S. cerevisiae

Curr Genet, 1995 Jan, 27(2), 131 - 4
Molecular genetic properties of the yeast Torulaspora pretoriensis: characterization of chromosomal DNA and genetic transformation by Saccharomyces cerevisiae-based plasmids; Oda Y et al.; Chromosomal DNA banding patterns were obtained for three strains of Torulaspora pretoriensis by contour-clamped homogeneous-electric-field gel electrophoresis . Chromosomes were resolved into six or seven bands in the range of 800 to 2000 kb, and a polymorphism of these lengths was observed . By Southern-blot analysis, the three strains were shown to lack the DNA sequences homologous to the URA3, LEU2, TRP1, and HO genes of Saccharomyces cerevisiae . A uracil auxotrophic mutant derived from T . pretoriensis was transformed with three plasmids (YEp24, YRpHI, and YCp50) carrying the URA3 gene of S . cerevisiae by the lithium acetate method.

Curr Genet, 1995 Jan, 27(2), 123 - 30
Molecular cloning of Rab-related genes in the yeast Yarrowia lipolytica . Analysis of RYL1, an essential gene encoding a SEC4 homologue; Pertuiset B et al.; Small GTP-binding proteins of the Rab family are involved in the vesicular traffic inside eukaryotic cells . A gene library from the yeast Yarrowia lipolytica was screened with an oligonucleotide deduced from a highly conserved sequence in the Rab family . Four different genes were isolated . One of them, RYL1, was shown to be essential for cell viability . RYL1p displayed a high similarity with and tight phylogenetic relationships to SEC4p . When placed under the control of the GAL10 promoter, RYL1 was able to specifically relieve the thermosensitivity of a sec4-8 mutant of Saccharomyces cerevisiae . Therefore, it is proposed that RYL1 is a functional homologue of the S . cerevisiae SEC4 gene and is involved in the fusion of secretory vesicles with the plasma membrane in the general protein secretion pathway.

DNA Res, 1995, 2(1), 15 - 9
Cloning and sequencing of sulfite reductase alpha subunit gene from Saccharomyces cerevisiae; Hosseini-Mazinani SM et al.; A DNA fragment of 2.1 kb was specifically amplified by PCR with primers based on the amino acid sequences obtained from the N-terminal region and the cyanogen bromide-derived peptide of the sulfite reductase alpha subunit in Saccharomyces cerevisiae . With this fragment as a probe, the gene coding for the sulfite reductase alpha subunit was isolated from a genomic library of S . cerevisiae . Sequencing analysis revealed that the gene contains a 3105-bp open reading frame, which is large enough to code for a protein of 1035 amino acid residues . The transcript of the sulfite reductase alpha subunit gene was detected by Northern analysis after methionine deprivation, but the amount of the transcript did not directly correlate with the enzyme activity . The DNA fragment containing the sulfite reductase alpha subunit gene rescued the met10 phenotype by complementation.

Micron, 1995, 26(2), 175 - 90
Ultracytochemical evidence of Golgi functions in microvesicles at all phases of cell cycle in Saccharomyces cerevisiae; Vorisek J; The topical question of Golgi compartment identity in the ascomycetous yeast Saccharomyces cerevisiae is illustrated by a multiple ultracytochemical approach . For this eucaryotic single-cell organism the established scheme of secretory transport via a cascade of cisternae housing different functions of Golgi apparatus has been deduced principally of genetic and molecular analyses ex situ and confirms the mammalian secretion scheme . Nevertheless, ultracytochemical in situ localizations of enzyme activities engaged in secretion represented evidence for localization of important steps of secretory glycoprotein maturation in two morphologically distinct populations of transport microvesicles formed from endoplasmic reticulum and Golgi cisternae . Both types of microvesicles function in exocytosis or transport into lysosomal vacuoles and have identical charge . However, their presence differs in interphase and in budding cells of S . cerevisiae . Smooth, larger membrane bound microvesicles are conspicuous at the onset of budding and at construction of scars, while the coated, smaller microvesicles of globular ultrastructure are present constitutively, throughout the cell cycle . Because the established model of the yeast secretory path considers only the part of the budding phase preceding the onset of mitosis, an alternative scheme for the cellular mechanism of glycoprotein secretion in S . cerevisiae that distinguishes interphase and budding yeast, has been established . The lumen of microvesicles contains proteases catalysing maturation of the mating pheromone alpha-factor (yscIV, yscF), vacuolar protease yscY, alkaline phosphohydrolase, polyphosphorylated components of the bud scar and glycoproteins . The in situ approach also reveals a minimum level of alpha-factor precursor processing proteolytic activity at the budding phase of cells, a transient presence of polyphosphorylated compounds in the bud scars and their transport by microvesicles . Ultracytochemical reactions suggest that the nuclear envelope lumen houses certain functions attributed to endoplasmic reticulum and that some steps of outer-chain glycosylation may occur in microvesicles . Microvesicles which contain proteases and polyphosphorylated intermediates also appear in juvenile vacuoles (lysosomes) . Ultracytochemical findings show the Golgi compartment of S . cerevisiae to consist not only of discrete endoplasmic cisternae, immunodetected by others as sites of outer chain alpha-1,6-mannosylation and of the Golgi membrane marker proteins Sec7p and Ypt1p, but also of microvesicles moving either to the cell plasma membrane or to vacuoles . The previously hypothesized hierarchy of segregated yeast Golgi cisternae was not revealed by ultracytochemical findings.(ABSTRACT TRUNCATED AT 400 WORDS)

Microbios, 1995, 81(328), 187 - 97
Introduction of flocculation into industrial yeast, Saccharomyces cerevisiae saké, by protoplast fusion; Lima N et al.; Protoplast fusion was applied to obtain intraspecific fusion in yeast strains in order to overcome restrictions imposed by the natural mating system . Acriflavine was used to construct petite mutants from non-flocculent industrial Saccharomyces cerevisiae sake strain . UV radiation was used to construct ura- mutants from a respiratory competent and highly flocculent S . cerevisiae NCYC869 strain . Fusion products were selected by complementation on minimal medium . The frequency of appearance of prototrophic hybrids was five per 10(5) protoplasts . The stable hybrids showed not only the S . cerevisiae sake characteristics but also flocculation ability, thereby confirming the success of the fusion.

Mol Microbiol, 1995 Jan, 15(2), 225 - 34
A recognition component of the ubiquitin system is required for peptide transport in Saccharomyces cerevisiae; Alagramam K et al.; Peptide transport in Saccharomyces cerevisiae is controlled by three genes: PTR1, PTR2, and PTR3, PTR1 was cloned and sequenced and found to be identical to UBR1, a gene previously described as encoding the recognition component of the N-end-rule pathway of the ubiquitin-dependent proteolytic system . Independently derived ubr1 mutants, like ptr1 mutants, were unable to transport small peptides into cells . Concomitantly, ptr1 mutants, like ubr1 mutants, were unable to degrade an engineered substrate of the N-end-rule pathway . Further, ptr1 mutants did not express PTR2, a gene encoding the integral membrane component required for peptide transport in S . cerevisiae . These results establish a physiological role for a protein previously known to be required for the degradation of N-end-rule substrates . Our findings show that peptide transport and the ubiquitin pathway--two dynamic phenomena universal to eukaryotic cells--share a common component, namely UBR1/PTR1.

Electrophoresis, 1995 Jan, 16(1), 149 - 56
Gene linkage of two-dimensional polyacrylamide gel electrophoresis resolved proteins from isogene families in Saccharomyces cerevisiae by microsequencing of in-gel trypsin generated peptides; Norbeck J et al.; The total cellular extract of proteins from the yeast Saccharomyces cerevisiae was resolved by preparative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) where 500 micrograms was loaded per gel, and a number of proteins in isogene families were selected for microsequencing analysis . Peptides were generated from resolved proteins by in-gel trypsin digestion, and fractionated by reversed phase-high performance liquid chromatography (RP-HPLC) . Subsequent sequencing of peptides yielded internal amino acid sequences which unambiguously identified the selected proteins spots as gene products from PCD1, ENO1, ENO2, ADH1, HXK2, TDH2, TDH3, SSB1 and SSB2 . The chromatograms obtained from RP-HPLC of related proteins were utilized to distinguish discriminating peptide fractions . With this approach two out of four amino acid differences between Ssb1p and Ssb2p were allocated . We estimate that by pooling five preparative gels, at least one hundred protein spots in the 2-D pattern of S . cerevisiae will be obtained in sequencable amounts.

Genetics, 1995 Jan, 139(1), 57 - 66
Isolation and genetic analysis of extragenic suppressors of the hyper-deletion phenotype of the Saccharomyces cerevisiae hpr1 delta mutation; Santos-Rosa H et al.; The HPR1 gene of Saccharomyces cerevisiae is involved in maintaining low levels of deletions between DNA repeats . To understand how deletions initiate in the absence of the Hpr1 protein and the mechanisms of recombination leading to deletions in S . cerevisiae, we have isolated mutations as suppressors of the hyper-deletion phenotype of the hpr1 delta mutation . The mutations defined five different genes called HRS for hyper-recombination suppression . They suppress the hyper-deletion phenotype of hpr1 delta strains for three direct repeat systems tested . The mutations eliminated the hyper-deletion phenotype of hpr1 delta strains either completely (hrs1-1 and hrs2-1) or significantly (hrs3-1, hrs4-1 and hrs5-1) . None of the mutations has a clear effect on the levels of spontaneous and double-strand break-induced deletions . Among other characteristics we have found are the following: (1) one mutation, hrs1-1, reduces the frequency of deletions in rad52-1 strains 20-fold, suggesting that the HRS1 gene is involved in the formation of RAD52-independent deletions; (2) the hrs2-1 hpr1 delta mutant is sensitive to methyl-methane-sulfonate and the single mutants hpr1 delta and hrs2-1 are resistant, which suggests that the HPR1 and HRS2 proteins may have redundant DNA repair functions; (3) the hrs4-1 mutation confers a hyper-mutator phenotype and (4) the phenotype of lack of activation of gene expression observed in hpr1 delta strains is only partially suppressed by the hrs2-1 mutation, which suggests that the possible functions of the Hpr1 protein in gene expression and recombination repair can be separated . We discuss the possible relationship between the HPR1 and the HRS genes and their involvement in initiation of the events responsible for deletion formation.

Methods Enzymol, 1995, 250, 51 - 68
Mutagenesis and biochemical analysis of recombinant yeast prenyltransferases; Caplin BE et al.; The use of the S . cerevisiae protein prenyltransferases as a model system for general prenyltransferase study is justified by the similarity of mechanism, substrate specificity, and evolutionarily conserved substrates with the mammalian prenyltransferases . Genetic identification of potential structural genes involved in prenyltransferase activity can be easily confirmed with biochemical assays using recombinant enzyme reconstitution . Yeast FTase and GGTase I produced in E . coli are indistinguishable from the native proteins and can be studied without interference from contaminating cellular protein prenyltransferases . Structure-function analysis of the yeast prenyltransferase subunits is also simplified by the rapidity with which mutant enzymes can be analyzed in E . coli and their biological activity characterized in yeast defective for the particular subunit gene.

Adv Biophys, 1995, 31, 77 - 91
Multi-site-specific endonucleases and the initiation of homologous genetic recombination in yeast; Shibata T et al.; The notion that homologous recombination is a regulated biological process is not a familiar one . In yeasts, homologous recombination and most site-specific ones are initiated by site-specific double-stranded breaks that are introduced within cis-acting elements for the recombination . On the other hand, yeasts have a group of site-specific endonucleases (multi-site-specific endonucleases) that have a number of cleavage sites on each DNA . One of them, Endo.SceI of S . cerevisiae, was shown to introduce double-stranded breaks at a number of well-defined sites on the mitochondrial DNA in vivo . An Endo.SceI-induced double-stranded break was demonstrated to induce homologous recombination in mitochondria . Like the case of homologous recombination of nuclear chromosomes, the double-stranded break induces gene conversion of both genetic markers flanking and in the proximity of the cleavage site, and the cleaved DNA acts as a recipient of genetic information from the uncleaved partner DNA . The 70 kDa-heat-shock protein (HSP70)-subunit of Endo.SceI and a general role of the HSP70 in the regulation of protein-folding suggest the regulation of nucleolytic activity of Endo.SceI.

Adv Biophys, 1995, 31, 67 - 76
Functions of the yeast meiotic recombination genes, MRE11 and MRE2; Ogawa H et al.; Mutants defective in meiotic recombination were isolated using a disomic haploid strain of S . cerevisiae, and were classified into 11 genes . Two, MRE2 and MRE11, are new genes and nine are previously identified genes . The mre2 and mre11 deletion mutants are proficient in mitotic recombination, but are defective in meiotic recombination and in formation of viable spores . The spore inviability, however, is alleviated by an additional mutation, spo13, which bypasses meiosis I . In addition, neither meiosis specific DSBs at recombination hot-spots nor formation of synaptonemal complex occur in either mutant . Therefore, these two genes are involved in the formation of DSBs in meiotic recombination . While a temperature sensitive mre11-1 mutant is able to form DSBs at a permissive temperature, the formed DSBs are unable to resect at non permissive temperature . Therefore, the MRE11 gene is also involved in some step of the repair process after the DSB formation . Analysis of properties of the mre11 disruption mutant as well as the xrs2 mutant showed a similarity to those of the rad50 disruptant . We found that the mre11 disruption mutation is epistatic to rad50S mutation, as the xrs2 deletion mutation is epistatic to rad50S with regard to DSBs . Therefore, these three genes form an epistatic group . Interaction of the Mre11 protein with the Rad50 and the Xrs2 protein as well as alone was shown in vivo using the two-hybrid system . The MRE2 gene encodes a protein containing two sets of RRM . Deficiency of recombination in a mre2 mutant that has an amino acid substitution in the N-terminal RRM can be suppressed by the MER2 gene on the multicopy plasmid . Further analysis showed that the Mre2 protein is involved in meiosis-specific splicing of the MER2 transcripts in cooperation with the Mer1 protein . In conclusion, MRE genes are involved in the initiation of meiotic recombination through the formation of DSBs at recombination hot-spots in S . cerevisiae.

DNA Res, 1995, 2(2), 89 - 93
Structural analysis of a recA-like gene in the genome of Arabidopsis thaliana; Sato S et al.; A recA-like gene was identified in the genome of Arabidopsis thaliana by means of PCR using primers designed on the basis of previously reported amino acid sequences of eukaryotic RecA-like proteins . The structure of the gene, termed ArLIM15, was investigated by comparing the primary structure of the genomic DNA with that of the corresponding cDNA . The open reading frame, which was split into 15 exons, was established to have the capacity for encoding a 37.3-kDa polypeptide . The amino acid sequence of the putative product of ArLIM15 showed a high degree of similarity to that of LIM15 in the monocotyledonous plant Lilium, including a 93% identity, and to those of other recA-like genes in yeasts and vertebrates with identities of 69-71% . Phylogenetic analysis indicated ArLIM15 to be much closer to meiosis-specific LIM15 and DMC1 in Saccharomyces cerevisiae than to RAD51 in S . cerevisiae and its homologues on an evolutionary scale.

Antonie Van Leeuwenhoek, 1995, 67(4), 351 - 5
A new genetically isolated population of the Saccharomyces sensu stricto complex from Brazil; Naumov GI et al.; Genetic and karyotypic studies of some Saccharomyces sensu stricto yeasts from Brazil revealed a genetically isolated population which apparently represents a new sibling species of S . cerevisiae.

Mol Biol Rep, 1995, 21(1), 3 - 10
Proteasomes of the yeast S . cerevisiae: genes, structure and functions; Hilt W et al.; Proteasomes are large multicatalytic protease complexes which fulfil central functions in major intracellular proteolytic pathways of the eukaryotic cell . 20S proteasomes are 700 kDa cylindrically shaped particles, found in the cytoplasm and the nucleus of all eukaryotes . They are composed of a pool of 14 different subunits (MW 22-25 kDa) arranged in a stack of 4 rings with 7-fold symmetry . In the yeast Saccharomyces cerevisiae a complete set of 14 genes coding for 20S proteasome subunits have been cloned and sequenced . 26S proteasomes are even larger proteinase complexes (about 1700 kDa) which degrade ubiquitinylated proteins in an ATP-dependent fashion in vitro . The 26S proteasome is build up from the 20S proteasome as core particle and two additional 19S complexes at both ends of the 20S cylinder . Recently existence of a 26S proteasome in yeast has been demonstrated . Several 26S proteasome specific genes have been cloned and sequenced . They share similarity with a novel defined family of ATPases . 20S and 26S proteasomes are essential for functioning of the eukaryotic cell . Chromosomal deletion of 20S and 26S proteasomal genes in the yeast S . cerevisiae caused lethality of the cell . The in vivo functions of proteasomes in major proteolytic pathways have been demonstrated by the use of 20S and 26S proteasomal mutants . Proteasomes are needed for stress dependent and ubiquitin mediated proteolysis . They are involved in the degradation of short-lived and regulatory proteins . Proteasomes are important for cell differentiation and adaptation to environmental changes . Proteasomes have also been shown to function in the control of the cell cycle.

Gene Expr, 1995, 5(1), 35 - 47
Recessive mutations in the second largest subunit of TFIIIC suggest a new step in RNA polymerase III transcription; Sethy I et al.; An analysis of mutant S . cerevisiae strains selected for their ability to increase transcription by RNA polymerase (pol) III has identified 14 isolates in which this phenotype is recessive . Genetic linkage and complementation studies suggest that all 14 isolates contain recessive alleles of PCF1 . PCF1 encodes the 131-kDa subunit of transcription factor IIIC (TFIIIC131) and was identified previously by dominant mutations that also increased transcription by pol III . The recessive mutation, pcf1-3, results in a conservative substitution (R728-->K) towards the carboxyl-terminus of the protein . This position is distinct from the site of the dominant mutation PCF1-1 (H190-->Y), which maps to a tetratricopeptide repeat (TPR) . Site-directed mutagenesis at amino acid 728 generated one allele, pcf1-4, with a stronger phenotype than pcf1-3 . Extracts from pcf1-3 and pcf1-4 strains increase pol III transcription two- to threefold and ninefold, respectively, over wild-type under conditions that permit either single or multiple rounds of initiation . The entire effect of these mutations in vitro can be accounted for by an increase in the amount of transcriptionally active TFIIIB . In contrast, PCF1-1 primarily affects the rate of preinitiation complex assembly . The genetic, molecular, and biochemical data suggest that amino acid 728 in TFIIIC131 constitutes part of a structural domain in this protein that affects TFIIIB activity by influencing a previously undefined step in transcription . This step is suggested to occur after the recruitment of TFIIIB to DNA.

J Basic Microbiol, 1995, 35(4), 207 - 15
{Effect of changes in the lipid composition on the plasma membrane of Saccharomyces cerevisiae through mutation of the phase transition and mixing behavior of the lipid fraction}; Dorfler HD et al.; Mutation of Saccharomyces cerevisiae leads to an alteration of the size and surface structure of the mutant cell . These phenomena are correlated with change in the lipid composition and hence membrane fluidity of the plasma membranes . Such alterations are in the fatty acyl constituents of phospholipids and glycolipids and include changes in the saturation or length of fatty acyl chains . Simultaneously, the "melting point" of phospholipid fractions, i.e . the temperature of the ordered-disordered phase transition and mixing behavior, changed . Consequently calorimetry on isolated phospholipid fractions extracted from plasma membranes is a useful analytical method to demonstrate directly such phase transitions and change in the mixing behavior of the cellular lipid fractions in connection with mutation . A correlation between the alteration of the lipid composition in the plasma membrane of strains of S . cerevisiae S 288 and its mutant VY 1160 is discussed.

Biochem Biophys Res Commun, 1994 Dec 30, 205(3), 1998 - 2007
The C-terminus of the B cell activator Oct-2 functions as an activation domain in yeast; Mead J et al.; Oct-1 and Oct-2 are human transcriptional activators that bind to the same DNA element but activate distinct sets of genes . We expressed these factors in S . cerevisiae and observed greater than 5-fold stimulation of a lacZ reporter gene only with Oct-2 . Transfer of the Oct-2 C-terminal domain onto either Oct-1 (Oct1.2) or a nonactivating DNA-binding domain from GAL4 created activators capable of greater than 15 and 10-fold stimulation of activity, respectively . Thus, the C-terminus of Oct-2 is sufficient to confer activation potential to nonactive DNA-binding fragments in yeast.

Nucleic Acids Res, 1994 Dec 25, 22(25), 5717 - 22
Expression of the E.coli ada gene in S.cerevisiae provides cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine in rad6 but not in rad52 mutants; Brozmanova J et al.; The Escherichia coli ada gene protein coding region under the control of the yeast alcohol dehydrogenase promoter in the extrachromosomally replicating yeast expression vectors pADHO6C and pVT103LO6C was introduced into the wild-type yeast strains, YNN-27 and FF-18733, and the repair deficient mutants LN-1 (rad1-1), VV-5 (rad6-1), C5-6 (rad52-1) and FF-18742 (rad52::URA3) . This resulted in the expression of 3950, 1900, 1870, 1620, 1320 and 1420 fmol ada-encoded ATase/mg protein respectively: transformation with the parent vectors resulted in ATase activities of 3-17 fmol/mg protein . The wild-types, rad1-1 and rad6-1 yeast expressing the bacterial ATase showed increased resistance to the toxic and mutagenic effects of N-methyl-N'-nitro-N- nitrosoguanidine (MNNG) . Expression of ATase in the rad52-1 and rad52::URA3 mutants neither complemented their sensitivity, nor reduced the mutagenic effects of this agent . These results suggest that whilst a portion of the toxic and mutagenic lesions induced by MNNG can be repaired in yeast by the E.coli Ada protein in a RAD1- and RAD6-independent manner, the RAD52 gene product may be essential for the complete functioning of the Ada ATase . This is the first suggestion of a possible cofactor requirement for ATase.

Proc Natl Acad Sci U S A, 1994 Dec 20, 91(26), 12554 - 8
Spontaneous and restriction enzyme-induced chromosomal recombination in mammalian cells; Godwin AR et al.; We have derived Chinese hamster ovary (CHO) cell hybrids containing herpes simplex virus thymidine kinase (tk) heteroalleles for the study of spontaneous and restriction enzyme-induced interchromosomal recombination . These lines allowed us to make a direct comparison between spontaneous intrachromosomal and interchromosomal recombination using the same tk heteroalleles at the same genomic insertion site . We find that the frequency of interchromosomal recombination is less by a factor of at least 5000 than that of intrachromosomal recombination . Our results with mammalian cells differ markedly from results with Saccharomyces cerevisiae, with which similar studies typically give only a 10-to 30-fold difference . Next, to inquire into the fate of double-strand breaks at either of the two different Xho I linker insertion mutations, we electroporated PaeR7I enzyme, an isoschizomer of Xho I, into these hybrids . A priori, these breaks can be repaired either by recombination from the homology or by end-joining . Despite a predicted bias against recovering end-joining products in our system, all cells characterized by enzyme-induced resistance to hypoxanthine/aminopterin/thymidine were, in fact, due to nonhomologous recombination or end-joining . These results are in agreement with other studies that used extrachromosomal sequences to examine the relative efficiencies of end-joining and homologous recombination in mammalian cells, but are in sharp contrast to results of analogous studies in S . cerevisiae, wherein only products of homologous events are detected.

FEBS Lett, 1994 Dec 19, 356(2-3), 249 - 54
Cdc25 is not the signal receiver for glucose induced cAMP response in S . cerevisiae; Goldberg D et al.; The Ras/cAMP pathway in the yeast S . cerevisiae couples the cell cycle of this unicellular organism to the availability of nutrients . Glucose derepressed S . cerevisiae cells respond to glucose addition by an intracellular rise in cAMP . In the prevailing model, yeast Ras plays a similar role to that of heterotrimeric G-proteins coupled to cell surface receptors . A crucial element of this model is that the exchanger, Cdc25 is activated by glucose . Such activation would result in a glucose-dependent rise in GTP-bound Ras concentration . We here show, in contrast to this view, that Cdc25 cannot be the receiver of the glucose signal . We suggest that the Ras-GTP/cyclase complex is the molecular element directly receiving the signal while Cdc25-dependent exchange constitutes a prerequisite for complex formation.

Cell, 1994 Dec 16, 79(6), 1069 - 80
Mutation of a meiosis-specific MutS homolog decreases crossing over but not mismatch correction; Ross-Macdonald P et al.; MSH4 is a novel meiosis-specific gene required for wild-type levels of spore viability in S . cerevisiae . The predicted product of the MSH4 gene is homologous to the MutS family of proteins; however, msh4-null mutants have no apparent defect in mismatch repair . msh4 mutant strains display wild-type levels of gene conversion and postmeiotic segregation, but they show a reduction in crossing over and a resultant increase in nondisjunction of homologous chromosomes at meiosis I . Immunofluorescence experiments demonstrate that the Msh4 protein is localized to discrete sites on pachytene chromosomes . We propose that Msh4 interacts with a recombination intermediate to influence its resolution.

Gene, 1994 Dec 15, 150(2), 401 - 2
The Saccharomyces cerevisiae homologue of ribosomal protein S26; Wu M et al.; The nucleotide sequence of RPS26, the gene encoding a homologue of ribosomal protein small subunit S26 in Saccharomyces cerevisiae, was determined . The deduced amino-acid sequence showed significant identity with its counterparts from Neurospora crassa, human, rat and Arabidopsis thaliana . Disruption of RPS26 resulted in the formation of micro-colonies, suggesting that it is important for the normal cell growth of S . cerevisiae.

Nucleic Acids Res, 1994 Dec 11, 22(24), 5223 - 8
Splicing factor SF3a60 is the mammalian homologue of PRP9 of S.cerevisiae: the conserved zinc finger-like motif is functionally exchangeable in vivo; Kramer A et al.; A cDNA encoding the 60 kDa subunit of mammalian splicing factor SF3a has been isolated . The deduced protein sequence reveals a 30% identity to the PRP9 splicing protein of the yeast S.cerevisiae . The highest homology is present in a zinc finger-like region in the C-terminal domain of both proteins . The PRP9 zinc finger-like motif has been replaced by the equivalent region of mammalian SF3a60 . The chimeric protein rescues the temperature-sensitive phenotype of the prp9-1 mutant strain demonstrating that not only the structure but also the function of this domain has been conserved during evolution.

Cell, 1994 Dec 2, 79(5), 829 - 39
Incomplete arrest in the outer membrane sorts NADH-cytochrome b5 reductase to two different submitochondrial compartments; Hahne K et al.; The S . cerevisiae gene MCR1 encodes two mitochondrial isoforms of NADH-cytochrome b5 reductase . The primary translation product has an amino-terminal matrix-targeting signal, followed by a stretch of 21 uncharged amino acids . This precursor protein is inserted into the outer membrane, but only about one-third of the molecules become firmly anchored to the outer face of that membrane . The remaining molecules pass through the outer membrane into the inner membrane, are cleaved by inner membrane protease 1, and are released into the intermembrane space . Incomplete translocation arrest in the outer membrane is a novel mechanism by which the product of a single gene is sorted into different compartments of the same organelle.

J Biol Chem, 1994 Dec 2, 269(48), 30510 - 6
Isolation and characterization of the THI6 gene encoding a bifunctional thiamin-phosphate pyrophosphorylase/hydroxyethylthiazole kinase from Saccharomyces cerevisiae; Nosaka K et al.; Thiamin-phosphate pyrophosphorylase (TMP-PPase; EC 2.5.1.3) involved in de novo synthesis of thiamin in Saccharomyces cerevisiae is a bifunctional enzyme with 4-methyl-5-beta-hydroxyethylthiazole kinase (Th-kinase; EC 2.7.1.50) activity, which is an octamer of identical 60-kDa subunits (Kawasaki, Y . (1993) J . Bacteriol . 175, 5153-5158) . Previous study demonstrated that the activities of both TMP-PPase and Th-kinase are reduced by the mutation of a single nuclear gene, designated THI6 . We have cloned the THI6 gene from a yeast genomic library by functional complementation of the thi6 mutant and determined by DNA blot analysis that THI6 is located on chromosome XVI . The nucleotide sequence of the THI6 gene contained an open reading frame of 1,620 base pairs encoding a 540-amino acid polypeptide with a calculated molecular weight of 58,058, which is similar to the determined molecular mass of the purified bifunctional enzyme . Gene disruption demonstrated that the thi6 null strain is auxotrophic for thiamin, indicating that the THI6 protein is essential for thiamin synthesis in yeast . A recently isolated thi6 mutant, thi6-3, bearing a replacement of Glu370 by Lys370, showed a decrease in only Th-kinase activity, proving that the THI6 gene of S . cerevisiae encodes a structural gene of the thiamin biosynthetic bifunctional enzyme . Furthermore, complementation analysis of the thi6 null strain with the modified THI6 DNAs by a 12-nucleotide linker insertion suggested that a region from amino acids 138 to 187 and that from amino acids 370 to 453 are involved in functional domains of TMP-PPase and Th-kinase, respectively, whereas the COOH-terminal region is necessary for both enzyme activities . Strains conferring no Th-kinase but slight TMP-PPase activity could grow in medium without thiamin, suggesting that 4-methyl-5-beta-hydroxyethylthiazole is not involved in the pathway of de novo synthesis of thiamin via 4-methyl-5-beta-hydroxyethylthiazole monophosphate . Northern blot analysis demonstrated that THI6 gene expression is regulated at the mRNA level by intracellular thiamin pyrophosphate, a coenzyme form of thiamin, and that it requires the positive regulatory factors encoded by the THI2 and THI3 genes.

Eur J Biochem, 1994 Dec 1, 226(2), 697 - 705
Heterologous expression of the human D2S dopamine receptor in protease-deficient Saccharomyces cerevisiae strains; Sander P et al.; The cDNA for the human D2S dopamine receptor has been functionally expressed in the unicellular yeast Saccharomyces cerevisiae . The original D2S gene and an elongated D2S gene with an N-terminal fusion to the first 24 amino acids of the STE2 gene from S . cerevisiae were introduced into the episomal yeast expression vector YEp51 under the control of the GAL10 promoter . Expression studies performed in a wild-type strain and in two protease-deficient strains of S . cerevisiae revealed that the receptor was functionally expressed with respect to its ligand-binding properties . The KD values for the binding of the dopamine antagonist {3H}spiperone were calculated to be 1.6 nM for the D2S receptor alone and 1.9 nM for the STE2-D2S chimaera . Both membrane proteins could be further characterized by ligand-displacement studies using certain dopamine agonists and antagonists . D2S dopamine-receptor-specific polyclonal antibodies were used to monitor the heterologous expression of the receptor . Western-blot analysis of membranes prepared from transformed yeast cells producing either the receptor protein alone or the receptor fusion protein revealed apparent molecular masses of 40 kDa (D2S receptor alone) and 42 kDa (STE2/D2S receptor fusion protein) . It could be shown that, in comparison to the expression in a wild-type S . cerevisiae strain, the amount of receptor degradation was drastically reduced in the protease-deficient strains . The localizations of the heterologously produced dopamine receptor and of the chimaera in the recombinant yeast were studied by immunogold electron microscopy and were found to be restricted mainly to the vacuole of the cells.

Mol Cell Biol, 1994 Dec, 14(12), 8155 - 65
Heat shock transcription factor activates yeast metallothionein gene expression in response to heat and glucose starvation via distinct signalling pathways; Tamai KT et al.; Metallothioneins constitute a class of low-molecular-weight, cysteine-rich metal-binding stress proteins which are biosynthetically regulated at the level of gene transcription in response to metals, hormones, cytokines, and other physiological and environmental stresses . In this report, we demonstrate that the Saccharomyces cerevisiae metallothionein gene, designated CUP1, is transcriptionally activated in response to heat shock and glucose starvation through the action of heat shock transcription factor (HSF) and a heat shock element located within the CUP1 promoter upstream regulatory region . CUP1 gene activation in response to both stresses occurs rapidly; however, heat shock activates CUP1 gene expression transiently, whereas glucose starvation activates CUP1 gene expression in a sustained manner for at least 2.5 h . Although a carboxyl-terminal HSF transcriptional activation domain is critical for the activation of CUP1 transcription in response to both heat shock stress and glucose starvation, this region is dispensable for transient heat shock activation of at least two genes encoding members of the S . cerevisiae hsp70 family . Furthermore, inactivation of the chromosomal SNF1 gene, encoding a serine-threonine protein kinase, or the SNF4 gene, encoding a SNF1 cofactor, abolishes CUP1 transcriptional activation in response to glucose starvation without altering heat shock-induced transcription . These studies demonstrate that the S . cerevisiae HSF responds to multiple, distinct stimuli to activate yeast metallothionein gene transcription and that these stimuli elicit responses through nonidentical, genetically separable signalling pathways.

