was decreased in comparison with that for stationary cells . For most mutant cells RBE was close to unity, i.e . cell radiosensitivity was almost identical for both sparsely and densely ionizing radiation . Possible reasons for the observed radiation responses are discussed.

Proc Natl Acad Sci U S A, 1995 Jan 31, 92(3), 920 - 4
The Saccharomyces Ty5 retrotransposon family is associated with origins of DNA replication at the telomeres and the silent mating locus HMR; Zou S et al.; We have characterized the genomic organization of the Ty5 retrotransposons among diverse strains of Saccharomyces cerevisiae and the related species Saccharomyces paradoxus . The S . cerevisiae strain S288C (or its derivatives) carries eight Ty5 insertions . Six of these are located near the telomeres, and five are found within 500 bp of autonomously replicating sequences present in the type X subtelomeric repeat . The remaining two S . cerevisiae elements are adjacent to the silent mating locus HMR and are located within 500 bp of the origin of replication present in the transcriptional silencer HMR-E . Although the S . cerevisiae Ty5 elements no longer appear capable of transposition, some strains of S . paradoxus have numerous Ty5 insertions, suggesting that transposition is occurring in this species . Most of these elements are adjacent to type X telomeric repeats, and regions flanking four of five characterized S . paradoxus insertions carry autonomously replicating sequences . The genomic organization of the Ty5 elements is in marked contrast to the other S . cerevisiae retrotransposon families (Ty1-4), which are typically located within 500 bp of tRNA genes . For Ty3, this association reflects an interaction between Ty3 and the RNA polymerase III transcription complex, which appears to direct integration {Chalker, D . L . & Sandmeyer, S . B . (1992) Genes Dev . 6, 117-128} . By analogy to Ty3, we predict that Ty5 target choice is specified by interactions with factors present at both the telomeres and HMR that are involved in DNA replication, transcription silencing, or the maintenance of the unique chromatin structure at these sites.

Biochem Biophys Res Commun, 1995 Jan 26, 206(3), 850 - 6
Biochemical and genetic characterization of a replication protein A dependent DNA helicase from the yeast, Saccharomyces cerevisiae; Biswas EE et al.; We have purified a DNA dependent ATPase/DNA helicase, DNA helicase B, from S . cerevisiae . Helicase B was a 129-kDa polypeptide . The ATPase activity of helicase B was strongly DNA dependent . The DNA helicase activity was stimulated by yeast replication protein A, indicating a probable function in DNA replication . Helicase B showed a 5'-->3' polarity of movement . Protein sequencing indicated that helicase B was identical to a hypothetical 127-kDa polypeptide encoded by yORF61, located 5' upstream of the BMH1 locus in chromosome V . The protein sequence contained a "type I ATP/GTP binding motif" and other helicase-like motifs and the expressed protein exhibited helicase activity . Thus, we concluded that yORF61 is the gene for helicase B and will be referred to as HCSB.

Gene, 1995 Jan 23, 152(2), 157 - 63
Expression of a putative ATPase suppresses the growth defect of a yeast potassium transport mutant: identification of a mammalian member of the Clp/HSP104 family; Perier F et al.; A cDNA encoding a novel mammalian member of the Clp/HSP104 family was isolated from a mouse macrophage-like cell line (J774.1) cDNA library by suppression of the growth defect of a Saccharomyces cerevisiae trk1 trk2 double mutant . The full-length version of this cDNA, termed SKD3, encodes a putative 76-kDa protein of 677 amino acids (aa) . The deduced aa sequence of the SKD3 polypeptide contains four ankyrin-like repeats in the N-terminal domain and a single ATP-binding consensus site in the C-terminal domain . The 378-aa C-terminal domain of SKD3 has 57-64% similarity (30-40% identity) with members of the Clp/HSP104 family, including the ClpA regulatory subunit of the Clp protease and S . cerevisiae heat-shock protein 104 . Northern analysis showed that the 2.3-kb SKD3 transcript is present in a wide variety of tissues, is abundant in mouse heart, skeletal muscle and kidney, and is most abundant in testis . Members of the Clp/HSP104 family have been identified previously from bacteria, yeast and chloroplasts, and are ATPases regulating Clp protease activity and specificity, or mediating cellular responses involved in thermotolerance . SKD3 is the first member of this protein family identified in a higher eukaryote.

