Infect Immun, 1994 Dec, 62(12), 5576 - 80 Saccharomyces cerevisiae recombinant Pfs25 adsorbed to alum elicits antibodies that block transmission of Plasmodium falciparum; Kaslow DC et al.; Antibodies to Pfs25, a cysteine-rich 25-kDa protein present on the surface of Plasmodium falciparum zygotes, can completely block the transmission of malaria parasites when mixed with infectious blood and fed to mosquitoes through a membrane feeding apparatus . Recently, a polypeptide analog, Pfs25-B, secreted from recombinant Saccharomyces cerevisiae was found to react with conformation-dependent, transmission-blocking monoclonal antibodies and to elicit transmission-blocking antibodies in experimental animals when emulsified in either Freund's or muramyl tripeptide adjuvant . In this study, Pfs25-B adsorbed to alum induced transmission-blocking antibodies in both rodents and primates . Bacterially produced Pfs25, however, did not elicit complete transmission-blocking antibodies in rodents . Furthermore, unlike monoclonal antibodies to Pfs25, which block transmission only after ookinete development, antisera to Pfs25-B adsorbed to alum appeared to block the in vivo development of zygotes to ookinetes as well. Genetics, 1994 Dec, 138(4), 1081 - 92 Control of adaptation to mating pheromone by G protein beta subunits of Saccharomyces cerevisiae; Grishin AV et al.; The STE4 gene of the yeast Saccharomyces cerevisiae encodes the beta subunit of a heterotrimeric G protein that mediates response to mating pheromones and influences recovery from pheromone-induced growth arrest . To explore how G beta subunits regulate response and recovery (adaptation), we isolated and characterized signaling-defective STE4 alleles (STE4sd) . STE4sd mutations resulted in amino acid substitutions in the N-terminal region of Ste4p, proximal to the first of seven repeat units conserved in G protein beta subunits . Genetic tests indicated that STE4sd mutations disrupted functions of Ste4p required for inducing pheromone responses . Wild-type cells that overexpressed STE4sd alleles displayed apparently normal initial responses to pheromone as judged by quantitative mating, G1 arrest and transcriptional assays . However, after undergoing initial G1 arrest, wild-type cells overexpressing STE4sd alleles recovered more quickly from division arrest, suggestive of a hyperadaptive phenotype . Because hyperadaptation occurred when STE4sd alleles were overexpressed in cells lacking Sst1p (Bar1p), Sst2p or the C-terminal domain of the alpha-factor receptor, this phenotype did not involve three principal modes of adaptation in yeast . However, hyperadaptation was abolished when STE4sd mutations were combined in cis with a deletion that removes a segment of Ste4p (residues 310-346) previously implicated in adaptation to pheromone . These results indicate that G beta subunits possess two independent activities, one required for triggering pheromone response and another that promotes adaptation . Potential models for G beta subunit-mediated adaptation are discussed. Genetics, 1994 Dec, 138(4), 1067 - 79 CHL12, a gene essential for the fidelity of chromosome transmission in the yeast Saccharomyces cerevisiae; Kouprina N et al.; We have analyzed the CHL12 gene, earlier identified in a screen for yeast mutants with increased rates of mitotic loss of chromosome III and circular centromeric plasmids . A genomic clone of CHL12 was isolated and used to map its physical position on the right arm of chromosome XIII near the ADH3 locus . Nucleotide sequence analysis of CHL12 revealed a 2.2-kb open reading frame with a 84-kD predicted protein sequence . Analysis of the sequence upstream of the CHL12 open reading frame revealed the presence of two imperfect copies of MluI motif, ACGCGT, a sequence associated with many DNA metabolism genes in yeast . Analysis of the amino acid sequence revealed that the protein contains a NTP-binding domain and shares a low degree of homology with subunits of replication factor C (RF-C) . A strain containing a null allele of CHL12 was viable under standard growth conditions, and as well as original mutants exhibited an increase in the level of spontaneous mitotic recombination, slow growth and cold-sensitive phenotypes . Most of cells carrying the null chl12 mutation arrested as large budded cells with the nucleus in the neck at nonpermissive temperature that typical for cell division cycle (cdc) mutants that arrest in the cell cycle at a point either immediately preceding M phase or during S phase . Cell cycle arrest of the chl12 mutant requires the RAD9 gene . We conclude that the CHL12 gene product has critical role in DNA metabolism. Genetics, 1994 Dec, 138(4), 1025 - 40 Mutational analysis defines a C-terminal tail domain of RAP1 essential for Telomeric silencing in Saccharomyces cerevisiae; Liu C et al.; Alleles specifically defective in telomeric silencing were generated by in vitro mutagenesis of the yeast RAP1 gene . The most severe phenotypes occur with three mutations in the C-terminal 28 amino acids . Two of the alleles are nonsense mutations resulting in truncated repressor/activator protein 1 (RAP1) species lacking the C-terminal 25-28 amino acids; the third allele is a missense mutation within this region . These alleles define a novel 28-amino acid region, termed the C-terminal tail domain, that is essential for telomeric and HML silencing . Using site-directed mutagenesis, an 8-amino acid region (amino acids 818-825) that is essential for telomeric silencing has been localized within this domain . Further characterization of these alleles has indicated that the C-terminal tail domain also plays a role in telomere size control . The function of the C-terminal tail in telomere maintenance is not mediated through the RAP1 interacting factor RIF1: rap1 alleles defective in both the C-terminal tail and RIF1 interaction domains have additive effects on telomere length . Overproduction of SIR3, a dose-dependent enhancer of telomeric silencing, suppresses the telomeric silencing, but not length, phenotypes of a subset of C-terminal tail alleles . In contrast, an allele that truncates the terminal 28 amino acids of RAP1 is refractory to SIR3 overproduction . These results indicate that the C-terminal tail domain is required for SIR3-dependent enhancement of telomeric silencing . These data also suggest a distinct set of C-terminal requirements for telomere size control and telomeric silencing. Genetics, 1994 Dec, 138(4), 1015 - 24 Three independent forms of regulation affect expression of HO, CLN1 and CLN2 during the cell cycle of Saccharomyces cerevisiae; Breeden L et al.; The G1 cyclins (CLNs) bind to and activate the CDC28 kinase during the G1 to S transition in Saccharomyces cerevisiae . Two G1 cyclins are regulated at the RNA level so that their RNAs peak at the G1/S boundary . In this report we show that the cell cycle regulation of CLN1 and CLN2 is partially determined by the restricted expression of SW14, a known trans-activator of SCB elements . When SWI4 is constitutively expressed or deleted, cell cycle regulation of CLN1/2 is reduced but not eliminated . In the absence of SwI6, another known regulator of both SCB and MCB elements, cell cycle regulation of the CLNs is also reduced, and the Start-dependence of HO transcription is eliminated . This indicates that SwI6 also plays an important role in the normal cell cycle regulation of all three promoters . When both SwI6 activity and the transcriptional regulation of SW14 are eliminated, cell cycle regulation is further reduced, indicating that these are two independent pathways of regulation . However, a twofold fluctuation in transcript levels still persists under these conditions . This reveals a third source of cell cycle control, which could affect SwI4 activity post-transcriptionally, or reflect the existence of another unidentified regulator of these promoters. Int J Biochem, 1994 Dec, 26(12), 1377 - 81 Inhibition of porphobilinogenase by porphyrins in Saccharomyces cerevisiae; Araujo LS et al.; The biosynthesis of uroporphyrinogen III, the precursor of hemes, chlorophylls, corrins and related structures, is catalyzed by the porphobilinogenase system (PBGase), a complex of two enzymes, PBG-Deaminase (PBG-D) and Isomerase . Although the separate enzymes have been studied in some detail less work has been performed on the properties of the complex . In this study the kinetic behaviour of the enzyme PBGase in a normal yeast strain, D273-10B, and its derivative B231 has been investigated . Uroporphyrinogen formation was linear with time up to 2 hr at 37 degrees C . The enzyme complex shows classical Michaelis-Menten kinetics . From the double reciprocal plots kinetic parameters were estimated for PBGase and PBG-D . Porphyrins were found to be competitive inhibitors with respect to porphobilinogen (PBG) and these compounds appeared to act as inhibitors by forming dead-end complexes with the free enzyme . 5-Aminolevulinic acid (ALA) also inhibited PBGase and this inhibition was overcome by addition of levulinic acid (2 microM) . These results indicate that ALA, is not an inhibitor but acts through its conversion into porphyrins which are the true inhibitors. Curr Opin Cell Biol, 1994 Dec, 6(6), 836 - 41 Signal transduction in the budding yeast Saccharomyces cerevisiae; Oehlen B et al.; As a key part of the mechanism which controls growth and division, cells are able to respond to a variety of intracellular and extracellular stimuli . Significant progress has been made in the understanding of the biochemical mechanisms underlying mating-factor signal transduction in Saccharomyces cerevisiae . Some of these mechanisms may be relevant to the regulation of other signal transduction pathways. J Cell Biol, 1994 Dec, 127(6 Pt 2), 1985 - 93 Daughter cells of Saccharomyces cerevisiae from old mothers display a reduced life span; Kennedy BK et al.; The yeast Saccharomyces cerevisiae typically divides asymmetrically to give a large mother cell and a smaller daughter cell . As mother cells become old, they enlarge and produce daughter cells that are larger than daughters derived from young mother cells . We found that occasional daughter cells were indistinguishable in size from their mothers, giving rise to a symmetric division . The frequency of symmetric divisions became greater as mother cells aged and reached a maximum occurrence of 30% in mothers undergoing their last cell division . Symmetric divisions occurred similarly in rad9 and ste12 mutants . Strikingly, daughters from old mothers, whether they arose from symmetric divisions or not, displayed reduced life spans relative to daughters from young mothers . Because daughters from old mothers were larger than daughters from young mothers, we investigated whether an increased size per se shortened life span and found that it did not . These findings are consistent with a model for aging that invokes a senescence substance which accumulates in old mother cells and is inherited by their daughters. Microbiologia, 1994 Dec, 10(4), 385 - 94 Genes involved in the regulation of invertase production in Saccharomyces cerevisiae; del Castillo Agudo L et al.; The expression of Saccharomyces cerevisiae SUC genes is exclusively regulated by catabolic repression, mediated by glucose . Genes involved in this process have been defined by means of mutants either unable to express invertase or with constitutive phenotype, although none of the genes is specific for invertase regulation . The affected genes in mutants unable to produce invertase are designated SNFX . These genes can be assorted into two groups considering either their function in regulation of gene expression or their epistatic relationships . Mutants with constitutive phenotype have been selected either by resistance to 2-deoxyglucose or by suppression of snf mutations . Among the different genes previously outlined, some of which code for transcription factors, only the MIG1 product, a "zinc finger" protein, shows a clear capacity of binding DNA in vitro . Besides the ON/OFF switch mechanism of the expression of SUC genes, some genes seem to play a role in modulating invertase expression, either hindering or stimulating transcription . A model to define the relationship between the different gene products involved in the regulation of transcription of the SUC genes is proposed. Appl Microbiol Biotechnol, 1994 Dec, 42(4), 587 - 94 Optimization of the expression system using galactose-inducible promoter for the production of anticoagulant hirudin in Saccharomyces cerevisiae; Choi ES et al.; We have tried to optimize the galactose-inducible gene expression system for the overproduction of the potent thrombin-specific inhibitor, hirudin, in a genetically engineered yeast, Saccharomyces cerevisiae . The expression and secretion of hirudin were directed by the galactose-inducible promoter, GAL10, and the mating factor alpha pre-pro leader sequence . The initial hirudin expression level in shake-flask culture was 2.3 mg 1-1 . Modification of the expression vector and optimization of culture conditions, including the induction conditions, improved the level of hirudin gene expression and secretion into the culture supernatant more than 20-fold (50 mg 1-1) in a 4-1 scale batch cultivation . The expression and secretion level of hirudin seemed to be partially dependent on cell growth when galactose was used as a carbon source . Overexpression of the transcriptional activator, GAL4, appeared to have only negative effects on the expression of the hirudin gene and lacZ directed by the GAL10 promoter in the strain used in this study, unlike the previously reported examples . The complex medium containing yeast extract used for the increase of the cell mass and hirudin level did not show any detrimental effect on plasmid stability and did not complicate the downstream purification of hirudin from the culture supernatant . Moreover, the complex medium could greatly improve the hirudin productivity and reduce the degradation of hirudin produced in the culture supernatants. Biosci Biotechnol Biochem, 1994 Dec, 58(12), 2228 - 31 A novel type of life cycle "delayed homothallism" in Saccharomyces cerevisiae wy2 showed slow interconversion of mating-type; Tani Y et al.; Saccharomyces cerevisiae wy2 segregated to 2 mater and 2 non-mater in relation to mating ability . The non-mater segregants behaved as the normal type of homothallic life cycle . On the other hand, the mater segregants gradually formed spores during successive subcultures, indicating that slow interconversion of mating-type happened to occur during subcultures . We termed this novel type of life cycle "delayed homothallism" . The results of complementation tests with standard ho strains and introduction of a wild type HO gene showed that delayed homothallism was caused by a defective HO gene . The amino acid sequence deduced from the nucleotide sequence of the wy2 HO gene differed from the wild type HO gene in three amino acid residues . In the carboxy terminus of HO protein, there are three repeats of cysteine and histidine that are postulated to play a role in binding of HO protein to DNA . However, wy2 HO protein lacked one such repeat at residues Cys470-His475, where His was replaced by Leu. Yeast, 1994 Dec, 10(12), 1653 - 6 Sequence of the PHO2-POL3 (CDC2) region of chromosome IV of Saccharomyces cerevisiae; Simon M et al.; The nucleotide sequence of a 5 kb EcoRI-NcoI fragment of chromosome IV, contiguous to gene POL3 (CDC2), has been determined . It contains three open reading frames: QRI1, QRI2 and QRI7 . Two of them are essential genes . QRI7 is homologous to the Escherichia coli orfx gene. Yeast, 1994 Dec, 10(12), 1591 - 9 The linkage of (1-3)-beta-glucan to chitin during cell wall assembly in Saccharomyces cerevisiae; Hartland RP et al.; Pulse-chase experiments with {14C}glucose demonstrated that in the cell wall of wild-type Saccharomyces cerevisiae alkali-soluble (1-3)-beta-glucan serves as a precursor for alkali-insoluble (1-3)-beta-glucan . The following observations support the notion that the insolubilization of the glucan is caused by linkage to chitin: (i) degradation of chitin by chitinase completely dissolved the glucan, and (ii) disruption of the gene for chitin synthase 3 prevented the formation of alkali-insoluble glucan . These cells, unable to form a glucan-chitin complex, were highly vulnerable to hypo-osmotic shock indicating that the linkage of the two polymers significantly contributes to the mechanical strength of the cell wall . Conversion of alkali-soluble glucan into alkali-insoluble glucan occurred both early and late during budding and also in the ts-mutant cdc24-1 in the absence of bud formation. J Biochem (Tokyo), 1994 Dec, 116(6), 1341 - 5 Uso1 protein contains a coiled-coil rod region essential for protein transport from the ER to the Golgi apparatus in Saccharomyces cerevisiae; Seog DH et al.; We have previously shown that the Saccharomyces cerevisiae USO1 gene required in the protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus encodes a 200-kDa protein (1,790 amino acids) which is present in a nonglobular high molecular mass complex . Antibodies against an N-terminal portion of Uso1 protein recognized a 100-kDa protein in Western blot of the temperature-sensitive uso1-1 mutant cell lysate . The nucleotide sequence of uso1-1 indicated the 951st codon was UAG (amber) in place of CAG (glutamine) in USO1 . Deletion study of USO1 gene indicated that such truncated Uso1 polypeptides are sufficiently functional at 25 degrees C but not at 37 degrees C . Mutant Uso1-1 protein displayed an apparent molecular mass of 400-500 kDa in gel filtration while it cosedimented with a globular 6S marker protein, horseradish peroxidase (44 kDa), in sucrose density gradient centrifugation . These results indicated that truncated Uso1-1 protein is still present in a nonglobular high molecular mass complex, similar to the wild-type Uso1 protein. J Biochem (Tokyo), 1994 Dec, 116(6), 1317 - 21 Cloning and sequence of the SCS3 gene which is required for inositol prototrophy in Saccharomyces cerevisiae; Hosaka K et al.; The SCS3 gene of Saccharomyces cerevisiae was cloned by functional complementation, using a conditional mutant exhibiting myo-inositol auxotrophy in the presence of choline, and sequenced . The sequence contained an open reading frame capable of encoding 380 amino acids with a calculated molecular weight of 42,734 . Disruption of the SCS3 locus caused myo-inositol auxotrophy . The gene appeared to be involved in the synthesis of inositol phospholipids from inositol but not in the control of inositol synthesis. Mol Cell Biol, 1994 Dec, 14(12), 8259 - 71 MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein, is required for Ca2+ influx and mating; Iida H et al.; By establishing a unique screening method, we have isolated yeast mutants that die only after differentiating into cells with a mating projection, and some of them are also defective in Ca2+ signaling . The mutants were classified into five complementation groups, one of which we studied extensively . This mutation defines a new gene, designated MID1, which encodes an N-glycosylated, integral plasma membrane protein with 548 amino acid residues . The mid1-1 mutant has low Ca2+ uptake activity, loses viability after receiving mating pheromones, and escapes death when incubated with high concentrations of CaCl2 . The MID1 gene is nonessential for vegetative growth . The efficiency of mating between MATa mid1-1 and MAT alpha mid1-1 cells is low . These results demonstrate that MID1 is required for Ca2+ influx and mating. J Biol Chem, 1994 Nov 25, 269(47), 29609 - 12 Cooperation between enzyme and transporter in the inner mitochondrial membrane of yeast . Requirement for mitochondrial citrate synthase for citrate and malate transport in Saccharomyces cerevisiae; Sandor A et al.; We have characterized 1,2,3-benzenetricarboxylic acid-sensitive, mersalyl-insensitive citrate uptake by mitochondria from two strains of Saccharomyces cerevisiae by describing the time course, Km and Vmax values, pH dependence, and response to inhibitors . In unloaded mitochondria from PSY142 CS1- cells, a mutant that lacks mitochondrial citrate synthase, both citrate uptake and efflux were reduced 7- and 8-fold, respectively, compared with the parental strain . No malate uptake was detectable in mitochondria from CS1- cells, while in the parental strain, uptake was 5.4 nmol/min/mg of protein . In contrast, mutations in peroxisomal citrate synthase (CS2-) or in other tricarboxylic acid cycle enzymes did not result in changes in mitochondrial citrate transport, suggesting a specific functional role for mitochondrial citrate synthase in citrate transport . More important, liposomes containing protein extracts from CS1- mitochondria showed the same citrate and malate transport rates as liposomes made from protein extracts of parental strain mitochondria . Thus, an apparently normal amount of both the citrate transporter and the dicarboxylate carrier is present in CS1- mitochondria, but both function abnormally in undisrupted mitochondria . We suggest that cooperation between the citrate transporter and mitochondrial citrate synthase is necessary for normal function of the transporter. J Biol Chem, 1994 Nov 25, 269(47), 29495 - 501 Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by nucleotides; Wu WI et al.; Regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by nucleotides was examined using pure enzyme and Triton X-100/phosphatidate-mixed micelles . Adenosine, guanosine, cytidine, and uridine nucleotides inhibited phosphatidate phosphatase activity in a dose-dependent manner . ATP and CTP were the most potent inhibitors of the enzyme . A kinetic analysis was performed to determine the mechanism of enzyme inhibition by nucleotides . The mechanism of inhibition by ATP and CTP with respect to phosphatidate (the substrate) was complex . The dependence of phosphatidate phosphatase activity on phosphatidate was cooperative, and nucleotides affected both Vmax and Km . ATP did not inhibit phosphatidate phosphatase activity by binding to the enzyme or to phosphatidate . Phosphatidate phosphatase dependence on Mg2+ ions (the cofactor) followed saturation kinetics, and the mechanism of nucleotide inhibition with respect to Mg2+ ions was competitive . Thus, the mechanism of enzyme inhibition by nucleotides was the chelation of Mg2+ ions . The inhibitor constant for ATP was lower than its cellular concentration in glucose-grown cells . However, the inhibitor constant for ATP was higher than its cellular concentration in glucose-starved cells . Changes in the cellular concentration of ATP affected the proportional synthesis of triacylglycerols and phospholipids . These results were consistent with the regulation of phosphatidate phosphatase activity by ATP through a Mg2+ ion chelation mechanism. Nucleic Acids Res, 1994 Nov 25, 22(23), 4906 - 13 Isolation and characterization of two Saccharomyces cerevisiae genes that encode proteins that bind to (TG1-3)n single strand telomeric DNA in vitro; Lin JJ et al.; By screening lambda gt11 libraries with a radiolabeled (TG1-3)n oligonucleotide, two Saccharomyces cerevisiae genes were identified that encode polypeptides that recognize the single-stranded telomeric repeat sequence (TG1-3)n . The first gene, NSR1, a previously identified gene, encodes a protein involved in ribosomal RNA maturation and possibly in transport of proteins into the nucleus . The second gene, GBP2 (G-strand Binding Protein), is an anonymous open reading frame from chromosome III . These two genes contain RNA recognition motifs (RRMs) that are found in proteins that interact with RNA . Both Nsr1p and Gbp2p bind specifically to yeast single strand (TG1-3)n DNA in vitro . To test whether these two proteins associate with telomeres in vivo, strains were constructed in which one or both of these genes were either disrupted or overexpressed . None of these alterations affected telomere length or telomere position effect . The potential role of these two (TG1-3)n binding proteins is discussed. Biochemistry, 1994 Nov 22, 33(46), 13801 - 7 Expression, purification, and characterization of the dihydrolipoamide dehydrogenase-binding protein of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae; Maeng CY et al.; Genes encoding dihydrolipoamide dehydrogenase (E3) and the E3-binding protein (E3BP, protein X), components of the Saccharomyces cerevisiae pyruvate dehydrogenase (PDH) complex, were coexpressed in Escherichia coli to produce an E3BP-E3 complex, thereby minimizing proteolysis of E3BP and facilitating its purification . The 2 genes were linked into a single transcriptional unit separated by a 31-nucleotide segment containing a ribosome-binding sequence . The E3BP-E3 complex was highly purified and then separated into E3 and E3BP by chromatography on hydroxylapatite in the presence of 5 M urea . The E3BP-E3 complex combined rapidly with a pyruvate dehydrogenase (E1)-dihydrolipoamide acetyltransferase (E2) subcomplex (E1-E2 subcomplex) to reconstitute a functional PDH complex, with pyruvate oxidation activity similar to that of PDH complex from bakers' yeast . The stoichiometry of binding of E3BP and E3BP-E3 complex to the 60-subunit pentagonal dodecahedron-like E2 was determined with a truncated form of E2 (tE2, residues 206-454) lacking the lipoyl domain and the E1-binding domain, and with E1-E2 subcomplex, which contains intact E2 . Mixtures containing tE2 or E1-E2 subcomplex and excess E3BP or E3BP-E3 complex were subjected to ultracentrifugation to separate the large complexes from unbound E3BP or E3BP-E3, and the complexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis . After staining with Coomassie brilliant blue and destaining, the gels were analyzed with a video area densitometer . The results showed that the E1-E2 subcomplex binds about 12 E3BP monomers attached to 12 E3 homodimers . Similar results were obtained by analysis of highly purified PDH complex from bakers' yeast.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1994 Nov 21, 355(1), 69 - 75 The 26S proteasome of the yeast Saccharomyces cerevisiae; Fischer M et al.; Proteasomes are large multicatalytic proteinase complexes found in all eukaryotic organisms investigated so far . They have been shown to play a central role in cytosolic and nuclear proteolysis . According to their sedimentation coefficients two types of these particles can be distinguished: 20S proteasomes and 26S proteasomes . In contrast to 20S proteasomes, which were mainly characterized on the basis of their ability to cleave small chromogenic peptide substrates and certain proteins in an ATP-independent manner, 26S proteasomes degrade ubiquitinylated proteins in an ATP-dependent reaction . 