Infect Immun, 1994 Dec, 62(12), 5447 - 55
Saccharomyces cerevisiae virulence phenotype as determined with CD-1 mice is associated with the ability to grow at 42 degrees C and form pseudohyphae; McCusker JH et al.; Saccharomyces cerevisiae isolates have been shown previously to exhibit a high degree of variation in their ability to proliferate and persist in CD-1 mice (K.V . Clemons, J.H . McCusker, R . W . Davis, and D.A . Stevens, J . Infect . Dis . 169:859-867, 1994) . Isolate origin was not a firm predictor of virulence phenotype, since the virulence phenotypes of clinical and nonclinical isolates ranged from virulent to avirulent and from intermediate to avirulent, respectively . Therefore, it was important to determine if there was any association between putative virulence traits and virulence that might help explain the variation in virulence phenotypes . S . cerevisiae isolates spanning a range of virulence phenotypes in experimental infections were examined for putative virulence traits: the ability to grow at supraoptimal temperatures (42, 39, and 37 degrees C), gelatin liquefaction, casein utilization, and pseudohyphal formation . Gelatin liquefaction appeared to be unrelated to pseudohyphal formation on casein or to virulence . Significant differences in the ability to grow at 39 and 42 degrees C were observed when the virulent and intermediate classes were compared with the avirulent class . Less extreme but still significant differences in pseudohyphal formation were observed when the virulent and intermediate classes were compared with the avirulent class . Therefore, two virulence traits, similar to those identified in other pathogenic fungi, the ability to grow at elevated temperatures and pseudohyphal formation, have been identified in S . cerevisiae.

J Lipid Res, 1994 Dec, 35(12), 2254 - 62
Regulation of phosphatidic acid biosynthetic enzymes in Saccharomyces cerevisiae; Minskoff SA et al.; Phosphatidic acid is the biosynthetic precursor of all glycerolipids . To understand how phosphatidic acid biosynthesis is controlled in Saccharomyces cerevisiae, we studied the regulation of three enzyme activities involved in the synthesis of this glycerolipid precursor, i.e., glycerophosphate acyltransferase (GPAT), dihydroxyacetone phosphate acyltransferase (DHAPAT), and acyl DHAP reductase . GPAT activity was increased 3-fold, while DHAPAT activity was increased up to 9-fold in wild type cells grown in a nonfermentable carbon source compared to that of glucose-grown cells . The ratio of GPAT/DHAPAT activity was 12 in glucose-grown cells but only 4 in cells grown in glycerol/ethanol . In the previously characterized tpa1 mutant, (T . S . Tillman and R . M . Bell . 1986 . J . Biol . Chem . 261: 9144-9149), GPAT was decreased 2-fold and DHAPAT 27-fold compared to activities in the wild type . Acyl DHAP reductase activity in both wild type and tpa1 cells grown on a nonfermentable carbon source was increased approximately 2-fold over that of glucose-grown cells . All three enzymatic activities increased as wild type cells grown on glucose entered the stationary phase of growth . Therefore, GPAT, DHAPAT, and acyl DHAP reductase activities appear to be regulated by the respiratory state of the cell . None of the activities was affected to a great extent by inositol, which is a key regulator of many enzymes involved in the synthesis of PtdOH-derived phospholipids in S . cerevisiae, nor by deletion of the mitochondrial genome.(ABSTRACT TRUNCATED AT 250 WORDS)

Microbiology, 1994 Dec, 140 ( Pt 12), 3277 - 83
Analysis of Saccharomyces cerevisiae proteins induced by peroxide and superoxide stress; Jamieson DJ et al.; Exponentially growing Saccharomyces cerevisiae cells are more sensitive to oxidants such as hydrogen peroxide and superoxides than stationary phase cells . Using disruption mutations in the genes encoding the two S . cerevisiae superoxide dismutases, we show that the principal mechanism of toxicity of redox-cycling compounds, such as menadione and plumbagin, is via the production of superoxide anions . Using two-dimensional polyacrylamide gel electrophoresis we have compared the pattern of protein expression in cells labelled with L-{35S}methionine and stressed with either H2O2 or menadione . Three groups of proteins were evident: those whose levels are elevated by both H2O2 and menadione, and those specifically induced by either H2O2 or menadione . Experiments with promoter fusions demonstrated that one of the heat inducible forms of HSP70 (SSA1) was inducible with H2O2 . Furthermore, induction of the yeast H2O2-responsive TRX2 promoter by menadione required the metabolism of menadione.

Biokhimiia, 1994 Dec, 59(12), 1882 - 91
{Characteristics of polyphosphatase activity of Saccharomyces cerevisiae cytosol}; Andreeva NA et al.; The cytosol fraction purified from cellular organelles was obtained from S . cerevisiae yeast cells . This cytosolic fraction contained a polyphosphatase activity comprising nearly 65% of such in the protoplast homogenate . The pH optimum of this activity was 6.5-7.5 . Bivalent metal cations stimulated the polyphosphatase activity 9-14-fold in the following order: Zn2+ > Co2+ > Mg2+, Mn2+ . Fe2+, Cu2+ and Ca2+ ions inhibited this activity at all concentrations used . The cytosolic polyphosphatase was effectively inhibited by molybdate and heparin . Heparin was effective only in the presence of Mg2+, Mn2+ and Co2+ but had no effect in the presence of Zn2+ . Heparin inhibited polyphosphatase competitively . The polyphosphatase activity was the same with poly (P)9-poly (P)208 . On poly (P)9, poly (P)15 and poly (P)208 the Km(app) values were equal to 19, 11, 1.2 microM, respectively . The molecular mass of cytosolic polyphosphatase determined by gel filtration on Sephacryl S-300 was 50 kDa.

Biopolymers, 1994 Dec, 34(12), 1627 - 35
Direct observation of cell wall glucans in whole cells of Saccharomyces cerevisiae by magic-angle spinning 13C-NMR; Krainer E et al.; Intact cells of Saccharomyces cerevisiae were examined as an aqueous paste by 13C-nmr spectroscopy with direct polarization and magic-angle spinning . The spectra obtained were highly resolved, showing numerous resonances in the 60-105 ppm range that were assigned to carbons of a liquid-like domain of the cell wall glucan . Assignments were confirmed by running the spectrum of S . cerevisiae in which the cell wall glucans were labeled with {13C} by feeding the cell {13C}galactose . The spectra indicate that the glucan in the cell wall of intact S . cerevisiae assumes a helical conformation and suggest that strain 17A fed with galactose preferentially incorporates the resulting glucose into beta (1-->3)-linkages.

Int J Radiat Biol, 1994 Dec, 66(6 Suppl), S133 - 9
Radiation checkpoints in model systems; Carr AM; The response to DNA damaging agents includes a delay to progression through the cell cycle . Irradiation of premitotic cells causes a delay to mitosis and irradiation of G1 and S phase cells causes a delay to DNA synthesis . These delays have become known as checkpoints . The mechanisms that mediate the mitotic (or G2) checkpoint delay have recently come under study in yeast model systems . Work in the eukaryotic organisms S . cerevisiae and S . pombe has identified at least seven proteins controlling the interactions between DNA damage and cell cycle progression . Genetic analysis of this checkpoint pathway has identified substantial overlap with the feedback controls that co-ordinate progression through the cell cycle . Molecular analysis has revealed structural conservation between these highly diverged yeasts, which suggests that similar proteins may act in related pathways in mammalian cells . In addition, the rad24 and rad25 genes of S . pombe (which are involved in the radiation checkpoint) encode functionally overlapping essential proteins that are highly conserved in mammalian cells . Studies of checkpoints in the yeasts may therefore help to define the signal pathways that control cell cycle delay in mammalian cells following irradiation, some of which have been proposed to be deficient in A-T cells.

J Cell Biol, 1994 Dec, 127(6 Pt 2), 1973 - 84
STU1, a suppressor of a beta-tubulin mutation, encodes a novel and essential component of the yeast mitotic spindle; Pasqualone D et al.; We have isolated a cold-sensitive allele of TUB2, the sole gene encoding beta-tubulin in S . cerevisiae, that confers a specific defect in spindle microtubule function . At 14 degrees C, tub2-406 cells lack a normal bipolar spindle but do assemble functional cytoplasmic microtubules . In an attempt to identify proteins that are important for spindle assembly, we screened for suppressors of the cold-sensitivity of tub2-406 and obtained four alleles of a novel gene, STU1 . Genetic interactions between stu1 alleles and alleles of TUB1 and TUB2 suggest that Stu1p specifically interacts with microtubules . STU1 is essential for growth and disruption of STU1 causes defects in spindle assembly that are similar to those produced by the tub2-406 mutation . The nucleotide sequence of the STU1 gene predicts a protein product of 174 kD with no significant similarity to known proteins . An epitope-tagged Stulp colocalizes with microtubules in the mitotic spindle of yeast . These results demonstrate that Stulp is an essential component of the yeast mitotic spindle.

Yeast, 1994 Dec, 10(13), 1793 - 808
New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae; Wach A et al.; We have constructed and tested a dominant resistance module, for selection of S . cerevisiae transformants, which entirely consists of heterologous DNA . This kanMX module contains the known kanr open reading-frame of the E . coli transposon Tn903 fused to transcriptional and translational control sequences of the TEF gene of the filamentous fungus Ashbya gossypii . This hybrid module permits efficient selection of transformants resistant against geneticin (G418) . We also constructed a lacZMT reporter module in which the open reading-frame of the E . coli lacZ gene (lacking the first 9 codons) is fused at its 3' end to the S . cerevisiae ADH1 terminator . KanMX and the lacZMT module, or both modules together, were cloned in the center of a new multiple cloning sequence comprising 18 unique restriction sites flanked by Not I sites . Using the double module for constructions of in-frame substitutions of genes, only one transformation experiment is necessary to test the activity of the promotor and to search for phenotypes due to inactivation of this gene . To allow for repeated use of the G418 selection some kanMX modules are flanked by 470 bp direct repeats, promoting in vivo excision with frequencies of 10(-3)-10(-4) . The 1.4 kb kanMX module was also shown to be very useful for PCR based gene disruptions . In an experiment in which a gene disruption was done with DNA molecules carrying PCR-added terminal sequences of only 35 bases homology to each target site, all twelve tested geneticin-resistant colonies carried the correctly integrated kanMX module.

Yeast, 1994 Dec, 10(12), 1581 - 9
The nucleotide sequence and initial characterization of pyruvate decarboxylase from the yeast Hanseniaspora uvarum; Holloway P et al.; We have isolated a pyruvate decarboxylase (PDC) gene from the yeast Hanseniaspora uvarum using the Saccharomyces cerevisiae PDC1 gene as a probe . The nucleotide sequence of this gene was determined and compared to PDC genes from yeast and other organisms . The H . uvarum PDC gene is more than 70% identical to the S . cerevisiae PDC isozymes and possesses a putative thiamine diphosphate binding site . The PDC enzyme was purified and partially characterized . The H . uvarum PDC was very similar to other known PDCs; the Km for pyruvate was 0.75 mM, and the enzyme is a homotetramer with subunits of M(r) = 57,000.

Yeast, 1994 Dec, 10(12), 1559 - 68
Consideration of the evolution of the Saccharomyces cerevisiae MEL gene family on the basis of the nucleotide sequences of the genes and their flanking regions; Turakainen H et al.; Analysis of the DNA sequences of new members of the Saccharomyces cerevisiae MEL1-MEL10 gene family showed high homology between the members . The MEL gene family, alpha-galactosidase-coding sequences, have diverged into two groups; one consisting of MEL1 and MEL2 and the other of MEL3-MEL10 . In two S . cerevisiae strains containing five or seven MEL genes each, all the genes are nearly identical, suggesting very rapid distribution of the gene to separate chromosomes . The sequence homology and the abrupt change to sequence heterogeneity at the centromere-proximal 3' end of the MEL genes suggest that the distribution of the genes to new chromosomal locations has occurred partly by reciprocal recombination at solo delta sequences . We identified a new open reading frame sufficient to code for a 554 amino acid long protein of unknown function . The new open reading frame (Accession number Z37509) is located in the 3' non-coding region of MEL3-MEL10 genes in opposite orientation to the MEL genes (Accession numbers Z37508, Z37510, Z37511) . Northern analysis of total RNA showed no hybridization to a homologous probe, suggesting that the gene is not expressed efficiently if at all.

Yeast, 1994 Dec, 10(12), 1553 - 8
Respiratory inhibitors affect incorporation of glucose into Saccharomyces cerevisiae cells, but not the activity of glucose transport; Walsh MC et al.; Incubation of starved galactose-grown S . cerevisiae cells with cyanide reduced glucose uptake as measured over a 5-s period . The Vmax for glucose uptake was decreased by over a factor of two but the apparent affinity for glucose doubled . When measured in the sub-second time scale, however, there was no significant inhibition of glucose uptake, by cyanide, up to 200-ms, clearly demonstrating that, in cyanide treated cells, glucose uptake was not linear for the first 5-s . After a 200-ms exposure of untreated cells to radio-labelled glucose, less than 10% of the intracellular label resided in soluble uncharged compounds . In cyanide-treated cells up to 43% of the labelled compounds were uncharged, with a concurrent reduction of intracellular label residing in anionic compounds . The results suggest that, in the presence of 10 mM cyanide when respiration is inhibited, a reduction in the cellular ATP concentration causes a reduction in hexose-kinase activity which results in an accumulation of internal free glucose, which in turn causes a reduction in net glucose transport.

Antimicrob Agents Chemother, 1994 Dec, 38(12), 2850 - 6
Characterization of the PNT1 pentamidine resistance gene of Saccharomyces cerevisiae; Ludewig G et al.; The Saccharomyces cerevisiae PNT1 gene was isolated and characterized . When present in high copy number in S . cerevisiae, PNT1 confers resistance to the anti-Pneumocystis carinii drug pentamidine . The PNT1 gene encodes a previously uncharacterized polypeptide of 409 amino acids . The predicted gene product is a very basic (pI 9.9) polypeptide with one potential membrane-associated region . PNT1 is located on chromosome XVR of S . cerevisiae . It is transcribed at a very low level . Overexpression of the gene increases resistance to the cytostatic and mitochondrial DNA-damaging effects of pentamidine and related cationic compounds . Disruption of the gene leads to slightly increased levels of susceptibility to pentamidine and some related compounds.

FEBS Lett, 1994 Nov 28, 355(2), 201 - 4
Germinating conidiospores of Aspergillus amino acid auxotrophs are hypersensitive to heat shock, oxidative stress and DNA damage; Donnelly E et al.; Germinating conidiospores (conidia) of Aspergillus nidulans amino acid-requiring strains are hypersensitive to heat, oxidative stress, UV radiation and chemical mutagens when compared with other strains . They also showed an increased mutation rate . Sensitivity to stress conditions has been correlated with an abnormal RAS/cAMP pathway in mutants of S . cerevisiae . We suggest that the RAS/cAMP pathway is defective in germinating conidia of Aspergillus amino acid auxotrophs and that this is responsible for suppressing DNA repair and conferring sensitivity to oxidative stress and heat shock.

EMBO J, 1994 Nov 15, 13(22), 5361 - 9
RAD26, the functional S . cerevisiae homolog of the Cockayne syndrome B gene ERCC6; van Gool AJ et al.; Transcription-coupled repair (TCR) is a universal sub-pathway of the nucleotide excision repair (NER) system that is limited to the transcribed strand of active structural genes . It accomplishes the preferential elimination of transcription-blocking DNA lesions and permits rapid resumption of the vital process of transcription . A defect in TCR is responsible for the rare hereditary disorder Cockayne syndrome (CS) . Recently we found that mutations in the ERCC6 repair gene, encoding a putative helicase, underly the repair defect of CS complementation group B . Here we report the cloning and characterization of the Saccharomyces cerevisiae homolog of CSB/ERCC6, which we designate RAD26 . A rad26 disruption mutant appears viable and grows normally, indicating that the gene does not have an essential function . In analogy with CS, preferential repair of UV-induced cyclobutane pyrimidine dimers in the transcribed strand of the active RBP2 gene is severely impaired . Surprisingly, in contrast to the human CS mutant, yeast RAD26 disruption does not induce any UV-, cisPt- or X-ray sensitivity, explaining why it was not isolated as a mutant before . Recovery of growth after UV exposure was somewhat delayed in rad26 . These findings suggest that TCR in lower eukaryotes is not very important for cell survival and that the global genome repair pathway of NER is the major determinant of cellular resistance to genotoxicity.

J Cell Biol, 1994 Nov, 127(4), 995 - 1008
Factors required for the binding of reassembled yeast kinetochores to microtubules in vitro; Sorger PK et al.; Kinetochores are structures that assemble on centromeric DNA and mediate the attachment of chromosomes to the microtubules of the mitotic spindle . The protein components of kinetochores are poorly understood, but the simplicity of the S . cerevisiae kinetochore makes it an attractive candidate for molecular dissection . Mutations in genes encoding CBF1 and CBF3, proteins that bind to yeast centromeres, interfere with chromosome segregation in vivo . To determine the roles played by these factors and by various regions of centromeric DNA in kinetochore function, we have developed a method to partially reassemble kinetochores on exogenous centromeric templates in vitro and to visualize the attachment of these reassembled kinetochore complexes to microtubules . In this assay, single reassembled complexes appear to mediate microtubule binding . We find that CBF3 is absolutely essential for this attachment but, contrary to previous reports (Hyman, A . A., K . Middleton, M . Centola, T.J . Mitchison, and J . Carbon . 1992 . Microtubule-motor activity of a yeast centromere-binding protein complex . Nature (Lond.) . 359:533-536) is not sufficient . Additional cellular factors interact with CBF3 to form active microtubule-binding complexes . This is mediated primarily by the CDEIII region of centromeric DNA but CDEII plays an essential modulatory role . Thus, the attachment of kinetochores to microtubules appears to involve a hierarchy of interactions by factors that assemble on a core complex consisting of DNA-bound CBF3.

J Bacteriol, 1994 Nov, 176(22), 7091 - 5
Cloning, sequence, and disruption of the Saccharomyces diastaticus DAR1 gene encoding a glycerol-3-phosphate dehydrogenase; Wang HT et al.; The Saccharomyces diastaticus DAR1 gene was cloned by complementation in an Escherichia coli strain auxogrophic for glycerol-3-phosphate . DAR1 encodes an NADH-dependent dihydroxyacetone phosphate reductase (sn-glycerol-3-phosphate dehydrogenase {G3PDase; EC 1.1.1.8}) homologous to several other eukaryotic G3PDases . DAR1 is distinct from GUT2, which encodes a glucose-repressed mitochondrial G3PDase, but is identical to GPD1 from S . cerevisiae, a close relative of S . diastaticus . The level of DAR1-encoded G3PDase was increased about threefold in a medium of high osmolarity . Disruption of DAR1 in a haploid S . cerevisiae was not lethal but led to a decrease in cytoplasmic NADH-dependent G3PDase activity, an increase in osmotic sensitivity, and a 25% reduction in glycerol secretion from cells grown anaerobically on glucose.

Mol Cell Biol, 1994 Nov, 14(11), 7643 - 51
Functional conservation of multiple elements in yeast chromosomal replicators; Rao H et al.; Replicators that control the initiation of DNA replication in the chromosomes of Saccharomyces cerevisiae retain their function when cloned into plasmids, where they are commonly referred to as autonomously replicating sequences (ARSs) . Previous studies of the structure of ARS1 in both plasmid and chromosome contexts have shown that it contains one essential DNA element, A, that includes a match to the ARS consensus sequence (ACS), and three additional elements, B1, B2, and B3, that are also important for ARS function . Elements A and B3 are bound by a candidate initiator protein called the origin recognition complex and ARS-binding factor 1, respectively . Although the A and B3 elements have been found in other ARSs, sequence comparisons among ARSs have failed to identify B1- and B2-like elements . To assess the generality of the modular nature of yeast replicators, linker substitution mutagenesis of another yeast chromosomal replicator, ARS307, was performed . Three DNA sequence elements were identified in ARS307, and they were demonstrated to be functionally equivalent to the A, B1, and B2 elements present in ARS1 . Despite the lack of DNA sequence similarity, the B1 and B2 elements at each ARS were functionally conserved . Single-base substitutions in the core of the ARS1 B1 and B2 elements identified critical nucleotides required for the function of the B1 element . In contrast, no single-point mutations were found to affect B2 function . The results suggest that multiple DNA sequence elements might be a general and conserved feature of replicator sequences in S . cerevisiae.

Mol Cell Biol, 1994 Nov, 14(11), 7611 - 20
Identification of a putative RNA helicase (HRH1), a human homolog of yeast Prp22; Ono Y et al.; In the budding yeast Saccharomyces cerevisiae, a number of PRP genes known to be involved in pre-mRNA processing have been genetically identified and cloned . Three PRP genes (PRP2, PRP16, and PRP22) were shown to encode putative RNA helicases of the family of proteins with DEAH boxes . However, any such splicing factor containing the helicase motifs in vertebrates has not been identified . To identify human homologs of this family, we designed PCR primers corresponding to the highly conserved region of the DEAH box protein family and successfully amplified five cDNA fragments, using HeLa poly(A)+ RNA as a substrate . One fragment, designated HRH1 (human RNA helicase 1), is highly homologous to Prp22, which was previously shown to be involved in the release of spliced mRNAs from the spliceosomes . Expression of HRH1 in a S . cerevisiae prp22 mutant can partially rescue its temperature-sensitive phenotype . These results strongly suggest that HRH1 is a functional human homolog of the yeast Prp22 protein . Interestingly, HRH1 but not Prp22 contains an arginine- and serine-rich domain (RS domain) which is characteristic of some splicing factors, such as members of the SR protein family . We could show that HRH1 can interact in vitro and in the yeast two-hybrid system with members of the SR protein family through its RS domain . We speculate that HRH1 might be targeted to the spliceosome through this interaction.

Mol Cell Biol, 1994 Nov, 14(11), 7037 - 45
The requirement for yeast superoxide dismutase is bypassed through mutations in BSD2, a novel metal homeostasis gene; Liu XF et al.; Oxygen toxicity in Saccharomyces cerevisiae strains lacking superoxide dismutase can be suppressed through mutations in either the BSD1 or BSD2 gene . In this report, we demonstrate that the BSD2 gene normally functions in the homeostasis of heavy metal ions . A mutation in BSD2 not only reverses the aerobic defects of yeast strains lacking superoxide dismutase but also is associated with an increased sensitivity to copper and cadmium toxicity and an elevation in copper ion accumulation . The BSD2 gene was cloned by functional complementation and is predicted to encode a novel 37.5-kDa protein with three potential transmembrane domains . The mutant bsd2-1 allele was isolated and found to contain a single C-to-T transition changing a centrally located proline to a serine . This substitution results in total inactivation of BSD2, since the bsd2-1 mutation is identical to a bsd2 delta gene deletion in phenotype . BSD2 is expressed in yeast cells as a 1.5-kb mRNA . Although the gene functions in copper detoxification, BSD2 is not induced by copper ions, as is the case with S . cerevisiae metallothioneins . A probable role for copper ions in the bsd2 reversal of oxidative damage is discussed.

J Virol, 1994 Nov, 68(11), 7628 - 33
Activation of the Epstein-Barr virus BMRF1 and BZLF1 promoters by ZEBRA in Saccharomyces cerevisiae; Countryman JK et al.; ZEBRA has been shown to activate model reporter genes consisting of synthetic oligomerized ZEBRA response elements upstream of a minimal CYC1 promoter fused to beta-galactosidase in the yeast Saccharomyces cerevisiae . Here it is shown that in S . cerevisiae ZEBRA activates transcription of natural Epstein-Barr virus promoters . Two Epstein-Barr virus promoters were shown to be activated by ZEBRA in S . cerevisiae: Zp, the promoter that regulates expression of BZLF1, which encodes ZEBRA; and EAp, the promoter controlling expression of BMRF1, which encodes diffuse early antigen, EA-D . These observations indicate that neither mammalian-specific nor virally encoded coactivators are obligatory for ZEBRA to stimulate expression from these two promoters . Zp was also strongly activated by endogenous yeast factors . EAp was not activated by yeast factors . The results show that in S . cerevisiae and in B cells, ZEBRA dominates the response of EAp; ZEBRA plus endogenous cell factors activate Zp.

Mikrobiol Z, 1994 Nov-Dec, 56(6), 3 - 16
A probabilistic approach to the analysis of the frequency of diploid formation in yeast (Saccharomyces cerevisiae) mating; Kopocinski B et al.; Conditions of optimizing mating efficiency in the yeast . Saccharomyces cerevisiae were tested and measured as a number of prototrophic diploids . It was found that the process of diploid formation could be controlled by suitably adjusting cell densities in liquid mating medium and alpha: a mating type cell ratio . When the first was about 5 x 10(8) per ml and the second 1:10, respectively, every alpha cell was able to find a partner of opposite mating type . Some mathematical models of the "courtship" in yeast were advanced and the conclusions are discussed in the light of recent data on mating pathway in S . cerevisiae.

Curr Genet, 1994 Nov-Dec, 26(5-6), 398 - 402
Cloning and molecular analysis of two different ILV5 genes from a brewing strain of Saccharomyces cerevisiae; Xie Q et al.; Two different ILV5 genes encoding acetohydroxy-acid isomeroreductases, and named ILV5G and ILV5X, were cloned and sequenced from a Saccharomyces cerevisiae brewing strain . The coding sequence of ILV5X shows a single nucleotide change with respect to that from the ILV5 gene of a S . cerevisiae laboratory strain . In addition, all promoter motifs which are, or are presumed to be, implicated in transcription regulatory functions are identical in ILV5 and ILV5X . In contrast, the coding sequence of ILV5G differs in 5.6% of its nucleotides from that of ILV5 and most of its promoter regulatory motifs show a single nucleotide change with respect to those from ILV5.

Curr Biol, 1994 Nov 1, 4(11), 990 - 5
Evidence against the existence of the purported Saccharomyces cerevisiae PKC2 gene; Levin DE et al.; BACKGROUND: The existence of a Saccharomyces cerevisiae gene encoding a novel isoform of protein kinase C was reported recently in this journal . RESULTS: We demonstrate here that, firstly, the purported PKC2 gene does not reside at the chromosomal location to which it was assigned; secondly, it does not exist as a contiguous sequence in the S . cerevisiae genome; thirdly, some of its reported sequences do exist within other yeast genes; and fourthly, some of its reported sequences, encoding regions of the predicted protein related to protein kinase C, do not exist in any context in the yeast genome . CONCLUSIONS: We conclude from these studies that the PKC2 gene is a composite construction of unrelated yeast and non-yeast sequences.

Biokhimiia, 1994 Nov, 59(11), 1675 - 88
{A prosegment of the yeast alpha-factor controls a heterologous protein (human growth factor) in Saccharomyces cerevisiae culture media}; Tsiomenko AB et al.; The role of the yeast pheromone prosegment--alpha-factor--in the export of the heterologous protein, the human growth hormone (hGH), in the culture medium of S . cerevisiae has been studied . Using genetic engineering constructions, it has been shown that different N-terminal signal peptides (SP) are not able to provide the hGH export . Transformant cells carrying the plasmid with a hybrid sequence encoding the prosegment-hGH (without SP) accumulate non-glycosylated pro-hGH in the cytosol . Only the combination of SP and the prosegment as the N-terminal fragment of the hGH precursor results in the processing and export of the mature form of the hormone . The origin (or type) of SP is of no significance . The glycosylation inhibitors--2-deoxy-D-glucose and tunicamycin--suppress the export but not the entry of hGH into the periplasm, thus indicating a critical role of the intactness of the prosegment polymannose chains for the efficient export of heterologous protein . A conclusion is drawn that the two preprosegment parts play different roles . The SP constituent of prepro-hGH introduces pro-GH into the general secretory pathway, while the prosegment resulting from the SP cleavage directs hGH from the cell into the culture medium.

Yeast, 1994 Nov, 10(11), 1421 - 8
Characterization of lipid particles of the yeast, Saccharomyces cerevisiae; Leber R et al.; Lipid particles of the yeast, Saccharomyces cerevisiae, were isolated to high purity and their components were analysed . The hydrophobic core of this organelle consists of triacylglycerols and steryl esters, which are almost exclusively located to that compartment . Lipid particles are stabilized by a surface membrane consisting of phospholipids and proteins . Electron microscopy confirmed the purity of the preparations and the proposed structure deduced from biochemical experiments . Major proteins of lipid particles have molecular weights of 72, 52, 43 and 34 kDa, respectively . The 43 kDa protein reacts with an antiserum against human apolipoprotein AII . In lipid particles of the yeast mutant strain S . cerevisiae erg6, which is deficient in sterol delta 24-methyltransferase, this protein is missing thereby identifying the protein and confirming our previous finding (Zinser et al., 1993) that sterol delta 24-methylation is associated with lipid particles . A possible involvement of surface proteins of lipid particles in the interaction with other organelles is discussed with respect to sterol translocation in yeast.

Microbiology, 1994 Nov, 140 ( Pt 11), 3039 - 46
Involvement of mitochondria in the assimilatory metabolism of anaerobic Saccharomyces cerevisiae cultures; Visser W et al.; The possible physiological role of mitochondria in anaerobically grown Saccharomyces cerevisiae was investigated via enzyme localization and inhibitor studies . Almost all of the activity of citrate synthase (EC 4.1.3.7) was recovered in the mitochondrial fraction after differential centrifugation of spheroplast lysates . The enzyme exhibited a high degree of latency which was demonstrated by sonication of the mitochondrial fractions . Since citrate synthase is an important enzyme in anabolic reactions, a consequence of this localization is the requirement for transport of metabolites across the mitochondrial membranes . Such transport is likely to require energy which, as a result of anaerobiosis, cannot be supplied by respiration . It was therefore investigated whether ATP translocation into the mitochondria by an ADP/ATP translocase might be involved in anaerobic mitochondrial energy metabolism . It was shown that addition of the ADP/ATP translocase inhibitor bongkrekic acid to anaerobic cultures indeed inhibited growth, although only partially . It is concluded that mitochondria of S . cerevisiae fulfil a vital role in anaerobic sugar metabolism.

Biol Chem Hoppe Seyler, 1994 Nov, 375(11), 759 - 63
Developmental regulation of DEAD box proteins and cloning of putative RNA helicase genes from Dictyostelium discoideum; Mahal B et al.; RNA secondary structure is essential for RNA function in pre-mRNA splicing, mRNA translation, ribosome assembly and RNA stability . The involvement of DEAD/H RNA helicases in the regulation of these processes has been demonstrated in some cases . To investigate the repertoire of DEAD box proteins expressed in Dictyostelium discoideum, we used PCR techniques to clone two cDNAs coding for DEAD box proteins with high similarity to known yeast proteins: Dictyostelium Hel2A is about 45% identical to Saccharomyces cerevisiae DBP2 and S . pombe dbp2, the yeast homologues of human p68 . Dictyostelium Hel2B is about 43% identical to the S . cerevisiae splicing factor PRP28, but has a different domain at the N-terminus, which is unique for Dictyostelium discoideum . Using a polyclonal antibody directed against a DEAD box peptide we show differential expression of three DEAD box proteins during the developmental cycle of Dictyostelium.