FEBS Lett, 1995 Jan 23, 358(2), 119 - 25
A mitochondrial elongation factor-like protein is over-expressed in tumours and differentially expressed in normal tissues; Wells J et al.; The tissue-specific expression of an antigen (P43) ubiquitously expressed at high levels in a variety of tumours of human and animal origin was investigated using a monoclonal antibody to P43 . Whereas low amounts of P43 are expressed in the spleen, skeletal muscle and pancreas, P43 is abundantly produced in the liver and in other tissues such as the kidney, heart and brain which have levels of oxidative metabolism . Interestingly, a related protein of higher molecular weight is abundantly expressed in the lung and in amounts which were higher than those observed with other tissues . The human cDNA for P43 was isolated from a human liver cDNA library and mapped to chromosome 16 between p11.2 and 12 and also to a position near the centromere on the long arm of chromosome 17 . The deduced amino acid sequence of P43 is remarkably similar to that of E . coli EF-Tu and the mitochondrial EF-Tu of S . cerevisiae with the structurally and functionally important amino acids of EF-Tu being completely conserved in P43 . A comparison of the distribution of P43 and a mitochondrial protein Hsp 60 among different cellular fractions indicated a likely mitochondrial localisation for P43 . Taken together these results suggest that P43 is a human mitochondrial elongation factor.

Biochem Biophys Res Commun, 1995 Jan 17, 206(2), 756 - 63
Nature and transcriptional role of catalytic subunits of yeast mitochondrial cAMP-dependent protein kinase; Rahman MU et al.; We have shown that mitochondrial (mt) transcription in yeast (S . cerevisiae) is governed in part by cAMP via a mt cAMP-dependent protein kinase (cAPK), and that the BCY1 gene product acts as regulatory subunit for that organellar enzyme, as it does for cytoplasmic cAPK . Here we assess mt cAPK activity and mt transcription in mutants for the TPK1, TPK2, and TPK3 genes, which encode catalytic subunits of cytoplasmic cAPK . Protein extracts from purified mitochondria from each of the three possible double TPK mutants show mt cAMP-dependent protein phosphorylation . Relative mt transcript levels in these mutants, however, suggest that TPK2 functions less well than does TPK1 or TPK3 in organellar transcriptional control . Thus, both mt and cytoplasmic cAPKs employ the same species of regulatory and catalytic proteins, and versions of the enzyme having various combinations of catalytic species function differentially in cAMP-dependent mt transcriptional control.

J Biol Chem, 1995 Jan 6, 270(1), 244 - 53
Saccharomyces cerevisiae CNE1 encodes an endoplasmic reticulum (ER) membrane protein with sequence similarity to calnexin and calreticulin and functions as a constituent of the ER quality control apparatus; Parlati F et al.; We have used a polymerase chain reaction strategy to identify in the yeast Saccharomyces cerevisiae genes of the mammalian calnexin/calreticulin family, and we have identified and isolated a single gene, CNE1 . The protein predicted from the CNE1 DNA sequence shares some of the motifs with calnexin and calreticulin, and it is 24% identical and 31% similar at the amino acid level with mammalian calnexin . On the basis of its solubility in detergents and its lack of extraction from membranes by 2.5 M urea, high salt, and sodium carbonate at pH 11.5, we have established that Cne1p is an integral membrane protein . However, unlike calnexins, the predicted carboxyl-terminal membrane-spanning domain of Cne1p terminates directly . Furthermore, based on its changed mobility from 76 to 60 kDa after endoglycosidase H digestion Cne1p was shown to be N-glycosylated . Localization of the Cne1p protein by differential and analytical subcellular fractionation as well as by confocal immunofluorescence microscopy showed that it was exclusively located in the endoplasmic reticulum (ER), despite the lack of known ER retention motifs . Although six Ca(2+)-binding proteins were detected in the ER fractions, they were all soluble proteins, and Ca2+ binding activity has not been detected for Cne1p . Disruption of the CNE1 gene did not lead to inviable cells or to gross effects on the levels of secreted proteins such as alpha-pheromone or acid phosphatase . However, in CNE1 disrupted cells, there was an increase of cell-surface expression of an ER retained temperature-sensitive mutant of the alpha-pheromone receptor, ste2-3p, and also an increase in the secretion of heterologously expressed mammalian alpha 1-antitrypsin . Hence, Cne1p appears to function as a constituent of the S . cerevisiae ER protein quality control apparatus.