20S proteasomes have been found in all eukaryotes from yeast to man . So far 26S proteasomes have only been discovered in higher eukaryotes . We now report the existence of the 26S proteasome in a lower eukaryote, the yeast Saccharomyces cerevisiae . Formation of the 26S proteasome could most effectively be induced in crude extracts of heat stressed yeast cells by incubation with ATP and Mg2+ ions . This treatment yielded a protein complex, which eluted from gel filtration columns at molecular masses higher than 1500 kDa . Besides chromogenic peptide substrates, this complex cleaves ubiquitinylated proteins in an ATP-dependent fashion . In non-denaturing-PAGE, the purified 26S proteasome disintegrated and migrated as four protein bands . One of these bands could be identified as the 20S proteasome . On SDS-PAGE, the 26S proteasome showed a complex pattern of subunit bands with molecular masses between 15 and 100 kDa . Further evidence for the 20S proteasome being the proteolytically active core of the 26S proteasome was obtained by following peptide cleaving activities in extracts of yeast strains carrying mutations in various subunits of the 20S proteasome. J Biol Chem, 1994 Nov 18, 269(46), 28769 - 76 Studies employing Saccharomyces cerevisiae cpt1 and ept1 null mutants implicate the CPT1 gene in coordinate regulation of phospholipid biosynthesis; Morash SC et al.; The Saccharomyces cerevisiae CPT1 and EPT1 genes are structural genes encoding sn-1,2-diacylglycerol choline phosphotransferase and sn-1,2-diacylglycerol choline/ethanolamine phosphotransferase, respectively . Incorporation of 32Pi into phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in wild type and ept1 strains was decreased in the presence of exogenous inositol . In contrast, inositol did not affect 32Pi incorporation into phospholipid in cpt1 or cpt1ept1 strains . In membranes isolated from wild type and ept1 strains grown in the presence of inositol or inositol/choline, the CPT1-derived cholinephosphotransferase activities were reduced 40-50 and 65%, respectively . Inositol-dependent reductions in CPT1 derived choline-phosphotransferase activity correlated with transcript levels in both wild type and ept- backgrounds . The ethanolaminephosphotransferase activity of the EPT1 gene product in wild type cells was reduced 40% by exogenous inositol alone and 50% by inositol/choline . In the cpt1 strain, however, the ethanolaminephosphotransferase activity was unaffected by exogenous inositol or inositol/choline . The inositol-dependent reduction of ethanolaminephosphotransferase activity observed in wild type cells correlated with reduced levels of EPT1 transcripts; in the cpt1 strain, EPT1 transcript levels were not affected by inositol . These results indicate that 1) a functional CPT1 gene or gene product is required for inositol-dependent regulation of phospholipid synthesis; 2) the enzyme activities of both the CPT1 and EPT1 gene products are repressed by inositol and inositol/choline, and require an intact CPT1 gene; 3) inositol mediates its regulatory effects on phospholipid synthesis via a transcriptional mechanism. FEMS Microbiol Lett, 1994 Nov 15, 124(1), 17 - 22 Relationship between ethanol tolerance, lipid composition and plasma membrane fluidity in Saccharomyces cerevisiae and Kloeckera apiculata; Alexandre H et al.; The lipid composition of a strain of each of two yeasts, Saccharomyces cerevisiae and Kloeckera apiculata, with different ethanol tolerances, was determined for cells grown with or without added ethanol . An increase in the proportion of ergosterol, unsaturated fatty acid levels and the maintenance of phospholipid biosynthesis seemed to be responsible for ethanol tolerance . The association of ethanol tolerance of yeast cells with plasma membrane fluidity, measured by fluorescence anisotropy, is discussed . We propose that an increase in plasma membrane fluidity may be correlated with a decrease in the sterol:phospholipid and sterol:protein ratios and an increase in unsaturation index. Biochem Biophys Res Commun, 1994 Nov 15, 204(3), 1016 - 22 The tetratricopeptide repeat-domain of the PAS10 protein of Saccharomyces cerevisiae is essential for binding the peroxisomal targeting signal-SKL; Brocard C et al.; The PAS10 gene was found in a two-hybrid screen for the isolation of genes encoding proteins which interact with the C-terminal peroxisomal targeting signal -SKL . The PAS10 protein is known to be involved in import of proteins into peroxisomes and to contain a tetratricopeptide repeat (TPR) domain . All TPR-containing proteins involved in diverse processes like mitosis or RNA-synthesis share the ability to interact with other proteins . Here we show that the PAS10 protein interacts in vivo with the C-terminal peroxisomal targeting signal . The part essential for this interaction contains the complete tetratricopeptide repeat domain. Eur J Biochem, 1994 Nov 15, 226(1), 133 - 40 Comparative analysis of ribosome-associated adenosinetriphosphatase (ATPase) from pig liver and the ATPase of elongation factor 3 from Saccharomyces cerevisiae; Kovalchuke O et al.; Elongation factor 3 (EF-3) is a unique and essential requirement of the fungal translational machinery . Non-fungal organisms do not have and do not require a soluble form of the third elongation factor for translation . To test whether non-fungal organisms have a direct analog of EF-3 incorporated in the structure of the ribosomes, a comparison of EF-3 adenosinetriphosphatase (ATPase) with ATPases associated with pig liver ribosomes was carried out . EF-3 function depends on ATP (GTP) hydrolysis . The hydrolytic activity of EF-3 is enhanced by two orders of magnitude by yeast ribosomes due to an increase in the turnover rate of EF-3 . The nucleotide hydrolytic activity of EF-3 is significantly constrained by the binding of aminoacylated tRNA(Phe) to poly(U)-programmed ribosomes . The translational inhibitors--neomycin and alpha-sarcin suppress the ATPase activity of EF-3 . These results reflect a direct correlation between EF-3 ATPase and the functional state of the ribosome . Four lines of evidence indicate that yeast EF-3 ATPase is functionally distinct from pig liver ribosome associated ATPases . The kinetic parameters of ATPases from these two sources are different . Poly(U) and tRNA have no effect on the ATPase activity associated with the pig liver ribosomes . The latter activity is unaffected by the translational inhibitors neomycin and alpha-sarcin . The translational activity of pig liver ribosomes is not influenced by ATP, ADP or adenosine 5'-{beta, gamma-imido}triphosphate . In an in vitro system, one can demonstrate a small but consistent stimulatory effect of yeast EF-3 on polyphenylalanine synthesis by pig liver ribosomes only when EF-1 alpha is present at a limited concentration . The EF-3 effect disappears when EF-1 alpha is added in a stoichiometric amount to the pig liver ribosomes . This result is in contrast to the yeast system where the ribosomes are completely dependent on EF-3 at all concentrations of EF-1 alpha. J Biol Chem, 1994 Nov 11, 269(45), 28420 - 8 Essential RNA binding and packaging domains of the Gag-Pol fusion protein of the L-A double-stranded RNA virus of Saccharomyces cerevisiae; Ribas JC et al.; The crucial process in the assembly of the L-A double-stranded RNA virus is the recognition of its (+) single-stranded RNA by the Gag-Pol protein . The Pol region of this protein has RNA binding activity and is necessary for RNA packaging . Here we show that there are actually two in vitro RNA-binding domains of Pol (residues 172-190 and 770-819), and both are necessary for viral propagation, (but not for particle assembly) . Furthermore, the N-terminal RNA-binding domain is necessary for in vivo packaging of viral (+) single-stranded RNA . We precisely define the extent of the Pol packaging domain (residues 67-213), which includes the N-terminal RNA-binding domain . This suggests that the N-terminal RNA-binding domain is responsible for binding the genomic RNA in the process of packaging and that additional surrounding residues are responsible for the specificity of binding. J Biol Chem, 1994 Nov 11, 269(45), 28335 - 46 Identification of the cis-acting DNA sequence elements regulating the transcription of the Saccharomyces cerevisiae gene encoding TBP, the TATA box binding protein; Schroeder SC et al.; TBP, the TATA-box binding protein, plays a key role in eukaryotic gene transcription since it is required for transcription initiation by all three eukaryotic nuclear DNA-dependent RNA polymerases . In order to gain insight into the mechanisms of regulation of this key basal transcription factor, we undertook a mutational analysis of the sequences involved in directing transcription of the gene encoding TBP in Saccharomyces cerevisiae . An extensive family of mutations in the promoter of the gene encoding TBP were fused to the Escherichia coli reporter gene lacZ, transferred back into yeast, and assayed for their ability to direct expression of beta-galactosidase . Levels of beta-galactosidase activity measured from yeast transformed with this family of constructs indicate that both positive- and negative-acting cis-elements located within 400 nucleotides of the transcription start site are involved in regulating transcription of the TBP-encoding gene . Analyses of RNA prepared from these same cells showed that specific transcription initiation is maintained in the mutant reporter constructs and that RNA levels mirror beta-galactosidase levels . In order to corroborate the results of these mutational analyses of the TBP-encoding gene, in vivo cis-element occupancy was examined using several different footprinting reagents . The patterns of protection observed demonstrated that the sequence elements implicated in the control of TBP gene transcription by reporter gene analyses appear to be bound by protein(s) in vivo . Interesting sequence similarities were noted between two TBP-gene regulatory elements and 5'-flanking sequences of genes encoding several other basal transcription factors. J Biol Chem, 1994 Nov 11, 269(45), 28106 - 17 Purification and characterization of a late Golgi compartment from Saccharomyces cerevisiae; Whitters EA et al.; We have devised a method for obtaining highly enriched membranes of a late yeast Golgi compartment, operationally defined by their containing the Kex2p protease, and generated four hybridoma cell lines that produced monoclonal antibodies directed against distinct Golgi membrane proteins (GMPs) (GMP36, GMP51, GMP77, and GMP95) . Immunofluorescence and subcellular fractionation data indicated that, of the four GMPs analyzed, only GMP51 exhibited essentially an absolute colocalization with Kex2p . Also, as in the case of Kex2p, retention of GMP51 in yeast Golgi membranes was dependent on clathrin function . In contrast, the remaining three GMPs exhibited substantial, but not absolute, colocalization with Kex2p . The collective data are most consistent with a model where GMP36, GMP77, and GMP95 are present in all Kex2p-containing membranes, but Kex2p is present in only a subpopulation of membranes that contain these GMPs, thereby suggesting that either these particular GMPs exhibit overlapping distributions in compartments of the yeast Golgi complex or are also present in non-Golgi compartments . These findings are not consistent with the view that resident yeast Golgi proteins are generally restricted to a specific Golgi subcompartment, but they are consistent with the view that Golgi compartmental identity is determined by the relative mixtures of Golgi proteins that reside within individual cisternae. J Biol Chem, 1994 Nov 11, 269(45), 28010 - 6 Phosphatidylcholine biosynthesis in Saccharomyces cerevisiae . Regulatory insights from studies employing null and chimeric sn-1,2-diacylglycerol choline- and ethanolaminephosphotransferases; McMaster CR et al.; The Saccharomyces cerevisiae CPT1 and EPT1 genes encode distinct choline- and choline/ethanolaminephosphotransferases, respectively . In vitro, each gene product accounts for 50% of the measurable choline-phosphotransferase activity . Strains containing null mutations in the CPT1 and EPT1 loci were used to investigate the function of each gene product in vivo . The CPT1 gene product was responsible for 95% of phosphatidylcholine (PC) synthesis via the CDP-choline pathway in vivo . The EPT1 gene product accounted for only 5% of PC synthesis in vivo . Chimeric CPT1/EPT1 enzymes with diacylglycerol and CDP-aminoalcohol specificities both similar and distinct from the parental enzymes were used to determine the specific segments of the CPT1/EPT1 gene products required to restore PC synthesis to cpt- cells in vivo . Only chimeras expressing the CDP-aminoalcohol specificity region of CPT1 were capable of PC synthesis via the CDP-choline pathway in vivo . Analysis of phospholipids extracted from wild type, cpt-, and ept- cells labeled with 32Pi indicated an intact CPT1 gene product was required for the pleiotropic regulation of phospholipid synthesis by inositol . Chimeric CPT1/EPT1 enzymes expressed in a cpt- background mapped the regulatory region of the CPT1 gene product required for the inositol-dependent regulation of phospholipid synthesis to the CDP-aminoalcohol binding domain of CPT1 . Strains harboring dysfunctional cholinephosphotransferase enzymes also displayed decreased levels of choline uptake, suggesting that a feedback loop exists to coordinate choline uptake with ongoing PC biosynthesis . The data also implicate the CPT1 gene product in PC biosynthesis from an endogenous source of choline derived from turnover of PC via the phosphatidylserine-dependent route for PC synthesis. J Biol Chem, 1994 Nov 11, 269(45), 27907 - 13 Interactions between cAMP-dependent and SNF1 protein kinases in the control of glycogen accumulation in Saccharomyces cerevisiae; Hardy TA et al.; The synthesis of glycogen in Saccharomyces cerevisiae is stimulated by nutrient limitation and requires both glycogen synthase and the glycogen branching enzyme . Of the two glycogen synthase genes present in yeast, GSY2 appears to be more important for the accumulation of glycogen upon entry into stationary phase . In cells grown on glucose, GSY2 mRNA levels increased approximately 10-fold during the transition from logarithmic to stationary phase . Growth of cells in glycerol, however, resulted in constitutive expression of GSY2 mRNA and the corresponding protein, GS-2, suggestive of glucose repression of GSY2 . Mutants defective in the SNF1 gene, which encodes a protein kinase important in glucose repression mechanisms, are known not to accumulate glycogen . A modest 2-4-fold decrease in total GS-2 level was observed, and upon entry into stationary phase, the enzyme was blocked in the inactive, phosphorylated state in snf1 strains . The GS-2 protein is thought to be regulated by covalent phosphorylation of three COOH-terminal sites (Hardy, T.A., and Roach, P.J . (1993) J . Biol . Chem . 