Biol Chem Hoppe Seyler, 1994 Nov, 375(11), 721 - 9
Antisense gene expression in yeast; Atkins D et al.; The use of antisense and ribozyme RNA to modulate gene expression is emerging as an effective genetic technique . A compilation of successful antisense gene suppression experiments reveals the absence of reports on the use of the yeast Saccharomyces cerevisiae as a host . We examine the field of antisense and ribozyme use in S . cerevisiae and discuss that this result is not due to any lack of attempts and may reflect unique features of S . cerevisiae biology . In an attempt to learn from cellular RNA physiology we review evidence for naturally occurring antisense RNA regulation . Although there are many examples of well characterised overlapping RNA transcripts there is, as yet, no clear evidence suggesting complementary RNA-dependent gene regulation in S . cerevisiae . The application of artificial antisense and ribozyme genes is then discussed with an emphasis on the role of yeast as a model system for the systematic and genetic analysis of antisense and ribozyme RNA function . In addition, potential reasons for the lack of attempts to use antisense or ribozyme genes to create pseudogenetic mutants are considered . We conclude that the application of successful antisense and ribozyme strategies in yeast may have to address features of S . cerevisiae RNA biology and offer experimental approaches that may identify some of these features.

Curr Genet, 1994 Nov-Dec, 26(5-6), 377 - 81
Homologous maturase-like proteins are encoded within the group I introns in different mitochondrial genes specifying Yarrowia lipolytica cytochrome c oxidase subunit 3 and Saccharomyces cerevisiae apocytochrome b; Matsuoka M et al.; A mitochondrial cox3 gene in the alkane yeast, Yarrowia lipolytica, encodes a subunit-3 protein of cytochrome c oxidase, and contains a 1044 base-pair-long intron, as compared with the corresponding intronless gene in Saccharomyces cerevisiae . The intron belongs to a group I intron as determined by the cDNA sequence for the splicing sites as well as the predicted RNA secondary structure . Remarkably, this intron could code for a protein of 206 amino-acid residues which showed 63% similarity with an RNA maturase encoded by the second intron of the mitochondrial apocytochrome b gene in S . cerevisiae . Both introns occurred within the conserved exon sequence, 5'-TT(G/C)AGGTGC-3', suggesting the possible transposition of a common ancestral intron.

J Cell Biol, 1994 Nov, 127(3), 623 - 39
A novel FK506- and rapamycin-binding protein (FPR3 gene product) in the yeast Saccharomyces cerevisiae is a proline rotamase localized to the nucleolus; Benton BM et al.; The gene (FPR3) encoding a novel type of peptidylpropyl-cis-trans-isomerase (PPIase) was isolated during a search for previously unidentified nuclear proteins in Saccharomyces cerevisiae . PPIases are thought to act in conjunction with protein chaperones because they accelerate the rate of conformational interconversions around proline residues in polypeptides . The FPR3 gene product (Fpr3) is 413 amino acids long . The 111 COOH-terminal residues of Fpr3 share greater than 40% amino acid identity with a particular class of PPIases, termed FK506-binding proteins (FKBPs) because they are the intracellular receptors for two immunosuppressive compounds, rapamycin and FK506 . When expressed in and purified from Escherichia coli, both full-length Fpr3 and its isolated COOH-terminal domain exhibit readily detectable PPIase activity . Both fpr3 delta null mutants and cells expressing FPR3 from its own promoter on a multicopy plasmid have no discernible growth phenotype and do not display any alteration in sensitivity to the growth-inhibitory effects of either FK506 or rapamycin . In S . cerevisiae, the gene for a 112-residue cytosolic FKBP (FPR1) and the gene for a 135-residue ER-associated FKBP (FPR2) have been described before . Even fpr1 fpr2 fpr3 triple mutants are viable . However, in cells carrying an fpr1 delta mutation (which confers resistance to rapamycin), overexpression from the GAL1 promoter of the C-terminal domain of Fpr3, but not full-length Fpr3, restored sensitivity to rapamycin . Conversely, overproduction from the GAL1 promoter of full-length Fpr3, but not its COOH-terminal domain, is growth inhibitory in both normal cells and fpr1 delta mutants . In fpr1 delta cells, the toxic effect of Fpr3 overproduction can be reversed by rapamycin . Overproduction of the NH2-terminal domain of Fpr3 is also growth inhibitory in normal cells and fpr1 delta mutants, but this toxicity is not ameliorated in fpr1 delta cells by rapamycin . The NH2-terminal domain of Fpr3 contains long stretches of acidic residues alternating with blocks of basic residues, a structure that resembles sequences found in nucleolar proteins, including S . cerevisiae NSR1 and mammalian nucleolin . Indirect immunofluorescence with polyclonal antibodies raised against either the NH2- or the COOH-terminal segments of Fpr3 expressed in E . coli demonstrated that Fpr3 is located exclusively in the nucleolus.

FEBS Lett, 1994 Oct 31, 354(1), 50 - 2
Degradation of the yeast MAT alpha 2 transcriptional regulator is mediated by the proteasome; Richter-Ruoff B et al.; Rapid degradation of specific regulatory proteins plays a role in a wide range of cellular phenomena, including cell cycle progression and the regulation of cell growth and differentiation . A major mechanism of selective protein turnover in vivo involves a large multi-subunit protease known as the proteasome or multi-catalytic proteinase . At the same time, the degradation of many cellular proteins requires their covalent ligation to the polypeptide ubiquitin . Here we show that the yeast S . cerevisiae MAT alpha 2 repressor, which is known to be ubiquitinylated in vivo, requires the proteasome for its rapid intracellular proteolysis.

FEBS Lett, 1994 Oct 31, 354(1), 117 - 22
Expression of the sarcoplasmic reticulum Ca(2+)-ATPase in yeast; Centeno F et al.; We describe here an easy system for the production of mg amounts of the rabbit Ca(2+)-ATPase SERCA 1a in the yeast S . cerevisiae . The protein is present in several membranes, including the plasma membrane of the yeast, in a native conformation . It can be purified by immunoprecipitation and can be phosphorylated from ATP in a Ca(2+)-dependent manner . Using a temperature-sensitive secretion mutant strain, the fully active protein can also be obtained in secretory vesicles.

Mol Gen Genet, 1994 Oct 28, 245(2), 167 - 76
Two subclasses of guanine exchange factor (GEF) domains revealed by comparison of activities of chimeric genes constructed from CDC25, SDC25 and BUD5 in Saccharomyces cerevisiae; Camus C et al.; Guanine Exchange Factor (GEF) activity for Ras proteins has been associated with a conserved domain in Cdc25p, Sdc25p in Saccharomyces cerevisiae and several other proteins recently found in other eukaryotes . We have assessed the structure-function relationships between three different members of this family in S . cerevisiae, Cdc25p, Sdc25p and Bud5p . Cdc25p controls the Ras pathway, whereas Bud5p controls bud site localization . We demonstrate that the GEF domain of Sdc25p is closely related to that of Cdc25p . We first constructed a thermosensitive allele of SDC25 by specifically altering amino acid positions known to be changed in the cdc25-1 mutation . Secondly, we constructed three chimeric genes from CDC25 and SDC25, the products of which are as active in the Ras pathway as are the wild-type proteins . In contrast, similar chimeras made between CDC25 and BUD5 lead to proteins that are inactive both in the Ras and budding control pathways . This difference in the ability of chimeric proteins to retain activity allows us to define two subclasses of structurally different GEFs: Cdc25p and Sdc25p are Ras-specific GEFs, and Bud5p is a putative GEF for the Rsr1/Bud1 Rap-like protein.

Cell, 1994 Oct 21, 79(2), 233 - 44
The B-type cyclin kinase inhibitor p40SIC1 controls the G1 to S transition in S . cerevisiae; Schwob E et al.; When yeast cells reach a critical size, they initiate bud formation, spindle pole body duplication, and DNA replication almost simultaneously . All three events depend on activation of Cdc28 protein kinase by the G1 cyclins Cln1, -2, and -3 . We show that DNA replication also requires activation of Cdc28 by B-type (Clb) cyclins . A sextuple clb1-6 mutant arrests as multibudded G1 cells that resemble cells lacking the Cdc34 ubiquitin-conjugating enzyme . cdc34 mutants cannot enter S phase because they fail to destroy p40SIC1, which is a potent inhibitor of Clb but not Cln forms of the Cdc28 kinase . In wild-type cells, p40SIC1 protein appears at the end of mitosis and disappears shortly before S phase . Proteolysis of a cyclin-specific inhibitor of Cdc28 is therefore an essential aspect of the G1 to S phase transition.

Science, 1994 Oct 21, 266(5184), 404 - 9
TLC1: template RNA component of Saccharomyces cerevisiae telomerase; Singer MS et al.; Telomeres, the natural ends of linear eukaryotic chromosomes, are essential for chromosome stability . Because of the nature of DNA replication, telomeres require a specialized mechanism to ensure their complete duplication . Telomeres are also capable of silencing the transcription of genes that are located near them . In order to identify genes in the budding yeast Saccharomyces cerevisiae that are important for telomere function, a screen was conducted for genes that, when expressed in high amounts, would suppress telomeric silencing . This screen lead to the identification of the gene TLC1 (telomerase component 1) . TLC1 encodes the template RNA of telomerase, a ribonucleoprotein required for telomere replication in a variety of organisms . The discovery of TLC1 confirms the existence of telomerase in S . cerevisiae and may facilitate both the analysis of this enzyme and an understanding of telomere structure and function.

EMBO J, 1994 Oct 17, 13(20), 4737 - 44
Mating type-specific cell-cell recognition of Saccharomyces cerevisiae: cell wall attachment and active sites of a- and alpha-agglutinin; Cappellaro C et al.; Mating type-specific agglutination of Saccharomyces cerevisiae a and alpha cells depends on the heterophilic interaction of two cell surface glycoproteins, the gene products of AG alpha 1 and AGA2 . Evidence is presented with immunogold labelling that the alpha-agglutinin is part of the outer fimbrial cell wall coat . The a-agglutinin is bound via two S-S bridges (Cys7 and Cys50) to a cell wall component, most probably the gene product of AGA1 . His273 of alpha-agglutinin has previously been shown to be essential for a- and alpha-agglutinin interaction and a model based on two opposing ion-pairs had been proposed . By site-directed mutagenesis this possibility has now been excluded . With the help of various peptides, either chemically synthesized, obtained by proteolysis of intact glycosylated a-agglutinin or prepared from a fusion protein expressed in Escherichia coli, the biologically active region of a-agglutinin was located at the C-terminus of the molecule . A peptide consisting of the C-terminal 10 amino acids (GSPIN-TQYVF) was active in nanomolar concentrations . Saccharide moieties, therefore, are not essential for the mating type-specific cell-cell interaction; glycosylated peptides are, however, four to five times more active than non-glycosylated ones . Comparisons of the recognition sequences of the S . cerevisiae agglutinins with that of the Dictyostelium contact site A glycoprotein (gp80), as well as with those of the various families of cell adhesion molecules of higher eucaryotes, have been made and are discussed.

EMBO J, 1994 Oct 17, 13(20), 4963 - 72
Homing of a group II intron in yeast mitochondrial DNA is accompanied by unidirectional co-conversion of upstream-located markers; Lazowska J et al.; Group II introns ai1 and ai2 of the Saccharomyces cerevisiae mitochondrial COXI gene encode proteins having a dual function (maturase and reverse transcriptase) and are mobile genetic elements . By construction of adequate donor genomes, we demonstrate that each of them is self-sufficient and practises homing in the absence of homing-type endonucleases encoded by either group I introns or the ENS2 gene . Each of the S . cerevisiae group II self-mobile introns was tested for its ability to invade mitochondrial DNA (mtDNA) from two related Saccharomyces species . Surprisingly, only ai2 was observed to integrate into both genomes . The non-mobility of ai1 was clearly correlated with some polymorphic changes occurring in sequences flanking its insertion sites in the recipient mtDNAs . Importantly, studies of the behaviour of these introns in interspecific crosses demonstrate that flanking marker co-conversion accompanying group II intron homing is unidirectional and efficient only in the 3' to 5' direction towards the upstream exon . Thus, the polar co-conversion and dependence of the splicing proficiency of the intron reported previously by us are hallmarks of group II intron homing, which significantly distinguish it from the strictly DNA-based group I intron homing and strictly RNA-based group II intron transposition.

FEMS Microbiol Lett, 1994 Oct 15, 123(1-2), 33 - 6
Physiological studies on the effect of Ca2+ on the duration of the lag phase of Saccharomyces cerevisiae; Friis J et al.; Cell multiplication and growth of Saccharomyces cerevisiae were followed in 2-ml test tubes containing Wickerham's synthetic medium or very dilute synthetic media supplemented in various ways . The ability of the cell cultures to leave the lag phase and enter the exponential phase of growth was investigated . Multiplication was assessed by microscopical observation . The results showed great differences in times required for the cultures to leave the lag phases and begin multiplication . In Wickerham's medium, all cultures grew well 6 h after inoculation . In the dilute medium, several days elapsed before all the cultures grew . These cultures went into exponential growth with approximately first order kinetics . In the unsupplemented medium, the 'half-lives' in the lag phase were about 28 h . Addition of either Ca2+ or Ca2+ plus A23187 (calcimycin) reduced the half-lives to 10 and 6 h, respectively . The doubling times in the exponential phases of growth were not shortened by these additions . We suggest that Ca2+ plays a crucial role as a signal to switch on the mode of cell proliferation in S . cerevisiae.

Eur J Biochem, 1994 Oct 15, 225(2), 565 - 72
Kex2p: a model for cellular endoprotease processing human immunodeficiency virus type 1 envelope glycoprotein precursor; Moulard M et al.; The endoproteolytic cleavage of the envelope glycoprotein precursor (gp160) of the human immunodeficiency virus type 1 (HIV-1) by a cellular protease is required for full activation of the virus . In this study, processing of gp160 was analyzed in vitro using the Kex2p endoprotease from the yeast Saccharomyces cerevisiae as a processing enzyme model . Endoproteolytic processing was examined using a synthetic peptide that mimics the cleavage site of HIV-1 glycoprotein, and a recombinant gp160 bearing the entire sequence of the env gene product, including the conserved cleavage site Arg508-Glu-Lys-Arg511 . Coexpression in BHK-21 of Kex2p and gp160 by recombinant vaccinia viruses demonstrates that Kex2p can correctly process the HIV-1 glycoprotein to gp120 and gp41 . Furthermore, recombinant gp160 and peptide were used as substrates and subjected to proteolysis with purified membranes from an S . cerevisiae strain overproducing the Kex2p endoprotease . Treatment of recombinant gp160, which has an apparent molecular mass of 127 kDa, with Kex2p and Western blot analysis showed that the precursor was cleaved into two products of about 101 and 34 kDa apparent molecular mass . Amino acid sequencing of the NH2-terminus of the 34-kDa product showed that the cleavage site of recombinant gp160 was between Arg511 and Ala512 . Recombinant gp160 mutated at the sequence coding for the potential cleavage site, and mature recombinant gp120, however, were not cleaved when treated with Kex2p . In summary, our results show that Kex2p cleaves both the HIV-1 envelope glycoprotein precursor and a synthetic peptide mimicking the cleavage site of HIV-1 gp160 at the dibasic site, suggesting functional analogy between yeast Kex2p and the cellular protease responsible for the maturation of HIV-1 envelope glycoproteins in infected human cells.

J Biol Chem, 1994 Oct 14, 269(41), 25783 - 7
Purification and characterization of Ag,Zn-superoxide dismutase from Saccharomyces cerevisiae exposed to silver; Ciriolo MR et al.; Cu,Zn-superoxide dismutase plays an important role in protecting cells from oxygen toxicity by catalyzing the dismutation of superoxide anion into hydrogen peroxide and oxygen . In Saccharomyces cerevisiae Cu,Zn-superoxide dismutase is coregulated with copper-thionein by copper via the transcription factor ACE 1 . We demonstrate here that presence of AgNO3 in the culture medium leads to a five times increase of Cu,Zn-superoxide dismutase mRNA, with a concomitant six times decrease of the enzyme activity . Susceptibility of yeast to silver was apparently inversely related to Cu,Zn-superoxide dismutase activity . From silver-treated yeast a Cu,Zn-superoxide dismutase with impaired dismutase function was purified and was shown to contain silver, which was located to the copper site . These data suggest that Cu,Zn-superoxide dismutase may play an additional direct role in the defense of S . cerevisiae against metal stress by functioning as metal chelator.

Cell, 1994 Oct 7, 79(1), 131 - 41
Cooperative interaction of S . pombe proteins required for mating and morphogenesis; Chang EC et al.; We isolated two S . pombe genes, scd1 and scd2, that are required for normal morphology and mating . scd1 and scd2 are homologous to CDC24 and BEM1, respectively, of S . cerevisiae . Epistasis analyses indicate that scd2 and ras1 converge upon scd1, which, in turn, interacts with cdc42sp, a RHO-like GTPase . Studies with the yeast two-hybrid system indicate that scd2 forms complexes with both scd1 and cdc42sp . Furthermore, biochemical studies indicate that the interaction between scd1 and scd2 is direct . The yeast two-hybrid data further suggest that scd1, scd2, cdc42sp, and ras1, in its GTP-bound state, act cooperatively to form a protein complex.

EMBO J, 1994 Oct 3, 13(19), 4636 - 44
Intron-dependent formation of pseudouridines in the anticodon of Saccharomyces cerevisiae minor tRNA(Ile); Szweykowska-Kulinska Z et al.; We have isolated and sequenced the minor species of tRNA(Ile) from Saccharomyces cerevisiae . This tRNA contains two unusual pseudouridines (psi s) in the first and third positions of the anticodon . As shown earlier by others, this tRNA derives from two genes having an identical 60 nt intron . We used in vitro procedures to study the structural requirements for the conversion of the anticodon uridines to psi 34 and psi 36 . We show here that psi 34/psi 36 modifications require the presence of the pre-tRNA(Ile) intron but are not dependent upon the particular base at any single position of the anticodon . The conversion of U34 to psi 34 occurs independently from psi 36 synthesis and vice versa . However, psi 34 is not formed when the middle and the third anticodon bases of pre-tRNA(Ile) are both substituted to yield ochre anticodon UUA . This ochre pre-tRNA(Ile) mutant has the central anticodon uridine modified to psi 35 as is the case for S.cerevisiae SUP6 tyrosine-inserting ochre suppressor tRNA . In contrast, neither the first nor the third anticodon pseudouridine is formed, when the ochre (UUA) anticodon in the pre-tRNA(Tyr) is substituted with the isoleucine UAU anticodon . A synthetic mini-substrate consisting of the anticodon stem and loop and the wild-type intron of pre-tRNA(Ile) is sufficient to fully modify the anticodon U34 and U36 into psi s . This is the first example of the tRNA intron sequence, rather than the whole tRNA or pre-tRNA domain, being the main determinant of nucleoside modification.

Microbiology, 1994 Oct, 140 ( Pt 10), 2625 - 32
Concomitant appearance of intrinsic thermotolerance and storage of trehalose in Saccharomyces cerevisiae during early respiratory phase of batch-culture is CIF1-dependent; Attfield PV et al.; Strains of Saccharomyces cerevisiae that exhibit varied capacities for accumulation of trehalose were tested for intrinsic thermotolerance . Yeast that accumulated trehalose rapidly in early respiratory phase showed equally rapid attainment of thermotolerance, whereas a strain unable to accumulate trehalose at this stage of culture showed markedly delayed appearance of thermotolerance . These results were obtained using closely related but non-isogenic diploids and so it is possible that variable factors other than trehalose were responsible for the observed thermotolerance effects . Therefore, a pair of isogenic diploid S . cerevisiae strains was generated to facilitate further testing of whether trehalose functions in intrinsic stress tolerance . Both isogenic strains inherited a partially reverted cif1 phenotype, designated CPR, from the trehalose-deficient progenitor that had been used in construction of the non-isogenic strains . The CPR phenotype permitted growth on glucose but not accumulation of trehalose, indicating that not all cif1-related deficiencies were suppressed in the CPR strains . However, one of the isogenic CPR pair was cif1/cif1 and failed to accumulate trehalose, whilst the other was cif1/CIF1 and was able to accumulate this sugar . The trehalose-proficient strain showed intrinsic stress tolerance whereas the trehalose-deficient strain was sensitive to heat stress during early respiratory growth . These results suggest that one or more functions of CIF1, not operating in the cif1/cif1(CPR) strains, are important for intrinsic thermotolerance of yeast in early respiratory phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Int J Syst Bacteriol, 1994 Oct, 44(4), 708 - 14
Rapid characterization of four species of the Saccharomyces sensu stricto complex according to mitochondrial DNA patterns; Guillamon JM et al.; Several strains of the four sibling species of the genus Saccharomyces (S . bayanus, S . cerevisiae, S . paradoxus, and S . pastorianus) were characterized by using a rapid and simple method of restriction analysis of mitochondrial DNA . Patterns obtained with four-cutter endonucleases (such as AluI, DdeI, HinfI, and RsaI) made it possible to differentiate each species . S . cerevisiae and S . paradoxus presented a greater number of large fragments than S . pastorianus and S . bayanus with all the assay enzymes . With AluI and DdeI, species-specific bands clearly permitted differentiation between S . pastorianus and S . bayanus . To test the resolution of this method, wild Saccharomyces strains were analyzed . The correct assignment of these strains to a known taxon by this rapid method was confirmed by means of electrophoretic karyotyping.

J Cell Biol, 1994 Oct, 127(2), 567 - 79
Characterization of the yeast (1-->6)-beta-glucan biosynthetic components, Kre6p and Skn1p, and genetic interactions between the PKC1 pathway and extracellular matrix assembly; Roemer T et al.; A characterization of the S . cerevisiae KRE6 and SKN1 gene products extends previous genetic studies on their role in (1-->6)-beta-glucan biosynthesis (Roemer, T., and H . Bussey . 1991 . Yeast beta-glucan synthesis: KRE6 encodes a predicted type II membrane protein required for glucan synthesis in vivo and for glucan synthase activity in vitro . Proc . Natl . Acad . Sci . USA . 88:11295-11299; Roemer, T., S . Delaney, and H . Bussey . 1993 . SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis . Mol . Cell . Biol . 13:4039-4048) . KRE6 and SKN1 are predicted to encode homologous proteins that participate in assembly of the cell wall polymer (1-->6)-beta-glucan . KRE6 and SKN1 encode phosphorylated integral-membrane glycoproteins, with Kre6p likely localized within a Golgi subcompartment . Deletion of both these genes is shown to result in a dramatic disorganization of cell wall ultrastructure . Consistent with their direct role in the assembly of this polymer, both Kre6p and Skn1p possess COOH-terminal domains with significant sequence similarity to two recently identified glucan-binding proteins . Deletion of the yeast protein kinase C homolog, PKC1, leads to a lysis defect (Levin, D . E., and E . Bartlett-Heubusch . 1992 . Mutants in the S . cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect . J . Cell Biol . 116:1221-1229) . Kre6p when even mildly overproduced, can suppress this pkc1 lysis defect . When mutated, several KRE pathway genes and members of the PKC1-mediated MAP kinase pathway have synthetic lethal interactions as double mutants . These suppression and synthetic lethal interactions, as well as reduced beta-glucan and mannan levels in the pkc1 null wall, support a role for the PKC1 pathway functioning in cell wall assembly . PKC1 potentially participates in cell wall assembly by regulating the synthesis of cell wall components, including (1-->6)-beta-glucan.

J Cell Biol, 1994 Oct, 127(1), 129 - 38
ACT3: a putative centractin homologue in S . cerevisiae is required for proper orientation of the mitotic spindle; Clark SW et al.; As part of our ongoing efforts to understand the functional role of vertebrate centractins, we have identified a new member of the actin-related family of proteins in the yeast Saccharomyces cerevisiae using a PCR-based approach . Consistent with the current nomenclature for actin-related proteins in yeast, we propose to denote this locus ACT3 . The primary amino acid sequence of Act3p is most similar to canine and human alpha-centractin (73% similarity/54% identity) . The sequence of a genomic clone indicates ACT3 lies adjacent to and is transcribed convergently with respect to FUR1 on chromosome VIII . Molecular genetic analysis indicates ACT3 is represented by a single gene from which the corresponding mRNA is expressed at a low level compared to ACT1 . Tetrad analysis of heterozygotes harboring a TRP1 replacement of the ACT3-coding region indicates ACT3 is nonessential for growth under normal conditions and at extremes of temperature and osmolarity . However, growth at 14 degrees C indicates a spindle orientation defect similar to phenotypes recently described for yeast harboring mutations in actin, tubulin, or cytoplasmic dynein . Taken together, our data suggest that ACT3 is the S . cerevisiae homologue of vertebrate centractins.

J Cell Biol, 1994 Oct, 127(1), 107 - 15
Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose; Machesky LM et al.; We identified four polypeptides of 47, 44, 40, and 35 kD that bind to profilin-Sepharose and elute with high salt . When purified by conventional chromatography using an antibody to the 47-kD polypeptide, these four polypeptides copurified as a stoichiometric complex together with three additional polypeptides of 19, 18, and 13 kD that varied in their proportions to the other polypeptides . Partial protein sequences showed that the 47-kD polypeptide is a homologue of S . pombe act2 and the 44-kD polypeptide is a homologue of S . cerevisiae ACT2, both unconventional actins . The 40-kD polypeptide contains a sequence similar to the WD40 motif of the G beta subunit of a trimeric G-protein from Dictyostelium discoideum . From partial sequences, the 35-, 19-, and 18-kD polypeptides appear to be novel proteins . On gel filtration the complex of purified polypeptides cochromatograph with a Stokes' radius of 4.8 nm, a value consistent with a globular particle of 220 kD containing one copy of each polypeptide . Cell extracts also contain components of the complex that do not bind the profilin column . Affinity purified antibodies localize 47- and 18/19-kD polypeptides in the cortex and filopodia of Acanthamoeba . Antibodies to the 47-kD unconventional actin cross-react on immunoblots with polypeptides of similar size in Dictyostelium, rabbit muscle, and conventional preparations of rabbit muscle actin but do not react with actin.

Yeast, 1994 Oct, 10(10), 1285 - 96
Genetic and molecular analysis of hybrids in the genus Saccharomyces involving S . cerevisiae, S . uvarum and a new species, S . douglasii; Hawthorne D et al.; We have studied the phenomenon of infertility of yeast hybrids obtained with physiological conditions under the control of compatible mating systems . The yeasts investigated are three Saccharomyces species: S . cerevisiae, S . uvarum and a new species, S . douglasii . The diploid hybrids from crosses between these species sporulate well but are essentially infertile . The rare viable spores, one per 10(4) to 10(5) asci, that have been examined carry a complete genome comprised of chromosomes contributed by both parents but invariably have extra chromosomes, i.e . they are generally disomic for at least two or three chromosomes . This observation is consistent with a failure, in meiosis I, of the pairing and disjunction of homologous chromosomes which in most cases results in spores with an incomplete set of chromosomes . This apparent lack of pairing of 'homeologous' chromosomes in meiosis I was analysed in most detail with S . cerevisiae/S . douglasii hybrids . As a genetic tool we studied frequencies of recombination, taking advantage of an S . douglasii breeding stock of some 50 identified mutations in non-switching haploids . Recombination, although markedly reduced, could be observed at both the chromosomal and allelic levels, implying a sporadic pairing in meiosis to allow genetic exchange . Meiotic recombination frequencies were studied for 14 gene pairs and generally found to be reduced ten-fold . Heteroallelic recombination (gene conversion) frequencies were measured at 22 loci and were judged to be reduced at least two- to 100-fold . DNA hybridization experiments with S . cerevisiae gene probes gave results consistent with low DNA sequence homologies between S . cerevisiae and S . douglasii . Moreover, by change, our experiments disclosed another Saccharomyces strain (CBS2908, originally classified as S . cerevisiae) with hybridization patterns identical to S . douglasii except for the hybridization with the Ty transposon probes . Crosses between CBS2908 and S . douglasii yielded diploid hybrids with 80-90% spore viability, thus establishing a second member of the S . douglasii species.

Biol Chem Hoppe Seyler, 1994 Oct, 375(10), 695 - 703
Recombinant leech-derived tryptase inhibitor: construction, production, protein chemical characterization and inhibition of HIV-1 replication; Auerswald EA et al.; A synthetic gene coding for leech-derived tryptase inhibitor, form C (LDTI-C), was designed, cloned and expressed . The gene assembled via 6 oligonucleotides contains linker sequences, stop codons and internal restriction recognition sites for cloning, expression and cassette mutagenesis . Periplasmatic expression products could not be detected in Escherichia coli (E . coli), but strong expression was found using Saccharomyces cerevisiae (S . cerevisiae) ( > 10 mg/l culture broth) if a variant of pVT102U/alpha was used as vector . The secreted material was isolated after cross-flow filtration and purified by cation exchange chromatography . The recombinant material proved to be pure and homogeneous by electrophoretic and chromatographic analyses . Amino acid sequencing and molecular mass determination (4737.6 +/- 0.77 Da) by electrospray ionization mass spectrometry confirmed that rLDTI-C was processed correctly and that it is indistinguishable from LDTI-C . The far UV-CD (circular dichroism) spectrum of the recombinant inhibitor is typical for a small folded protein . rLDTI-C is inhibitorily fully active, its complexes with bovine trypsin and human mast cell tryptase display equilibrium dissociation constants which are nearly identical to those with the natural inhibitor . Remarkably, the inhibitor blocked replication of HIV-1 in HUT-78 cells at a concentration of 20 microM.

Glycobiology, 1994 Oct, 4(5), 697 - 702
Production, purification and characterization of recombinant yeast processing alpha 1,2-mannosidase; Lipari F et al.; The Saccharomyces cerevisiae processing alpha 1,2-mannosidase, which trims Man9GlcNAc to Man8GlcNAc, has a lumenally oriented catalytic domain and an N-terminal transmembrane domain . To obtain sufficient protein to study the structure and mechanism of action of this enzyme, the sequence encoding the catalytic domain was inserted downstream of the alpha-factor promoter and signal peptide in a high-copy vector for expression in S . cerevisiae as a secreted protein . Using oligosaccharide substrate (Glc1Man9GlcNAc or Man9GlcNAc), the medium of cells transformed with this plasmid showed an increase in alpha-mannosidase activity that was directly related to the increase in cell density, whereas no alpha-mannosidase activity was detected in cells transformed with vector alone . SDS-PAGE of the medium showed the presence of a doublet of 63 and 60 kDa that was revealed by Coomassie Blue staining and by Western blotting with antibodies to the endogenous solubilized alpha-mannosidase . The recombinant alpha-mannosidase was present in the medium at a level of approximately 1 mg/l and was purified in a single step by chromatography on S-Sepharose . High-resolution 1H NMR analysis of the Man8GlcNAc formed from Man9GlcNAc in the presence of the recombinant enzyme proved that it retained its specificity and removed only one specific alpha 1,2-mannose residue of the alpha 1,3 branch . Endoglycosidase H treatment decreased the molecular mass of both components of the doublet by approximately 5 kDa, showing that the heterogeneity is not due to differential N-glycosylation . EDTA inhibited the activity of the recombinant enzyme, but the inhibition was reversed by the addition of divalent cations.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Mol Biol Int, 1994 Oct, 34(4), 745 - 53
Regulation of mitochondrial cAMP-dependent protein kinase activity in yeast; Rahman MU et al.; We have shown that transcription of the yeast (S . cerevisiae) mitochondrial (mt) genome is cAMP-sensitive, via a mt cAMP-dependent protein kinase (cAPK) . In relation to that work, we examined whether the BCY 1 gene product functions as regulatory subunit for mt cAPK, as it does for the cytoplasmic enzyme . We demonstrate that mt protein extracts from a bcy 1 strain show no cAPK activity, whereas similar extracts from an otherwise isochromosomal BCY 1 strain show high levels of such activity . Partial purification of mt cAPK from each strain confirms this difference . Photoaffinity labeling with 8-N3{32P}cAMP and highly-purified mt protein extracts from the BCY 1 strain identifies one cAMP-binding protein (M(r) approximately 47000), while similar mt extracts from the bcy 1 strain lack all cAMP-binding proteins . These data suggest that BCY 1 regulates yeast mt cAPK, and that inactivation of BCY 1 removes that mt activity from cAMP control.

Genetics, 1994 Oct, 138(2), 283 - 7
Mapping of DBR1 and YPK1 suggests a major revision of the genetic map of the left arm of Saccharomyces cerevisiae Chromosome XI; Simchen G et al.; The Saccharomyces cerevisiae dbr1 mutation has been mapped on the left arm of chromosome XI . XIL is a chromosome arm that was until now rather sparsely populated with accurately mapped markers . On the basis of physical data, the overall order of markers is inverted relative to the existing genetic map of XI . We present tetrad analyses using a variety of markers on XI that indicate that the existing genetic map of XIL should be inverted, at least for the strains in which our mapping was carried out, and probably for other S . cerevisiae strains.