Biochem Biophys Res Commun, 1995 Jan 5, 206(1), 253 - 9
The minimal active domain of the mouse ras exchange factor CDC25Mm; Coccetti P et al.; The minimal active domain of the mouse CDC25Mm, a GDP/GTP exchange factor (GEF) active on H-ras protein, was determined by constructing several deletion mutants of the C-terminal domain of the protein . The functional activity of these fragments was analyzed for the ability to complement the yeast temperature sensitive mutation cdc25-1 and to catalyze the GDP/GTP exchange on Ras proteins in vitro . A C-terminal domain of 256 residues (CDC25Mm 1005-1260) was sufficient for full biological activity in vivo . Deletion of 27 C-terminal amino acids (CDC25Mm 1005-1233) abolished the complementing activity while deletion of 25 N-terminal residues (CDC25Mm 1030-1260 corresponding to the most conserved domain) led to a complete loss of expression . The results in vivo were supported by experiments in vitro . Highly purified CDC25Mm 1005-1260, expressed in E . coli using the pMAL system, enhanced the GDP release from both H-ras p21 and S . cerevisiae Ras2p and its activity was nearly as high as that of CDC25Mm 974-1260 . Comparison with the Cdc25p protein yielded further evidence that the minimal active domain of CDC25Mm is shorter than the yeast one.

Folia Microbiol (Praha), 1995, 40(3), 257 - 62
Expression of Escherichia coli recA and ada genes in Saccharomyces cerevisiae using a vector with geneticin resistance; Slaninova M et al.; Construction of E . coli-yeast shuttle plasmids containing the neo selection gene is described . The protein-coding regions of the E . coli ada or recA genes under the control of the ADH1 promoter and terminator were ligated into the SphI unique site of pNF2 to produce pMSada and pMSrecA, respectively . The plasmids were used for transformation of the haploid and diploid pso4-1 strains of S . cerevisiae and their corresponding wild types . Transformants were obtained by selection for geneticin (G418) resistance . Crude protein samples were extracted from the individual transformants . Both the RecA and Ada proteins were present in all strains containing the recA and ada genes on plasmids, respectively . Thus the geneticin selection system was successfully used for the preparation of model yeast strains.

Nucleic Acids Symp Ser, 1995, (34), 45 - 6
The construction of mobilizable YACs and their direct conjugative transfer from E . coli to yeasts; Mahmood A et al.; YAC, yeast artificial chromosome, consists of Saccharomyces cerevisiae's ARS, CEN and nutrient requirement genes and TEL from Tetrahymena with ori and ampicillin resistant genes of pBR322 . we have constructed two mobilizable pAY-YACs plasmids by inserting oriT at different positions . pAY-YAC-B (12.2 kbp) containes oriT in between two telomeres, whereas in pAY-YAC-E (14 kbp) oriT has been inserted at the cloning site of pYAC4 . Both plasmids were mediated from E.coli to S.cerevisiae and S.kluyveri with the aid of helper plasmid pRH220 which resides mob and tra genes . The results of conjugal transmission, restriction enzyme digestion and Southern blotting analyses indicated that both plasmids were able to maintained their size and structure, thus reflected the faithfull accomplishment of nicking and resealing of plasmids during conjugation . The direct transformation of plasmid DNA into yeast showed that pAY-YACs plasmids, may be due to their small size, have tendency of circularization.

Mol Biol Rep, 1995, 21(3), 147 - 58
A complex interplay of positive and negative elements is responsible for the different transcriptional activity of liver NF1 variants; Monaci P et al.; A full-length cDNA of the rat liver Nuclear Factor 1 (NF1L21) has been cloned and expressed in S . cerevisiae to analyse the architecture of its activation domain . NF1L21 displays a specific DNA-binding activity, as well as the ability to activate transcription from an artificial NF 1-responsive promoter in yeast . Interaction of two or more NF1L21 molecules with multiple sites on the same promoter activated transcription in a synergistic fashion . Functional analysis of the activation domain of NF1L21 reveals a tripartite structure . Two distinct positive elements are required for NF1L21 -mediated transcription activation . A proline-rich element sandwiched between these two positive domains attenuates their transactivation potential . A shorter NF1L variant (NFlL4) in which the distal positive element is replaced by a different sequence was also isolated . NF1L4 displays the same DNA-binding activity and dimerisation properties as NF1L21, but is unable to activate transcription in yeast.