268, 23799-23805), removal of which results in constitutively active glycogen synthase that bypasses phosphorylation controls . Expression of COOH-terminally truncated GS-2 in snf1 cells restored glycogen accumulation, and so we propose that the SNF1 kinase controls the phosphorylation state of GS-2 . Cyclic AMP pathways also exert control over glycogen accumulation . In bcy1 cells, which have constitutively active cyclic AMP-dependent protein kinase, greatly reduced levels of both GS-2 message and protein were observed . With wild type GSY2 placed under control of the ADH1 promoter, bcy1 cells did not accumulate glycogen despite increased GS-2 . Overexpression of truncated GS-2, however, resulted in definite though reduced glycogen accumulation; the glycogen synthesized was structurally distinct from wild type with properties characteristic of less branched polysaccharide . We conclude that the cAMP pathway controls both the expression and the phosphorylation state of GS-2 . Furthermore, other factor(s) necessary for glycogen biosynthesis, such as the branching enzyme GLC3, must also be under negative control by the cAMP pathway . The results demonstrate interactive controls of GS-2 by the cAMP-dependent and SNF1 protein kinases. Biochim Biophys Acta, 1994 Nov 11, 1201(2), 235 - 44 Mitochondrial gene expression in Saccharomyces cerevisiae . IV . Effects of yeast cytosol on mitochondrial protein synthesis, degradation, and respiration; McKee EE; It has been known for some time that the addition of a crude yeast cytosolic fraction to isolated mitochondria stimulates the rate of amino acid incorporation into protein in the isolated organelles . However, the mechanism and importance of this phenomenon relative to mitochondrial function has not been established . While it has been assumed that this effect is at the level of translation, the recognition that newly synthesized mitochondrial translation products are rapidly degraded in isolated yeast mitochondria raises the possibility that cytosol affects amino acid incorporation by inhibiting proteolysis . Using pulse-chase experiments we demonstrate that the rate constants of degradation of the nascent products are not affected by yeast cytosol . Further, not only is proteolysis not inhibited by cytosol, but the loss of label caused by proteolysis is actually increased . This increase is directly related to an increase in the size of the nascent product pool which increases simply as a consequence of increasing the rate of translation . By utilizing an approach in which the loss of label due to proteolysis is minimized, the true stimulatory activity of the cytosolic fraction on synthesis was determined (2.1-fold vs . 1.3-fold by the previous method) . Pulse-chase experiments in the presence of pactamycin, an initiation inhibitor, demonstrate that yeast cytosol causes an initial increase in the rate of translational initiation without increasing the rate of elongation . However, at later intervals the yeast cytosol acts primarily to maintain the rate of elongation which falls steadily in the controls . Finally, the presence of yeast cytosol dramatically increases the length of incubation time in which the mitochondrial preparation consumes oxygen and maintains coupled respiration, parameters that fall rapidly in the controls . Thus, a yeast cytosolic fraction may function to promote the stability of the mitochondrial preparation, which in turn may account for the increase in rates of translation, particularly with regard to maintaining rates of elongation. Appl Environ Microbiol, 1994 Nov, 60(11), 3926 - 30 Analysis of the raw starch-binding domain by mutation of a glucoamylase from Aspergillus awamori var . kawachi expressed in Saccharomyces cerevisiae; Goto M et al.; Carboxy-terminal deletions were introduced into the raw starch-binding domain (A-515 to R-615) encoded by the gene for glucoamylase I (GAI) from Aspergillus awamori var . kawachi . Genes coding for proteins designated GA596 (A-1 to E-596), GA570 (A-1 to A-570), and GA559 (A-1 to N-559) were constructed and resulted in truncated proteins . All of the mutant genes were expressed heterologously in Saccharomyces cerevisiae . GA596 adsorbed to raw starch and digested it . GA570 and GA559 did not adsorb to raw starch or to an alpha-cyclodextrin-Sepharose CL-4B gel under our experimental conditions . However, GA570 was able to digest raw starch, and the digestion of raw starch by GA570 was inhibited by beta-cyclodextrin . Residue Trp-562 of GAI, which was suggested previously to contribute to formation of an inclusion complex with raw starch, was replaced by Leu (GAW562L), Phe (GAW562F), and Gly (GAW562G) . GAW562L and GAW562F adsorbed to raw starch and an alpha-cyclodextrin gel, but GAW562G did not . Although GAW562L digested raw starch to the same extent as wild-type GAI (designated GAY), GAW562F and GAW562G exhibited less ability to digest raw starch . On the basis of our results, it appears that the sequence around Trp-562, PL(W-562)YVTVTLPA, is the minimal sequence necessary for digestion of raw starch and that hydrophobic residue Trp-562 contributes to formation of an inclusion complex . The sequence near Trp-589, which has abundant hydrogen bond-forming residues and the charged amino acid residues needed for stable adsorption to raw starch, probably assists in the formation of the inclusion complex. J Cell Biol, 1994 Nov, 127(3), 667 - 78 Clathrin-dependent localization of alpha 1,3 mannosyltransferase to the Golgi complex of Saccharomyces cerevisiae; Graham TR et al.; Posttranslational modification of yeast glycoproteins with alpha 1,3-linked mannose is initiated within a Golgi compartment analogous to the medial Golgi cisternae of higher eukaryotes . We have characterized the synthesis, posttranslational modification, and localization of the yeast alpha 1,3 mannosyltransferase (Mnn1p) using antibodies prepared against a segment of this protein expressed in bacteria . Mnn1p is initially synthesized as a 98.5-kD, type II integral membrane glycoprotein that is modified with both N- and O-linked oligosaccharides . It is subject to a slow, incremental increase in molecular mass that is dependent upon protein transport to the Golgi complex . Self-modification of Mnn1p with alpha 1,3 mannose epitopes, primarily on O-linked oligosaccharides, is at least partly responsible for the incremental increase in molecular mass . Mnn1p is a resident protein of the Golgi complex and colocalizes with guanosine diphosphatase to at least two physically distinct Golgi compartments by sucrose gradient fractionation, one of which may be a late Golgi compartment that also contains the Kex2 endopeptidase . Surprisingly, we found that a significant fraction of Mnn1p is mislocalized to the plasma membrane in a clathrin heavy chain temperature sensitive mutant while guanosine diphosphatase remains intracellular . A mutant Mnn1p that lacks the NH2-terminal cytoplasmic tail is properly localized to the Golgi complex, indicating that clathrin does not mediate Mnnlp Golgi retention by a direct interaction with the Mnn1p cytoplasmic tail . These results indicate that clathrin plays a broader role in the localization of Golgi proteins than anticipated. J Bacteriol, 1994 Nov, 176(22), 7126 - 8 SPE1 and SPE2: two essential genes in the biosynthesis of polyamines that modulate +1 ribosomal frameshifting in Saccharomyces cerevisiae; Balasundaram D et al.; We previously showed that a mutant of Saccharomyces cerevisiae, which cannot make spermidine as a result of a deletion in the SPE2 gene (spe2 delta), exhibits a marked elevation in +1 ribosomal frameshifting efficiency in response to the Ty1 frameshift sequence, CUU AGG C . In the present study, we found that spermidine deprivation alone does not result in increased +1 ribosomal frameshifting efficiency . The high level of +1 ribosomal frameshifting efficiency in spe2 delta cells is the result of the combined effects of both spermidine deprivation and the large increase in the level of intracellular putrescine resulting from the derepression of the gene for ornithine decarboxylase (SPE1) in spermidine-deficient strains. Genes Dev, 1994 Nov 1, 8(21), 2629 - 40 Efficient translation of poly(A)-deficient mRNAs in Saccharomyces cerevisiae; Proweller A et al.; The polyadenylate tail of eukaryotic mRNAs is thought to influence various metabolic phenomena including mRNA stability, translation initiation, and nucleo-cytoplasmic transport . We have analyzed the fate of mRNAs following inactivation of poly(A) polymerase in Saccharomyces cerevisiae containing a temperature-sensitive, lethal mutation (pap1-1) in the gene for poly(A) polymerase (PAP1) . Inactivation of poly(A) polymerase (Pap1) by shifting cells to the nonpermissive temperature resulted in the loss of at least 80% of measurable poly(A) within 60 min . Northern blot analysis revealed the disappearance of some mRNAs (CYH2 and HIS4) consistent with a role for poly(A) tails in mRNA stability . However, other mRNAs (TCM1, PAB1, ACT1, and HTB2) accumulate as poly(A)-deficient (A < approximately 25) transcripts as defined by an inability to bind oligo(dT)-cellulose . Sucrose density gradient analysis of polyribosomes revealed a twofold reduction in the amount of each size class of polyribosomes in shifted cells and a commensurate increase in free ribosomes . However, poly(A)-deficient mRNAs in shifted cells remain associated with the same size polyribosomes as poly(A)+ mRNAs in unshifted cells, indicating normal initiation of translation . RNase mapping of transcripts from pap1-1 cells revealed PAB1 mRNA to be poly(A)- whereas TCM1 exists as equal amounts of poly(A)- and poly(A)+ mRNA 60 min after shift . Interestingly, both of these classes of TCM1 mRNA appear in similar amounts in each polyribosome fraction indicating that ribosomes may not distinguish between them . These findings suggest that under conditions of excess translational capacity, poly(A)- and poly(A)+ mRNAs may initiate translation with comparable efficiencies. EMBO J, 1994 Nov 1, 13(21), 5203 - 11 A zinc finger protein, essential for chromosome segregation, constitutes a putative DNA binding subunit of the Saccharomyces cerevisiae kinetochore complex, Cbf3; Lechner J; A multisubunit protein complex, Cbf3, is a component of the Saccharomyces cerevisiae kinetochore . Cbf3 was recently shown to be essential for chromosome segregation in vivo and for movement of centromere DNA (CEN) along microtubules in vitro . Cbf3 contains three proteins, Cbf3a, Cbf3b and Cbf3c . Here the characterization of Cbf3b is described . Cbf3b contains an N-terminal Zn2Cys6 type zinc finger domain, a C-terminal acidic domain and a putative coiled coil dimerization domain . Cbf3b is essential for growth . Mutations within the zinc finger domain result in cells that exhibit a G2-M cell cycle delay and increased chromosome loss in each mitotic cell division . Therefore, Cbf3b has an essential function in chromosome segregation and the zinc finger domain executes part of this function presumably by providing the specific interaction between Cbf3 and CEN . Finally, data are provided to show that Cbf3c is encoded by CTF13, a gene that had been cloned recently by complementing a temperature sensitive mutant that exhibits chromosome loss as a result of a defective centromere. Mol Cell Biol, 1994 Nov, 14(11), 7455 - 65 Differential effects of Cdc68 on cell cycle-regulated promoters in Saccharomyces cerevisiae; Lycan D et al.; Swi4 and Swi6 form a complex which is required for Start-dependent activation of HO and for high-level expression of G1 cyclin genes CLN1 and CLN2 . To identify other regulators of this pathway, we screened for dominant, recessive, conditional, and allele-specific suppressors of swi4 mutants . We isolated 16 recessive suppressors that define three genes, SSF1, SSF5, and SSF9 (suppressor of swi four) . Mutations in all three genes bypass the requirement for both Swi4 and Swi6 for HO transcription and activate transcription from reporter genes lacking upstream activating sequences (UASs) . SSF5 is allelic with SIN4 (TSF3), a gene implicated in global repression of transcription and chromatin structure, and SSF9 is likely to be a new global repressor of transcription . SSF1 is allelic with CDC68 (SPT16) . cdc68 mutations have been shown to increase expression from defective promoters, while preventing transcription from other intact promoters, including CLN1 and CLN2 . We find that CDC68 is a required activator of both SWI4 and SWI6, suggesting that CDC68's role at the CLN promoters may be indirect . The target of CDC68 within the SWI4 promoter is complex in that known activating elements (MluI cell cycle boxes) in the SWI4 promoter are required for CDC68 dependence but only within the context of the full-length promoter . This result suggests that there may be both a chromatin structure and a UAS-specific component to Cdc68 function at SWI4 . We suggest that Cdc68 functions both in the assembly of repressive complexes that form on many intact promoters in vivo and in the relief of this repression during gene activation. Mol Cell Biol, 1994 Nov, 14(11), 7322 - 30 Cap-dependent and cap-independent translation by internal initiation of mRNAs in cell extracts prepared from Saccharomyces cerevisiae; Iizuka N et al.; Translation extracts were prepared from various strains of Saccharomyces cerevisiae . The translation of mRNA molecules in these extracts were cooperatively enhanced by the presence of 5'-terminal cap structures and 3'-terminal poly(A) sequences . These cooperative effects could not be observed in other translation systems such as those prepared from rabbit reticulocytes, wheat germ, and human HeLa cells . Because the yeast translation system mimicked the effects of the cap structure and poly(A) tail on translational