Braz J Med Biol Res, 1994 Oct, 27(10), 2359 - 64
The gene encoding eIF-2 beta in S . cerevisiae maps to chromosome XVI; Hsin CY et al.; The gene encoding the beta subunit of the translation initiation factor eIF-2 in the yeast Saccharomyces cerevisiae was mapped by physical methods to the distal part of the left arm of chromosome XVI, adjacent to the HSP90 locus . This assignment was confirmed by genetic linkage data with the GAL4 locus.

J Mol Biol, 1994 Sep 30, 242(4), 595 - 8
A voltage-gated chloride channel in the yeast Saccharomyces cerevisiae; Huang ME et al.; We report the sequencing and identification on chromosome X of Saccharomyces cerevisiae of an open reading frame whose product, designated yClC-1, displays significant structural similarity to a voltage-gated Cl- channel family . This putative protein contains 13 hydrophobic domains very similar to transmembrane domains exhibited by known members of this family . Some amino acids in the domains and at the loops between them are well conserved among all members . This is the first voltage-gated Cl- channel described in the yeast S . cerevisiae . The identification of yClC-1 will facilitate the functional analysis of Cl- channels in general, and should also assist in the identification of other ClC genes in higher eukaryotes.

Cell, 1994 Sep 23, 78(6), 1027 - 37
A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins; Rouse J et al.; MAPK-activated protein kinase-2 (MAPKAP kinase-2) is activated in vitro by the p42 and p44 isoforms of MAPK (p42/p44MAPK) . In several cell lines, however, MAPKAP kinase-2 is activated by sodium arsenite, heat shock, or osmotic stress and not by agonists that activate p42/p44MAPK . We have identified a MAPK-like enzyme that acts as a MAPKAP kinase-2 reactivating kinase (RK) . RK is recognized by an antiserum raised against a Xenopus MAPK (Mpk2), which is most similar to HOG1 from S . cerevisiae . We also identified a RK kinase (RKK) on the basis of its ability to activate either RK or a GST-Mpk2 fusion protein . The RKK, RK, and MAPKAP kinase-2 constitute a new stress-activated signal transduction pathway in vertebrates that is distinct from the classical MAPK cascade.

J Immunol, 1994 Sep 15, 153(6), 2544 - 53
Immunogenicity of the C-terminal 19-kDa fragment of the Plasmodium falciparum merozoite surface protein 1 (MSP1), YMSP1(19) expressed in S . cerevisiae; Hui GS et al.; The immunogenicity of the C-terminal 19-kDa fragment of Plasmodium falciparum MSP1 expressed in yeast as a nonfusion product, YMSP1(19), was studied . Immunization with YMSP1(19) in rabbits induced high titers of Abs specific for native conformational epitopes on parasite MSP1 . In mice, immunogenicity was dependent on the mouse strain and the adjuvant formulation . This suggests that different adjuvants may alter the immunogenicity of MSP1(19) in a genetically diverse population . Although YMSP1(19) induced anti-MSP1 Abs, they did not inhibit in vitro parasite growth . This contrasts with the strong inhibitory activities of Abs produced against a recombinant MSP1(42) (BVp42), which contains the entire MSP1(19) coding sequence . Further analyses showed that YMSP1(19) was the target of the inhibitory, anti-BVp42 Abs because YMSP1(19) could completely block binding of anti-BVp42 Abs to parasite MSP1 or BVp42 . Moreover, depletion of YMSP1(19)-specific Abs completely abolished the parasite inhibitory activities of anti-BVp42 sera . Anti-YMSP1(19) sera did not block the inhibitory activities of anti-BVp42 sera, suggesting that inhibitory epitopes were not in close structural proximity with noninhibitory epitopes . The finding that YMSP1(19) possessed inhibitory epitopes but induced anti-MSP1 Abs that were not inhibitory suggests that although the T-epitope(s) produced by immunization with YMSP1(19) could provide help for Ab production, it did not induce an effective inhibitory Ab response . We hypothesize that the nature/specificity of T helper epitopes on MSP1 may be crucial in efficient induction of biologically relevant and/or protective Abs.

Biochem J, 1994 Sep 15, 302 ( Pt 3), 709 - 16
Inositol trisphosphate metabolism in Saccharomyces cerevisiae: identification, purification and properties of inositol 1,4,5-trisphosphate 6-kinase; Estevez F et al.; Ins(1,4,5)P3 metabolism was examined in Saccharomyces cerevisiae extracts . S . cerevisiae contains readily detectable Ins(1,4,5)P3 kinase activity that is predominantly soluble, but phosphomonoesterase activity acting on Ins(1,4,5)P3 was not detected in either soluble or particulate preparations from this organism . We have purified the kinase activity approximately 685-fold in a rapid four-step process, and obtained a stable preparation . The enzyme has an apparent native molecular mass of approximately 40 kDa, and displays Michaelis-Menten kinetics with respect to its two substrates, ATP and Ins(1,4,5)P3 . The Km for ATP was 2.1 mM, and that for Ins(1,4,5)P3 was 7.1 microM . The enzyme appeared to be the first step in the conversion of Ins(1,4,5)P3 into an InsP5, and the partially purified preparation contained another activity that converted the InsP4 product into an InsP5 . The InsP4 product of the partially purified kinase was not metabolized by human erythrocyte ghosts and co-chromatographed with an Ins(3,4,5,6)P4 {L-Ins(1,4,5,6)P4} standard, identifying it as D-Ins(1,4,5,6)P4 . The yeast enzyme is thus an Ins(1,4,5)P3 6-kinase . This activity may be an important step in the production of inositol polyphosphates such as InsP5 and InsP6 in S . cerevisiae.

FEBS Lett, 1994 Sep 5, 351(2), 263 - 6
A 70-kDa heat shock cognate protein suppresses the defects caused by a proteasome mutation in Saccharomyces cerevisiae; Ohba M; An allele of mutation in the proteasome subunit gene Y7, y7-1, caused a temperature-sensitive growth in S . cerevisiae . One of the multi-copy suppressor genes for this growth defect was identical to SSB1, which encodes a 70-kDa heat shock cognate protein of the yeast . Introduction of the multi-copy SSB1 gene into the y7-1 mutant cells suppressed defects in the degradation of X-beta-galactosidase (X = Arg or Pro) observed in the mutant cells . Thus, the SSB1 protein, one of the chaperons of the yeast, facilitated intracellular protein degradation.

Gene, 1994 Sep 2, 146(2), 215 - 9
The STL1 gene of Saccharomyces cerevisiae is predicted to encode a sugar transporter-like protein; Zhao S et al.; A gene has been cloned from the yeast Saccharomyces cerevisiae which, on the basis of the deduced translation product, encodes a sugar transporter-like protein . This gene, STL1, was identified as an open reading frame (ORF) closely linked to the cinnamic-acid-resistance gene POF1 on chromosome IV . The putative translation product of STL1 (STL1) contains 536 amino acids, with a M(r) of 60,507 . Hydropathy analysis of STL1 suggests that it contains the twelve transmembrane (TM) domains characteristic of a family of sugar transporters from S . cerevisiae and other organisms . STL1 displays greatest homology (28% identity) to the products of the yeast HXT2 (hexose transporter) and GAL2 (galactose transporter) genes . Disruption of STL1 had no detectable effect on yeast growth on glucose, galactose, mannose, maltose or glycerol as sole carbon source . The transport function of the gene product remains unknown at present.

Biochem J, 1994 Sep 1, 302 ( Pt 2), 479 - 85
Yeast acyl-CoA-binding protein: acyl-CoA-binding affinity and effect on intracellular acyl-CoA pool size; Knudsen J et al.; Acyl-CoA-binding protein (ACBP) is a 10 kDa protein characterized in vertebrates . We have isolated two ACBP homologues from the yeast Saccharomyces carlsbergensis, named yeast ACBP types 1 and 2 . Both proteins contain 86 amino acid residues and are identical except for four conservative substitutions . In comparison with human ACBP, yeast ACBPs exhibit 48% (type 1) and 49% (type 2) conservation of amino acid residues . The amino acid sequence of S . carlsbergensis ACBP type 1 was found to be identical with the one ACBP present in Saccharomyces cerevisiae . A recombinant form of this protein was expressed in Escherichia coli and S . cerevisiae, purified, and its acyl-CoA-binding properties were characterized by isoelectric focusing and microcalorimetric analyses . The yeast ACBP was found to bind acyl-CoA esters with high affinity (Kd 0.55 x 10(-10) M) . Overexpression of yeast ACBP in S . cerevisiae resulted in a significant expansion of the intracellular acyl-CoA pool . Finally, Southern-blotting analysis of the two genes encoding ACBP types 1 and 2 in S . carlsbergensis strongly indicated that this species is a hybrid between S . cerevisiae and Saccharomyces monacensis.

Mol Cell Biol, 1994 Sep, 14(9), 6164 - 70
C25, an essential RNA polymerase III subunit related to the RNA polymerase II subunit RPB7; Sadhale PP et al.; We identified a partially sequenced Saccharomyces cerevisiae gene which encodes a protein related to the S . cerevisiae RNA polymerase II subunit, RPB7 . Several lines of evidence suggest that this related gene, YKL1, encodes the RNA polymerase III subunit C25 . C25, like RPB7, is present in submolar ratios, easily dissociates from the enzyme, is essential for cell growth and viability, but is not required in certain transcription assays in vitro . YKL1 has ABF-1 and PAC upstream sequences often present in RNA polymerase subunit genes . The sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of the YKL1 gene product is equivalent to that of the RNA polymerase III subunit C25 . Finally, a C25 conditional mutant grown at the nonpermissive temperature synthesizes tRNA at reduced rates relative to 5.8S rRNA, a hallmark of all characterized RNA polymerase III mutants.

Mol Cell Biol, 1994 Sep, 14(9), 6135 - 42
The RAD7 and RAD16 genes, which are essential for pyrimidine dimer removal from the silent mating type loci, are also required for repair of the nontranscribed strand of an active gene in Saccharomyces cerevisiae; Verhage R et al.; The rad16 mutant of Saccharomyces cerevisiae was previously shown to be impaired in removal of UV-induced pyrimidine dimers from the silent mating-type loci (D . D . Bang, R . A . Verhage, N . Goosen, J . Brouwer, and P . van de Putte, Nucleic Acids Res . 20:3925-3931, 1992) . Here we show that rad7 as well as rad7 rad16 double mutants have the same repair phenotype, indicating that the RAD7 and RAD16 gene products might operate in the same nucleotide excision repair subpathway . Dimer removal from the genome overall is essentially incomplete in these mutants, leaving about 20 to 30% of the DNA unrepaired . Repair analysis of the transcribed RPB2 gene shows that the nontranscribed strand is not repaired at all in rad7 and rad16 mutants, whereas the transcribed strand is repaired in these mutants at a fast rate similar to that in RAD+ cells . When the results obtained with the RPB2 gene can be generalized, the RAD7 and RAD16 proteins not only are essential for repair of silenced regions but also function in repair of nontranscribed strands of active genes in S . cerevisiae . The phenotype of rad7 and rad16 mutants closely resembles that of human xeroderma pigmentosum complementation group C (XP-C) cells, suggesting that RAD7 and RAD16 in S . cerevisiae function in the same pathway as the XPC gene in human cells . RAD4, which on the basis of sequence homology has been proposed to be the yeast XPC counterpart, seems to be involved in repair of both inactive and active yeast DNA, challenging the hypothesis that RAD4 and XPC are functional homologs.

Microbiology, 1994 Sep, 140 ( Pt 9), 2217 - 23
Sexual agglutination substances require a 'carrier' glycoprotein for integration into the cell wall of Saccharomyces cerevisiae; Yamaguchi M et al.; Sexual agglutination, caused by agglutination substance (AS) on a and alpha cell walls, is the first indispensable step of the mating reaction in ascosporogenous yeasts including Saccharomyces cerevisiae . The AS biosynthetic process in S . cerevisiae was investigated by pulse label-chase experiments with analysis by polyacrylamide gel electrophoresis (PAGE) for 16 h in the presence of urea . Because of its low mobility, AS can be separated from other proteins by prolonged PAGE . Nascent AS was integrated into cell walls after it linked covalently to a 'carrier' glycoprotein . The results suggest that the 'carrier' is synthesized stepwise through three distinct precursors (III-->II-->I) . The 'carrier' glycoprotein (I) and its precursors (II, III) were synthesized in both a, alpha haploid and a/alpha diploid cells . The N-glycosylation linkage inhibitor, tunicamycin, and protein synthesis inhibitor, puromycin, inhibited the III to I maturation . The results indicated that both the 'carrier' and the nascent active site of AS linked to the 'carrier' are integrated into the wall in a haploid cell while the 'carrier' alone is integrated in a diploid cell.

Neuron, 1994 Sep, 13(3), 555 - 66
Mutations in the Drosophila Rop gene suggest a function in general secretion and synaptic transmission; Harrison SD et al.; The Drosophila protein Rop shows similarity with the Sec1p protein of S . cerevisiae . Sec1p has an essential role in secretion, whereas most related proteins from higher organisms are hypothesized to function in neurotransmitter release . We show that, like the latter proteins, Rop is expressed in the nervous system, but it is expressed in other tissues as well, many of which are actively engaged in secretion . We have isolated mutations in the Rop gene and find that the extracellular accumulation of a number of normally secreted cellular products fails to occur in null mutant animals, which subsequently die at a late embryonic stage . Electrophysiological recordings on temperature-sensitive Rop mutants show that reductions in Rop activity result in a loss of the normal synaptic response to a light stimulus . These data suggest that a member of the Sec1p class of proteins has an in vivo function in both general secretion and synaptic transmission.

Cell Signal, 1994 Sep, 6(7), 793 - 812
Molecular cloning and expression, in both COS-1 cells and S . cerevisiae, of a human cytosolic type-IVA, cyclic AMP specific phosphodiesterase (hPDE-IVA-h6.1); Sullivan M et al.; Screening a human T lymphocyte cDNA library with a phosphodiesterase (PDE) specific probe resulted in the isolation of two overlapping cDNA clones, h2.2 and h6.1, that encode a type IV, rolipram inhibited cAMP-specific PDE . Clones h2.2 and h6.1 were 1015 bp and 2288 bp in length, respectively, and overlapped for 984 bp with only one nucleotide difference . The h6.1 cDNA was extended at the 5'-end by 1304 bp, with respect to h2.2, and encoded an incomplete ORF (lacking an initiation codon) of 668 amino acids . The merged nucleotide sequence of h6.1/h2.2 exhibited 99.5% homology in the ORF (ten nucleotide changes resulting in six amino acid changes), and 95% homology in the 3'-untranslated region, with the previously reported human PDE-IVA cDNA {Livi G . P., Kmetz P., Mchale M . M., Cieslinski L . B., Sathe G . M., Taylor D . P., Davis R . L., Torphy T . J . and Balcarek J . M . (1990) Mol . Cell Biol . 10, 2678-2686} . The sequence reported for h6.1/h2.2 matched that found for IVA clones isolated from three other human cDNA libraries, a human genomic cosmid clone and pcr amplified products of the exon covering these differences in two individuals . The h6.1 cDNA was engineered to generate a complete ORF by building in the 56 bp, including the initiation codon, present in hPDE-IVA-Livi and missing from the 5'-end of h6.1, producing a cognate ORF encoding a protein of 687 amino acids but differing in five amino acids which lay in or adjacent to the putative catalytic domain . The complete h6.1 ORF was engineered for expression in both Saccharomyces cerevisiae and in COS-1 cells . Integration of a single copy of the engineered ORF of h6.1, under the transcriptional control of a constitutive yeast promoter, at the pep4 locus of a S . cerevisiae strain lacking both yeast PDE genes resulted in functional complementation of the yeast pde-phenotype . Yeast strains with functional PDE were a light creamy white colour, while strains devoid of PDE activity were a dull brown colour . Expression of h6.1 in COS-1 cells led to the production of a typical type IV PDE activity in that cAMP, but not cGMP, served as substrate and its activity was insensitive to either Ca2+/CaM or cGMP but was inhibited by low concentrations of rolipram.(ABSTRACT TRUNCATED AT 400 WORDS)

Mol Biol Cell, 1994 Sep, 5(9), 933 - 42
Nonlethal sec71-1 and sec72-1 mutations eliminate proteins associated with the Sec63p-BiP complex from S . cerevisiae; Fang H et al.; The sec71-1 and sec72-1 mutations were identified by a genetic assay that monitored membrane protein integration into the endoplasmic reticulum (ER) membrane of the yeast Saccharomyces cerevisiae . The mutations inhibited integration of various chimeric membrane proteins and translocation of a subset of water soluble proteins . In this paper we show that SEC71 encodes the 31.5-kDa transmembrane glycoprotein (p31.5) and SEC72 encodes the 23-kDa protein (p23) of the Sec63p-BiP complex . SEC71 is therefore identical to SEC66 (HSS1), which was previously shown to encode p31.5 . DNA sequence analyses reveal that sec71-1 cells contain a nonsense mutation that removes approximately two-thirds of the cytoplasmic C-terminal domain of p31.5 . The sec72-1 mutation shifts the reading frame of the gene encoding p23 . Unexpectedly, the sec71-1 mutant lacks p31.5 and p23 . Neither mutation is lethal, although sec71-1 cells exhibit a growth defect at 37 degrees C . These results show that p31.5 and p23 are important for the trafficking of a subset of proteins to the ER membrane.

Cell Mol Biol (Noisy-le-grand), 1994 Sep, 40(6), 833 - 42
L-leucine transport systems in Saccharomyces cerevisiae participation of GAP1, S1 and S2 transport systems; Kotliar N et al.; L-leucine uptake in Saccharomyces cerevisiae is mediated by three different transport systems, S1, S2 and GAP1 . Their activities are dependent on the nitrogen source of the culture media . Wild type cells grown in L-proline exhibit a single transport system with high affinity and high Vmax that is partially inhibited by L-citrulline . A gap1 mutant shows two transport systems with Km and Vmax values similar to those previously described as S1 and S2, this transport activity is not inhibited by D-leucine, D-isoleucine or D-valine . Two systems can be also determined in wild type cells grown in rich medium containing a mixed nitrogen source where decreased GAP1 function is observed . In either wild type or gap1 cells grown in medium containing ammonium ions as sole nitrogen source, L-leucine uptake kinetics shows two systems with lower Vmax and similar Km values to those of the S1 and S2 systems . These results show that in S . cerevisiae GAP1, S1 and S2 participate in L-leucine entrance in cells grown in a poor nitrogen source, and that S1 and S2 are two ammonia-sensitive permeases that mediate the uptake in cells grown in a rich nitrogen source.

Anat Rec, 1994 Sep, 240(1), 32 - 41
Ultrastructural modifications of vesicular and Golgi elements in the Saccharomyces cerevisiae sec21 mutant at permissive and non-permissive temperatures; Rambourg A et al.; BACKGROUND: The secretory protein transit between cisternae of endoplasmic reticulum (ER) and Golgi elements is blocked when the yeast Saccharomyces cerevisiae sec21 mutant is shifted from the permissive (24 degrees C) to a non-permissive (37 degrees C) temperature, but 30-50 nm vesicles accumulate in the cytoplasm . At the semi-permissive temperature of 33 degrees C there is no complete block but rather a slowdown of the protein transport between ER and Golgi . The purpose of the present investigation is to analyze the structural expression of these events . METHODS: S . cerevisiae sec21 mutants were maintained for 90 min at semi-restrictive (33 degrees C) or restrictive (37 degrees C) temperatures and then progressively returned to 24 degrees C . Following fixation in glutaraldehyde and a postfixation in potassium ferrocyanide reduced osmium, 0.08 to 0.2 microns thick sections were cut from Epon embedded yeasts . Using the thicker sections, stereopairs of electron microscope photographs were prepared and used to visualize the three-dimensional configuration of the organelles . RESULTS: At permissive temperature, the Golgi elements appeared as isolated networks of membranous tubules dispersed throughout the cytoplasm . The diameter of these membranous tubules varied considerably from one Golgi element to another . Larger tubules showed at their intersections distensions with size and staining intensity comparable with that of the secretory granules seen at proximity of the Golgi networks or at the cell periphery . Small vesicles in the 30-50 nm size range were rarely if ever observed in cells grown at permissive temperature . Golgi networks and secretion granules were less conspicuous in mutant cells maintained at 33 degrees C and completely disappeared at 37 degrees C . In both cases, the main structural feature was the presence in the cytoplasm of numerous small vesicles and of short membranous tubules with a diameter identical to that of the small vesicles . As soon as 5 minutes after shifting mutants from 33 degrees C to 24 degrees C, the small vesicles disappeared from the cytoplasm, while secretory granules were actively produced in extensively developed Golgi network . When mutants were returned from 37 degrees C to 24 degrees C, the disappearance of small vesicles was more progressive and concomitant with the progressive reconstruction of Golgi networks . CONCLUSIONS: It is thus postulated that, in the above mentioned conditions, the small vesicles of the sec21 mutant did not act as intermediate carriers between the endoplasmic reticulum and a pre-existing Golgi apparatus, but rather fused together to produce newly formed Golgi networks.

Biosci Biotechnol Biochem, 1994 Sep, 58(9), 1685 - 93
Isolation and characterization of two chitin synthase genes of Rhizopus oligosporus; Motoyama T et al.; Two chitin synthase genes (chs1 and chs2) were isolated from Rhizopus oligosporus by plaque hybridization probed with the chitin synthase 2 gene of Saccharomyces cerevisiae . From their deduced amino acid sequences, they were both class II chitin synthases according to the classification proposed by Bowen et al . The expression of these genes was controlled differently in each stage of differentiation . It was suggested that the gene products of chs1 and chs2 function mainly in the hyphae growing stage but not in the late stage of spore formation . When each of these genes was expressed in S . cerevisiae, elevation of chitin synthase activity was observed in both cases.

Cell, 1994 Aug 26, 78(4), 669 - 79
A yeast actin-related protein homologous to that in vertebrate dynactin complex is important for spindle orientation and nuclear migration; Muhua L et al.; Spindle orientation controls nuclear migration and segregation during mitosis . In yeast, defects in dynein and astral microtubules lead to abnormal spindle orientation and nuclear migration . Dynactin complex is necessary for dynein-mediated vesicle motility in vitro . The major polypeptide of dynactin complex is an actin-related protein in the family Arp1 . We have identified in S . cerevisiae a novel actin-related gene, ACT5, in the Arp1 family . An act5 null mutant has defects in spindle orientation and nuclear migration, as does overexpression of Act5p . The phenotype of a double mutant lacking dynein and Act5p is similar to that of single mutants . Therefore, dynactin complex is in the same pathway as dynein and may be necessary for the action of dynein in vivo.

Proc Natl Acad Sci U S A, 1994 Aug 16, 91(17), 8165 - 9
Specificity of the mutator caused by deletion of the yeast structural gene (APN1) for the major apurinic endonuclease; Kunz BA et al.; The loss of bases from cellular DNA occurs via both spontaneous and mutagen-induced reactions . The resulting apurinic/apyrimidinic (AP) sites are cytotoxic and mutagenic but are counteracted by repair initiated by AP endonucleases . Previously, in vitro and bacterial transfection studies suggested that AP sites often prompt insertion of dAMP residues during replication, the A-rule . Dissimilar results have been obtained by transfecting DNA into eukaryotic cells . It seemed possible that these differences might be due to idiosyncrasies of transfection or aberrant replication of the transecting DNA . The observation that AP endonuclease-deficient strains of the yeast Saccharomyces cerevisiae have elevated spontaneous mutation rates allowed us to determine the mutational specificity of endogenously generated AP sites in nuclear DNA . With the yeast SUP4-o gene as a mutational target, we found that a deficiency in the major yeast AP endonuclease, Apn1, provoked mainly single base-pair substitution; the rate of transposon Ty insertion was also enhanced . The rate of transversion to a G.C pair was increased 10-fold in Apn1-deficient yeast, including a 59-fold increase in the rate of A.T-->C.G events . In contrast, the rate of transversion to an A.T pair was increased by only 3-fold . A deficiency in N3-methyladenine glycosylase offset these substitution rate increases, indicating that they are due primarily to AP sites resulting from glycosylase action . Thus, the A-rule does not seem to apply to the mutagenic processing of endogenous abasic sites in S . cerevisiae . Other results presented here show that AP endonuclease-deficient Escherichia coli exhibit a mutator phenotype consistent with the A-rule.

Eur J Biochem, 1994 Aug 15, 224(1), 71 - 9
Isolation of the ALG5 locus encoding the UDP-glucose:dolichyl-phosphate glucosyltransferase from Saccharomyces cerevisiae; Heesen S et al.; UDP-glucose:dolichyl-phosphate glucosyltransferase is a transmembrane-bound enzyme of the endoplasmic reticulum involved in protein N-linked glycosylation . This enzyme catalyzes the transfer of glucose from UDP-glucose to dolichyl phosphate . The structural gene encoding this transferase from Saccharomyces cerevisiae was isolated by complementation of an alg5-1 mutation . DNA sequencing of ALG5 revealed an open-reading frame of 1002 bases encoding a transmembrane protein of molecular mass 38.3 kDa . Overexpression of Alg5p in both yeast and Escherichia coli results in an increase of UDP-glucose:dolichyl-phosphate glucosyltransferase activity, whereas a deletion of the yeast gene leads to a loss of this activity and a concomitant underglycosylation of carboxypeptidase Y . The ALG5 protein has sequence similarity to the GDP-mannose:dolichyl-phosphate mannosyltransferase (Dpm1p) from S . cerevisiae . Topological studies indicate that UDP-glucose:dolichyl-phosphate glucosyltransferase is a transmembrane protein that spans the membrane several times.

FEBS Lett, 1994 Aug 15, 350(1), 1 - 4
The human ubiquitin-conjugating enzyme UbcH1 is involved in the repair of UV-damaged, alkylated and cross-linked DNA; Kaiser P et al.; The human ubiquitin-conjugating enzyme UbcH1 shows 69% identity to the Saccharomyces cerevisiae RAD6/UBC2 which plays a key role in DNA repair . To examine the function of UbcH1 (formerly named E2, M(r) 17,000), {(1990) EMBO J . 9, 1431-1435}) we tested its ability to functionally substitute for yeast RAD6/UBC2 in the recovery of cells from various DNA damage . Complementation by expression of the human UbcH1 cDNA revealed that the UbcH1 carries out the function of S . cerevisiae RAD6/UBC2 in the repair of UV-damaged, alkylated and cross-linked DNA.

Cell, 1994 Aug 12, 78(3), 499 - 512
Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S . cerevisiae; Choi KY et al.; Ste5 is a Zn2+ finger-like protein thought to function before three kinases, Ste11 (a MEKK), Ste7 (a MEK), and Fus3 (a MAPK), in a conserved MAP kinase cascade required for mating in S . cerevisiae . Here, we present evidence that Ste5 forms a multikinase complex that joins these kinases for efficient Fus3 activation . By two-hybrid analysis, Ste11, Ste7, and Fus3 associate with different domains of Ste5, while Kss1, another MAPK, associates with the same domain as Fus3, thus implying that Ste5 simultaneously binds a MEKK, MEK, and MAPK . Ste5 copurifies with Ste11, Fus3, and a hypophosphorylated form of Ste7, and all four proteins cosediment in a glycerol gradient as if in a large complex . Ste5 also increases the amount of Ste11 complexed to Ste7 and Fus3 and is required for Ste11 to function . These results substantiate a novel signal transduction component that physically links multiple kinases within a single cascade.

Nucleic Acids Res, 1994 Aug 11, 22(15), 3187 - 93
PRP28, a 'DEAD-box' protein, is required for the first step of mRNA splicing in vitro; Strauss EJ et al.; We previously reported the isolation of PRP28, a gene in Saccharomyces cerevisiae whose activity is required for the first step of nuclear mRNA splicing in vivo . Sequence analysis revealed that PRP28 is included in the 'DEAD-box' gene family, members of which are thought to function as ATP-dependent RNA helicases . Genetic interactions led us to suggest that PRP28 is functionally associated with the U4/U5/U6 snRNP . We have now purified the PRP28 protein from S . cerevisiae and demonstrated that it is required for the first step of splicing in vitro . Interestingly, PRP28 is not a stably associated snRNP protein . Strand displacement assays indicate that PRP28 does not exhibit RNA helicase activity, suggesting that an additional factor or factors may be required for its activation.

Gene, 1994 Aug 5, 145(2), 299 - 303
Efficient intra- and extracellular production of human beta-1,4-galactosyltransferase in Saccharomyces cerevisiae is mediated by yeast secretion leaders; Schwientek T et al.; We have compared the function of homologous and heterologous secretion leaders to mediate production of human beta-1,4-galactosyltransferase (Gal-Tf) in the yeast Saccharomyces cerevisiae . Although all genes encoding leader/Gal-Tf fusions were transcribed by strong yeast promoters, only low production levels were obtained for full-length Gal-Tf containing the human membrane-anchor region . In contrast, a gene fusion encoding the membrane-anchor region of the yeast alpha-1,2-mannosyltransferase (Mnt1) fused to soluble Gal-Tf yielded high mRNA and intracellular protein levels . Gal-Tf could also be produced extracellularly using a fusion of the pre-pro region of the yeast Mf alpha 1 precursor (MF alpha 1) to soluble Gal-Tf; a fusion containing only the pre-region of Mf alpha 1 was synthesized intracellularly, but did not lead to Gal-Tf activity in the culture medium . All yeast-produced Gal-Tf proteins were enzymatically active . These results demonstrate that yeast secretion leaders are advantageous to achieve efficient production of active Gal-Tf in S . cerevisiae.

Biochim Biophys Acta, 1994 Aug 3, 1193(2), 255 - 62
Constitutive expression of the human D2S-dopamine receptor in the unicellular yeast Saccharomyces cerevisiae; Sander P et al.; The cDNA for the human D2S-dopamine receptor has been functionally expressed in the unicellular yeast Saccharomyces cerevisiae . Two expression plasmids pRS421D2 (original D2S-gene coding region) and pRS421D2S (the first 24 aa of the yeast STE2-gene are fused to the N-terminus of the D2S-gene) were constructed and transformed into the protease deficient S . cerevisiae strain cI3-ABYS-86 . Northern blot analysis of total RNA from transformed yeast clones revealed that for both constructs the D2S-gene was constitutively transcribed from the plasmids PMA1 promoter . Membranes prepared from recombinant S . cerevisiae exhibited saturable binding with the antagonist {3H}methylspiperone . Competition studies revealed pharmacological properties for these sites which were comparable to those reported for the D2-receptor heterologously expressed in mammalian cells . The expression of the receptor was monitored by Western blot analysis using an antiserum raised against a peptide from the third intracellular domain of the receptor protein and by ligand binding assay.

Appl Environ Microbiol, 1994 Aug, 60(8), 2786 - 92
Molecular cloning, sequence analysis, and expression of the yeast alcohol acetyltransferase gene; Fujii T et al.; The ATF1 gene, which encodes alcohol acetyltransferase (AATase), was cloned from Saccharomyces cerevisiae and brewery lager yeast (Saccharomyces uvarum) . The nucleotide sequence of the ATF1 gene isolated from S . cerevisiae was determined . The structural gene consists of a 1,575-bp open reading frame that encodes 525 amino acids with a calculated molecular weight of 61,059 . Although the yeast AATase is considered a membrane-bound enzyme, the results of a hydrophobicity analysis suggested that this gene product does not have a membrane-spanning region that is significantly hydrophobic . A Southern analysis of the yeast genomes in which the ATF1 gene was used as a probe revealed that S . cerevisiae has one ATF1 gene, while brewery lager yeast has one ATF1 gene and another, homologous gene (Lg-ATF1) . Transformants carrying multiple copies of the ATF1 gene or the Lg-ATF1 gene exhibited high AATase activity in static cultures and produced greater concentrations of acetate esters than the control.