Cell Mol Biol (Noisy-le-grand), 1995, 41 Suppl 1, S73 - 81
A mutant approach and molecular strategy to study fungal cell walls; Pramanik A et al.; The current study describes recombinant plasmids which complement the hypersusceptibility to killing bleomycin of blm1-1 mutant cells of Saccharomyces cerevisiae, and a strategy developed and used to recover active clones from a stable yeast genomic library . The resistance of a spontaneous revertant isolated from the original blm1-1 mutant strain and of mutant cells transformed with each of several recombinant plasmids which complemented the recessive blm1-1 mutation was comparable to the resistance of the parental (non-mutant) strain from which the original blm1-1 mutant was derived . The strategy for cloning S . cerevisiae DNA was based on complementation and in situ hybridization . This strategy employed 32P-labelled 6.6-kb BamHI and 3.8-kb BamHI-ClaI probes from a cloned DNA fragment to recover clones which either fully or partially complemented the hypersensitivity of mutant cells to killing by bleomycin . This method considerably reduced the time and effort required to recover biologically active clones from a genomic library.

Nippon Rinsho, 1995 Jan, 53(1), 239 - 49
{Structure and function of RAD51 gene in eukaryote}; Shinohara A et al.; RAD51 gene of S . cerevisiae is involved in DNA repair of double-strand breaks, mitotic and meiotic recombination . Rad51 protein is structurally similar to bacterial RecA proteins, which play crucial roles in recombination and DNA repair . Like RecA, Rad51 protein forms nucleofilament with a right handed helical structure, in which the DNA is extended 1.5 times than B-form DNA . Structural homologs have been cloned from various organisms including human and mouse . These results suggest that Rad51 is an essential recombination protein conserved from yeasts to human . Different from RecA, Rad51 protein requires at least an another protein, Rad52, to carry out recombination through the interaction between them.

Anat Rec, 1995 Jan, 241(1), 1 - 9
Effects of brefeldin A on the three-dimensional structure of the Golgi apparatus in a sensitive strain of Saccharomyces cerevisiae; Rambourg A et al.; BACKGROUND: Brefeldin A (BFA), when added to the medium of cultured mammalian cells, induces a reversible block of secretion and disrupts the Golgi apparatus whereas Golgi enzyme markers appear to redistribute into the endoplasmic reticulum (ER) . It has been shown in addition that in mammalian cells, BFA would prevent the assembly of coatomer proteins (COP) onto membranes by inhibiting the GTP-dependent interaction of the ADP-ribosylation factor (ARF) with such membranes . The purpose of the present study is to analyze, by stereoelectron microscopy, the structural modifications of Golgi elements and of the ER-Golgi relationship in a BFA-sensitive yeast mutant, S . cerevisiae erg6 . METHODS: S . cerevisiae erg6 cells were placed in a medium containing 100 micrograms/ml BFA dissolved in 1% alcohol and collected after exposures of 0.5, 1.5, 5, 10, 15, 20, 30, and 70 min to the drug . Yeasts placed in a BFA-free medium but containing 1% alcohol served as controls . After fixation in 2% glutaraldehyde, the cells were postfixed in reduced osmium and embedded in Epon . Then 0.08-0.2 microns thick sections stained with lead citrate were examined with the electron microscope . Photographs of the thicker sections, tilted at +/- 15 degrees from the 0 degree position of the goniometric stage, were used to prepare stereopairs from which the three-dimensional configuration of the organelles was visualized . Since BFA is known to prevent the interaction of ARF with membranes, the phenotype of the arf1 mutant deficient in this protein was also examined for comparative purposes . RESULTS: In control cells, as in wild-type strains, two types of Golgi elements were observed: small networks of fine tubules seen close and occasionally connected to ER cisternae and coarser tubular networks showing nodular distensions having a size comparable to that of secretion granules . The latter networks were considered as trans-Golgi elements and the former as cis-Golgi elements . Several networks of both types were distributed throughout the cytoplasm . At short time intervals (0.5-5 min) of BFA treatment, the trans-Golgi elements disappeared from the cytoplasm, while the ER-connected cis-Golgi elements developed and formed large spheroidal masses frequently showing concentrically arranged fine tubular networks . Such spheroidal, cage-like structures later disappeared, and after 30 min Golgi elements were no longer identifiable, while ER cisternae assumed pleomorphic configurations as the cells showed signs of degeneration . S . cerevisiae arf1 mutants presented a phenotype similar to that of BFA-treated S . cerevisiae erg6 . CONCLUSIONS: It is therefore concluded that soon after exposure to BFA there is, in this sensitive yeast mutant, a transitory hypertrophy of the ER-connected cis-Golgi network presumably resulting from a block at the exit end of this compartment . At longer time intervals (i.e., after 30 min) the Golgi elements are no longer formed, and the cells present signs of cell degeneration.