efficiency seen in vivo, this system was used to study cap-dependent and cap-independent translation of viral and cellular mRNA molecules . Both the 5' noncoding regions of hepatitis C virus and those of coxsackievirus B1 conferred cap-independent translation to a reporter coding region during translation in the yeast extracts; thus, the yeast translational apparatus is capable of initiating cap-independent translation . Although the translation of most yeast mRNAs was cap dependent, the unusually long 5' noncoding regions of mRNAs encoding cellular transcription factors TFIID and HAP4 were shown to mediate cap-independent translation in these extracts . Furthermore, both TFIID and HAP4 5' noncoding regions mediated translation of a second cistron when placed into the intercistronic spacer region of a dicistronic mRNA, indicating that these leader sequences can initiate translation by an internal ribosome binding mechanism in this in vitro translation system . This finding raises the possibility that an internal translation initiation mechanism exists in yeast cells for regulated translation of endogenous mRNAs. Electrophoresis, 1994 Nov, 15(11), 1466 - 86 Protein identifications for a Saccharomyces cerevisiae protein database; Garrels JI et al.; The rapid progress in understanding the genes of the yeast Saccharomyces cerevisiae can be supplemented by two-dimensional (2-D) gel studies to understand global patterns of protein synthesis, protein modification, and protein degradation . The first step in building a protein database for yeast is to identify many of the spots on 2-D gels . We are using protein sequencing, overexpression of genes on high-copy number plasmids, and amino acid analysis to identify the proteins from 2-D gels of yeast . The amino acid analysis technique involves labeling yeast samples with different amino acids and using quantitative image analysis to determine the relative amino acid abundances . The observed amino acid abundances are then searched against the current database of 2600 known yeast protein sequences . At present about 90 proteins on our yeast maps have been identified, and the number is rising rapidly . With many known proteins on the map, it will soon be possible to use 2-D gel analysis to study regulatory pathways in normal and mutant yeast, with knowledge of many the protein products that respond to each genetic or environmental manipulation. Curr Genet, 1994 Nov-Dec, 26(5-6), 564 - 6 Molecular characterisation of GTP1, a Saccharomyces cerevisiae gene encoding a small GTP-binding protein; Wolter R et al.; DNA sequence analysis upstream of the yeast DNA repair gene SNM1 revealed gene GTP1 with an ORF of 573 bp on chromosome XIII . The putative amino-acid sequence of the encoded protein shows homology to proteins of the ARF-class of small GTP-binding proteins . Homology within GTP-binding motifs is highly conserved . Gene disruption showed that GTP1 is not an essential gene and that it has no influence on the expression of the DNA repair gene SNM1 with which it shares a 191-bp promoter region. Curr Genet, 1994 Nov-Dec, 26(5-6), 546 - 52 Sequence analysis of three deficient mutants of cytochrome oxidase subunit I of Saccharomyces cerevisiae and their revertants; Lemarre P et al.; Three respiratory-deficient mutants of cytochrome oxidase subunit I in the yeast mitochondrion have been sequenced . They are located in, or near, transmembrane segment VI, the catalytic core of the enzyme . Respiratory-competent revertants have been selected and studied . The mutant V244M was found to revert at the same site in valine (wild-type), isoleucine or threonine . The revertants of the mutant G251R were of three types: glycine (wild-type), serine and threonine at position 251 . A search for second-site mutations was carried out but none were found . Among 60 revertants tested, the mutant K265M was found to revert only to the wild-type allele. Yeast, 1994 Nov, 10(11), 1531 - 4 ATP1 and ATP2, the F1F0-ATPase alpha and beta subunit genes of Saccharomyces cerevisiae, are respectively located on chromosomes II and X; Takeda M et al.; Southern blot analysis showed that ATP1 and ATP2 map on chromosomes II and X, respectively . Physical mapping of ATP1 and ATP2 by chromosome fragmentation showed that ATP1 is at the left end of chromosome II and ATP2 is at the right end of chromosome X . Both are located close to telomere sequences of each chromosome; ATP1 and ATP2 being approximately 30 kb and 85 kb from the respective telomeres. Yeast, 1994 Nov, 10(11), 1503 - 9 Cloning and sequence of REV7, a gene whose function is required for DNA damage-induced mutagenesis in Saccharomyces cerevisiae; Torpey LE et al.; The function of the REV7 gene is required for DNA damage-induced mutagenesis in budding yeast, Saccharomyces cerevisiae, and is therefore thought to promote replication past sites of mutagen damage in the DNA template . We have cloned this gene by complementation of the rev7-2 mutant defect, and determined its sequence . REV7 encodes a predicted protein of M(r) 28,759 which is unlikely any other protein in the NCBI non-redundant protein sequence data base, and which is inessential for viability. Yeast, 1994 Nov, 10(11), 1481 - 8 Sequence and function analysis of a 9.74 kb fragment of Saccharomyces cerevisiae chromosome X including the BCK1 gene; Miosga T et al.; In the framework of the European BIOTECH project for sequencing the Saccharomyces cerevisiae genome, we have determined the nucleotide sequence of the cosmid clone 233 provided by F . Galibert (Rennes Cedex, France) . We present here 9743 base pairs of sequence derived from the left arm of chromosome X . This sequence reveals three new open reading frames and includes the published sequence (5' end and open reading frame) of the gene BCK1/SLK1/SSP31 also identified as ORFAA . Deletion mutants of two earlier unknown open reading frames J0840 and J0904 are viable and the open reading frame J0902 is essential for yeast growth. Yeast, 1994 Nov, 10(11), 1439 - 46 The in vivo activation of Saccharomyces cerevisiae plasma membrane H(+)-ATPase by ethanol depends on the expression of the PMA1 gene, but not of the PMA2 gene; Monteiro GA et al.; The expression of the PMA1 and PMA2 genes during Saccharomyces cerevisiae growth in medium with glucose plus increasing concentrations of ethanol was monitored by using PMA1-lacZ and PMA2-lacZ fusions and Northern blot hybridizations of total RNA probed with PMA1 gene . The presence of sub-lethal concentrations of ethanol enhanced the expression of PMA2 whereas it reduced the expression of PMA1 . The inhibition of PMA1 expression by ethanol corresponded to a decrease in the content of plasma membrane ATPase as quantified by immunoassays . Although an apparent correspondence could exist between the increase of plasma membrane ATPase activity and the level of PMA2 expression, the maximal level of PMA2 expression remained about 200 times lower than PMA1 . On the other hand, ethanol activated the plasma membrane H(+)-ATPase activity from a strain expressing only the PMA1 ATPase but did not activate that from a strain expressing only the PMA2 ATPase . These results provide evidence that in the presence of ethanol it is the PMA1 ATPase which is activated, probably by a post-translational mechanism and that the PMA2 ATPase is not involved. Yeast, 1994 Nov, 10(11), 1429 - 37 Generation of glycerophospholipid molecular species in the yeast Saccharomyces cerevisiae . Fatty acid pattern of phospholipid classes and selective acyl turnover at sn-1 and sn-2 positions; Wagner S et al.; Acyl chains linked to phospholipids of the yeast, Saccharomyces cerevisiae, are mainly C16:1 and C18:1 accompanied by minor amounts of C14:0, C16:0 and C18:0 . In view of this rather simple fatty acid composition, the question arose whether in yeast, as in higher eukaryotes, fatty acyl groups were characteristically distributed among the sn-1 and sn-2 positions of distinct phospholipid classes . Analysis of fatty acids linked to the sn-1 and sn-2 positions of the major phospholipids showed that indeed saturated fatty acyl groups predominated in the sn-1 positions . While the percentage of saturated fatty acids was low (10%) in phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) from cells grown on rich medium, it was higher in phosphatidylserine (PtdSer) (25%) and highest in phosphatidylinositol (PtdIns) (41%) . Oleate was mainly linked to position sn-2, while palmitoleate predominated in position sn-1 . Striking differences in the fatty acid distribution of phospholipids that are metabolically closely related (e.g . PtdSer and PtdEtn, PtdEtn and PtdCho, and PtdIns and PtdSer) suggest that pathways must exist for the generation of distinct phospholipid molecular species within the different phospholipid classes . The highly selective incorporation of exogenous {14C}palmitic acid (90%) and {3H}oleic acid (99%) into the sn-2 position of PtdCho, and the preferential incorporation of these fatty acids into the sn-2 position of PtdEtn (70 and 90%, respectively, for palmitic and oleic acid) are compatible with the postulate that phospholipase A2-mediated deacylation followed by reacylation of the lysophospholipids is involved in the generation of phospholipid species in yeast. Yeast, 1994 Nov, 10(11), 1403 - 13 Transcript map of two regions from chromosome XI of Saccharomyces cerevisiae for interpretation of systematic sequencing results; Fairhead C et al.; A detailed and systematic transcript map is a first and necessary step to characterize new genes revealed by systematic sequencing . Chromosome XI of Saccharomyces cerevisiae contains 331 open reading frames (ORFs) of which 44% are of unknown function (Dujon et al., 1994) . As a first study towards complete transcript analysis of chromosome XI, we have extracted RNA from three isogenic strains (a, alpha and 2n) grown in three standard laboratory media, and have analysed them using contiguous probes covering two regions of 17 and 19 kilobases, respectively . All 20 predicted ORFs in the sequences correspond to expressed genes, six of which have no predicted function . Four short ORFs which were suspected as not being real genes on the basis of their sequence are not expressed in our growth conditions . An additional transcript which does not correspond to a large ORF was found . Steady-state RNA level of most ORFs is 10 to 100 times than that of the actin gene, only three are transcribed in comparable amounts . Three ORFs show variable levels of transcripts in the different growth conditions, all patterns being different from one another . Extrapolation of these results to systematic transcript analysis of chromosome XI and other yeast chromosomes is presented. Mol Biol Cell, 1994 Nov, 5(11), 1185 - 98 Metabolic instability and constitutive endocytosis of STE6, the a-factor transporter of Saccharomyces cerevisiae; Berkower C et al.; STE6, a member of the ATP binding cassette (ABC) transporter superfamily, is a membrane protein required for the export of the a-factor mating pheromone in Saccharomyces cerevisiae . To initiate a study of the intracellular trafficking of STE6, we have examined its half-life and localization . We report here that STE6 is metabolically unstable in a wild-type strain, and that this instability is blocked in a pep4 mutant, suggesting that degradation of STE6 occurs in the vacuole and is dependent upon vacuolar proteases . In agreement with a model whereby STE6 is routed to the vacuole via endocytosis from the plasma membrane, we show that degradation of STE6 is substantially reduced at nonpermissive temperature in mutants defective in delivery of proteins to the plasma membrane (sec6) or in endocytosis (end3 and end4) . Whereas STE6 appears to undergo constitutive internalization from the plasma membrane, as do the pheromone receptors STE2 and STE3, we show that two other proteins, the plasma membrane ATPase (PMA1) and the general amino acid permease (GAP1), are significantly more stable than STE6, indicating that rapid turnover in the vacuole is not a fate common to all plasma membrane proteins in yeast . Investigation of STE6 partial molecules (half- and quarter-molecules) indicates that both halves of STE6 contain sufficient information to mediate internalization . Examination of STE6 localization by indirect immunofluorescence indicates that STE6 is found in a punctate, possibly vesicular, intracellular pattern, distinct from the rim-staining pattern characteristic of PMA1 . The punctate pattern is consistent with the view that most of the STE6 molecules present in a cell at any given moment could be en route either to or from the plasma membrane . In a pep4 mutant, STE6 is concentrated in the vacuole, providing further evidence that the vacuole is the site of STE6 degradation, while in an end4 mutant STE6 exhibits rim-staining, indicating that it can accumulate in the plasma membrane when internalization is blocked . Taken together, the results presented here suggest that STE6 first travels to the plasma membrane and subsequently undergoes endocytosis and degradation in the vacuole, with perhaps only a transient residence at the plasma membrane; an alternative model, in which STE6 circumvents the plasma membrane, is also discussed. Poult Sci, 1994 Nov, 73(11), 1766 - 70 The effects of supplementing diets with Saccharomyces cerevisiae var . boulardii on male poult performance and ileal morphology; Bradley GL et al.; This study determined the effects of three levels of supplemental yeast of Saccharomyces cerevisiae var . boulardii (SCB) on commercial male poult performance and ileum morphology . One hundred and sixty Nicholas poults were randomly assigned to 16 battery cages (10 poults per cage, 4 cages per diet) from 1 to 21 d of age (DOA) . Poults were fed diets (26% CP) consisting of corn-soybean (CS, control), CS + .01% SCB, CS + .02% SCB, and CS + .06% SCB . At 21 DOA, 30 poults