Appl Environ Microbiol, 1994 Aug, 60(8), 2754 - 8
Purification and partial characterization of a flocculin from brewer's yeast; Straver MH et al.; Analysis of a shear supernatant from flocculent, "fimbriated" Saccharomyces cerevisiae brewer's yeast cells revealed the presence of a protein involved in flocculation of the yeast cells and therefore designated a flocculin . The molecular mass of the flocculin was estimated to be over 300 kDa, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Gel permeation chromatography of the flocculin yielded an aggregate with an apparent molecular weight of > 2,000 . The flocculin was found to be protease sensitive, and the sequence of its 16 N-terminal amino acids revealed at least 69% identity with the predicted N terminus of the putative protein encoded by the flocculation gene FLO1 . The flocculin was isolated from flocculent S . cerevisiae cells, whereas only a low amount of flocculin, if any, could be isolated from nonflocculent cells . The flocculin was found to stimulate the flocculation ability of flocculent yeast cells without displaying lectinlike activity (that is, the ability to agglutinate yeast cells).

Mol Cell Biol, 1994 Aug, 14(8), 5501 - 9
Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant; Finley D et al.; The degradation of many proteins requires their prior attachment to ubiquitin . Proteolytic substrates are characteristically multiubiquitinated through the formation of ubiquitin-ubiquitin linkages . Lys-48 of ubiquitin can serve as a linkage site in the formation of such chains and is required for the degradation of some substrates of this pathway in vitro . We have characterized the recessive and dominant effects of a Lys-48-to-Arg mutant of ubiquitin (UbK48R) in Saccharomyces cerevisiae . Although UbK48R is expected to terminate the growth of Lys-48 multiubiquitin chains and thus to exert a dominant negative effect on protein turnover, overproduction of UbK48R in wild-type cells results in only a weak inhibition of protein turnover, apparently because the mutant ubiquitin can be removed from multiubiquitin chains . Surprisingly, expression of UbK48R complements several phenotypes of polyubiquitin gene (UB14) deletion mutants . However, UbK48R cannot serve as a sole source of ubiquitin in S . cerevisiae, as evidenced by its inability to rescue the growth of ubi1 ubi2 ubi3 ubi4 quadruple mutants . When provided solely with UbK48R, cells undergo cell cycle arrest with a terminal phenotype characterized by replicated DNA, mitotic spindles, and two-lobed nuclei . Under these conditions, degradation of amino acid analog-containing proteins is severely inhibited . Thus, multiubiquitin chains containing Lys-48 linkages play a critical role in protein degradation in vivo.

Mol Cell Biol, 1994 Aug, 14(8), 5223 - 8
SPT10 and SPT21 are required for transcription of particular histone genes in Saccharomyces cerevisiae; Dollard C et al.; The Saccharomyces cerevisiae genome contains four loci that encode histone proteins . Two of these loci, HTA1-HTB1 and HTA2-HTB2, each encode histones H2A and H2B . The other two loci, HHT1-HHF1 and HHT2-HHF2, each encode histones H3 and H4 . Because of their redundancy, deletion of any one histone locus does not cause lethality . Previous experiments demonstrated that mutations at one histone locus, HTA1-HTB1, do cause lethality when in conjunction with mutations in the SPT10 gene . SPT10 has been shown to be required for normal levels of transcription of several genes in S . cerevisiae . Motivated by this double-mutant lethality, we have now investigated the interactions of mutations in SPT10 and in a functionally related gene, SPT21, with mutations at each of the four histone loci . These experiments have demonstrated that both SPT10 and SPT21 are required for transcription at two particular histone loci, HTA2-HTB2 and HHF2-HHT2, but not at the other two histone loci . These results suggest that under some conditions, S . cerevisiae may control the level of histone proteins by differential expression of its histone genes.

Mol Cell Biol, 1994 Aug, 14(8), 5139 - 53
Casein kinase II mediates multiple phosphorylation of Saccharomyces cerevisiae eIF-2 alpha (encoded by SUI2), which is required for optimal eIF-2 function in S . cerevisiae; Feng L et al.; Previous studies have demonstrated that the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha), encoded by the SUI2 gene in the yeast Saccharomyces cerevisiae, is phosphorylated at Ser-51 by the GCN2 kinase in response to general amino acid control . Here we describe that yeast eIF-2 alpha is a constitutively phosphorylated protein species that is multiply phosphorylated by a GCN2-independent mechanism . 32Pi labeling and isoelectric focusing analysis of a SUI2+ delta gcn2 strain identifies eIF-2 alpha as radiolabeled and a single isoelectric protein species . Treatment of SUI2+ delta gcn2 strain extracts with phosphatase results in the identification of three additional isoelectric forms of eIF-2 alpha that correspond to the stepwise removal of three phosphates from the protein . Mutational analysis of SUI2 coupled with biochemical analysis of eIF-2 alpha maps the sites to the carboxyl region of SUI2 that correspond to Ser residues at amino acid positions 292, 294, and 301 that compose consensus casein kinase II sequences . 32Pi labeling or isoelectric focusing analysis of eIF-2 alpha from conditional casein kinase II mutants indicated that phosphorylation of eIF-2 alpha is abolished or dephosphorylated forms of eIF-2 alpha are detected when these strains are grown at the restrictive growth conditions . Furthermore, yeast casein kinase II phosphorylates recombinant wild-type eIF-2 alpha protein in vitro but does not phosphorylate recombinant eIF-2 alpha that contains Ser-to-Ala mutations at all three consensus casein kinase II sequences . These data strongly support the conclusion that casein kinase II directly phosphorylates eIF-2 alpha at one or all of these Ser amino acids in vivo . Although substitution of SUI2 genes mutated at these sites for the wild-type gene have no obvious effect on cell growth, one test that we have used appears to demonstrate that the inability to phosphorylate these sites has a physiological consequence on eIF-2 function in S . cerevisiae . Haploid strains constructed to contain Ser-to-Ala mutations at the consensus casein kinase II sequences in SUI2 in combination with a mutated allele of either the GCN2, GCN3, or GCD7 gene have synthetic growth defects . These genetic data appear to indicate that the modifications that we describe at the carboxyl end of the eIF-2 alpha protein are required for optimal eIF-2 function in S . cerevisiae.

Biochem Mol Biol Int, 1994 Aug, 33(5), 917 - 25
Expression cloning, purification and characterization of a beta-1,4-mannanase from Aspergillus aculeatus; Christgau S et al.; A cDNA library from the filamentous fungus Aspergillus aculeatus was constructed in the yeast expression vector pYES2.0 and used to isolate 57 full length cDNA's encoding beta-1,4-mannanase by expression in S . cerevisiae . The positive clones were identified on agar plates containing 0.2% azurine dyed cross-linked mannan by the formation of blue halos around the colonies . All clones represented transcripts of the same mannanase gene (man1) . The gene was sub-cloned into an Aspergillus expression vector and transformed into Aspergillus oryzae for overexpression and purification of the enzyme . The recombinant enzyme had a molecular weight of 45 kDa, an isoelectric point of pH 4.5, a pH optimum of pH 5.0 and a temperature optimum of 60-70 degrees.

Biochem Mol Biol Int, 1994 Aug, 33(5), 901 - 7
Accurate transcription initiation by RNA polymerase II from Candida utilis; Patturajan M et al.; An in vitro transcription system from Candida utilis is described . The template used is a hybrid plasmid containing Saccharomyces cerevisiae CYC1 promoter linked to a synthetic 377-bp G-minus casette (1) . In vitro transcriptions are carried out in the presence of RNase . T1 . Under these conditions only the transcripts that are resistant to RNase T1 accumulate . Using this protocol, it has been shown that in the absence of cytosolic factors RNA polymerase II (pol II) from C . utilis initiated RNA synthesis randomly . But both C . utilis and S . cerevisiae cell-free extracts could direct pol II from C . utilis to initiate transcription accurately . Results also indicated that the general transcription factors are functionally interchangeable between S . cerevisiae and C . utilis.

Cryobiology, 1994 Aug, 31(4), 383 - 92
Effect of moisture content on the invertase activity of freeze-dried S . cerevisiae; Pitombo RN et al.; The invertase activity of intact Saccharomyces cerevisiae submitted to freezing-thawing was affected by pH, the chemical nature of the buffer, and the freezing cooling rate (CR), leading in some cases to a complete invertase inactivation (acetate buffer, pH 4.0, CR = 0.5 degree C/min) . Once established under adequate freezing conditions the invertase activity remained unchanged after freeze-drying . Nevertheless, in some cases the cell-growing capability after freeze-drying diminished around 70%, mainly if the frozen cell suspension was attained through freezing carried out at CR = 0.5 degree C/min . Water sorption isotherms of freeze-dried samples (freeze-dryer Edwards L-4KR; 30 degrees C and 0.1 mB) were determined at 10 and 25 degrees C . The monolayer moisture content (MMC) at each temperature (12.7 and 3.71 for 10 and 25 degrees C, respectively) was calculated from isotherms by applying BET and GAB models . Freeze-dried yeast with water activity (Aw) between 0 and 0.33 (about the MMC value) maintained at 25 degrees C for 235 days and at 89 degrees C for 15 min retained at least 85% of its original invertase activity (IA), whereas samples with Aw > MMC lost at least 60% of its IA . X ray diffraction showed that the freeze-dried cake before and after storage presented an amorphous structure.

Protein Eng, 1994 Aug, 7(8), 1005 - 12
Thermosensitive mutants of Aspergillus awamori glucoamylase by random mutagenesis: inactivation kinetics and structural interpretation; Flory N et al.; Seven thermosensitive glucoamylase mutants generated by random mutagenesis and expressed in Saccharomyces cerevisiae were sequenced and their inactivation kinetics were determined . Wild-type glucoamylase expressed in S . cerevisiae was more glycosylated and more stable than the native Aspergillus niger enzyme . All mutants had lower free energies of inactivation than wild-type glucoamylase . In the Ala39-->Val, Ala302-->Val and Leu410-->Phe mutants, small hydrophobic residues were replaced by larger ones, showing that increases in size and hydrophobicity of residues included in hydrophobic clusters were destabilizing . The Gly396-->Ser and Gly407-->Asp mutants had very flexible residues replaced by more rigid ones, and this probably induced changes in the backbone conformation that destabilized the protein . The Pro128-->Ser mutation changed a rigid residue in an alpha-helix to a more flexible one, and destabilized the protein by increasing the entropy of the unfolded state . The Ala residue in the Ala442-->Thr mutation is in the highly O-glycosylated region surrounded by hydrophilic residues, where it may be a hydrophobic anchor linking the O-glycosylated arm to the catalytic core . It was replaced by a residue that potentially is O-glycosylated . In five of the seven mutations, residues that were part of hydrophobic microdomains were changed, confirming the importance of the latter in protein stability and structure.

Cell Signal, 1994 Aug, 6(6), 681 - 94
Interactions between adenylyl cyclase, CAP and RAS from Saccharomyces cerevisiae; Mintzer KA et al.; The adenylyl cyclase complex from Saccharomyces cerevisiae contains at least two subunits, a catalytic subunit of M(r) 200,000, encoded by CYR1 and a cyclase associated subunit, of apparent M(r) 70,000, encoded by CAP . The complex is a major effector of RAS proteins in S . cerevisiae . The interactions between CAP, adenylyl cyclase and RAS were explored in a strain of yeast that lacked CAP and contained an epitope tagged adenylyl cyclase . Adenylyl cyclase activity in this strain was not immunoprecipitated with anti-CAP antibodies, but was immunoprecipitated with anti-epitope antibodies . Two anti-CAP polyclonal antisera and five anti-CAP monoclonal antibodies were used in these studies . Like CAP-bound adenylyl cyclase, the CAP-free adenylyl cyclase was fully activated by yeast RAS2 . Transformation of cap strains with plasmids expressing portions of CAP allowed the adenylyl cyclase binding sites on CAP to be mapped by immunoprecipitation experiments . In other experiments, deletion mutations of adenylyl cyclase were used to map the CAP binding site on adenylyl cyclase . The adenylyl cyclase binding site localized to the amino one third of CAP (amino acids 1-168), and the CAP binding site localized to the carboxyl terminus of adenylyl cyclase (amino acids 1768-2026).

FEBS Lett, 1994 Jul 18, 348(3), 301 - 4
X-ray crystallographic studies of recombinant inorganic pyrophosphatase from Escherichia coli; Oganessyan VYu et al.; An E . coli inorganic pyrophosphatase overproducer and a method for a large-scale production of the homogeneous enzyme are described . The inorganic pyrophosphatase was crystallized in the form containing one subunit of a homohexameric molecule per asymmetric unit: space group R32, a = 110.4 A, c = 76.8 A . The electron density map to 2.5 A resolution phased with Eu- and Hg-derivatives (figure of merit, <m> = 0.51) was improved by the solvent flattening procedure (<m> = 0.77) . The course of the polypeptide chain and the secondary structure elements, intersubunit contacts and positions of the active sites were characterized . Homology with S . cerevisiae inorganic pyrophosphatase structure was found.

EMBO J, 1994 Jul 15, 13(14), 3395 - 400
Replicator dominance in a eukaryotic chromosome; Marahrens Y et al.; Replicators are genetic elements that control initiation at an origin of DNA replication (ori) . They were first identified in the yeast Saccharomyces cerevisiae as autonomously replicating sequences (ARSs) that confer on a plasmid the ability to replicate in the S phase of the cell cycle . The DNA sequences required for ARS function on a plasmid have been defined, but because many sequences that participate in ARS activity are not components of chromosomal replicators, a mutational analysis of the ARS1 replicator located on chromosome IV of S . cerevisiae was performed . The results of this analysis indicate that four DNA elements (A, B1, B2 and B3) are either essential or important for ori activation in the chromosome . In a yeast strain containing two closely spaced and identical copies of the ARS1 replicator in the chromosome, only one is active . The mechanism of replicator repression requires the essential A element of the active replicator . This element is the binding site for the origin recognition complex (ORC), a putative initiator protein . The process that determines which replicator is used, however, depends entirely upon flanking DNA sequences.

Biochim Biophys Acta, 1994 Jul 13, 1193(1), 107 - 17
Mutations in LIS1 (ERG6) gene confer increased sodium and lithium uptake in Saccharomyces cerevisiae; Welihinda AA et al.; A Saccharomyces cerevisiae mutant, lis1-1, hypersensitive to Li+ and Na+ was isolated from a wild-type strain after ethylmethane sulfonate mutagenesis . The rates of Li+ and Na+ uptake of the mutant are about 3-4-times higher than that of the wild-type; while the rates of cation efflux from the mutant and wild-type strains are indistinguishable . The LIS1 was isolated from a yeast genomic library by complementation of the cation hypersensitivity of the lis1-1 strain . LIS1 is a single copy, nonessential gene . However, the deletion of LIS1 from the wild-type results in a growth defect in addition to the cation hypersensitive phenotype . The order of increasing cation uptake rates of the wild-type and mutant strains, LIS1 < lis1-1 < lis1-delta 1::LEU2, correlates perfectly with the degree of cation hypersensitivity, suggesting that the cation hypersensitivity is primarily due to increased rates of cation influx . LIS1 encodes a membrane associated protein 384 amino acids long . Data base searches indicate that LIS1 is identical to ERG6 in S . cerevisiae which encodes a putative S-adenosylmethionine-dependent methyltransferase in the ergosterol biosynthetic pathway . Cell membranes of lis1 (erg6) mutants are known to be devoid of ergosterol and have altered sterol composition . Since membrane sterols can influence the activity of cation transporters, the increased cation uptake of the lis1 mutants may stem from an altered function of one or many different membrane transporters.

Mol Gen Genet, 1994 Jul 8, 244(1), 90 - 6
Cerulenin-resistant mutants of Saccharomyces cerevisiae with an altered fatty acid synthase gene; Inokoshi J et al.; Cerulenin, an antifungal antibiotic produced by Cephalosporium caerulens, is a potent inhibitor of fatty acid synthase in various organisms, including Saccharomyces cerevisiae . The antibiotic inhibits the enzyme by binding covalently to the active center cysteine of the condensing enzyme domain . We isolated 12 cerulenin-resistant mutants of S . cerevisiae following treatment with ethyl methanesulfonate . The mechanism of cerulenin resistance in one of the mutants, KNCR-1, was studied . Growth of the mutant was over 20 times more resistant to cerulenin than that of the wild-type strain . Tetrad analysis suggested that all mutants mapped at the same locus, FAS2, the gene encoding the alpha subunit of the fatty acid synthase . The isolated fatty acid synthase, purified from the mutant KNCR-1, was highly resistant to cerulenin . The cerulenin concentration causing 50% inhibition (IC50) of the enzyme activity was measured to be 400 microM, whereas the IC50 value was 15 microM for the enzyme isolated from the wild-type strain, indicating a 30-fold increase in resistance to cerulenin . The FAS2 gene was cloned from the mutant . Sequence replacement experiments suggested that an 0.8 kb EcoRV-HindIII fragment closely correlated with cerulenin resistance . Sequence analysis of this region revealed that the GGT codon encoding Gly-1257 of the FAS2 gene was altered to AGT in the mutant, resulting in the codon for Ser . Furthermore, a recombinant FAS2 gene, in which the 0.8 Kb EcoRV-HindIII fragment of the wild-type FAS2 gene was replaced with the same region from the mutant, when introduced into FAS2-defective S . cerevisiae complemented the FAS2 phenotype and showed cerulenin resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1994 Jul 8, 269(27), 18090 - 5
Probing close DNA contacts of RNA polymerase III transcription complexes with the photoactive nucleoside 4-thiodeoxythymidine; Bartholomew B et al.; Photoactive 4-thiodeoxythymidine 5'-triphosphate (4-S-dTTP) has been synthesized and used to enzymatically incorporate the corresponding nucleotide, 4-thiodeoxythymidine 5'-phosphate (4-S-dTMP), at specific positions of the Saccharomyces cerevisiae 5 S rRNA and SUP4 tRNA(Tyr) genes . RNA polymerase III transcription complexes have been assembled on this DNA and analyzed by photocrosslinking for proteins making close contact with DNA . Comparison DNA probes with a long-tether photoactive nucleotide 5-{N-(p-azidobenzoyl)-3-aminoallyl}-dUMP (N3RdUMP) incorporated at the same positions have also been analyzed, in order to compare the properties of these two crosslinking reagents . At least 10 of the 16 different S . cerevisiae polymerase III subunits make direct contact with DNA . The 120-kDa subunit of transcription factor (TF)IIIC, which is thought to play the key role in positioning TFIIIB upstream of the transcriptional start site, also contacts DNA near the transcriptional start site in TFIII(C+B) complexes with a SUP4 tRNA(Tyr) gene . The photocrosslinking patterns generated by 4-S-dTMP and N3RdUMP are distinctive, implying that these two reagents can yield complementary information about the structures of complex protein assemblies on DNA . Surprisingly, some subunits of the S . cerevisiae RNA polymerase III are crosslinked by 4-S-dTMP but not by N3RdUMP.

J Biol Chem, 1994 Jul 8, 269(27), 18037 - 46
Genetic analysis of the role of Saccharomyces cerevisiae acyl-CoA synthetase genes in regulating protein N-myristoylation; Johnson DR et al.; NMT1 is an essential Saccharomyces cerevisiae gene which encodes myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) . Nmt1p transfers myristate (C14:0), from myristoyl-CoA to the amino-terminal Gly residue of several essential cellular proteins . Little information is available about how myristoyl-CoA metabolism is regulated in eukaryotic cells . We have isolated and characterized three unlinked Fatty Acid Activation genes from S . cerevisiae, FAA1, FAA2, and FAA3 . In vitro biochemical assays reveal that the myristoyl-CoA synthetase activity of purified Faa2p is approximately equal to that of Faa1p, and two orders of magnitude greater than that of Faa3p . Analysis of NMT1 strains containing faa1, faa2, and/or faa3 null alleles indicates that Faa1p, Faa2p, and Faa3p are not essential for vegetative growth when de novo acyl-CoA synthesis by fatty acid synthetase (Fas) is active . S . cerevisiae strains containing nmt1-181 exhibit temperature-sensitive growth arrest and myristic acid auxotrophy due to the reduced affinity of its mutant protein product (nmtGly451-->Asp) for myristoyl-CoA . Comparison of the growth characteristics of isogenic NMT1 and nmt1-181 strains with all possible combinations of faa1, faa2, and faa3 null alleles, in the presence or absence of an active Fas complex, indicates that (i) Faa1p is responsible for activation of imported fatty acids to their CoA derivatives; (ii) Faa2p and Faa3p are able to access endogenous but not imported fatty acid substrates; (iii) nmt181p requires myristoyl-CoA production from both Fas and Faas for cells to remain viable at nonpermissive temperatures; (iv) Faa2p is unique among the three Faas in its ability, when overproduced, to partially rescue growth of a nmt1-181 strain at nonpermissive temperatures on yeast/peptone/dextrose (YPD) media without C14:0 supplementation; (v) acyl-CoAs produced by Faa1p, Faa2p, or Faa3p are not specifically targeted for beta-oxidation; and (vi) the ability of NMT1, faa1 delta, faa2 delta, faa3 delta strains to remain viable in the absence of an active Fas complex on YPD plus C14:0, or on media that contains fatty acids as the sole carbon source, suggests that S . cerevisiae contains other acyl-CoA synthetases which can activate imported fatty acids.

Oncogene, 1994 Jul, 9(7), 1891 - 8
Characterization of recombinant Xenopus MAP kinase kinases mutated at potential phosphorylation sites; Gotoh Y et al.; Xenopus mitogen-activated protein kinase kinase (MAPKK) previously inactivated with protein phosphatase 2A can be reactivated by serine phosphorylation catalyzed by a partially purified MAPKK kinase (MAPKK-K), and is phosphorylated by MAPK on a threonine residue . The sequence analysis of a threonine-phosphorylated tryptic peptide of Xenopus MAPKK from mature oocytes suggested that Thr388 is phosphorylated in vivo . A mutant MAPKK that has Thr388 changed to Ala (T388A-MAPKK) was not phosphorylated by purified MAPK, indicating that Thr388 is phosphorylated by MAPK . We then produced and analysed MAPKKs mutated at potential serine phosphorylation sites (S218A-MAPKK and S222A-MAPKK) . The wild-type MAPKK (WT-MAPKKK), T388A-MAPKK and a kinase-deficient (K97S)-MAPKK were phosphorylated efficiently by MAPKK-Ks purified from Xenopus eggs, and WT-MAPKK and T388A-MAPKK became activated . In contrast, neither S218A-MAPKK nor S222A-MAPKK was phosphorylated and activated efficiently by the Xenopus MAPKK-Ks . Similarly, WT-MAPKK, but not S218A-MAPKK or S222A-MAPKK, was activated efficiently by an active Raf-1 immunoprecipitate . However, when the recombinant STE11, a putative MAPKK-K in S . cerevisiae, was used as a source of MAPKK-K, S218A-MAPKK as well as WT-MAPKK, but not S222A-MAPKK, was phosphorylated and activated . Furthermore, replacement of Ser222 with an acidic residue (S222E) elevated substantially the basal kinase activity of MAPKK, while replacement of Ser218 (S218E) did not . These results may suggest an essential role for Ser222 phosphorylation in activating Xenopus MAPKK.

Plant Mol Biol, 1994 Jul, 25(4), 649 - 58
Isolation of an Arabidopsis thaliana casein kinase II beta subunit by complementation in Saccharomyces cerevisiae; Collinge MA et al.; Casein kinase II is thought to play an essential role in the control of cell division and differentiation in all eukaryotes . Through complementation of a defective casein kinase II catalytic subunit gene from Saccharomyces cerevisiae, we isolated an Arabidopsis thaliana casein kinase II regulatory subunit homologue, CKB1 . A second regulatory subunit was identified by low-stringency hybridization with CKB1 . Casein kinase II from S . cerevisiae is composed of two catalytic (alpha) and two regulatory (beta) subunits . Simultaneous disruption of the genes for the alpha and alpha' subunits, CKA1 and CKA2, respectively, is lethal . Strain YDH8 has disruptions of CKA1 and CKA2; its viability depends on a temperature-sensitive allele of CKA2, cka2-8, carried on a centromeric plasmid . We screened an A . thaliana cDNA library, whose inserts are under the control of the galactose-inducible GAL10 promoter, for cDNAs which enabled YDH8 cells to grow at the restrictive temperature . One cDNA, CKB1, was isolated by this screen which had homology to cDNAs of casein kinase II beta subunits . A second cDNA, CKB2, was isolated by hybridization and was also able to suppress the YDH8 mutant phenotype . The proteins encoded by CKB1 and CKB2 are 80% identical . The carboxy-terminal two thirds of both proteins is ca . 54% identical to the regulatory beta subunits of casein kinase II from other species . The amino termini are unrelated to any other known proteins . CKB1 and CKB2 lack the conserved autophosphorylation site characteristic of animal beta subunits, but have potential casein kinase II phosphorylation sites in the same region . Suppression of the cka1 delta cka2-8 mutant phenotype occurs by interaction of CKB1 with the defective, cka2-8-encoded, catalytic subunit . Cells with disruptions in CKA1 and CKA2 are not rescued by expression of CKB1.

Plant Physiol, 1994 Jul, 105(3), 897 - 902
Cloning of a cDNA encoding ATP sulfurylase from Arabidopsis thaliana by functional expression in Saccharomyces cerevisiae; Leustek T et al.; ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate . Here we report the cloning of a cDNA encoding ATP sulfurylase (APS1) from Arabidopsis thaliana . APS1 was isolated by its ability to alleviate the methionine requirement of an ATP sulfurylase mutant strain of Saccharomyces cerevisiae (yeast) . Expression of APS1 correlated with the presence of ATP sulfurylase enzyme activity in cell extracts . APS1 is a 1748-bp cDNA with an open reading frame predicted to encode a 463-amino acid, 51,372-D protein . The predicted amino acid sequence of APS1 is similar to ATP sulfurylase of S . cerevisiae, with which it is 25% identical . Two lines of evidence indicate that APS1 encodes a chloroplast form of ATP sulfurylase . Its predicted amino-terminal sequence resembles a chloroplast transit peptide; and the APS1 polypeptide, synthesized in vitro, is capable of entering isolated intact chloroplasts . Several genomic DNA fragments that hybridize with the APS1 probe were identified . The APS1 cDNA hybridizes to three species of mRNA in leaves (1.85, 1.60, and 1.20 kb) and to a single species of mRNA in roots (1.85 kb).

Cell, 1994 Jul 1, 77(7), 1083 - 92
The yeast KEM1 gene encodes a nuclease specific for G4 tetraplex DNA: implication of in vivo functions for this novel DNA structure; Liu Z et al.; We have previously reported the identification of a G4-DNA-dependent nuclease from S . cerevisiae that recognizes a tetrastranded G4-DNA structure and cuts in a single-stranded region 5' to the G4 structure . We purify this activity to homogeneity and show it to be the product of the S . cerevisiae KEM1 gene, which is also known as SEP1, DST2, XRN1, and RAR5 . Since a homozygous deletion of the KEM1 gene blocks meiotic cells at the 4N stage, the finding of these G4-dependent DNA binding and cleavage activities for the KEM1 gene product supports the hypothesis that G4-DNA may play a role in meiosis.

Cell, 1994 Jul 1, 77(7), 1027 - 36
Mutations in Hsp83 and cdc37 impair signaling by the sevenless receptor tyrosine kinase in Drosophila; Cutforth T et al.; A highly conserved signal cascade functions subsequent to receptor tyrosine kinase activation . Signaling by the sevenless receptor, required for differentiation of the R7 photoreceptor neuron in Drosophila, is reduced by mutations in E(sev)3A and E(sev)3B . We show here that E(sev)3A is a member of the Hsp90 family of stress proteins and that E(sev)3B encodes a homolog of the cell cycle control protein Cdc37 from S . cerevisiae . Mutations in E(sev)3B also dominantly enhance mutations in Dmcdc2, the gene encoding the p34 protein kinase that regulates the G2/M transition . Together, these data support a role for Hsp90 proteins in tyrosine kinase regulation and suggest that signals promoting neuronal differentiation may involve cell cycle control.

Yeast, 1994 Jul, 10(7), 953 - 8
LTE1 of Saccharomyces cerevisiae is a 1435 codon open reading frame that has sequence similarities to guanine nucleotide releasing factors; Keng T et al.; The DNA sequence of the LTE1 gene on the left arm of chromosome I of Saccharomyces cerevisiae has been determined . The LTE1 open reading frame comprises 4305 bp that can be translated into 1435 amino acid residues . The position of this open reading frame corresponds well to that of a 4.7 kb transcript that has been mapped to this position . The derived amino acid sequence has significant similarities to the amino acid sequence of the guanine nucleotide releasing factor isolated from a rat brain library . The carboxy-terminus of the LTE1 protein also shows similarities to other guanine nucleotide exchange factors of the S . cerevisiae CDC25 family.

Hum Mol Genet, 1994 Jul, 3(7), 1061 - 7
Isolation of new genes in distal Xq28: transcriptional map and identification of a human homologue of the ARD1 N-acetyl transferase of Saccharomyces cerevisiae; Tribioli C et al.; In this paper, we describe the physical and transcriptional organization of a region of 140 kb in Xq28, 5' to the L1CAM gene . By isolation and mapping of CpG islands to the physical map of the region, isolation of cDNAs, determination of partial nucleotide sequences and study of the pattern of expression and of the orientation of the transcripts identified we have established a transcriptional map of this region . In this map, previously identified genes (L1CAM, V2R, HCF1 and RnBP) have been positioned as well as 3 new genes . All genes in the region are rather small, ranging in size from 2 to 30 kb, and very close to one another . With the exception of the V2R gene, they are housekeeping, have a CpG island at their 5' end and the same orientation of transcription . This kind of organization is consistent with the one previously described for the more distal portion of Xq28, between the Color Vision (CV) and the G6PD genes and indicates that genes with housekeeping and tissue specific pattern of expression are interspersed in the genome but they are probably found in different 'transcriptional domains' . Among the new genes, TE2 demonstrated 40% identity with the protein N-acetyl transferase ARD1 of S . cerevisiae: TE2 may be the human homologue of the S . cerevisiae gene.

Biochem Mol Biol Int, 1994 Jul, 33(4), 769 - 74
Origin of the N-1, C-2 and C-2' atoms of the pyrimidine moiety of thiamin in Saccharomyces cerevisiae; Tazuya K et al.; The precursor of the pyrimidine moiety of thiamin in S . cerevisiae was studied . The tracers {15N}- and {2'-13C}-pyridoxine were chemically synthesized, and incorporated efficiently into the pyrimidine . The mass fragmentation pattern showed that the nitrogen atom of pyridoxine was incorporated into the N-1 of the pyrimidine . In addition, the C-2' atom of pyridoxine was incorporated into the C-2' atom of the pyrimidine . These results and the structural resemblance between the pyrimidine and pyridoxine revealed that the C-2, C-2' and N-1 unit of the pyrimidine are derived from the C-2, C-2' and N-1 unit of pyridoxine.

Anal Biochem, 1994 Jul, 220(1), 39 - 45
Inorganic pyrophosphatase-based detection systems . I . Detection and enumeration of cells; Nyren P et al.; A novel technique, useful for detection and enumeration of both pro- and eukaryotic cells, has been developed . The method relies on the detection by a very sensitive assay of an enzyme, the inorganic pyrophosphatase, which is constitutively expressed in all cells . This technique has been used to demonstrate detection and enumeration of both Escherichia coli and Saccharomyces cerevisiae cells . Cells were incubated, for a chosen time period and at a specific temperature, in a specific lysis buffer which contained both lysing activity and inorganic pyrophosphate . The activity of the inorganic pyrophosphatase released from the cells after lysis was determined by an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA; P . Nyren and A . Lundin (1985) Anal . Biochem . 151, 504-509) . The amount of PPi hydrolyzed was proportional to the logarithm of the cell number . The sensitivity of the assay was dependent on several factors, such as cell type, incubation time, and incubation temperature . E . coli cells at concentrations of 1 x 10(4) cells/ml could be detected in an assay performed at 58 degrees C for 10 min and S . cerevisiae cells at concentrations of 2 x 10(4) cells/ml at 49 degrees C within 10 min . The assay could be designed both as a present/absent test, e.g., for determination of bacteriuria, or as a quantitative assay . Determination and enumeration of all types of cells is in principle possible by the appropriate design of the experimental parameters . Possible applications for the approach in a wide variety of areas, such as fundamental and medical sciences, clinical laboratories, food and dairy industry, pharmaceutical industry, water sanitary plants, and biomass determination in general, are discussed.