Biochem J, 1995 Jan 1, 305 ( Pt 1), 125 - 32
Molecular cloning, expression and characterization of a ubiquitin conjugation enzyme (E2(17)kB) highly expressed in rat testis; Wing SS et al.; Ubiquitin-conjugating enzymes (E2s) play a key role in ubiquitin-mediated proteolysis by catalysing the conjugation of ubiquitin to protein substrates . We have previously reported the cDNA cloning of a 14 kDa conjugating enzyme {E2(14)k; Wing, Dumas and Banville (1992) J . Biol . Chem . 267, 6495-6501} that efficiently supported ubiquitination and protein degradation in reticulocyte extracts . Surprisingly, the structure of this E2 was markedly more similar to the Saccharomyces cerevisiae DNA repair gene RAD6, than to the S . cerevisiae UBC4/UBC5 genes which are required for the degradation of short-lived proteins and support much of the ubiquitination of yeast proteins . This suggested that mammalian homologues of UBC4/UBC5 remained to be identified . Using oligonucleotides derived from the S . cerevisiae UBC4 sequence as primers in a PCR reaction with rat muscle cDNA as a template, a 390 bp DNA fragment was amplified which predicted an amino acid sequence that was 83% identical to yeast UBC4 . Screening a rat testes cDNA library identified a family of cDNAs which predicted two very similar proteins with basic pIs and molecular masses of approx . 16,700 Da . Isoform 2E was expressed in Escherichia coli and purified to homogeneity . It supported ubiquitination to reticulocyte and testis proteins more rapidly in vitro and produced larger conjugates than E2(14)k . Examination of RNA from different tissues indicated that this type of E2 was expressed in a broad spectrum of tissues but at particularly high levels in the testis . Fractionation of a testis extract by anion-exchange chromatography identified several putative ubiquitin protein ligase activities with which this E2 could interact in promoting conjugation of ubiquitin to proteins . One of these activities supported conjugation of ubiquitin to histone H2A, a substrate degraded in the ubiquitin system by a non-N-end rule mechanism . This paper reports the first cloning of a apparent mammalian homologue of S . cerevisiae UBC4/UBC5 . Its high expression in testis and ability to efficiently support conjugation to testis proteins suggest that this family of E2s may play a role in the proteolysis that occurs during spermatogenesis.

Mol Cell Biol, 1995 Jan, 15(1), 69 - 75
Isoform-specific complementation of the yeast sac6 null mutation by human fimbrin; Adams AE et al.; The actin cytoskeleton is a fundamental component of eukaryotic cells, with both structural and motile roles . Actin and many of the actin-binding proteins found in different cell types are highly conserved, showing considerable similarity in both primary structure and biochemical properties . To make detailed comparisons between homologous proteins, it is necessary to know whether the various proteins are functionally, as well as structurally, conserved . Fimbrin is an example of a cytoskeletal component that, as shown by sequence determinations and biochemical characterizations, is conserved between organisms as diverse as Saccharomyces cerevisiae and humans . In this study, we examined whether the human homolog can substitute for the yeast protein in vivo . We report here that two isoforms of human fimbrin, also referred to as T- and L-plastin, can both substitute in vivo for yeast fimbrin, also known as Sac6p, whereas a third isoform, I-fimbrin (or I-plastin), cannot . We demonstrate that the human T- and L-fimbrins, in addition to complementing the temperature-sensitive growth defect of the sac6 null mutant, restore both normal cytoskeletal organization and cell shape to the mutant cells . In addition, we show that human T- and L-fimbrins can complement a sporulation defect caused by the sac6 null mutation . These findings indicate that there is a high degree of functional conservation in the cytoskeleton, even between organisms as diverse as S . cerevisiae and humans.