fed the CS and CS + .02% SCB diets (from 1 to 21 DOA) were randomly selected within each diet, placed in 1 of 6 cages (5 poults per cage, 3 cages per diet), and fed their respective diet to 35 DOA . Body weights and feed consumptions (FC) were measured at 21 and 35 DOA and morphological comparisons of ileal tissues were conducted at 35 DOA . Increased BW (P < .004) at 7, 14, and 21 DOA were observed for poults fed diets containing SCB at .01, .02, and .06% of the diet . No dietary differences (P > .05) were observed in FC or feed:gain ratios from 1 to 21 DOA . Increased (P < .03) BW were maintained from 21 to 35 DOA for poults fed .02% SCB, whereas no dietary differences (P > .05) in FC or feed:gain were observed . Histological examination of ileal sections from poults (35 DOA) fed the CS and CS + .02% SCB diets revealed a decrease (P < .04) in the number of goblet cells per millimeter of villus height and a decreased (P < .02) crypt depth in poults receiving .02% SCB . No dietary differences (P > .05) were observed for either villus height or width. Genetics, 1994 Nov, 138(3), 633 - 47 Nonhomologous synapsis and reduced crossing over in a heterozygous paracentric inversion in Saccharomyces cerevisiae; Dresser ME et al.; Homologous chromosome synapsis ("homosynapsis") and crossing over are well-conserved aspects of meiotic chromosome behavior . The long-standing assumption that these two processes are causally related has been challenged recently by observations in Saccharomyces cerevisiae of significant levels of crossing over (1) between small sequences at nonhomologous locations and (2) in mutants where synapsis is abnormal or absent . In order to avoid problems of local sequence effects and of mutation pleiotropy, we have perturbed synapsis by making a set of isogenic strains that are heterozygous and homozygous for a large chromosomal paracentric inversion covering a well marked genetic interval and then measured recombination . We find that reciprocal recombination in the marked interval in heterozygotes is reduced variably across the interval, on average to approximately 55% of that in the homozygotes, and that positive interference still modulates crossing over . Cytologically, stable synapsis across the interval is apparently heterologous rather than homologous, consistent with the interpretation that stable homosynapsis is required to initiate or consummate a large fraction of the crossing over observed in wild-type strains . When crossing over does occur in heterozygotes, dicentric and acentric chromosomes are formed and can be visualized and quantitated on blots though not demonstrated in viable spores . We find that there is no loss of dicentric chromosomes during the two meiotic divisions and that the acentric chromosome is recovered at only 1/3 to 1/2 of the expected level. Genetics, 1994 Nov, 138(3), 587 - 95 Use of a chromosomal inverted repeat to demonstrate that the RAD51 and RAD52 genes of Saccharomyces cerevisiae have different roles in mitotic recombination; Rattray AJ et al.; An intrachromosomal recombination assay that monitors events between alleles of the ade2 gene oriented as inverted repeats was developed . Recombination to adenine prototrophy occurred at a rate of 9.3 x 10(-5)/cell/generation . Of the total recombinants, 50% occurred by gene conversion without crossing over, 35% by crossover and 15% by crossover associated with conversion . The rate of recombination was reduced 3,000-fold in a rad52 mutant, but the distribution of residual recombination events remained similar to that seen in the wild type strain . In rad51 mutants the rate of recombination was reduced only 4-fold . In this case, gene conversion events unassociated with a crossover were reduced 18-fold, whereas crossover events were reduced only 2.5-fold . A rad51 rad52 double mutant strain showed the same reduction in the rate of recombination as the rad52 mutant, but the distribution of events resembled that seen in rad51 . From these observations it is concluded that (i) RAD52 is required for high levels of both gene conversions and reciprocal crossovers, (ii) that RAD51 is not required for intrachromosomal crossovers, and (iii) that RAD51 and RAD52 have different functions, or that RAD52 has functions in addition to those of the Rad51/Rad52 protein complex. Genetics, 1994 Nov, 138(3), 577 - 86 Requirement for RGR1 and SIN4 in RME1-dependent repression in Saccharomyces cerevisiae; Covitz PA et al.; RME1 is a zinc-finger protein homolog that functions as a repressor of the meiotic activator IME1 . RME1 is unusual among yeast repressors in two respects: it acts over a considerable distance (2 kbp) and it can activate transcription from a binding site separated from its natural flanking region . To identify genes required for RME1 to exert repression, we have selected mutants with improved RME1-dependent activation . One rare mutant was defective in RME1-dependent repression of an artificial reporter gene as well as the native IME1 gene . The mutation permits sporulation of a/a diploids, which express RME1 from its natural promoter, and of a/alpha diploids constructed to express RME1 from the GAL1 promoter . The mutation also causes temperature-sensitive growth and a methionine or cysteine requirement . Analysis of a complementing genomic clone indicates that the mutation lies in a known essential gene, RGR1 . Prior studies have indicated a functional relationship between RGR1 and SIN4 (also called TSF3); we have found that a sin4 null mutation also causes a defect in RME1-dependent repression and a methionine or cysteine requirement . The rgr1 and sin4 mutations do not cause a reduction of RME1 polypeptide levels . The defect in RME1-dependent repression may result from effects of sin4 and, presumably, rgr1 on chromatin structure. Genetics, 1994 Nov, 138(3), 565 - 75 Suppressor analyses of temperature-sensitive cbp1 strains of Saccharomyces cerevisiae: the product of the nuclear gene SOC1 affects mitochondrial cytochrome b mRNA post-transcriptionally; Staples RR et al.; The induction of mitochondrial function is dependent upon both nuclearly encoded and mitochondrially encoded gene products . To understand nuclear-mitochondrial interactions, we must first understand gene-specific interactions . The accumulation of mitochondrial cytochrome b (COB) RNA is dependent upon Cbp1p, encoded by the nuclear gene CBP1 . Thus, respiration is dependent upon Cbp1p . In this study, suppressors of temperature-sensitive cbp1 (cbp1ts) strains were selected for restoration of respiratory capability at the restrictive temperature Ts+) . One nuclearly encoded suppressor, extragenic to CBP1, is recessive with respect to the wild-type suppressor allele and is unlinked to other known genetic loci whose gene products are necessary for expression of COB mRNA . The suppressor, called soc1 for Suppressor of cbp1, suppresses several other cbp1ts alleles but does not operate via a bypass mechanism . Molecular analyses indicate that soc1 allows the steady-state level of COB mRNA to increase at high temperature but has little or no effect on the levels of COB pre-mRNA . These data have led us to propose that the product of the nuclear gene SOC1 is required for normal turnover of COB mRNA. J Exp Biol, 1994 Nov, 196, 157 - 66 Ca2+ transport in Saccharomyces cerevisiae; Cunningham KW et al.; Cytosolic free Ca2+ is maintained at submicromolar levels in budding yeast by the activity of Ca2+ pumps and antiporters . We have recently identified the structural genes for two Ca2+ pumps, PMC1 {correction of PCM1} and PMR1, which are required for Ca2+ sequestration into the vacuole and secretory organelles, respectively . The function of either Ca2+ pump is sufficient for yeast viability, but deletion of both genes is lethal because of elevation of cytosolic {Ca2+} and activation of calcineurin, a Ca(2+)- and calmodulin-dependent protein phosphatase . Calcineurin activation decreases Ca2+ sequestration in the vacuole by a putative Ca2+ antiporter and may also increase Ca2+ pump activity . These regulatory processes can affect the ability of yeast strains to tolerate high extracellular {Ca2+} . We propose a model in which the cellular response to changes in the environmental levels of Ca2+ is mediated by calmodulin and calcineurin which, in turn, modulate the various types of Ca2+ transporters. Mol Gen Genet, 1994 Nov 1, 245(3), 323 - 33 Inactivation of SSM4, a new Saccharomyces cerevisiae gene, suppresses mRNA instability due to rna14 mutations; Mandart E et al.; Decay rates of mRNAs depend on many elements and among these, the role of the poly(A) tail is now well established . In the yeast Saccharomyces cerevisiae, thermosensitive mutations in two genes, RNA14 and RNA15, result in mRNAs having shorter poly(A) tails and reduced half-life . To identify other components interacting in the same process, we have used a genetic approach to isolate mutations that suppress the thermosensitivity of an rna14 mutant strain . Mutations in a single locus, named SSM4, not only suppress the cell growth phenotype but also the mRNA instability and extend the short mRNA poly(A) tails . The frequency of appearance and the recessive nature of these mutations suggested that the suppressor effect was probably due to a loss of function . We failed to clone the SSM4 gene directly by complementation, owing to its absence from gene banks; it later emerged that the gene is toxic to Escherichia coli, but we have nevertheless been able to clone the SSM4 sequence by Ty element transposition tagging . Disruption of the SSM4 gene does not affect cell viability and suppresses the rna14 mutant phenotypes . The protein encoded by the SSM4 gene has a calculated molecular mass of 151 kDa and does not contain any known motif or show homology with known proteins . The toxicity of the SSM4 gene in E . coli suggests that a direct biochemical activity is associated with the corresponding protein. Can J Microbiol, 1994 Nov, 40(11), 974 - 7 Production and partial characterization of an endopolygalacturonase from Saccharomyces cerevisiae; Blanco P et al.; Saccharomyces cerevisiae CECT1389 secreted an extracellular endopolygalacturonase (EC 3.2.1.15) when grown in shake flasks in medium containing galactose alone, or either galactose and polygalacturonic acid or galactose and galacturonic acid as the carbon sources . The synthesis of the enzyme was repressed by glucose and by high oxygen tensions . The enzyme was partially purified by gel exclusion chromatography over Sephacryl S-200, where it showed an apparent molecular mass of 39 kDa; the value determined by high-performance liquid chromatography (HPLC) was 65 kDa . The optimal temperature and pH for enzyme activity were 45 degrees C and 5.5, respectively . The Km and Vmax values for polygalacturonic acid were 4.7 mg.mL-1 and 6.4 nmol.mL-1.min-1 . The Ki for HgCl2 was 6.8 x 10(-5) M . The enzyme exhibited an endo-splitting mechanism as deduced from viscosimetry experiments as well as from an HPLC study of the end products. Appl Microbiol Biotechnol, 1994 Nov, 42(2-3), 353 - 7 Construction of squalene-accumulating Saccharomyces cerevisiae mutants by gene disruption through homologous recombination; Kamimura N et al.; Saccharomyces cerevisiae synthesizes ergosterol via squalene, but squalene is hardly detected in aerobically grown cells . To obtain a stable squalene-accumulating yeast strain, we attempted to disrupt a gene required in the conversion of squalene to ergosterol, by homologous recombination with a short piece of the gene fragment conjugated with an integration plasmid vector carrying the LEU2 gene . Two mutants that required ergosterol at least for fast growth were isolated . In an aerobic cultivation and with ergosterol supplementation, the two mutants accumulated squalene up to 5 mg/g dry cells . Southern hybridization analysis indicated that both mutants had acquired the vector DNA integrated in the same gene, or nearby genes, on chromosome 12. Mycoses, 1994 Nov-Dec, 37(11-12), 405 - 10 Effects of insulin and glucose tolerance factor (GTF) on growth of Saccharomyces cerevisiae; Berdicevsky I et al.; Addition of mammalian insulin to Saccharomyces cerevisiae enhanced the growth of the cells in several glucose concentrations . The enhancement of growth was dependent on insulin concentrations . Morphological changes were also observed depending on the presence of insulin: the cells were almost round, whereas elongated forms appeared under depletion of the hormone . The effect of insulin was very similar to the increase in yeast growth observed by the addition of GTF (glucose tolerance factor) to the medium . Our findings support the view of a common mechanism regulating metabolic and growth processes in lower and higher organisms. Yeast, 1994 Nov, 10(11), 1477 - 9 Sequence of MKT1, needed for propagation of M2 satellite dsRNA of the L-A virus of Saccharomyces cerevisiae; Vermut M et al.; MKT1 is required for maintenance of K2 above 30 degrees C in strains with the L-A-HN variant of the L-A double-stranded RNA virus of Saccharomyces cerevisiae . We report that MKT1 encodes a 92,979 Da protein with serine-rich regions and the retroviral protease signature, DTG, but with no substantial homology to proteins presently in the databases. Mutat Res, 1994 Nov, 315(3), 275 - 9 A single amino acid change in SNM1-encoded protein leads to thermoconditional deficiency for DNA cross-link repair in Saccharomyces cerevisiae; Niegemann E et al.; Molecular characterization of a thermoconditional mutant snm1-2ts shows that the coding sequence contains three mutations, two of which are silent . The third changes amino acid glycine to arginine at position 256 thereby altering a hydrophilic domain of the protein . While sensitivity to nitrogen mustard of the mutant at 36 degrees C is very similar to that of the non-leaky allele snm1-1, multi-copy vector-mediated overexpression of snm1-2ts