Curr Genet, 1994 Jul, 26(1), 1 - 7
An analysis of the sequence of part of the right arm of chromosome II of S . cerevisiae reveals new genes encoding an amino-acid permease and a carboxypeptidase; Nasr F et al.; We have analysed two new genes, YBR1007 and YBR1015, discovered during the systematic sequencing of chromosome II of S . cerevisiae . YBR1007 shows strong similarities to amino-acid permeases, in particular the high-affinity proline permeases of S . cerevisiae and A . nidulans . The number and position of the predicted membrane-spanning domains suggest a conserved structure for these proteins, with 12 trans-membrane domains . YBR1015 shows strong similarities to serine carboxypeptidases; all three residues of the "catalytic triad" typical of this family of enzymes are conserved in the YBR1015 protein . In a preliminary functional analysis we have created a null allele of the YBR1015 gene, and shown that it is not essential for cellular viability.

Virus Res, 1994 Jul, 33(1), 11 - 25
Expression of the outer capsid proteins VP2 and VP5 of bluetongue virus in Saccharomyces cerevisiae; Martyn JC et al.; cDNAs transcribed from bluetongue virus serotype 1 (Australia) ds RNA 2 and ds RNA 6 coding for the major neutralising antigen VP2 and the outer capsid protein VP5, respectively, were amplified in polymerase chain reactions and ligated downstream of the copper-inducible metallothionein promoter in the yeast expression plasmid pYELC5 . Saccharomyces cerevisiae transformed with the recombinant plasmid pYELC5-VP2 expressed full-length VP2 only following induction with 1 mM CuSO4 and reached the maximum level after 6 h . In contrast, S . cerevisiae transformants harboring the recombinant plasmid pYELC5-VP5 expressed VP5 constitutively, although induction increased the level to a maximum after 4 h . A sheep trial was done testing the recombinant proteins, however it was shown that none of these were effective immunogens for eliciting a protective response against a subsequent challenge with bluetongue virus . An analysis of the yeast expression products for the VP2 outer coat protein using a panel of monoclonal antibodies showed that the yeast expressed VP2 was in a conformation different from native VP2 and hence probably unable to elicite an appropriate protective immune response.

J Cell Biol, 1994 Jul, 126(2), 305 - 15
Role of the chaperonin cofactor Hsp10 in protein folding and sorting in yeast mitochondria; Hohfeld J et al.; Protein folding in mitochondria is mediated by the chaperonin Hsp60, the homologue of E . coli GroEL . Mitochondria also contain a homologue of the cochaperonin GroES, called Hsp10, which is a functional regulator of the chaperonin . To define the in vivo role of the co-chaperonin, we have used the genetic and biochemical potential of the yeast S . cerevisiae . The HSP10 gene was cloned and sequenced and temperature-sensitive lethal hsp10 mutants were generated . Our results identify Hsp10 as an essential component of the mitochondrial protein folding apparatus, participating in various aspects of Hsp60 function . Hsp10 is required for the folding and assembly of proteins imported into the matrix compartment, and is involved in the sorting of certain proteins, such as the Rieske Fe/S protein, passing through the matrix en route to the intermembrane space . The folding of the precursor of cytosolic dihydrofolate reductase (DHFR), imported into mitochondria as a fusion protein, is apparently independent of Hsp10 function consistent with observations made for the chaperonin-mediated folding of DHFR in vitro . The temperature-sensitive mutations in Hsp10 map to a domain (residues 25-40) that corresponds to a previously identified mobile loop region of bacterial GroES and result in a reduced binding affinity of hsp10 for the chaperonin at the non-permissive temperature.

Mol Cell Biol, 1994 Jul, 14(7), 4633 - 42
Flexibility and interchangeability of polyadenylation signals in Saccharomyces cerevisiae; Heidmann S et al.; Various signal motifs have been reported to be essential for proper mRNA 3'-end formation in the yeast Saccharomyces cerevisiae . However, none of these motifs has been shown to be sufficient to direct 3'-end processing and/or transcription termination . Therefore, several structural motifs have to act in concert for efficient 3'-end formation . In the region upstream of the three polyadenylation sites of the yeast gene for alcohol dehydrogenase I (ADH1), we have identified a hitherto unknown signal sequence contained within the octamer AAAAAAAA . This motif, located 11 nucleotides upstream of the first ADH1 polyadenylation site, is responsible for the utilization of this site in vitro and in vivo, since mutational alteration drastically reduced 3'-end formation at this position . Insertion of 38 ADH1-derived nucleotides encompassing the (A)8 motif into the 3'-end formation-deficient cyc1-512 deletion mutant restored full processing capacity in vitro . Insertion of the octamer alone did not restore 3'-end formation, although mutation of the (A)8 motif in the functional construct had abolished 3'-end processing activity almost completely . This demonstrates that the sequence AAAAAAAA is a necessary, although not sufficient, signal for efficient mRNA 3'-end formation in S . cerevisiae.

Biosci Biotechnol Biochem, 1994 Jul, 58(7), 1292 - 6
A novel culture method for high level production of heterologous protein in Saccharomyces cerevisiae; Nagashima T et al.; A high level production system for heterologous protein by cold culture of yeast transformants at 15 degrees C was developed . The yeast transformants, carrying a plasmid containing cDNA for Aspergillus oryzae alpha-amylase (Taka-amylase A) or human lysozyme synthetic DNA, were cultivated in a selective medium for 1 or 2 days until full growth at 30 degrees C . The yeast cells were harvested by centrifugation from the culture fluid and then were transferred to YPD medium . These inoculated broths were incubated for 2 days at 15 degrees C and then for another 2 days at 30 degrees C . By the cold culture method described above, higher amounts of Taka-amylase A (28.6 mg/liter) and human lysozyme (6.1 mg/liter) were produced by the yeast transformants compared to those by conventional methods . Heterologous protein productions using YEp, YCp, and YIp types of yeast expression vectors with ADH1 or GAPDH promoter by the cold culture method showed effective productivity of about 2-fold compared to those by the conventional method of culture at 30 degrees C . The high level production of heterologous protein by this method was not specific to the S . cerevisiae strains examined.

Antimicrob Agents Chemother, 1994 Jul, 38(7), 1615 - 9
Potentiation of bleomycin cytotoxicity in Saccharomyces cerevisiae; Moore CW; Lesions introduced into cellular DNAs prelabeled with {2-14C}thymidine or {6-3H}thymidine, as well as cell killing, were inhibited by the presence of EDTA during 20-min reactions of Saccharomyces cerevisiae cells with the low-molecular-weight bleomycin family of anticancer antibiotics . In contrast, the level of killing by low concentrations of bleomycin was higher among cells which had grown for three generations in defined synthetic complete medium supplemented with ferrous sulfate than among cells grown without iron supplementation . In S . cerevisiae, the uptake of iron is facilitated by a plasma membrane ferric reductase activity and a high-affinity (Km = 5 x 10(-6) M) ferrous uptake system . Lethal effects of 1.3 x 10(-6) M bleomycin increased approximately 50% with 10(-5) M Fe(II), nearly twofold with 10(-4) M Fe(II), and 2.8 times with 10(-3) M Fe(II) . Thus, iron preloading is a new experimental approach to increasing and studying the effects of the glycopeptides on cellular DNAs and other cellular targets . This approach could also be used for studying and better understanding DNA repair genes and could serve as a model for studies of redox active chemicals in biological systems.

Mutat Res, 1994 Jul 1, 308(1), 43 - 51
Mutagenicity of 5-bromouracil and N6-hydroxyadenine studied by yeast oligonucleotide transformation assay; Noskov V et al.; The mutagenicity of 5-bromouracil (BrU) and N6-hydroxyadenine (HA) was tested by means of the yeast oligonucleotide transformation procedure . BrU-containing oligonucleotide was not mutagenic; although two mutants (per 200 micrograms oligonucleotide) were obtained, they were attributed to base insertion or base substitution at positions different from BrU . This result supports the view that BrU mutagenesis is dependent on intracellular nucleotide pool imbalance . In contrast, HA-containing oligonucleotide was highly mutagenic; 56 mutants (per 140 micrograms oligonucleotide) were obtained . Of 21 induced mutants examined, 20 had G and one had C at the HA position, a result indicating that HA-->G changes took place . To provide back-up evidence, we carried out a general reversion assay for base HA using a set of yeast tester strains, and the results showed that HA induces exclusively AT-to-GC and GC-to-AT transitions . We conclude that in S . cerevisiae HA is a classic base analog mutagen, causing AT-to-GC and GC-to-AT transitions by ambiguous base pairing . The present work has clearly demonstrated the usefulness of the oligonucleotide transformation procedure for elucidating mutagenicity of modified bases.

FEBS Lett, 1994 Jun 27, 347(2-3), 143 - 6
Yeast serine isoacceptor tRNAs: variations of their content as a function of growth conditions and primary structure of the minor tRNA(Ser)GCU; Heyman T et al.; The primary structure of Saccharomyces cerevisiae tRNA(Ser)GCU is presented (EMBL database accession No . X74268 S . cerevisiae tRNA-Ser) . In addition, quantitation of the relative amounts of serine isoaccepting tRNAs in yeast grown on different media showed that the minor tRNA(Ser)GCU decreased while the major tRNA(Ser)AGA increased as the growth rate and the cellular protein content increased . The minor species, tRNA(Ser)CGA and tRNA(Ser)UGA, were not separated by our gel system, however, taken together they appeared to vary in the same way as tRNA(Ser)GCU . These data suggest a growth rate dependence of yeast tRNAs similar to that previously described for E . coli tRNAs.

Biochemistry, 1994 Jun 21, 33(24), 7647 - 53
Copper amine oxidase: heterologous expression, purification, and characterization of an active enzyme in Saccharomyces cerevisiae; Cai D et al.; A copper amine oxidase gene from a methylotrophic yeast Hansenula polymorpha has been expressed in Saccharomyces cerevisiae under the control of the ADHI promoter and the recombinant protein purified to near homogeneity . The recombinant enzyme is as active as the native enzyme in catalyzing methylamine oxidation . We demonstrate that it is a quinoprotein by redox-cycling staining and titrations with carbonyl reagents . The absorption spectral properties of the recombinant amine oxidase and its phenylhydrazine derivative are very similar to those of other copper amine oxidases . The cofactor in the enzyme is 2,4,5-trihydroxyphenylalanine (topa) quinone, as demonstrated by the pH-dependent shift in the lambda max of the p-nitrophenylhydrazone adduct . Alignment of an active-site peptide and DNA-derived protein sequences reveals a tyrosine residue as the precursor to topa quinone, consistent with findings with other copper amine oxidases . All evidence presented herein indicates that the heterologously expressed copper amine oxidase protein is processed posttranslationally in S . cerevisiae to form an active enzyme with an intact cofactor . This occurs despite an inability of S . cerevisiae to utilize amines as a nitrogen source . The implications of this study for the mechanism of topa quinone biogenesis are discussed.

Nature, 1994 Jun 16, 369(6481), 578 - 81
RAD25 is a DNA helicase required for DNA repair and RNA polymerase II transcription; Guzder SN et al.; The RAD25 gene of Saccharomyces cerevisiae functions in nucleotide excision repair of ultraviolet-damaged DNA and is also required for cell viability . The RAD25 protein shows remarkable homology to the protein encoded by the human nucleotide-excision-repair gene XPB (ERCC3), mutations in which cause the cancer-prone disease xeroderma pigmentosum and also Cockayne's syndrome . Here we purify RAD25 protein from S . cerevisiae and show that it contains single-stranded DNA-dependent ATPase and DNA helicase activities . Extract from the conditional lethal mutant rad25-ts24 exhibits a thermolabile transcriptional defect which can be corrected by the addition of RAD25 protein, indicating a direct and essential role of RAD25 in RNA polymerase II transcription . The protein encoded by the rad25799am allele is defective in DNA repair but is proficient in RNA polymerase II transcription, indicating that RAD25 DNA-repair activity is separable from its transcription function . The rad25 Arg-392 encoded product, which contains a mutation in the ATP-binding motif, is defective in RNA polymerase II transcription, suggesting that the RAD25-encoded DNA helicase functions in DNA duplex opening during transcription initiation.

Eur J Biochem, 1994 Jun 15, 222(3), 843 - 50
Characterization of recombinant plant cinnamate 4-hydroxylase produced in yeast . Kinetic and spectral properties of the major plant P450 of the phenylpropanoid pathway; Urban P et al.; Helianthus tuberosus cinnamate 4-hydroxylase (CYP73 or CA4H), a member of the P450 superfamily which catalyses the first oxidative step of the phenylpropanoid pathway in higher plants by transforming cinnamate into p-coumarate, was expressed in the yeast Saccharomyces cerevisiae . The PCR-amplified CA4H open reading frame was inserted into pYeDP60 under the transcriptional control of a galactose-inducible artificial promoter . Engineered S . cerevisiae strains producing human P450 reductase or normal or overproduced amounts of yeast P450 reductase were transformed to express recombinant CA4H . When grown on galactose, yeast cells produced CA4H holoprotein bound to the endoplasmic reticulum membrane as judged from the reduced iron/carbon monoxide difference spectrum centered at 452 nm and from typical cinnamate 4-hydroxylase activity upon coupling with the different P450 reductases and NADPH . Some CA4H protein was found also addressed to the yeast mitochondria but as a low-activity form . The spectral and kinetic characterizations of the yeast-produced CA4H in different redox protein environments are presented using both assays on yeast microsomal fractions and bioconversions on living cells . Results indicate that the microsomal system constituted by the overexpressed yeast P450 reductase and CA4H is characterized by a 1:1 coupling between NADPH oxidation and cinnamate hydroxylation and by one of the highest turnover numbers reported for an NADPH-dependent P450 reaction . Based on spectral perturbation and inhibition studies, coumarate appeared to have no detectable affinity for the enzyme . A possible geometry of the substrate recognition pocket is discussed in the light of these data.

Nucleic Acids Res, 1994 Jun 11, 22(11), 1981 - 7
Interaction between the first and last nucleotides of pre-mRNA introns is a determinant of 3' splice site selection in S . cerevisiae; Chanfreau G et al.; The splicing of group II and nuclear pre-mRNAs introns occurs via a similar splicing pathway and some of the RNA-RNA interactions involved in these splicing reactions show structural similarities . Recently, genetic analyses performed in a group II intron and the yeast nuclear actin gene suggested that non Watson-Crick interactions between intron boundaries are important for the second splicing step efficiency in both classes of introns . We here show that, in the yeast nuclear rp51A intron, a G to A mutation at the first position activates cryptic 3' splice sites with the sequences UAC/ or UAA/ . Moreover, the natural 3' splice site could be reactivated by a G to C substitution of the last intron nucleotide . These results demonstrate that the interaction between the first and last intron nucleotides is a conserved feature of nuclear pre-mRNA splicing in yeast and is involved in the mechanism of 3' splice site selection.

J Biol Chem, 1994 Jun 10, 269(23), 16348 - 56
Biochemical studies of three Saccharomyces cerevisiae acyl-CoA synthetases, Faa1p, Faa2p, and Faa3p; Knoll LJ et al.; The efficiency and specificity of protein N-myristoylation appear to be influenced by the availability of myristoyl-CoA and other potential acyl-CoA substrates of myristoyl-CoA:protein N-myristoyltransferase . Recent studies have revealed that Saccharomyces cerevisiae contains at least three acyl-CoA synthetase genes (FAA for fatty acid activation) . We have expressed Faa1p, Faa2p, and Faa3p in a strain of Escherichia coli that lacks its own endogenous acyl-CoA synthetase (FadD) . Each S . cerevisiae acyl-CoA synthetase contained a carboxyl-terminal His tag so that it could be purified to homogeneity in a single step using nickel chelate affinity chromatography . In vitro assays of C3:0-C24:0 fatty acids indicate that Faa1p prefers C12:0-C16:0, with myristic and pentadecanoic acid (C15:0) having the highest activities . Faa2p can accommodate a wider range of acyl chain lengths: C9:0-C13:0 are preferred and have equivalent activities, although C7:0-C17:0 fatty acids are tolerated as substrates with no greater than a 2-fold variation in specific activity . The myristoyl-CoA synthetase activities of Faa1p and Faa2p are 2 orders of magnitude greater than that of Faa3p in vitro . Faa3p has a preference for C16 and C18 fatty acids with a cis-double bond at C-9-C-10 . The temperature optimum for Faa1p is 30 degrees C, while Faa2p and Faa3p have the greatest activities at 25 degrees C . These in vitro observations were confirmed using two in vivo assays: (i) measurement of the ability of each S . cerevisiae acyl-CoA synthetase to direct the incorporation of exogenously derived tritiated myristate, palmitate, or oleate into cellular phospholipids produced in a fadD- strain of E . coli during exponential growth at 24 or 37 degrees C and (ii) measurement of the incorporation of {3H}myristate into a yeast N-myristoylprotein coexpressed with Nmt1p and Faa1p, Faa2p, or Faa3p in the fadD- strain.

J Bacteriol, 1994 Jun, 176(12), 3730 - 7
Expression of high-affinity glucose transport protein Hxt2p of Saccharomyces cerevisiae is both repressed and induced by glucose and appears to be regulated posttranslationally; Wendell DL et al.; Expression of putative high-affinity glucose transport protein Hxt2p of Saccharomyces cerevisiae was repressed 15- to 20-fold in high concentrations of glucose or fructose . S . cerevisiae with either the ssn6-delta 9 or the hxk2-delta 1::URA3 mutation, each of which relieves glucose repression, exhibited high Hxt2p expression in both 2.0% glucose (normally repressing) and 0.05% glucose (normally derepressing) while S . cerevisiae with the snf1-delta 10 mutation, which causes constitutive repression, did not detectably express Hxt2p in either glucose concentration . In addition to repressing at high concentrations, glucose or fructose is required for induction of Hxt2p expression . Hxt2p was not expressed by wild-type S . cerevisiae in media containing only ethanol or galactose as carbon and energy source but was expressed if glucose was added . An hxk2-delta 1::URA3 mutant did not detectably express Hxt2p in ethanol or galactose, but an ssn6-delta9 mutant did highly express Hxt2p in both carbon sources . Thus, simple relief of glucose repression as occurs with hxk2 null mutants is insufficient for high-level Hxt2p expression . Mutation of ssn6, a general transcriptional repressor, does lead to Hxt2p expression in the absence of glucose induction, suggesting relief of an additional negative regulatory system . High expression of Hxt2p does not always result in HXT2-dependent high-affinity transport, implying that Hxt2p activity is regulated posttranslationally . In the high glucose condition for the ssn6 mutant, high-affinity glucose transport is derepressed . Deletion of the HXT2 locus does not diminish this level of transport . However, high-affinity glucose transport is diminished in the ssn6-delta9 hxt2 delta1 double mutant compared with ssn6-delta9 alone in low glucose . Thus, while constitutively expressed in ssn6 mutants, Hxt2p only appears to be active as a transporter under low-glucose conditions . Similarly, Hxt2p was found to be expressed under low-glucose conditions in an snf3 mutant which does not display high-affinity uptake . This finding suggests that SNF3 may be involved in the posttranslational regulation of Hxt2p.

Biochim Biophys Acta, 1994 Jun 1, 1192(1), 7 - 13
Trehalose lowers membrane phase transitions in dry yeast cells; Leslie SB et al.; Recent work has clearly demonstrated a direct correlation between the amount of trehalose present in the yeast Saccharomyces cerevisiae and its ability to tolerate dehydration, but has failed to elucidate the specific role played by trehalose . By using Fourier transform infrared spectroscopy we measured the transition temperature of phospholipids in both intact S . cerevisiae and isolated plasma membranes dried in the presence and absence of trehalose . Our results show that trehalose lowers the temperature of the dry gel to liquid crystal phase transition in yeast from around 60 degrees C to about 40 degrees C, thus allowing yeast rehydrated above 40 degrees C to avoid the damaging effects of passing through a phase transition . These results explain both the need for trehalose and the observation that yeast must be rehydrated with warm water if they are to remain viable . Only when trehalose is present is the dry transition within a physiologically tolerable range and only when the cells are rehydrated above 40 degrees C will they avoid passing through a phase transition.

Mol Cell Biol, 1994 Jun, 14(6), 3719 - 28
Equivalent mutations in the two repeats of yeast TATA-binding protein confer distinct TATA recognition specificities; Arndt KM et al.; To investigate the process of TATA box recognition by the TATA box-binding protein (TBP), we have performed a detailed genetic and biochemical analysis of two Saccharomyces cerevisiae TBP mutants with altered DNA-binding specificity . The mutant proteins have amino acid substitutions (Leu-205 to Phe and Leu-114 to Phe) at equivalent positions within the two repeats of TBP that are involved in TATA element binding . In an in vivo assay that employs a nearly complete set of single point mutations of the consensus TATAAA sequence, one of the TBP mutants (TBP-L114F) recognizes the sequence TATAAG, while the other TBP mutant (TBP-L205F) recognizes one substitution at the first position of the TATA element, CATAAA, and three substitutions at the 3' end of the TATA box . Specificity patterns determined from in vitro transcription experiments with purified recombinant wild-type TBP and TBP-L205F agree closely with those observed in vivo, indicating that altered TATA utilization in the mutant strains is a direct consequence of altered TATA recognition by the mutant TBPs . The distinct TATA recognition patterns exhibited by TBP-L114F and TBP-L205F strongly suggest that in vivo, TBP binds to the TATA element in a specific orientation . The orientation predicted from these studies is further supported by the identification of intragenic suppressors that correct the defect of TBP-L205F . This orientation is consistent with that observed in vitro by crystallographic analyses of TBP-TATA box complexes . Finally, the importance of altered DNA-binding specificity in transcriptional regulation at the S . cerevisiae his4-912 delta promoter was addressed for TBP-L205F . A mutational analysis of this promoter region demonstrates that the nonconsensus TATA element CATAAA is required for a transcriptional effect of TBP-L205F in vivo . This finding suggests that the interaction of TBP with nonconsensus TATA elements may play an important regulatory role in transcription initiation.

Curr Genet, 1994 Jun, 25(6), 504 - 7
Comparative analysis of the region of the mitochondrial genome containing the ATPase subunit 9 gene in the two related yeast species Saccharomyces douglasii and Saccharomyces cerevisiae; Nicoletti L et al.; We have determined the nucleotide sequence of a region of the mitochondrial genome of the yeast Saccharomyces douglasii which contains the ATPase subunit 9 gene and part of the intergenic sequences that surround it . The gene is 228 nucleotides long and encodes a polypeptide of 76 aa . A comparison of the coding sequence with that of S . cerevisiae reveals the presence of three silent transitions . A high level of similarity is also found between regions involved in the initiation of transcription and mRNA processing . More interestingly, a region of similarity situated outside the known regulatory regions has been identified . As the intergenic regions are generally highly divergent, the remarkable conservation of these non-coding sequences suggests that their structure may be relevant to the expression of this region of the mitochondrial DNA.

Genetics, 1994 Jun, 137(2), 423 - 37
BFR1, a multicopy suppressor of brefeldin A-induced lethality, is implicated in secretion and nuclear segregation in Saccharomyces cerevisiae; Jackson CL et al.; Brefeldin A (BFA) blocks protein transport out of the Golgi apparatus and causes disassembly of this organelle in mammalian cells . The primary effect of BFA is the release of the non-clathrin coat from Golgi membranes and vesicles . We sought to elucidate the mechanism of BFA action using a genetic approach in Saccharomyces cerevisiae . When an erg6 S . cerevisiae strain is treated with BFA, cell growth is arrested, cells lose viability and secretory proteins are accumulated in the endoplasmic reticulum (ER) and early Golgi compartments . We demonstrate that the mutant sec21 (defective in the S . cerevisiae homolog of gamma-COP, a non-clathrin coat protein) is supersensitive to BFA . Hence BFA probably affects the same processes in S . cerevisiae as in mammalian cells . We used a multicopy genomic DNA library to search for multicopy suppressors of BFA-induced lethality . We identified one such gene, BFR1, that, in addition, partially suppresses the growth and secretion defects of the ER-to-Golgi secretion mutant sec17 . A bfr1-delta 1::URA3 deletion strain is viable, but has defects in cell morphology and nuclear segregation, and the mutation accentuates the growth and secretion defects of a sec21 mutant.

Genetics, 1994 Jun, 137(2), 381 - 92
Overexpression of yeast homologs of the mammalian checkpoint gene RCC1 suppresses the class of alpha-tubulin mutations that arrest with excess microtubules; Kirkpatrick D et al.; Microtubules in eukaryotic cells participate in a variety of nuclear and cytoplasmic structures, reflecting functional requirements and cell cycle position . We are studying the cellular regulation of microtubule assembly and organization in the yeast Saccharomyces cerevisiae . We screened for genes that when overexpressed suppress the growth phenotype of conditional mutants in alpha-tubulin that arrest with excess microtubules at the nonpermissive temperature (class 2 mutations) . Here we describe one such suppressing element, called ATS1 (for Alpha Tubulin Suppressor) . Overexpression of this gene rescues both the growth and microtubule phenotypes of all class 2 mutations, but not the cold-sensitive mutations that arrest with no microtubules (class 1 mutations) . Deletion of ATS1 confers a modest slow growth phenotype which is slightly enhanced in strains containing both a deletion of ATS1 and a class 2 tub 1 mutation . The predicted ATS1 protein contains 333 amino acids and has considerable structural homology to the products of both the mammalian mitotic control gene RCC1 and the S . cerevisiae gene SRM1/PRP20 . Overexpression of SRM1/PRP20 also suppresses class 2 mutants . The results suggest that this family of genes may participate in regulatory interactions between microtubules and the cell cycle.

Biotechnol Appl Biochem, 1994 Jun, 19 ( Pt 3), 265 - 9
Heterologous gene expression and protein secretion from Candida glabrata; Macreadie IG et al.; We have examined heterologous protein secretion from Candida glabrata with the aid of a stable C . glabrata vector and a secretion reporter cassette comprising the Saccharomyces cerevisiae PGK-gene promoter and a Kluveromyces Iactis secretion signal to drive secretion of Escherichia coli beta-lactamase . Abundant secretion of beta-lactamase from C . glabrata indicates that the S . cerevisiae PGK promoter functions in C . glabrata . Furthermore, we show that C . glabrata processes the secreted beta-lactamase in a manner similar to, but not identical with, S . cerevisiae and K . lactis . C . glabrata may be a suitable new host for the expression of foreign genes.

J Cell Sci, 1994 Jun, 107 ( Pt 6), 1509 - 17
Cell cycle analysis and chromosomal localization of human Plk1, a putative homologue of the mitotic kinases Drosophila polo and Saccharomyces cerevisiae Cdc5; Golsteyn RM et al.; polo and CDC5 are two genes required for passage through mitosis in Drosophila melanogaster and Saccharomyces cerevisiae, respectively . Both genes encode structurally related protein kinases that have been implicated in regulating the function of the mitotic spindle . Here, we report the characterization of a human protein kinase that displays extensive sequence similarity to Drosophila polo and S . cerevisiae Cdc5; we refer to this kinase as Plk1 (for polo-like kinase 1) . The largest open reading frame of the Plk1 cDNA encodes a protein of 68,254 daltons, and a protein of this size is detected by immunoblotting of HeLa cell extracts with monoclonal antibodies raised against the C-terminal part of Plk1 expressed in Escherichia coli . Northern blot analysis of RNA isolated from human cells and mouse tissues shows that a single Plk1 mRNA of 2.3 kb is highly expressed in tissues with a high mitotic index, consistent with a possible function of Plk1 in cell proliferation . The Plk1 gene maps to position p12 on chromosome 16, a locus for which no associations with neoplastic malignancies are known . The Plk1 protein levels and its distribution change during the cell cycle, in a manner consistent with a role of Plk1 in mitosis . Thus, like Drosophila polo and S . cerevisiae Cdc5, human Plk1 is likely to function in cell cycle progression.

Biosci Biotechnol Biochem, 1994 Jun, 58(6), 1112 - 4
Metabolic engineering for production of beta-carotene and lycopene in Saccharomyces cerevisiae; Yamano S et al.; We have engineered a conventional yeast, Saccharomyces cerevisiae, to confer a novel biosynthetic pathway for the production of beta-carotene and lycopene by introducing the bacterial carotenoid biosynthesis genes, which are individually surrounded by the promoters and terminators derived from S . cerevisiae . beta-Carotene and lycopene accumulated in the cells of this yeast, which was considered to be a result of the carbon flow for the ergosterol biosynthetic pathway being partially directed to the pathway for the carotenoid production.

Biochem Biophys Res Commun, 1994 May 30, 201(1), 310 - 7
A possible yeast homolog of human active-gene-repairing helicase ERCC6+; Huang ME et al.; We report here the sequencing and identification on the chromosome X of S . cerevisiae of an open reading frame, designated GTA1085, encoding a protein 1085 amino acids in size that displays significant homology to a of helicase subfamily . The highest similarity score is with ERCC6, a human putative helicase involved in the repair of active genes, with 53.3% identity over a stretch of 589 amino acids . This putative protein contains all seven consecutive domains conserved among DNA and RNA helicases . Thus, it apparently constitutes a novel member of this subfamily and might be involved, like ERCC6, in the preferential repair of active genes in yeast.

Cell, 1994 May 20, 77(4), 579 - 86
The sorting receptor for yeast vacuolar carboxypeptidase Y is encoded by the VPS10 gene; Marcusson EG et al.; The S . cerevisiae VPS10 (vacuolar protein sorting) gene encodes a type I transmembrane protein of 1577 amino acids required for the sorting of the soluble vacuolar protein carboxypeptidase Y (CPY) . Mutations in VPS10 result in the selective missorting and secretion of CPY; all other vacuolar proteins tested are delivered to the vacuole in vps10 mutants . Chemical cross-linking studies demonstrate that Vps10p and the Golgi-modified precursor form of CPY directly interact . A single amino acid change in the CPY vacuolar sorting signal prevents this interaction . Vps10p also interacts with a hybrid protein containing the CPY sorting signal fused to the normally secreted enzyme invertase . Subcellular fractionation indicates that the majority of Vps10p is localized to a late Golgi compartment where vacuolar proteins are sorted . We propose that VPS10 encodes a CPY sorting receptor that executes multiple rounds of sorting by cycling between the late Golgi and a prevacuolar endosome-like compartment.

J Biol Chem, 1994 May 20, 269(20), 14776 - 83
Phosphatidylcholine biosynthesis via the CDP-choline pathway in Saccharomyces cerevisiae . Multiple mechanisms of regulation; McMaster CR et al.; Multiple mechanisms of regulation in the CDP-choline pathway for phosphatidylcholine (PC) synthesis were revealed by exploring the effects of choline and inositol on this pathway in Saccharomyces cerevisiae . At exogenous choline concentrations below 100 microM, phosphocholine cytidylyltransferase was rate-limiting; at higher choline concentrations the conversion of choline to phosphocholine by choline kinase became rate-limiting . Choline and inositol were found to regulate choline uptake; this established another regulatory mechanism by which PC synthesis is regulated in yeast . Inositol addition did not immediately affect labeled choline uptake or its incorporation into PC in actively dividing cells; however, preculturing the cells in the presence of choline decreased the rate of choline uptake, and this effect was amplified by the concomitant addition of inositol and choline . Additionally, a growth phase dependent effect of inositol supplementation was observed . Inositol addition to stationary phase cells resulted in an increase in choline uptake and subsequent PC production in these cells . This increase was shown to be due to an increase in the rate of choline transport into the cell . In the presence of inositol, choline transport is the main regulatory mechanism controlling flux through the CDP-choline pathway in S . cerevisiae . Inositol supplementation resulted in changes in the levels of enzyme activity detected in vitro . However, the effects observed in vivo correlated exclusively with changes in choline uptake . Choline transporter assays were consistent with these results . Since both the CPT1 and EPT1 gene products catalyze the cholinephosphotransferase reaction in vitro (Hjelmstad, R . H., and Bell, R . M . (1991) J . Biol . Chem . 266, 4357-4365), the effect of inositol on these two separate routes for PC biosynthesis was investigated . The data revealed that only cells harboring a functional CPT1 gene synthesized PC in vivo . These cells (ept1-delta 1::URA3) also displayed an identical mode of regulation in response to inositol as did cells containing an intact EPT1 gene (wild type) indicating there is no requirement for an alternate functional CDP-amino-alcohol pathway for inositol to regulate PC synthesis via the CDP-choline pathway.