Mol Cell Biol, 1995 Jan, 15(1), 445 - 55
Structurally related but functionally distinct yeast Sm D core small nuclear ribonucleoprotein particle proteins; Roy J et al.; Spliceosome assembly during pre-mRNA splicing requires the correct positioning of the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) on the precursor mRNA . The structure and integrity of these snRNPs are maintained in part by the association of the snRNAs with core snRNP (Sm) proteins . The Sm proteins also play a pivotal role in metazoan snRNP biogenesis . We have characterized a Saccharomyces cerevisiae gene, SMD3, that encodes the core snRNP protein Smd3 . The Smd3 protein is required for pre-mRNA splicing in vivo . Depletion of this protein from yeast cells affects the levels of U snRNAs and their cap modification, indicating that Smd3 is required for snRNP biogenesis . Smd3 is structurally and functionally distinct from the previously described yeast core polypeptide Smd1 . Although Smd3 and Smd1 are both associated with the spliceosomal snRNPs, overexpression of one cannot compensate for the loss of the other . Thus, these two proteins have distinct functions . A pool of Smd3 exists in the yeast cytoplasm . This is consistent with the possibility that snRNP assembly in S . cerevisiae, as in metazoans, is initiated in the cytoplasm from a pool of RNA-free core snRNP protein complexes.

Mol Cell Biol, 1995 Jan, 15(1), 365 - 78
Structural requirements for double-stranded RNA binding, dimerization, and activation of the human eIF-2 alpha kinase DAI in Saccharomyces cerevisiae; Romano PR et al.; The protein kinase DAI is activated upon viral infection of mammalian cells and inhibits protein synthesis by phosphorylation of the alpha subunit of translation initiation factor 2 (eIF-2 alpha) . DAI is activated in vitro by double-stranded RNAs (dsRNAs), and binding of dsRNA is dependent on two copies of a conserved sequence motif located N terminal to the kinase domain in DAI . High-level expression of DAI in Saccharomyces cerevisiae cells is lethal because of hyperphosphorylation of eIF-2 alpha; at lower levels, DAI can functionally replace the protein kinase GCN2 and stimulate translation of GCN4 mRNA . These two phenotypes were used to characterize structural requirements for DAI function in vivo, by examining the effects of amino acid substitutions at matching positions in the two dsRNA-binding motifs and of replacing one copy of the motif with the other . We found that both copies of the dsRNA-binding motif are required for high-level kinase function and that the N-terminal copy is more important than the C-terminal copy for activation of DAI in S . cerevisiae . On the basis of these findings, we conclude that the requirements for dsRNA binding in vitro and for activation of DAI kinase function in vivo closely coincide . Two mutant alleles containing deletions of the first or second binding motif functionally complemented when coexpressed in yeast cells, strongly suggesting that the active form of DAI is a dimer . In accord with this conclusion, overexpression of four catalytically inactive alleles containing different deletions in the protein kinase domain interfered with wild-type DAI produced in the same cells . Interestingly, three inactivating point mutations in the kinase domain were all recessive, suggesting that dominant interference involves the formation of defective heterodimers rather than sequestration of dsRNA activators by mutant enzymes . We suggest that large structural alterations in the kinase domain impair an interaction between the two protomers in a DAI dimer that is necessary for activation by dsRNA or for catalysis of eIF-2 alpha phosphorylation.