leads to a significantly reduced sensitivity to nitrogen mustard. Mutat Res, 1994 Nov, 315(3), 261 - 73 Repair of 6-4 photoproducts and cyclobutane pyrimidine dimers in rad mutants of Saccharomyces cerevisiae; McCready S; Repair rates of both pyrimidine-pyrimidone (6-4) photoproducts and cyclobutane pyrimidine dimers have been measured in the UV-sensitive mutants of Saccharomyces cerevisiae: rad1 to rad12 and rad14 to rad24 . A dot blot immunoassay for UV photoproducts was used which measures lesions in the genome as a whole and which distinguishes 6-4 photoproducts from cyclobutane dimers . The principal findings are: (1) Wild-type yeast cells, like normal mammalian cells, repair 6-4 photoproducts more rapidly than cyclobutane dimers . (2) All mutants that are defective in repair are defective in repair of both lesions . (3) The most sensitive alleles of rad1, rad2, rad3, rad4 and rad10 show no repair of either lesion . (4) Leaky alleles of rad1, rad3 and rad14 show a very marked difference in repair rates of the two lesions, rather like the human XPA revertant cell line XP129 and the Chinese hamster mutants UV61 and V-H1 . (5) No mutant repairs cyclobutane dimers more rapidly than 6-4 photoproducts. J Biol Chem, 1994 Oct 28, 269(43), 27143 - 8 The glycosylation of phosphoglucomutase is modulated by carbon source and heat shock in Saccharomyces cerevisiae; Dey NB et al.; Phosphoglucomutase is the acceptor for UDP-glucose: glycoprotein glucose-1-phosphotransferase and contains Glc in a phosphodiester linkage to O-linked Man . In this study, we have characterized the glycosylation of phosphoglucomutase by Saccharomyces cerevisiae in response to heat shock and growth in media containing carbon sources other than Glc . Phosphoglucomutase synthesized under these conditions is underglucosylated relative to that synthesized during logarithmic growth in Glc . The underglucosylation results in increased UDP-glucose:glycoprotein glucose-1-phosphotransferase acceptor activity in in vitro assays and a newly appearing less negatively charged form of phosphoglucomutase resolvable by anion exchange chromatography . Utilizing a yeast strain in which phosphoglucomutase is overexpressed via a multicopy plasmid, metabolic labeling of the enzyme with {35S}Met and {3H}Man increased in response to heat shock, whereas {3H}Glc labeling decreased . The glucosylation state of phosphoglucomutase was also compared in cells grown in media containing various carbon sources and was found to be lowest in cells utilizing Gal as the sole carbon source compared with Glc or lactate . In mammalian cells, the glucosylation of phosphoglucomutase has been shown to be sensitive to changes in cytoplasmic Ca2+ and to correlate with a change in its membrane association . The change in phosphoglucomutase's oligosaccharide in Saccharomyces cerevisiae may be important to alterations in its distribution under conditions of nutrient deprivation or metabolic stress. Mol Gen Genet, 1994 Oct 28, 245(2), 224 - 36 Increase in incidence of chromosome instability and non-conservative recombination between repeats in Saccharomyces cerevisiae hpr1 delta strains; Santos-Rosa H et al.; Null hpr1 delta strains show a large increase (up to 2000-fold) over wild type in the frequency of occurrence of deletions between direct repeats on three different chromosomes . However, we show that hpr1 delta mutations have little or no effect on reciprocal exchange, gene conversion or unequal sister chromatid exchange, as determined using intrachromosomal, interchromosomal and plasmid-chromosome assay systems . A novel intrachromosomal recombination system has allowed us to determine that over 95% of deletions in hpr1 delta strains do not occur by reciprocal exchange . On the other hand, hpr1 delta strains show chromosome loss frequencies of up to 100 times the wild-type level . Our results suggest that yeast cells have a very efficient non-conservative recombination mechanism, dependent on RAD1 and RAD52, that causes deletions between direct DNA repeats, and this mechanism is strongly stimulated in hpr1 delta strains . The results indicate that the Hpr1 protein is required for stability of DNA repeats and chromosomes . We propose that in the absence of the Hpr1 protein the cell destabilizes the genome by allowing the initiation of events that lead to deletions of sequences between repeats, and to chromosome instability . We discuss the roles that proteins such as Hpr1 have in maintaining direct repeats and in preventing non-conservative recombination and consider the importance of these functions for chromosome stability. Proc Natl Acad Sci U S A, 1994 Oct 25, 91(22), 10541 - 5 Saccharomyces cerevisiae peroxisomal thiolase is imported as a dimer; Glover JR et al.; The active conformation of native peroxisomal 3-ketoacyl-CoA thiolases (EC 2.3.1.16) is homodimeric . We have previously shown that a truncated Saccharomyces cerevisiae thiolase lacking its first 16 N-terminal amino acids fails to be translocated into peroxisomes but assembles into an enzymatically active form in the cytoplasm of a strain with a disrupted nuclear thiolase gene . We now report that when truncated thiolase is cosynthesized with full-length thiolase, approximately 50% of truncated thiolase cofractionates with the full-length thiolase to fractions enriched for peroxisomes and is translocated into peroxisomes as shown by its protection from the action of external proteases . We constructed an immunologically distinct cytosolic variant of thiolase by adding an influenza hemagglutinin epitope tag to the N terminus of the truncated thiolase . In a strain simultaneously expressing the full-length, truncated, and epitope-tagged truncated thiolases, we demonstrated that normally untargeted thiolase subunits are efficiently translocated into peroxisomes by dimerization with full-length thiolase subunits . Even though truncated and epitope-tagged truncated thiolase subunits are translocated into peroxisomes in this strain, only the full-length thiolase subunit can be coimmunoprecipitated with the epitope-tagged truncated thiolase subunit from the peroxisomal matrix . This observation suggests that interactions between thiolase subunits are not disrupted during translocation. Proc Natl Acad Sci U S A, 1994 Oct 25, 91(22), 10531 - 5 Dominant lethal mutations in the plasma membrane H(+)-ATPase gene of Saccharomyces cerevisiae; Harris SL et al.; The plasma membrane H(+)-ATPase of Saccharomyces cerevisiae is an essential protein that is required to establish cellular membrane potential and maintain a normal internal pH . An Asp-378 to Asn substitution at the residue phosphorylated during catalysis is dominant lethal when the pma1-D378N mutation is expressed along with a wild-type plasma membrane H(+)-ATPase (PMA1) gene . Several mutations in the first two putative transmembrane domains are also dominant lethal . However, these dominant lethal mutants often appear to be innocuous, because they are frequently lost by gene conversion to the wild-type sequence during the process of introducing the mutant sequence and subsequently removing the wild-type gene . Loss of the mutation by gene conversion does not occur while introducing recessive lethal mutations . Cells carrying the wild-type PMA1 gene on the chromosome and a dominant lethal mutation under the control of a GAL1 promoter on a centromere-containing plasmid exhibit a galactose-dependent lethality . Indirect immunofluorescence staining using anti-Pma1 antibodies shows that induction of dominant lethal PMA1 mutations leads to the accumulation of a number of intensely staining cytoplasmic structures that are not coincident with the nucleus and its immediately surrounding endoplasmic reticulum . These structures also accumulate the endoplasmic reticulum protein Kar2 . Expression of the dominant lethal protein also prevents transport of the wild-type ATPase to the plasma membrane. J Biol Chem, 1994 Oct 21, 269(42), 26144 - 51 A new member of a family of ATPases is essential for assembly of mitochondrial respiratory chain and ATP synthetase complexes in Saccharomyces cerevisiae; Tzagoloff A et al.; Respiration-defective pet mutants of Saccharomyces cerevisiae, assigned to complementation group G25, are grossly deficient in mitochondrial respiratory and ATPase complexes . This phenotype is usually found in strains impaired in mitochondrial protein synthesis . The G25 mutants, however, synthesize all of the proteins encoded by mitochondrial DNA . The mutants are also able to import and process cytoplasmically derived subunits of these enzymes . These results are most compatible with the idea that the gene defined by G25 mutants (RCA1) codes for a protein essential for the assembly of functional respiratory and ATPase complexes . The RCA1 gene has been cloned by complementation of an rca1 mutant with a yeast genomic library . The sequence of the encoded product shows Rca1 protein to be a new member of a recently described family of ATPases . The Rca1 protein is a mitochondrial membrane protein and is the third known member of this family implicated to function in the biogenesis of mitochondria . The primary structure of Rca1 protein indicates several distinct domains in addition to the common purine nucleotide binding region shared by all members of this protein family . One, located in the amino-terminal half, contains two hydrophobic stretches of sufficient length to span a membrane lipid bilayer. J Biol Chem, 1994 Oct 21, 269(42), 26092 - 9 The FET4 gene encodes the low affinity Fe(II) transport protein of Saccharomyces cerevisiae; Dix DR et al.; Previous studies on Fe(II) uptake in Saccharomyces cerevisiae suggested the presence of two uptake systems with different affinities for this substrate . We demonstrate that the FET3 gene is required for high affinity uptake but not for the low affinity system . This requirement has enabled a characterization of the low affinity system . Low affinity uptake is time-, temperature-, and concentration-dependent and prefers Fe(II) over Fe(III) as substrate . We have isolated a new gene, FET4, that is required for low affinity uptake, and our results suggest that FET4 encodes an Fe(II) transporter protein . FET4's predicted amino acid sequence contains six potential transmembrane domains . Overexpressing FET4 increased low affinity uptake, whereas disrupting this gene eliminated that activity . In contrast, overexpressing FET4 decreased high affinity activity, while disrupting FET4 increased that activity . Therefore, the high affinity system may be regulated to compensate for alterations in low affinity activity . These analyses, and the analysis of the iron-dependent regulation of the plasma membrane Fe(III) reductase, demonstrate that the low affinity system is a biologically relevant mechanism of iron uptake in yeast . Furthermore, our results indicate that the high and low affinity systems are separate uptake pathways. J Biol Chem, 1994 Oct 21, 269(42), 25974 - 7 VMA7 encodes a novel 14-kDa subunit of the Saccharomyces cerevisiae vacuolar H(+)-ATPase complex; Graham LA et al.; The Saccharomyces cerevisiae vacuolar proton-translocating ATPase (V-ATPase) is composed of at least 10 subunits belonging to either the peripheral V1 or integral membrane V0 subcomplex . We have characterized a novel 14-kDa V-ATPase subunit (Vma7p), encoded by the VMA7 gene, which exhibits features common to both V1 and V0 subunit proteins . Vma7p is a hydrophilic protein of 118 amino acids with a predicted molecular mass of 13,452 Da . Vacuolar membranes isolated from a vma7 delta null mutant contained no V-ATPase activity . Western analysis of vma7 delta cells revealed greatly reduced levels of the remaining V0 complex V-ATPase subunits, but normal levels of the V1 subunits . However, the V1 subunits failed to associate with the vacuolar membrane . Unlike the integral membrane subunits of the V0 complex, Vma7p was easily stripped from vacuolar membranes . Density gradient fractionation revealed that Vma7p associated only with the fully assembled V-ATPase and did not associate with a separate lower density V0 subcomplex fraction . The unique properties of the Vma7p may reflect a critical role in stabilizing the V0 complex and bridging the V1 and V0 complexes to form a functional V-ATPase complex. Mol Gen Genet, 1994 Oct 17, 245(1), 69 - 77 Mutational spectrum induced in Saccharomyces cerevisiae by the carcinogen N-2-acetylaminofluorene; Roy A et al.; The spectrum of mutations induced by the carcinogen N-2-acetylaminofluorene (AAF) was analysed in Saccharomyces cerevisiae using a forward mutation assay, namely the inactivation of the URA3 gene . The URA3 gene, carried on a yeast/bacterial shuttle vector, was randomly modified in vitro using N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) as a model reactive metabolite of the carcinogen AAF . The binding spectrum of AAF to the URA3 gene was determined and found to be essentially random, as all guanine residues reacted about equally well with N-AcO-AAF . Independent Ura- mutants were selected in vivo after transformation of the modified plasmid into a ura3 delta yeast strain . Plasmid survival decreased as a function of AAF modification, leading to one lethal hit (37% relative survival) for an average of approximately 50 AAF adducts per plasmid molecule . At this level of modification the mutation frequency was equal to approximately 70 x 10(-4), i.e . approximately 50-fold above the background mutation frequency . UV irradiation of the yeast cells did not further stimulate the mutagenic response, indicating the lack of an SOS-like mutagenic response in yeast . Sequence analysis of the URA3 mutants revealed approximately 48% frameshifts, approximately 44% base substitutions and approximately 8% complex events . While most base substitutions (74%) were found to be targeted at G residues where AAF is known to form covalent C8 adducts, frameshift mutations were observed at GC base pairs in only approximately 24% of cases.