FEMS Microbiol Lett, 1994 May 15, 118(3), 297 - 304
A murine monoclonal antibody directed against a yeast cell wall glycoprotein antigen of the yeast genus Saccharomyces; Broker M et al.; Murine monoclonal antibodies (mAbs) were selected against a cell wall glycoprotein of Saccharomyces cerevisiae . One of the mAbs (92-276/018) specifically identified S . cerevisiae and the sibling species S . paradoxus, S . pastorianus and S . bayanus in immunofluorescence studies and immunoblot analyses, while no other yeast genera except Saccharomyces were recognized . Further analysis indicated that the mAb 92-276/018 reacts with an epitope in the carbohydrate chain of the cell wall glycoproteins.

Nucleic Acids Res, 1994 May 11, 22(9), 1527 - 35
The RFC2 gene encoding a subunit of replication factor C of Saccharomyces cerevisiae; Noskov V et al.; Replication Factor C (RF-C) of Saccharomyces cerevisiae is a complex that consists of several different polypeptides ranging from 120- to 37 kDa (Yoder and Burgers, 1991; Fien and Stillman, 1992), similar to human RF-C . We have isolated a gene, RFC2, that appears to be a component of the yeast RF-C . The RFC2 gene is located on chromosome X of S . cerevisiae and is essential for cell growth . Disruption of the RFC2 gene led to a dumbbell-shaped terminal morphology, common to mutants having a defect in chromosomal DNA replication . The steady-state levels of RFC2 mRNA fluctuated less during the cell cycle than other genes involved in DNA replication . Nucleotide sequence of the gene revealed an open reading frame corresponding to a polypeptide with a calculated Mr of 39,716 and a high degree of amino acid sequence homology to the 37-kDa subunit of human RF-C . Polyclonal antibodies against bacterially expressed Rfc2 protein specifically reduced RF-C activity in the RF-C-dependent reaction catalyzed by yeast DNA polymerase III . Furthermore, the Rfc2 protein was copurified with RF-C activity throughout RF-C purification . These results strongly suggest that the RFC2 gene product is a component of yeast RF-C . The bacterially expressed Rfc2 protein preferentially bound to primed single-strand DNA and weakly to ATP.

Mol Gen Genet, 1994 May 10, 243(3), 253 - 60
A novel method for efficient expression cloning of fungal enzyme genes; Dalboge H et al.; We have developed a method for fast and efficient isolation of enzyme genes from filamentous fungi by combining the ability of Saccharomyces cerevisiae to express heterologous genes with the utilisation of sensitive and reliable enzyme assays . A cDNA library from the fungus Humicola insolens was constructed in a S . cerevisiae/Escherichia coli shuttle vector in E . coli . Sub-pools of the library were subsequently screened for enzyme activity in S . cerevisiae . More than 130 clones were identified as positive in either an endo-beta-glucanase or an endo-xylanase assay . Based on a partial characterization of the DNA sequence of the individual clones, they could be grouped into five distinct types of endo-beta-glucanases and three types of endo-xylanases . A representative cDNA from each type was sub-cloned in an Aspergillus vector and expressed in A . oryzae . The new cloning method may be an important alternative to traditional cloning methods based on amino acid sequence information.

Cell, 1994 May 6, 77(3), 381 - 90
The UV response involving the Ras signaling pathway and AP-1 transcription factors is conserved between yeast and mammals; Engelberg D et al.; UV irradiation of mammalian cells activates AP-1 through a Ras-dependent pathway, independently of DNA damage . We show that the yeast S . cerevisiae has a remarkably similar UV response involving the AP-1 factor Gcn4, which is distinct from the DNA damage response . Transcriptional activation of HIS3 and HIS4 by Gcn4 is triggered by UV irradiation in a Ras-dependent fashion . Moreover, resistance of yeast to UV irradiation is correlated with the level of Ras activity and Gcn4 function . Like mammalian cells in which activated Ras leads to increased c-Jun synthesis and phosphorylation, the effects in yeast involve increased translation of GCN4 mRNA and a posttranslational event . However, this effect on GCN4 translation is different from the response to amino acid or purine starvation . Therefore, a UV signaling pathway involving Ras and AP-1 is an ancient and universal mechanism involved in protection against damage to cellular components other than DNA.

Cell, 1994 May 6, 77(3), 371 - 9
The Drosophila peanut gene is required for cytokinesis and encodes a protein similar to yeast putative bud neck filament proteins; Neufeld TP et al.; We have identified a Drosophila gene, peanut (pnut), that is related in sequence to the CDC3, CDC10, CDC11, and CDC12 genes of S . cerevisiae . These genes are required for cytokinesis, and their products are present at the bud neck during cell division . We find that pnut is also required for cytokinesis: in pnut mutants, imaginal tissues fail to proliferate and instead develop clusters of large, multinucleate cells . Pnut protein is localized to the cleavage furrow of dividing cells during cytokinesis and to the intercellular bridge connecting postmitotic daughter cells . In addition to its role in cytokinesis, pnut displays genetic interactions with seven in absentia, a gene required for neuronal fate determination in the compound eye, suggesting that pnut may have pleiotropic functions . Our results suggest that this class of proteins is involved in aspects of cytokinesis that have been conserved between flies and yeast.

Gene, 1994 May 3, 142(1), 135 - 40
A system for gene cloning and manipulation in the yeast Candida glabrata; Zhou P et al.; The opportunistic pathogenic yeast, Candida (Torulopsis) glabrata, is an asexual imperfect fungus that exists largely as a haploid . Besides being a clinically important pathogen, this yeast also provides a model system for understanding basic biological mechanisms such as metal-activated metallothionein-encoding gene transcription . To facilitate molecular genetic studies in C . glabrata, we isolated a strain auxotrophic for uracil biosynthesis . The ura- mutation could be functionally complemented by the URA3 gene of Saccharomyces cerevisiae, consistent with a defect in the C . glabrata URA3 gene in this strain . We also found that the centromere-based S . cerevisiae plasmid pRS316 could stably transform and replicate in multiple copies in C . glabrata . In contrast, high-copy-number S . cerevisiae plasmids containing the 2 mu circle autonomous replication sequence were not able to replicate productively in C . glabrata . We cloned the C . glabrata URA3 gene, encoding orotidine-5'-phosphate decarboxylase, by complementation of a ura3- strain of S . cerevisiae . The deduced amino-acid sequence is highly similar to that of the URA3 protein from S . cerevisiae . C . glabrata URA3 provides a genetic locus for targeted gene integration in C . glabrata . Integrative plasmids were constructed based on the cloned C . glabrata URA3 and are applicable for directed insertions of genes of interest at the ura3 locus through homologous recombination.

J Bacteriol, 1994 May, 176(10), 3021 - 32
Molecular analysis of the Trichosporon cutaneum DSM 70698 argA gene and its use for DNA-mediated transformations; Reiser J et al.; Genomic clones capable of complementing a previously isolated arginine auxotrophic mutant strain of the filamentous yeast Trichosporon cutaneum DSM 70698 have been identified by DNA-mediated transformation, and a complementing 4,082-bp subfragment was sequenced . This analysis revealed an intact gene (arg4) showing a high degree of homology with the Saccharomyces cerevisiae CPA2 gene encoding the large subunit of carbamoyl-phosphate synthetase (CPS-A) . The inferred amino acid sequence of the T . cutaneum argA-encoded protein contains 1,168 residues showing 62% identity with the sequence of the S . cerevisiae CPA2 protein, and the comparison of the two sequences uncovered a putative intron sequence of 81 nucleotides close to the 5' end of the coding region of the T . cutaneum argA gene . The presence of this intron was confirmed by nuclease protection studies and by direct DNA sequence analysis of a cDNA fragment which had been obtained by PCR amplification . The T . cutaneum intron shares the general characteristics of introns found in yeasts and filamentous fungi . A major transcript of around 4 kb was found in Northern (RNA) blots . The T . cutaneum argA coding region was expressed in Escherichia coli under the control of the regulatable tac promoter . A roughly 130-kDa protein which was found to cross-react with an anti-rat CPS antibody in Western blots (immunoblots) was observed . Two putative ATP-binding domains were identified, one in the amino-terminal half of the argA-encoded protein and the other in the carboxy-terminal half . These domains are highly conserved among the known CPS-A sequences from S . cerevisiae, E . coli, and the rat . From these results we conclude that the T . cutaneum argA gene encodes the large subunit of CPS . This is the first gene to be identified and analyzed in the T . cutaneum DSM 70698 strain.

Mol Cell Biol, 1994 May, 14(5), 3022 - 9
The Ubc3 (Cdc34) ubiquitin-conjugating enzyme is ubiquitinated and phosphorylated in vivo; Goebl MG et al.; The transition from G1 to S phase of the cell cycle in Saccharomyces cerevisiae requires the activity of the Ubc3 (Cdc34) ubiquitin-conjugating enzyme . S . cerevisiae cells lacking a functional UBC3 (CDC34) gene are able to execute the Start function that initiates the cell cycle but fail to form a mitotic spindle or enter S phase . The Ubc3 (Cdc34) enzyme has previously been shown to catalyze the attachment of multiple ubiquitin molecules to model substrates, suggesting that the role of this enzyme in cell cycle progression depends on its targeting an endogenous protein(s) for degradation . In this report, we demonstrate that the Ubc3 (Cdc34) protein is itself a substrate for both ubiquitination and phosphorylation . Immunochemical localization of the gene product to the nucleus renders it likely that the relevant substrates similarly reside within the nucleus.

Mol Cell Biol, 1994 May, 14(5), 2958 - 65
Characterization of tobacco protein kinase NPK5, a homolog of Saccharomyces cerevisiae SNF1 that constitutively activates expression of the glucose-repressible SUC2 gene for a secreted invertase of S . cerevisiae; Muranaka T et al.; We have isolated a cDNA (cNPK5) that encodes a protein kinase of 511 amino acids from suspension cultures of tobacco cells . The predicted kinase domain of NPK5 is 65% identical in terms of amino acid sequence to that of the SNF1 serine/threonine protein kinase of Saccharomyces cerevisiae, which plays a central role in catabolite repression in yeast cells . SNF1 positively regulates transcription of various glucose-repressible genes of the yeast, such as the SUC2 gene for a secreted invertase, in response to glucose deprivation: snf1 mutants cannot utilize sucrose as a carbon source . Expression of cNPK5 in yeast cells allowed the snf1 mutant cells to utilize sucrose for growth and caused constitutive expression of the SUC2 gene in wild-type cells even in the presence of glucose, an indication that the NPK5 protein is present in a constitutively active form in S . cerevisiae . On the other hand, expression of cNPK5 failed to suppress the growth defect of the snf4 mutant cells in the presence of sucrose and to induce expression of the SUC2 gene . These results indicate that SNF4 is required for the induction of SUC2 expression by NPK5, as by SNF1, even if NPK5 is constitutively active in S . cerevisiae . The recombinant NPK5 protein is capable of autophosphorylation in vitro in a reaction that requires Mn2+ rather than Mg2+ ions but is inhibited by Ca2+ ions . Both dicotyledonous and monocotyledonous plants have several copies of the NPK5-related gene, which probably constitute a small gene family . NPK5-related genes were found to be expressed in the roots, leaves, and stems of tobacco plants . The high degree of structural conservation and the functional similarity of NPK5 to SNF1 lead us to speculate that NPK5 (or a related kinase) also plays a role in sugar metabolism in higher plants.

Microbiology, 1994 May, 140 ( Pt 5), 1097 - 101
A surface lectin associated with flocculation in brewing strains of Saccharomyces cerevisiae; Shankar CS et al.; A cell-wall-surface protein purified from the cells of Saccharomyces cerevisiae NCYC 227 was found to be involved in the non-sexual flocculation of this yeast . This 13 kDa protein was found to bind specifically to mannose . The protein bound to mannans isolated from yeast as well as in situ to intact cells, but only in the presence of calcium ions . The protein, a mannoprotein, formed aggregates as revealed in SDS-PAGE . Urea and higher temperatures prevented protein aggregation, suggesting that the flocculation of S . cerevisiae is primarily due to hydrogen bonding between mannan and protein.

Microbiology, 1994 May, 140 ( Pt 5), 1079 - 84
Iron regulation of triosephosphate isomerase transcript stability in the yeast Saccharomyces cerevisiae; Krieger K et al.; By differential hybridization we have identified cDNA clones that are derived from iron-regulated genes in Saccharomyces cerevisiae . Sequencing of seven cDNA clones revealed that five clones correspond to TPI1 encoding triosephosphate isomerase (Tpi1p) and one corresponds to TDH3 encoding glyceraldehyde-3-phosphate dehydrogenase (Tdh3p) . During iron-limited growth mRNA levels for Tpi1p and Tdh3p were at least 3-fold lower than during iron-saturated growth; as shown with a hem1 mutant strain this regulation does not require haem synthesis . mRNA half-lives of TPI1 (TDH3) were 11.5 min (18 min) in low-iron medium and 30 min (32.5 min) in high-iron medium, indicating iron-regulation of transcript half-lives; the stabilities of the ACT1 and PDC1 transcripts were not influenced by iron . Increased glycerol production during growth in low-iron, as compared to high-iron medium, is consistent with a modification of the glycolytic flux during iron-limited growth in S . cerevisiae.

Appl Environ Microbiol, 1994 May, 60(5), 1467 - 72
Energy-dependent, carrier-mediated extrusion of carboxyfluorescein from Saccharomyces cerevisiae allows rapid assessment of cell viability by flow cytometry; Breeuwer P et al.; Carboxyfluorescein diacetate is a nonfluorescent compound which can be used in combination with flow cytometry for vital staining of yeasts and bacteria . The basis of this method is the assumption that, once inside the cell, carboxyfluorescein diacetate is hydrolyzed by nonspecific esterases to produce the fluorescent carboxyfluorescein (cF) . cF is retained by cells with intact membranes (viable cells) and lost by cells with damaged membranes . In this report, we show that Saccharomyces cerevisiae extrudes cF in an energy-dependent manner . This efflux was studied in detail, and several indications that a transport system is involved were found . Efflux of cF was stimulated by the addition of glucose and displayed Michaelis-Menten kinetics . A Km for cF transport of 0.25 mM could be determined . The transport of cF was inhibited by the plasma membrane H(+)-ATPase inhibitors N,N'-dicyclohexylcarbodiimide and diethylstilbestrol and by high concentrations of tetraphenylphosphonium ions . These treatments resulted in a dissipation of the proton motive force, whereas the intracellular ATP concentration remained high . Transport of cF is therefore most probably driven by the membrane potential and/or the pH gradient . The viability of S . cerevisiae was determined by a two-step procedure consisting of loading the cells with cF followed by incubation at 40 degrees C in the presence of glucose . Subsequently, the fluorescence intensity of the cells was analyzed by flow cytometry . The efflux experiments showed an excellent correlation between the viability of S . cerevisiae cells and the ability to translocate cF . This method should prove of general utility for the rapid assessment of yeast vitality and viability.

Int J Biochem, 1994 May, 26(5), 721 - 34
The relationship between the trimethylation of lysine 77 and cytochrome c metabolism in Saccharomyces cerevisiae; Ceesay KJ et al.; 1 . Site directed mutations were constructed in the yeast iso-1-cytochrome c gene adjacent to the lysine 77 (methylation site) codon . 2 . These mutant genes were then cloned and transformed into the S . cerevisiae strain B-6642 which contains a deficiency in the iso-1-cytochrome c gene . 3 . The resulting transformants were screened for cytochrome c production using gel electrophoresis . 4 . Amino acid analysis of the mutated cytochromes c demonstrated varying levels of trimethyllysine formation, depending on the nature of the site directed mutation . 5 . The resulting transformants were then used as tools in order to investigate the relationship between trimethyllysine formation and various aspects of cytochrome c metabolism including protein stability and heme conjugation.

Yeast, 1994 May, 10(5), 659 - 62
A Saccharomyces cerevisiae gene encoding a potential adenine phosphoribosyltransferase; Yuryev A et al.; The nucleotide sequence of a Saccharomyces cerevisiae gene encoding a potential adenine phosphoribosyltransferase (APRT) has been determined . The protein encoded by this gene shows a high degree of similarity with APRTs from a variety of other species . The S . cerevisiae gene, named APT2, has been mapped to chromosome IV . The sequence has been deposited in the GenBank data library under Accession Number L14434.

Yeast, 1994 May, 10(5), 567 - 78
The Saccharomyces cerevisiae gene PPH3 encodes a protein phosphatase with properties different from PPX, PP1 and PP2A; Hoffmann R et al.; A clone encoding the catalytic subunit of a protein phosphatase from Saccharomyces cerevisiae was isolated . Except for replacement of IIe-245 by Met the structure of the phosphatase was identical to that encoded by PPH3 (Ronne, H., Carlberg, M., Hu, G . Z . and Nehlin, J . O . (1991) . Mol . Cell . Biochem . 11, 4876-4884) and exhibited 63% sequence identity to PPX cloned from a rabbit liver cDNA library (Brewis, N.D., Street, A.J., Prescott, A.R . and Cohen, P.T.W . (1993) . EMBO J . 12, 987-996) . Expression of active enzyme was achieved in Escherichia coli mutants which were generated by a genetic selection based on functional complementation of bacterial phosphoserine phosphatase . Though some of the properties of PPH3 resembled those of protein phosphatase 2A and PPX, others were different . PPH3 exhibited lower sensitivity against inhibition by okadaic acid, showed different substrate specificity and required a divalent cation (Mn2+ was preferred before Mg2+ and Ca2+) for activity when assayed with phospho-histone as a substrate . However, 25% of maximum activity was observed in the absence of divalent cations when the peptide LRRAS(P)LG was used as substrate . The PPH3-protein was also identified by chromatography of extracts from S . cerevisiae on DEAE-cellulose . Protein immunoreactive with an antiserum raised against the non-conserved N-terminal 53 amino acids of PPH3 was coeluted with a single peak of LRRAS(P)LG dephosphorylating activity.

Mol Cell Biol, 1994 May, 14(5), 3364 - 75
Specific DNA recognition and intersite spacing are critical for action of the bicoid morphogen; Hanes SD et al.; We examined DNA site recognition by Bicoid and its importance for pattern formation in developing Drosophila embryos . Using altered DNA specificity Bicoid mutants and appropriate reporter genes, we show that Bicoid distinguishes among related DNA-binding sites in vivo by a specific contact between amino acid 9 of its recognition alpha-helix (lysine 50 of the homeodomain) and bp 7 of the site . This result is consistent with our earlier results using Saccharomyces cerevisiae but differs from that predicted by crystallographic analysis of another homeodomain-DNA interaction . Our results also demonstrate that Bicoid binds directly to those genes whose transcription it regulates and that the amino acid 9 contact is necessary for Bicoid to direct anterior pattern formation . In both Drosophila embryos and yeast cells, Bicoid requires multiple binding sites to activate transcription of target genes . We find that the distance between binding sites is critical for Bicoid activation but that, unexpectedly, this critical distance differs between Drosophila and S . cerevisiae . This result suggests that Bicoid activation in Drosophila might require an ancillary protein(s) not present in S . cerevisiae.

Biosci Biotechnol Biochem, 1994 May, 58(5), 954 - 6
Expression of the cellulase (FI-CMCase) gene of Aspergillus aculeatus in Saccharomyces cerevisiae; Ooi T et al.; As a step to breed a Saccharomyces cerevisiae strain able to produce ethanol directly from cellulose, we combined cDNA for Aspergillus aculeatus FI-CMCase (FI-carboxymethyl cellulase) with the GAP (glyceraldehyde-3-phosphate dehydrogenase) promoter of S . cerevisiae and used the resultant plasmid, pYEC91, to transform S . cerevisiae . The transformed cells produced active FI-CMCase within the cytoplasm . Western-blot analysis following SDS-polyacrylamide gel electrophoresis demonstrated that the cells contained a peptide having the same molecular mass and immunological identity as A . aculeatus FI-CMCase.

Mutat Res, 1994 May, 314(3), 209 - 20
The E . coli recA gene can restore the defect in mutagenesis of the pso4-1 mutant of S . cerevisiae; Morais Junior MA et al.; The E . coli recA gene was introduced into the pso4-1 mutant of S . cerevisiae and transformants were treated with 8-MOP+UVA and 254-nm UV light . The results showed that the recA gene increased the resistance to the toxic effect of 8-MOP+UVA and restored the frequency of reversion of the pso4-1 mutants after both treatments . The presence of the recA gene stimulated expression of the small subunit of the ribonucleotide reductase (Rnr2) in the pso4-1 mutants . Thus the E . coli recA gene is functional in yeast . Moreover, it was shown that the pso4-1 mutant is epistatic to pso1-1 and rad6-1, which belong to a mutagenic repair pathway . We propose here that the PSO4 gene has some role in the control of mutagenic repair in yeast.

FEBS Lett, 1994 Apr 25, 343(2), 160 - 4
Level of ATP synthase activity required for yeast Saccharomyces cerevisiae to grow on glycerol media; Mukhopadhyay A et al.; Two independent cold-sensitive pet mutants in the gene (ATP5) coding for the oligomycin sensitivity conferring protein (OSCP) have been isolated in the yeast Saccharomyces cerevisiae . The mutations in both strains alter the initiating methionine codon in the ATP5 gene: ATG to ATA (Ile) and AAG (Lys) . Western blot analysis of total yeast protein after the cells were grown at 18 degrees C, 30 degrees C, and 37 degrees C, indicates that the level of OSCP decreased 80% relative to the wild type strain . In addition, the level of the oligomycin-sensitive ATPase decreased 85% relative to the wild type strain, after growth at 30 degrees C . These findings indicate that for S . cerevisiae, the level of oxidative phosphorylation can decrease 85% without showing a large growth defect on media containing glycerol at 30 degrees C, but not at 18 degrees C.

Eur J Biochem, 1994 Apr 15, 221(2), 741 - 7
Characterization of the essential yeast gene encoding N-acetylglucosamine-phosphate mutase; Hofmann M et al.; A previously cloned gene of Saccharomyces cerevisiae, which complements the growth defect of a phosphoglucomutase (pgm1 delta/pgm2 delta) double deletion mutant on a pure galactose medium {Boles, E., Liebetrau, W., Hofmann, M . & Zimmermann, F . K . (1994) Eur . J . Biochem . 220, 83-96}, was identified as the structural gene encoding N-acetylglucosamine-phosphate mutase . The complete nucleotide sequence of the gene, AGM1, and surrounding regions were determined . AGM1 codes for a predicted 62-kDa protein with 557 amino acids and is located on chromosome V adjacent to the known gene PRB1 encoding protease B . No extended nucleotide or amino acid sequence similarities could be found in the databases, except for a small region of amino acids with high similarity to the active-site consensus sequence of hexosephosphate mutases . Three putative pheromone-responsive elements have been identified in the upstream region of the AGM1 gene . The gene is essential for cell viability . An agm1 deletion mutant progresses through only approximately five cell cycles to form a 'string' of undivided cells with an abnormal cell morphology resembling glucosamine auxotrophic mutants . Expression of the AGM1 gene on a multi-copy plasmid led to a significantly increased N-acetylglucosamine-phosphate mutase activity . Unlike over-expression of the AGM1 gene in a pgm1/pgm2 double deletion mutant which could restore phosphoglucomutase activity, over-expression of the PGM2 gene encoding the major isoenzyme of phosphoglucomutase did not increase N-acetylglucosamine-phosphate-mutase activity and did not restore growth of agm1 deletion mutant cells . Our observations indicate that the different hexosephosphate mutases of S . cerevisiae have partially overlapping substrate specificities but, nevertheless, distinct physiological functions.

Nucleic Acids Res, 1994 Apr 11, 22(7), 1265 - 71
SRD1, a S . cerevisiae gene affecting pre-rRNA processing contains a C2/C2 zinc finger motif; Hess SM et al.; The Saccharomyces cerevisiae genes, RRP1 and SRD1, are involved in processing rRNA precursor species to mature rRNAs . We reported previously that the rrp1-1 mutation caused temperature-sensitive lethality, hypersensitivity to aminoglycoside antibiotics, and defective processing of 27S pre-rRNA to 25S and 5.8S mature rRNAs . A second-site suppressor of the rrp1-1 mutation, srd1, corrects all three rrp1 mutant phenotypes . In order to learn more about the roles of the SRD1 and RRP1 genes in rRNA processing, we cloned and characterized the SRD1 gene . We identified an ORF, YCR18C, that complements srd1-2 suppression of rrp1-1 . The DNA is physically located at the region of chromosome III where SRD1 has been genetically mapped . SRD1 encodes a putative 225 amino acid, 26 kDa protein containing a C2/C2 zinc finger motif that is also found in some transcription regulators and the eIF-2 beta translation initiating factors . The similarity of SRD1 to transcription regulators led us to test the model that srd1 mutations suppress rrp1 defects by altering the level of the RRP1 transcript . However, we found that SRD1 has no detectable effect on the steady state levels of RRP1 mRNA . We describe alternative models to explain the role of Srd1p in pre-rRNA processing.

Science, 1994 Apr 8, 264(5156), 261 - 5
Isolation of S . cerevisiae snRNPs: comparison of U1 and U4/U6.U5 to their human counterparts; Fabrizio P et al.; Small nuclear ribonucleoprotein (snRNP) particles are essential for pre-messenger RNA splicing . In human HeLa cells, 40 proteins associated with snRNPs have been identified . Yet, the function of many of these proteins remains unknown . Here, the immunoaffinity purification of the spliceosomal snRNPs U1, U2, U4/U6.U5, and several nucleolar snRNP species from the yeast Saccharomyces cerevisiae is presented . The U1 and U4/U6.U5 snRNPs were purified extensively and their protein composition and ultrastructure analyzed . The yeast U1 snRNP is larger and contains three times more specific proteins than its human counterpart . In contrast, the size, protein composition, and morphology of the yeast and the human U4/U6.U5 snRNPs are significantly similar . The preparative isolation of yeast snRNPs will allow the cloning as well as genetic and phylogenetic analysis of snRNP proteins which will accelerate our understanding of their function.

J Biol Chem, 1994 Apr 8, 269(14), 10193 - 6
A conditionally lethal yeast mutant blocked at the first step in glycosyl phosphatidylinositol anchor synthesis; Leidich SD et al.; Glycosyl phosphatidylinositols (GPIs) anchor many proteins to the surface of eukaryotic cells and may also serve as sorting signals on proteins and participate in signal transduction . We have isolated a Saccharomyces cerevisiae GPI anchoring mutant, gpi1, using a colony screen for cells blocked in {3H}inositol incorporation into protein . The gpi1 mutant is defective in vitro in the synthesis of N-acetylglucosaminyl phosphatidylinositol, the first intermediate in GPI synthesis, and is also temperature-sensitive for growth . Completion of the first step in GPI assembly is therefore required for growth of the unicellular eukaryote S . cerevisiae . GPI synthesis could therefore be exploited as a target for antifungal or antiparasitic agents.

J Biol Chem, 1994 Apr 1, 269(13), 9556 - 61
The prosequence of Rhizopus niveus aspartic proteinase-I supports correct folding and secretion of its mature part in Saccharomyces cerevisiae; Fukuda R et al.; Extracellular Rhizopus niveus aspartic proteinase-I (RNAP-I) was secreted effectively by Saccharomyces cerevisiae when RNAP-I with its preprosequence was synthesized in this organism (Horiuchi, H., Ashikari, T., Amachi, T., Yoshizumi, H., Takagi, M., and Yano, K . (1990) Agric . Biol . Chem . 54, 1771-1779) . Certain deletions (delta pro, delta 1, delta 2), and amino acid substitutions (M1) in the prosequence blocked secretion of RNAP-I, although the protease protection assay revealed that even delta pro could be translocated across the membrane of the endoplasmic reticulum . When delta pro or M1 was synthesized simultaneously with the wild-type preprosequence in S . cerevisiae, secretion of RNAP-I was recovered . Therefore, the physical linkage of the prosequence to the mature region is not a prerequisite for secretion of active RNAP-I . Purified RNAP-I with the prosequence once denatured in 6 M guanidine HCl could be renatured and activated to have its enzymatic activity by removing guanidine HCl in vitro, but RNAP-I without the prosequence could not . Furthermore, the wild-type prosequence helped the recovery of the activity of the denatured RNAP-I in trans, but the prosequences of M1 with which secretion of RNAP-I was not observed in vivo, did not . From these results we concluded that the prosequence of RNAP-I supports correct folding of RNAP-I in the endoplasmic reticulum lumen and its subsequent secretion in S . cerevisiae . The functional role of the prosequence of an aspartic proteinase was elucidated.

Mol Cell Biol, 1994 Apr, 14(4), 2822 - 35
Histone H1 expressed in Saccharomyces cerevisiae binds to chromatin and affects survival, growth, transcription, and plasmid stability but does not change nucleosomal spacing; Linder C et al.; Histone H1 is proposed to serve a structural role in nucleosomes and chromatin fibers, to affect the spacing of nucleosomes, and to act as a general repressor of transcription . To test these hypotheses, a gene coding for a sea urchin histone H1 was expressed from the inducible GAL1 promoter in Saccharomyces cerevisiae by use of a YEp vector for high expression levels (strain YCL7) and a centromere vector for low expression levels (strain YCL1) . The H1 protein was identified by its inducibility in galactose, its apparent molecular weight, and its solubility in 5% perchloric acid . When YCL7 was shifted from glucose to galactose for more than 40 h to achieve maximal levels of H1, H1 could be copurified in approximately stoichiometric amounts with core histones of Nonidet P-40-washed nuclei and with soluble chromatin fractionated on sucrose gradients . While S . cerevisiae tolerated the expression of low levels of H1 in YCL1 without an obvious phenotype, the expression of high levels of H1 correlated with greatly reduced survival, inhibition of growth, and increased plasmid loss but no obvious change in the nucleosomal repeat length . After an initial induction, RNA levels for GAL1 and H1 were drastically reduced, suggesting that H1 acts by the repression of galactose-induced genes . Similar effects, but to a lower extent, were observed when the C-terminal tail of H1 was expressed.

J Infect Dis, 1994 Apr, 169(4), 859 - 67
Comparative pathogenesis of clinical and nonclinical isolates of Saccharomyces cerevisiae; Clemons KV et al.; Although considered nonpathogenic, Saccharomyces cerevisiae is being encountered more frequently in the clinical setting . To assess pathogenic potential, 13 clinical isolates, 10 nonclinical isolates, and 5 constructed strains of S . cerevisiae were analyzed . All were S . cerevisiae by biochemical profiles, sporulation, or genetic evidence . Intravenous inoculation of yeasts into CD-1 mice showed that some clinical isolates proliferated in the brain (5-fold) but nonclinical isolates were cleared (1000-fold) by day 7 after infection . Comparison of burdens with those of YJM128 (clinical) and Y55 (laboratory strain) revealed three virulence groupings: virulent, those greater than or equal to YJM128 (5 clinical and 2 genetic constructs); intermediate virulent, those less than YJM128 and greater than Y55 (5 clinical, 3 genetic constructs, and 4 nonclinical); and avirulent, those less than or equal to Y55 (1 clinical and 6 nonclinical) . Genetic crosses indicated that virulence was a dominant trait . Growth of various isolates at 37 degrees C and 39 degrees C indicated that temperature is associated with but not solely responsible for differences in virulence . These data demonstrate that some clinical isolates of S . cerevisiae can proliferate and resist clearance in vivo and support the potential of S . cerevisiae as a cause of clinical disease.

Yeast, 1994 Apr, 10 Suppl A, S75 - 80
The complete sequence of a 33 kb fragment on the right arm of chromosome II from Saccharomyces cerevisiae reveals 16 open reading frames, including ten new open reading frames, five previously identified genes and a homologue of the SCO1 gene; Smits PH et al.; We report here the sequence of a 33,117 bp DNA fragment located approximately 30 kb from the centromere on the right arm of Saccharomyces cerevisiae chromosome II . We have detected 16 open reading frames (ORFs) longer than 450 bp, provisionally called YBR0301 to YBR0322, covering 70.4% of the entire sequence . The ORFs YBR0301, YBR0302, YBR0303, YBR0305 and YBR0315 correspond to previously sequenced S . cerevisiae genes GAL10, GAL1, FUR4, CAL1 and L2B, respectively . Translation products of two other ORFs, YBR0308 and YBR0312 exhibit similarity to previously known S . cerevisiae proteins: the mitochondrially associated protein SCO1 and the protein kinase YKR2 . The predicted protein product of the ORF YBR0321 shows a 41.6% identity score with the Escherichia coli pyroxamine 5'-phosphate oxidase . The nine other ORFs show no significant homology to known proteins.