Mol Cell Biol, 1995 Jan, 15(1), 264 - 71
Cloning and characterization of FAD1, the structural gene for flavin adenine dinucleotide synthetase of Saccharomyces cerevisiae; Wu M et al.; The FAD1 gene of Saccharomyces cerevisiae has been selected from a genomic library on the basis of its ability to partially correct the respiratory defect of pet mutants previously assigned to complementation group G178 . Mutants in this group display a reduced level of flavin adenine dinucleotide (FAD) and an increased level of flavin mononucleotide (FMN) in mitochondria . The restoration of respiratory capability by FAD1 is shown to be due to extragenic suppression . FAD1 codes for an essential yeast protein, since disruption of the gene induces a lethal phenotype . The FAD1 product has been inferred to be yeast FAD synthetase, an enzyme that adenylates FMN to FAD . This conclusion is based on the following evidence . S . cerevisiae transformed with FAD1 on a multicopy plasmid displays an increase in FAD synthetase activity . This is also true when the gene is expressed in Escherichia coli . Lastly, the FAD1 product exhibits low but significant primary sequence similarity to sulfate adenyltransferase, which catalyzes a transfer reaction analogous to that of FAD synthetase . The lower mitochondrial concentration of FAD in G178 mutants is proposed to be caused by an inefficient exchange of external FAD for internal FMN . This is supported by the absence of FAD synthetase activity in yeast mitochondria and the presence of both extramitochondrial and mitochondrial riboflavin kinase, the preceding enzyme in the biosynthetic pathway . A lesion in mitochondrial import of FAD would account for the higher concentration of mitochondrial FMN in the mutant if the transport is catalyzed by an exchange carrier . The ability of FAD1 to suppress impaired transport of FAD is explained by mislocalization of the synthetase in cells harboring multiple copies of the gene . This mechanism of suppression is supported by the presence of mitochondrial FAD synthetase activity in S . cerevisiae transformed with FAD1 on a high-copy-number plasmid but not in mitochondrial of a wild-type strain.

Mol Cell Biol, 1995 Jan, 15(1), 246 - 54
The maf proto-oncogene stimulates transcription from multiple sites in a promoter that directs Purkinje neuron-specific gene expression; Kurschner C et al.; L7 is expressed in all adult cerebellar Purkinje cells, although during development it appears in a stereotyped spatial and temporal pattern that is manifested as parasagittal domains of neurons . Mutations of the L7 promoter in transgenic mice have established that these domains represent functional compartments of Purkinje neurons . Therefore, it is hoped that by defining the transcriptional control of the L7 gene insights into the mechanisms that control functional fate and organization in the nervous system can be gained . Fragments of the L7 promoter were introduced into a selectable reporter gene in Saccharomyces cerevisiae, and these strains were used to select for cerebellar cDNAs encoding proteins that can bind to, and activate transcription from, these elements . This assay identified the c-Maf proto-oncogene as activating transcription from two sites in the L7 promoter . We did a functional domain analysis of vertebrate c-Maf based upon transcriptional activation in S . cerevisiae and showed the requirement for a transactivation domain, leucine zipper, and DNA-binding region in c-Maf . The c-Maf interaction site was mapped to the sequence G/TGG/CNG/TNCT CAGNN in the L7 promoter, which represents an atypical 12-O-tetradecanoate-13-acetate-responsive element-type Maf-responsive element . However, neither Fos nor Jun, either alone or in combination with each other or c-Maf, altered transcription from this element . In contrast, a Maf-related protein, Nrl, completely mimicked c-Maf actions . These data suggest that Maf may interact with additional basic-zipper proteins that determine a subtype of Maf-responsive element binding.

Cell Motil Cytoskeleton, 1995, 30(4), 285 - 300
beta-Tubulin mutation suppresses microtubule dynamics in vitro and slows mitosis in vivo; Sage CR et al.; Microtubule (MT) dynamics vary both spatially and temporally within cells and are thought to be important for proper MT cellular function . Because MT dynamics appear to be closely tied to the guanosine triphosphatase (GTPase) activity of beta-tubulin subunits, we examined the importance of MT dynamics in the budding yeast S . cerevisiae by introducing a T107K point mutation into a region of the single beta-tubulin gene, TUB2, known to affect the assembly-dependent GTPase activity of MTs in vitro . Analysis of MT dynamic behavior by video-enhanced differential interference contrast microscopy, revealed that T107K subunits slowed both the growth rates and catastrophic disassembly rates of individual MTs in vitro . In haploid cells tub2-T107K is lethal; but in tub2-T107K/tub2-590 heterozygotes the mutation is viable, dominant, and slows cell-cycle progression through mitosis, without causing wholesale disruption of cellular MTs . The correlation between the slower growing and shortening rates of MTs in vitro, and the slower mitosis in vivo suggests that MT dynamics are important in budding yeast and may regulate the rate of nuclear movement and segregation . The slower mitosis in mutant cells did not result in premature cytokinesis and cell death, further suggesting that cell-cycle control mechanisms "sense" the mitotic slowdown, possibly by monitoring MT dynamics directly.

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