(ABSTRACT TRUNCATED AT 250 WORDS) Biochem J, 1994 Oct 15, 303 ( Pt 2), 461 - 5 Comparison of the effects of Ca2+, adenine nucleotides and pH on the kinetic properties of mitochondrial NAD(+)-isocitrate dehydrogenase and oxoglutarate dehydrogenase from the yeast Saccharomyces cerevisiae and rat heart; Nichols BJ et al.; The regulatory properties of NAD(+)-isocitrate dehydrogenase and oxoglutarate dehydrogenase in extracts of yeast and rat heart mitochondria were studied under identical conditions . Yeast NAD(+)-isocitrate dehydrogenase exhibits a low K0.5 for isocitrate and is activated by AMP and ADP, but is insensitive to ATP and Ca2+ . In contrast, the rat heart NAD(+)-isocitrate dehydrogenase was insensitive to AMP, but was activated by ADP and by Ca2+ in the presence of ADP or ATP . Both yeast and rat heart oxoglutarate dehydrogenase were stimulated by ADP, but only the heart enzyme was activated by Ca2+ . All the enzymes studied were activated by decreases in pH, but to differing extents . The effects of Ca2+, adenine nucleotides and pH were through K0.5 for isocitrate or 2-oxoglutarate . These observations are discussed with reference to the deduced amino acid sequences of the constituent subunits of the enzymes, where they are available. Eur J Biochem, 1994 Oct 15, 225(2), 677 - 85 Expression, assembly and secretion of a fully active plant ferredoxin-NADP+ reductase by Saccharomyces cerevisiae; Ottado J et al.; The flavoprotein ferredoxin-NADP+ reductase catalyzes the final step of the photosynthetic electron transport i.e., the reduction of NADP+ by ferredoxin . Expression and secretion of this enzyme was examined in Saccharomyces cerevisiae using a cDNA cloned from a pea library {Newman, B . J . & Gray, J . C . (1988) Plant Mol . Biol . 10, 511-520} . Two pea library cDNA sequences were employed, one corresponding to the mature enzyme and the other containing, in addition, the sequence of the transit peptide that directs ferredoxin-NADP+ reductase to the chloroplast . These sequences were introduced into a yeast shuttle vector in frame with the mating factor alpha 1 secretion-signal coding region under the control of its natural mating factor alpha 1 promoter . Saccharomyces cerevisiae cells transformed with the recombinant plasmids were able to synthesize and secrete fully active pea ferredoxin-NADP+ reductase . In both cases, a 35-kDa polypeptide was the major product . N-terminal sequencing of the secreted proteins indicates processing at position -1 with respect to the N-terminus of the pea mature enzyme . Yeast cells transformed with plasmid encoding the ferredoxin-NADP+ reductase precursor secrete four-times more ferredoxin-NADP+ reductase to the medium than cells transformed with the plasmid encoding the mature form of the enzyme . Ferredoxin-NADP+ reductases purified from culture medium showed structural and enzymatic properties that were identical, within the experimental error, to those of native plant ferredoxin-NADP+ reductase . The overall results indicate that pea ferredoxin-NADP+ reductase can be properly folded and its prosthetic group assembled in the yeast endoplasmic reticulum, and that its natural transit peptide favors its secretion. Eur J Biochem, 1994 Oct 15, 225(2), 641 - 9 Characterization, quantification and subcellular localization of inositol-containing sphingolipids of the yeast, Saccharomyces cerevisiae; Hechtberger P et al.; In yeast, as in higher eukaryotic cells, sphingolipids are essential membrane components . The yeast, Saccharomyces cerevisiae, contains three classes of sphingolipids, inositolphosphorylceramide (InsPCer), mannosylinositolphosphorylceramide (ManInsPCer) and mannosyldiinositolphosphorylceramide (ManPIns2PCer) . As a prerequisite to localize these sphingolipids in subcellular membranes, authentic standards of the respective lipids were isolaed and characterized using biochemical methods and electrospray ionization mass spectrometry . The complete set of yeast subcellular membranes was isolated at high purity, and sphingolipids were extracted . InsPCer, ManInsPCer, and ManPIns2PCer were separated by thin-layer chromatography, stained and densitometrically scanned along with the respective standards . These methods enable a complete overview of the subcellular distribution of yeast sphingolipids to be obtained, as far as is known, for the first time . InsPCer was highly enriched in Golgi and vacuolar membranes, whereas the largest amounts of ManInsPCer and ManPIns2PCer were found in the plasma membrane . The presence of inositol-containing sphingolipids in organelles of the protein-secretory pathway strongly supports the notion that protein secretion and intracellular trafficking of sphingolipids are linked processes. J Biol Chem, 1994 Oct 14, 269(41), 25660 - 7 The Saccharomyces cerevisiae copper transport protein (Ctr1p) . Biochemical characterization, regulation by copper, and physiologic role in copper uptake; Dancis A et al.; The CTR1 gene of Saccharomyces cerevisiae encodes a protein required for high affinity copper uptake . The protein is expressed on the plasma membrane, is heavily glycosylated with O-linkages, and exists as an oligomer in vivo . The transcript abundance is strongly regulated by copper availability, being induced by copper deprivation and repressed by copper excess . Regulation occurs at very low, nontoxic levels of available copper and is independent of ACE1, the trans-inducer of yeast metallothionein . Expression of Ctr1p is limiting for copper uptake, since overexpression from a 2 mu high copy number plasmid increases copper uptake . Mutations in CTR1 result in altered cellular responses to extracellular copper, demonstrating a physiologic role for CTR1 in the delivery of copper to the cytosol . A copper-dependent reporter gene construct, CUP1-lacZ, is not expressed in CTR1 mutants to the same level as in wild-type strains, and Cu,Zn superoxide dismutase activity is deficient in these mutants . The growth arrest that occurs in CTR1 mutants grown aerobically in copper-deficient media is attributable to the defect in Cu,Zn superoxide dismutase activity. J Biol Chem, 1994 Oct 14, 269(41), 25295 - 302 CRS5 encodes a metallothionein-like protein in Saccharomyces cerevisiae; Culotta VC et al.; Protection from copper toxicity in the bakers' yeast Saccharomyces cerevisiae involves the action of a copper binding metallothionein encoded by the CUP1 locus . To identify additional factors contributing to copper ion homeostasis and detoxification, we screened a genomic library for genes that confer high levels of copper resistance to yeast strains lacking CUP1 . This screen led to the identification of the CRS5 (copper-resistant suppressor) gene . By sequence analyses, CRS5 encodes a small molecular weight cysteine-rich protein with an amino acid sequence bearing all the features of a eukaryotic metallothionein . The CRS5 polypeptide exhibits a striking similarity to a number of mammalian and invertebrate metallothioneins, yet shares surprisingly little homology with CUP1 . In yeast, CRS5 is expressed as a 0.5-kilobase mRNA that is regulated both by copper ions and by oxidative stress, and expression is dependent upon ACE1, a copper and DNA binding transcription factor also known to regulate CUP1 . Deletion of the chromosomal CRS5 locus was found to increase cellular sensitivity to copper, but not cadmium, toxicity . These studies support an important role for the CRS5 metallothionein-like protein in copper homeostasis and detoxification. Proc Natl Acad Sci U S A, 1994 Oct 11, 91(21), 10158 - 62 Suppressors of nmtl-181, a conditional lethal allele of the Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene, reveal proteins involved in regulating protein N-myristoylation; Johnson DR et al.; Several essential Saccharomyces cerevisiae proteins require myristate to be covalently bound to their amino-terminal glycine for biological activity . Protein N-myristoylation is catalyzed by myristoyl-CoA:protein N-myristoyl-transferase, Nmt1p . nmt1-181 encodes a mutant enzyme with a Gly451-->Asp substitution . nmt181p has a reduced affinity for myristoyl-CoA and produces global defects in protein N-myristoylation at > or = 30 degrees C . nmt1-181 results in growth arrest at various stages of the cell cycle within 1 hr after cells are shifted to > or = 30 degrees C and lethality within 8 hr . The growth-arrest phenotype and loss of viability do not require components of the mating pathway and are associated with lysis sensitivity that may be related to undermyristoylation of two protein phosphatases, Ppz1p and Ppz2p . Growth can be rescued at 30 degrees C by adding myristate or sorbitol to the medium or by removing inosine . Cells can be rescued at 37 degrees C by overexpressing nmt1-181p or Nmt1p or by adding myristate to the medium . Selection of high-copy suppressors of the myristate auxotrophy and lethality observed at 37 degrees C yielded only NMT1, whereas six unlinked suppressors of the myristoylation defect (SMD1-6) were obtained when the screen was conducted at 30 degrees C . The protein products of three SMD loci were identified: (i) cdc39-delta 1.7p, which transactivates NMT1; (ii) Fas1p, the beta subunit of the fatty acid synthetase complex, activates FAS2's promoter and increases myristoylation of Gpa1p; and (iii) Pho5p, the major secreted acid phosphatase produced by this yeast . PHO5 is normally induced when yeast are grown in phosphate-depleted medium . Removal of inorganic phosphate from the medium also rescues nmt1-181 cells at 30 degrees C . PHO5's mechanism of suppression of nmt1-181 appears to involve, at least in part, activation of FAS2 transcription and a resulting effect on FAS1 expression . There is an inverse relationship between cellular N-myristoyltransferase and secreted acid phosphatase activities . These observations provide a potential mechanism for coupling phosphate metabolism with the regulation of myristoyl-CoA synthesis and protein N-myristoylation. Proc Natl Acad Sci U S A, 1994 Oct 11, 91(21), 10124 - 8 Colocalization of vertebrate lamin B and lamin B receptor (LBR) in nuclear envelopes and in LBR-induced membrane stacks of the yeast Saccharomyces cerevisiae; Smith S et al.; We have expressed human lamin B and the chicken lamin B receptor (LBR), either separately or together, in yeast and have monitored the subcellular location of the expressed proteins by immunofluorescence microscopy, immunoelectron microscopy, and cell fractionation . At the light microscopic level, the heterologous lamin B localized to the yeast nuclear rim and at electron microscopic resolution was found subjacent to the yeast inner nuclear membrane . These data indicate that vertebrate lamin B was correctly targeted in yeast . Expression of the heterologous LBR, either alone or together with the heterologous lamin B, resulted in the formation of membrane stacks primarily adjacent to the nuclear envelope, but also projecting from the nuclear envelope into the cytoplasm or under the plasma membrane . Double immunoelectron microscopy showed colocalization of the heterologous lamin B and LBR in the yeast nuclear envelope and in the LBR-induced membrane stacks . Cell fractionation showed the presence of the heterologous lamin B and LBR in a subnuclear fraction enriched in nuclear envelopes . The heterologous lamin B was extracted at 8 M urea, but not at 4 M urea, thus behaving as a peripheral membrane protein and indistinguishable from assembled lamins . The heterologous LBR was not extracted by 8 M urea, indicating that it was integrated into the membrane . The observed colocalization and cofractionation are consistent with previously reported in vitro binding data and suggest that heterologous lamin B and LBR interact with each other when coexpressed in yeast. Gene, 1994 Oct 11, 148(1), 149 - 53 Seripauperins of Saccharomyces cerevisiae: a new multigene family encoding serine-poor relatives of serine-rich proteins; Viswanathan M et al.; A gene, PAU1, has been cloned from Saccharomyces cerevisiae and sequenced . It is located in a telomeric region, probably on chromosome IV, and contains an open reading frame encoding a protein of 120 amino acids (aa) (approx . 13 kDa) . The deduced sequence is nearly identical to two other genes found in GenBank (named PAU2 and PAU3 by us), which are located close to the ends of chromosomes V and III, respectively . Blotting of separated chromosomes with a PAU1 probe at high stringency revealed that at least six chromosomes in addition to III, IV and V possessed related sequences, suggesting a large gene family . Probing of an ordered array of phage lambda clones containing yeast genomic DNA inserts ('Olson filters') revealed ten additional hybridizing sequences, located close to the ends of the left and/or right arms of chromosomes I, II, VII, VIII, X, XII, XIV and XV . Transcription of these sequences could not be demonstrated, however, under a wide variety of growth and culture conditions . The deduced PAU1, PAU2 and PAU3 aa sequences are all highly homologous with the SRP1 aa sequences, which contains eight serine-rich tandem repeats of 12 aa each, at its C terminus . This homology is limited, however, to the N-terminal half of SRP1, and does not include the repeats . In fact, PAU1 is quite serine-poor (5.8%), leading to the suggested name of seripauperins for this family of genes. Gene, 1994 Oct 11, 148(1), 143 - 7 Differential transcription of the two Saccharomyces cerevisiae genes encoding elongation factor 2; Veldman S et al.; The single polypeptide chain of elongation factor 2 (EF-2) is encoded by two Saccharomyces cerevisiae genes (EFT1 and EFT2) with unique flanking sequences . One gene is necessary and either is sufficient for cell viability . In the present work, we have analyzed the transcription of EFT1 and EFT2 . Although both genes