Glycobiology, 1994 Apr, 4(2), 135 - 40
Glycoprotein biosynthesis in Saccharomyces cerevisiae . Partial purification of the alpha-1,6-mannosyltransferase that initiates outer chain synthesis; Romero PA et al.; The alpha-1,6-mannosyltransferase (alpha-1,6-ManT) that initiates outer chain synthesis in Saccharomyces cerevisiae was partially purified along with an alpha-1,2-mannosyltransferase (alpha-1,2-ManT) that acts on alpha-methylmannoside . The enzymes were solubilized by extracting a 145,000 g pellet of S.cerevisiae mnn1 mutant with 1% Triton X-100 . The extract was then passed through a concanavalin A-Sepharose column and the bound material was eluted with alpha-methylmannoside . After exhaustive dialysis, the fractions containing both mannosyltransferase activities were chromatographed on DEAE-Trisacryl which removed approximately 90% of the alpha-1,2-ManT . The fractions containing alpha-1,6-ManT and residual alpha-1,2-ManT were further purified by sequential chromatography on Sephacryl S-200 and CM-Trisacryl . Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of individual fractions eluted from Sephacryl S-200 and from CM-Trisacryl, followed by silver staining of the gels, showed two major bands whose intensity corresponded to the enzyme activities . A protein band of approximately 62 kDa corresponded to the alpha-1,6-ManT and another band of approximately 66 kDa, which was eluted from the Sephacryl S-200 column slightly earlier, corresponded to the alpha-1,2-ManT.

Genetics, 1994 Apr, 136(4), 1261 - 9
Genetic characterization of pathogenic Saccharomyces cerevisiae isolates; McCusker JH et al.; Saccharomyces cerevisiae isolates from human patients have been genetically analyzed . Some of the characteristics of these isolates are very different from laboratory and industrial strains of S . cerevisiae and, for this reason, stringent genetic tests have been used to confirm their identity as S . cerevisiae . Most of these clinical isolates are able to grow at 42 degrees, a temperature that completely inhibits the growth of most other S . cerevisiae strains . This property can be considered a virulence trait and may help explain the presence of these isolates in human hosts . The ability to grow at 42 degrees is shown to be polygenic with primarily additive effects between loci . S . cerevisiae will be a useful model for the evolution and genetic analysis of fungal virulence and the study of polygenic traits.

Cryobiology, 1994 Apr, 31(2), 193 - 8
Cryoprotection of yeast by alcohols during rapid freezing; Lewis JG et al.; We have investigated the cryoprotective effect of alcohols on Saccharomyces cerevisiae and other yeast under various freezing conditions . For S . cerevisiae, at a cooling rate of 3 degrees C min-1 methanol and ethanol acted as cryosensitizers . However, at a cooling rate of 200 degrees C min-1, both methanol and ethanol proved superior to all other cryoprotectants tested, including glycerol, dimethyl sulfoxide, lactose, trehalose, polyethylene glycol, and polyvinyl pyrrolidone . Propan-2-ol also demonstrated a small but measurable effect although propan-1-ol and butan-1-ol demonstrated no cryoprotective effect . A minimum cooling rate of 25 degrees C min-1 to elicit the cryoprotective effect of ethanol was necessary; below this rate it acted as a cryosensitizer . At cooling rates up to 650 degrees C min-1 substantial cryoprotective effect was still evident . Although the effect of ethanol was variable for other yeast genera tested, ethanol acted positively for all strains of S . cerevisiae . We hypothesize that the cryoprotective effect of alcohols during rapid cooling is a result of their ability to induce increased membrane permeability, allowing rapid water equilibration during extracellular freezing and avoidance of intracellular ice crystal formation.

Yeast, 1994 Apr, 10(4), 509 - 13
Isolation and characterization of the LEU2 gene of Hansenula polymorpha; Agaphonov MO et al.; A DNA fragment carrying the LEU2 gene of methylotrophic yeast Hansenula polymorpha was isolated by complementation of the leuB mutation of Escherichia coli . The nucleotide sequence of the isolated DNA fragment contains an open reading frame of 363 codons, coding for a protein 80% identical to the LEU2 gene product of Saccharomyces cerevisiae . Further downstream, there is a partial reading frame with no obvious similarity to known proteins . The LEU2 gene of H . polymorpha cannot complement the leu2 mutation of S . cerevisiae.

Microbiology, 1994 Apr, 140 ( Pt 4), 717 - 22
Propionate metabolism in Saccharomyces cerevisiae: implications for the metabolon hypothesis; Pronk JT et al.; Aerobic, glucose-limited chemostat of Saccharomyces cerevisiae CBS 8066 co-metabolized propionate when this compound was added to the reservoir medium . Co-metabolism of propionate led to an increase of the biomass and protein yields . Attempts to grow S . cerevisiae on propionate as a sole source of carbon and energy were not successful . Activities of propionyl-CoA synthetase in cell-free extracts were sufficient to account for the rates of propionate consumption observed in the chemostat cultures . Activities of propionyl-CoA carboxylase, a key enzyme of the methylmalonyl-CoA pathway of propionate metabolism, were negligible . In contrast, activities of 2-methylcitrate synthase, a key enzyme activity of the 2-methylcitrate pathway of propionate metabolism, increased substantially with increasing propionate-to-glucose ratios in the reservoir media, and were sufficient to account for the propionate consumption rates observed in the chemostat cultures . This suggested that the 2-methylcitrate pathway is the major pathway of propionate metabolism in S . cerevisiae . In the literature, labelling patterns observed after incubation of this yeast with {3-13C}propionate have been interpreted as evidence for channelling of tricarboxylic acid (TCA) cycle intermediates, possibly as a consequence of the organization of TCA cycle enzymes in a metabolon . However, this interpretation of 13C-labelling patterns rested on the assumption that propionate metabolism in S . cerevisiae occurs via the methylmalonyl-CoA pathway . Since the distribution of 13C in alanine reported in the literature is fully compatible with a major role of the 2-methylcitrate pathway in propionate metabolism, it cannot be interpreted as evidence for the existence of a TCA cycle metabolon in S . cerevisiae.

Mutat Res, 1994 Apr, 321(1-2), 19 - 26
The genetic activity of 6-N-hydroxylaminopurine in Aspergillus nidulans; Babudri N et al.; The activity of a base analog (6-N-hydroxylaminopurine, HAP) has been tested on Aspergillus nidulans . In germinating haploid conidia HAP is a strong mutagen, while it does not have any activity in resting conidia . Moreover, HAP does not increase the frequency of recombination in germinating conidia . The mutagenic activity of this base analog has also been tested in diploid conidia of A . nidulans; in fact, it has been shown (Pavlov et al., 1991) that the HAP-induced frequency of heteroallelic recessive mutations in diploid cells of the yeast S . cerevisiae is higher than expected . In A . nidulans, we did not observe any increase in the frequency of recessive homozygous fpaA/fpaA (p-fluorophenylalanine-resistant) mutants over the expected one, which has been calculated on the basis of the observed mutation frequency in the haploid strain.

Biochim Biophys Acta, 1994 Mar 16, 1205(1), 39 - 48
Substrate characterization of the Saccharomyces cerevisiae protein farnesyltransferase and type-I protein geranylgeranyltransferase; Caplin BE et al.; The in vitro substrate preferences of recombinant S . cerevisiae protein farnesyltransferase and type-I protein geranylgeranyl-transferase were determined . Proteins ending in 16 different CaaX sequences (C = cysteine, a = aliphatic amino acid, X = variable amino acids) were used to determine the protein substrate preferences of these S . cerevisiae prenyltransferases . The identities of the attached prenyl groups were confirmed by iodomethane treatment of prenylated substrates and reverse-phase HPLC . The CaaX preference of each recombinant yeast enzyme was found to be nearly identical to the reported preferences of purified mammalian protein farnesyltransferase and type-I protein geranylgeranyltransferase . S . cerevisiae farnesyltransferase preferentially farnesylated CaaX sequences ending in methionine, cysteine or serine . The farnesyltransferase also attached geranylgeranyl to some CaaX sequences ending in methionine, leucine and cysteine . The S . cerevisiae type-I geranylgeranyltransferase preferentially geranylgeranylated CaaX sequences ending in leucine and to a lesser degree methionine . Yeast extracts were found to contain prenylating activities identical to those observed with the recombinant enzymes . Farnesyltransferase activity in yeast extracts exceeded type-I geranylgeranyltransferase activity by at least 3-fold, resulting in prenylation of leucine-ending CaaX substrates with a mixture of 75% geranylgeranyl and 25% farnesyl . These results suggest that some substrate overlap may occur between the S . cerevisiae protein farnesyltransferase and the type-I protein geranylgeranyltransferase in vivo.

Eur J Biochem, 1994 Mar 15, 220(3), 809 - 17
Human beta 1,4 galactosyltransferase and alpha 2,6 sialyltransferase expressed in Saccharomyces cerevisiae are retained as active enzymes in the endoplasmic reticulum; Krezdorn CH et al.; Biosynthesis and intracellular transport of recombinant human full-length beta 1,4 galactosyltransferase (GT) and full-length alpha 2,6 sialyltransferase (ST) were investigated in Saccharomyces cerevisiae . Recently, enzymic activity of recombinant GT (rGT) in crude homogenates of S . cerevisiae could successfully be demonstrated {Krezdorn, C., Watzele, G., Kleene, R . B., Ivanov, S . X . & Berger, E . G . (1993) Eur . J . Biochem . 212, 113-120} . In the present work, we show that, in yeast strains transformed with plasmid pDPSIA containing the cDNA coding for human ST, rST enzymic activity using asialo-fetuin or N-acetyllactosamine as acceptor substrates could readily be detected . Analysis by 1H-NMR spectroscopy of the disaccharide product of rGT, as recently reported, and the trisaccharide product of rST demonstrated that only the expected glycosidic linkages were formed . Following mechanical disruption of yeast cells, both enzymes sedimented with a fraction enriched in membranes of the endoplasmic reticulum (ER) and were activated by Triton X-100 3-5-fold . rGT and rST could be immunoprecipitated from their {35S}Met-labelled transformed yeast extracts using polyclonal antibodies raised against fusion proteins consisting of beta-galactosidase-GT or beta-galactosidase-ST, respectively, expressed in Escherichia coli . For rGT a single glycosylated form of apparent molecular mass 48 kDa was reported, but for rST two main bands corresponding to apparent molecular masses of 48 kDa and 44 kDa, respectively, were detected . Immunoprecipitation from either tunicamycin-treated {35S}Met-labelled transformed yeast cells or labelling with radio-active sugars both indicated that the 44-kDa form of rST was non-glycosylated and that the 48-kDa form of rST was core N-glycosylated . In addition, core glycosylation of both recombinant enzymes demonstrated that they were competent for translocation across the ER membranes . However, the 44-kDa form of rST was converted to the 48-kDa glycosylated form only slowly, suggesting a mechanism of posttranslational translocation . Absence of hyperglycosylation of rST and rGT in wild type and lack of the Golgi-specific man-alpha 1,6-man epitope suggest that the recombinant enzymes did not enter the yeast Golgi apparatus . These results indicated that both rGT and rST are retained as enzymically active enzymes in the ER of yeast and suggest a ribonucleoprotein-independent import of rST into the ER.

Biochem Biophys Res Commun, 1994 Mar 15, 199(2), 497 - 503
Properties of the catalytic domain of CDC25, a Saccharomyces cerevisiae GDP/GTP exchange factor: comparison of its activity on full-length and C-terminal truncated RAS2 proteins; Jacquet E et al.; Two C-terminal fragments (334 and 509 amino acid residues) of CDC25, a Saccharomyces cerevisiae GDP/GTP exchange factor, and the RAS2 protein were purified from E . coli, using the pGEX system . With this method it was possible to avoid in part the proteolytic phenomena that usually convert full-length RAS2 (42kDa) into 37 and 30kDa forms . Of the two CDC25 fragments containing the conserved catalytic domain, only CDC25-509 could enhance the guanine nucleotide exchange on RAS2 . Comparison of the activities of RAS2-42/37kDa and RAS2-30kDa showed that the C-terminal region (112 residues) influences neither the intrinsic GDP/GTP exchange nor its stimulation by CDC25-509 . RAS2-42/37kDa was somewhat more effective in enhancing the adenylylcyclase activity of a yeast membrane reconstituted system . CDC25-509 displayed a higher specific activity than the catalytic domains of the two CDC25-like proteins: S . cerevisiae SDC25 and mouse CDC25Mm.

J Biol Chem, 1994 Mar 11, 269(10), 7558 - 63
Mutagenesis of the amino targeting signal of Saccharomyces cerevisiae 3-ketoacyl-CoA thiolase reveals conserved amino acids required for import into peroxisomes in vivo; Glover JR et al.; Saccharomyces cerevisiae peroxisomal 3-ketoacyl-CoA thiolase is a soluble matrix protein that does not end in a consensus peroxisomal targeting signal-1 . The amino terminus of S . cerevisiae peroxisomal thiolase is conserved in 6 of 11 residues with the amino terminus of rat thiolase B, shown to act as a peroxisomal targeting signal-2 (Swinkels, B.W., Gould, S.J., Bodnar, A.G., Rachubinski, R.A., and Subramani, S . (1991) EMBO J . 10, 3255-3262) . Unlike mammalian peroxisomal thiolases, there is no extensive cleavage of S . cerevisiae thiolase upon import into peroxisomes . We demonstrate by in vivo expression that the amino-terminal 16 amino acids of S . cerevisiae thiolase are necessary and sufficient for targeting to peroxisomes . This result implies that yeast, like mammalian cells, can target proteins to the peroxisomal matrix by at least two different routes . We also demonstrate by targeted mutagenesis and in vivo expression of mutated thiolase genes that three amino acids conserved in the amino termini of all known thiolases are critical for efficient targeting of S . cerevisiae thiolase to peroxisomes.

Gene, 1994 Mar 11, 140(1), 33 - 40
Saccharomyces carlsbergensis contains two functional MET2 alleles similar to homologues from S . cerevisiae and S . monacensis; Hansen J et al.; The brewing yeast, Saccharomyces, carlsbergensis, is allopolyploid, derived from two diverged genomes . To obtain information about the possible origin of this yeast, we cloned two different S . carlsbergensis MET2 genes (encoding homoserine acetyltransferase) . One has a nucleotide (nt) sequence identical or very similar to MET2 of Saccharomyces cerevisiae . The other has a different sequence, but was functional in S . cerevisiae . This allele was sequenced and revealed a coding region of 486 amino acids (aa) . The nt sequence of the coding region showed 82% homology to S . cerevisiae MET2, while the derived aa sequences were 94% identical . Hybridization experiments to genomic DNA of different yeast strains revealed that the divergent MET2 gene had higher sequence homology to segments from type strains of S . monacensis, S . bayanus and S . uvarum than to MET2 from S . cerevisiae . Sequencing of 330 bp of a PCR-amplified fragment of MET2 from these organisms shows that the non-S . cerevisiae-like sequence from S . carlsbergensis is identical to the corresponding sequence in S . monacensis, while it is 93% homologous with S . bayanus and S . uvarum . Our results are consistent with the proposal that S . carlsbergensis originated as a hybrid between S . monacensis and S . cerevisiae . The complete identity of the MET2 fragments from S . monacensis and the S . carlsbergensis-specific MET2 allele suggests that the hybridization must have been a quite recent event.

Science, 1994 Mar 4, 263(5151), 1273 - 6
Heat-inducible degron: a method for constructing temperature-sensitive mutants; Dohmen RJ et al.; A temperature-sensitive (ts) mutant retains the function of a gene at a low (permissive) temperature but not at a high (nonpermissive) temperature . Arg-DHFR, a dihydrofolate reductase bearing an amino-terminal (N-terminal) arginine, is long-lived in the yeast Saccharomyces cerevisiae, even though arginine is a destabilizing residue in the N-end rule of protein degradation . A ts derivative of Arg-DHFR was identified that is long-lived at 23 degrees C but rapidly degraded by the N-end rule pathway at 37 degrees C . Fusions of ts Arg-DHFR to either Ura3 or Cdc28 of S . cerevisiae confer ts phenotypes specific for these gene products . Thus, Arg-DHFRts is a heat-inducible degradation signal that can be used to produce ts mutants without a search for ts mutations.

Arch Biochem Biophys, 1994 Mar, 309(2), 348 - 55
Casein kinase II of Saccharomyces cerevisiae contains two distinct regulatory subunits, beta and beta'; Bidwai AP et al.; The subunit composition of casein kinase II (CKII) from S . cerevisiae has been difficult to define, particularly with respect to the existence and number of regulatory (beta) subunits . A single, integral beta subunit, a loosely associated beta subunit, two distinct beta subunits, and a complete absence of beta subunits have all been proposed . Our laboratory reported yeast CKII to be composed of four polypeptides of 42, 41, 35, and 32 kDa (R . Padmanabha and C . V . C . Glover, 1987, J . Biol . Chem . 262, 1829-1835) . The 42- and 35-kDa polypeptides were identified as distinct catalytic subunits, alpha and alpha', on the basis of N-terminal sequencing and subsequent molecular cloning . The 41- and 32-kDa polypeptides were found to undergo autophosphorylation, a characteristic of the beta subunit in other species, but antibodies raised against the beta subunit of Drosophila CKII crossreacted only with the 41-kDa polypeptide . In order to clarify the subunit composition of yeast CKII, particularly with regard to the 32-kDa polypeptide, we have purified the enzyme to homogeneity using a modified procedure . Based on the results of autophosphorylation studies, Western blotting, peptide mapping of the 41- and 32-kDa peptides, and sequencing of subunit-specific peptides, we demonstrate that the 32-kDa polypeptide is an additional beta subunit (beta') distinct from the 41-kDa beta subunit . This represents the first demonstration of beta subunit heterogeneity in purified CKII from any species.

J Cell Biol, 1994 Mar, 124(6), 903 - 13
Ultrastructural analysis of the autophagic process in yeast: detection of autophagosomes and their characterization; Baba M et al.; Under nutrient-deficient conditions, the yeast S . cerevisiae sequesters its own cytoplasmic components into vacuoles in the form of "autophagic bodies" (Takeshige, K., M . Baba, S . Tsuboi, T . Noda, and Y . Ohsumi . 1992 . J . Cell Biol . 119:301-311) . Immunoelectron microscopy showed that two cytosolic marker enzymes, alcohol dehydrogenase and phosphoglycerate kinase, are present in the autophagic bodies at the same densities as in the cytosol, but are not present in vacuolar sap, suggesting that cytosolic enzymes are also taken up into the autophagic bodies . To understand this process, we performed morphological analyses by transmission and immunological electron microscopies using a freeze-substitution fixation method . Spherical structures completely enclosed in a double membrane were found near the vacuoles of protease-deficient mutant cells when the cells were shifted to nutrient-starvation media . Their size, membrane thickness, and contents of double membrane-structures corresponded well with those of autophagic bodies . Sometimes these double membrane structures were found to be in contact with the vacuolar membrane . Furthermore their outer membrane was occasionally seen to be continuous with the vacuolar membrane . Histochemical staining of carbohydrate strongly suggested that the structures with double membranes fused with the vacuoles . These results indicated that these structures are precursors of autophagic bodies, "autophagosomes" in yeast . All the data obtained suggested that the autophagic process in yeast is essentially similar to that of the lysosomal system in mammalian cells.

Mol Cell Biol, 1994 Mar, 14(3), 2180 - 90
Architecture of the U5 small nuclear RNA; Frank DN et al.; We have used comparative sequence analysis and deletion analysis to examine the secondary structure of the U5 small nuclear RNA (snRNA), an essential component of the pre-mRNA splicing apparatus . The secondary structure of Saccharomyces cerevisiae U5 snRNA was studied in detail, while sequences from six other fungal species were included in the phylogenetic analysis . Our results indicate that fungal U5 snRNAs, like their counterparts from other taxa, can be folded into a secondary structure characterized by a highly conserved stem-loop (stem-loop 1) that is flanked by a moderately conserved internal loop (internal loop 1) . In addition, several of the fungal U5 snRNAs include a novel stem-loop structure (ca . 30 nucleotides) that is adjacent to stem-loop 1 . By deletion analysis of the S . cerevisiae snRNA, we have demonstrated that the minimal U5 snRNA that can complement the lethal phenotype of a U5 gene disruption consists of (i) stem-loop 1, (ii) internal loop 1, (iii) a stem-closing internal loop 1, and (iv) the conserved Sm protein binding site . Remarkably, all essential, U5-specific primary sequence elements are encoded by a 39-nucleotide domain consisting of stem-loop 1 and internal loop 1 . This domain must, therefore, contain all U5-specific sequences that are essential for splicing activity, including binding sites for U5-specific proteins.

Mol Cell Biol, 1994 Mar, 14(3), 2100 - 12
Induction of pseudohyphal growth by overexpression of PHD1, a Saccharomyces cerevisiae gene related to transcriptional regulators of fungal development; Gimeno CJ et al.; When starved for nitrogen, MATa/MAT alpha cells of the budding yeast Saccharomyces cerevisiae undergo a dimorphic transition to pseudohyphal growth . A visual genetic screen, called PHD (pseudohyphal determinant), for S . cerevisiae pseudohyphal growth mutants was developed . The PHD screen was used to identify seven S . cerevisiae genes that when overexpressed in MATa/MAT alpha cells growing on nitrogen starvation medium cause precocious and unusually vigorous pseudohyphal growth . PHD1, a gene whose overexpression induced invasive pseudohyphal growth on a nutritionally rich medium, was characterized . PHD1 maps to chromosome XI and is predicted to encode a 366-amino-acid protein . PHD1 has a SWI4- and MBP1-like DNA binding motif that is 73% identical over 100 amino acids to a region of Aspergillus nidulans StuA . StuA regulates two pseudohyphal growth-like cell divisions during conidiophore morphogenesis . Epitope-tagged PHD1 was localized to the nucleus by indirect immunofluorescence . These facts suggest that PHD1 may function as a transcriptional regulatory protein . Overexpression of PHD1 in wild-type haploid strains does not induce pseudohyphal growth . Interestingly, PHD1 overexpression enhances pseudohyphal growth in a haploid strain that has the diploid polar budding pattern because of a mutation in the BUD4 gene . In addition, wild-type diploid strains lacking PHD1 undergo pseudohyphal growth when starved for nitrogen . The possible functions of PHD1 in pseudohyphal growth and the uses of the PHD screen to identify morphogenetic regulatory genes from heterologous organisms are discussed.

J Bacteriol, 1994 Mar, 176(5), 1488 - 99
Cloning of the Saccharomyces cerevisiae gene whose overexpression overcomes the effects of HM-1 killer toxin, which inhibits beta-glucan synthesis; Kasahara S et al.; A gene whose overexpression can endow Saccharomyces cerevisiae cells with resistance to HM-1 killer toxin was cloned from an S . cerevisiae genomic library . This gene, designated HKR1 (Hansenula mrakii killer toxin-resistant gene 1), contains a 5.4-kb open reading frame . The predicted amino acid sequence of the protein specified by HKR1 indicates that the protein consists of 1,802 amino acids and is very rich in serine and threonine, which could serve as O-glycosylation sites . The protein also contains two hydrophobic domains at the N-terminal end and in the C-terminal half, which could function as a signal peptide and transmembrane domain, respectively . Hkr1p is found to contain an EF hand motif of the calcium-binding consensus sequence in the C-terminal cytoplasmic domain . Thus, Hkr1p is expected to be a calcium-binding, glycosylated type I membrane protein . Southern and Northern (RNA) analyses demonstrated that there is a single copy of the HKR1 gene in the S . cerevisiae genome, and the transcriptional level of HKR1 is extremely low . Gene disruption followed by tetrad analysis showed that HKR1 is an essential gene . Overexpression of the truncated HKR1 encoding the C-terminal half of Hkr1p made the cells more resistant to HM-1 killer toxin than the full-length HKR1 did, demonstrating that the C-terminal half of Hkr1p is essential for overcoming the effect of HM-1 killer toxin . Furthermore, overexpression of HKR1 increased the beta-glucan content in the cell wall without affecting in vitro beta-glucan synthase activity, suggesting that HKR1 regulates beta-glucan synthesis in vivo.

Biochim Biophys Acta, 1994 Mar 1, 1217(2), 214 - 8
Cloning and sequencing of a gene coding for an actin binding protein of Saccharomyces exiguus; Lange U et al.; The actin binding protein Abp1p of the yeast Saccharomyces cervisiae is thought to be involved in the spatial organisation of cell surface growth . It contains a potential actin binding domain and an SH-3 region, a common motif of many signal transduction proteins {1} . We have cloned and sequenced an ABP1 homologous gene of Saccharomyces exiguus, a yeast which is only distantly related to S . cerevisiae . The protein encoded by this gene is slightly larger than the respective S . cerevisiae protein (617 versus 592 amino acids) . The two genes are 67.4% identical and the deduced amino acid sequences share an overall identity of 59.8% . The most conserved regions are the 148 N-terminal amino acids containing the potential actin binding site and the 58 C-terminal amino acids including the SH3 domain . In addition, both proteins contain a repeated motif of unknown function which is rich in glutamic acids with the sequence EEEEEEEAPAPSLPSR in the S . exiguus Abp1p.

Yeast, 1994 Mar, 10(3), 391 - 8
Genes of the linear mitochondrial DNA of Williopsis mrakii: coding sequences for a maturase-like protein, a ribosomal protein VAR1 homologue, cytochrome oxidase subunit 2 and methionyl tRNA; Drissi R et al.; The mitochondrial DNA (mtDNA) in some yeasts has a linear structure with inverted terminal repeats closed by a single-stranded loop . These mtDNAs have generally a constant gene order, beginning with a small ribosomal RNA gene at the right end and terminating with a cytochrome oxidase subunit 2 gene (COX2) at the left end, independently of the wide variation in genome size . In the mtDNAs from several species of the genus Williopsis, we found an additional open reading frame, ORF1, which was homologous to the Saccharomyces cerevisiae RF1 gene encoding a group I intron maturase-like protein . ORF1 genes from W . mrakii and W . suaveolens were mapped and sequenced . Next to ORF1, COX2 and methionyl tRNA genes were present on the opposite strand . The same relative positions of genes in the mtDNAs so far examined suggests that the constancy of gene order is generally conserved also at the level of individual tRNA genes . We identified another open reading frame, ORF2, in W . mrakii mtDNA . It was mapped next to the cytochrome oxidase subunit 3 gene . Rich in adenine-thymine bases, ORF2 appears to be a homologue of the VAR1 gene which codes for a small ribosomal subunit protein in S . cerevisiae mitochondria . Nucleotide sequences data have been deposited in the EmBL data library under the following Accession Numbers: X66594 (Apocytochrome b and ORF2 genes of W . mrakii), X66595 (ORF1, tRNA-Met and COX2 genes of W . mrakii), X73415 (tRNA-Met and COX2 genes of W . suaveolens), X73416 (ORF1 gene of W . suaveolens) and X73414 (tRNA-Met and COX2 genes of P . jadinii).

Yeast, 1994 Mar, 10(3), 309 - 317
Sequence comparison of the ARG4 chromosomal regions from the two related yeasts, Saccharomyces cerevisiae and Saccharomyces douglasii; Adjiri A et al.; A 3.6 kb DNA fragment from Saccharomyces douglasii, containing the ARG4 gene, has been cloned, sequenced and compared to the corresponding region from Saccharomyces cerevisiae . The organization of this region is identical in both yeasts . It contains besides the ARG4 gene, another complete open reading frame (ORF) (YSD83) and a third incomplete one (DED81) . The ARG4 and the YSD83 coding regions differ from their S . cerevisiae homologs by 8.1% and 12.5%, respectively, of base substitutions . The encoded proteins have evolved differently: amino acid replacements are significantly less frequent in Arg4 (2.8%) than in Ysc83 (12.4%) and most of the changes in Arg4 are conservative, which is not the case for Ysc83 . The non-coding regions are less conserved, with small AT-rich insertions/deletions and 20% base substitutions . However, the level of divergence is smaller in the aligned sequences of these regions than in silent sites of the ORFs, probably revealing a higher degree of constraints . The Gcn4 binding site and the region where meiotic double-strand breaks occur, are fully conserved . The data confirm that these two yeasts are evolutionarily closely related and that comparisons of their sequences might reveal conserved protein and DNA domains not expected to be found in sequence comparisons between more diverged organisms.

Microbiology, 1994 Mar, 140 ( Pt 3), 601 - 10
Energetic aspects of glucose metabolism in a pyruvate-dehydrogenase-negative mutant of Saccharomyces cerevisiae; Pronk JT et al.; Saccharomyces cerevisiae T23C (pda1::Tn5ble) is an isogenic gene replacement mutant of the wild-type strain S . cerevisiae T23D . The mutation causes a complete loss of pyruvate dehydrogenase activity . Pyruvate metabolism in this pyruvate-dehydrogenase-negative (Pdh-) strain was investigated in aerobic glucose-limited chemostat cultures, grown at a dilution rate of 0.10 h-1, and compared with the metabolism in the isogenic wild-type strain . Under these conditions, growth of the Pdh- strain was fully respiratory . Enzyme activities in cell-free extracts indicated that the enzymes pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-coenzyme A (acetyl-CoA) synthetase could provide a functional bypass of the pyruvate dehydrogenase complex . Since this metabolic sequence involves ATP hydrolysis in the acetyl-CoA synthetase reaction, a negative effect of the pda1::Tn5ble mutation on the growth efficiency was anticipated . Indeed, the biomass yield of the Pdh- strain {0.44 g biomass (g glucose)-1} was significantly lower than that of wild-type S . cerevisiae {0.52 g biomass (g glucose)-1} . The effect of the mutation on biomass yield could be quantitatively explained in terms of a lower ATP yield from glucose catabolism and an increased ATP requirement for the synthesis of acetyl-CoA used in anabolism . Control experiments showed that the pda1::Tn5ble mutation did not affect biomass yield in ethanol-limited chemostat cultures . The results support the view that, during aerobic glucose-limited growth of S . cerevisiae at low growth rates, the pyruvate dehydrogenase complex accounts for the major part of the pyruvate flux . Moreover, it is concluded that hydrolysis of pyrophosphate formed in the acetyl-CoA synthetase reaction does not contribute significantly to energy transduction in this yeast . Respiratory-deficient cells did not contribute to glucose metabolism in the chemostat cultures and were probably formed upon plating.

Genetics, 1994 Mar, 136(3), 789 - 802
The chromosome end in yeast: its mosaic nature and influence on recombinational dynamics; Louis EJ et al.; Yeast chromosome ends are composed of several different repeated elements . Among six clones of chromosome ends from two strains of Saccharomyces cerevisiae, at least seven different repeated sequence families were found . These included the previously identified Y' and X elements . Some families are highly variable in copy number and location between strains of S . cerevisiae, while other elements appear constant in copy number and location . Three repeated sequence elements are specific to S . cerevisiae and are not found in its evolutionarily close relative, Saccharomyces paradoxus . Two other repeated sequences are found in both S . cerevisiae and S . paradoxus . None of those described here is found (by low stringency DNA hybridization) in the next closest species, Saccharomyces bayanus . The loosely characterized X element is now more precisely defined . X is a composite of at least four small (ca . 45-140 bp) sequences found at some, but not all, ends . There is also a potential "core" X element of approximately 560 bp which may be found at all ends . Distal to X, only one of six clones had (TG1-3)n telomere sequence at the junction between X and Y' . The presence of these internal (TG1-3)n sequences correlates with the ability of a single Y' to expand into a tandem array of Y's by unequal sister chromatid exchange . The presence of shared repeated elements proximal to the X region can override the strong preference of Y's to recombine ectopically with other Y's of the same size class . The chromosome ends in yeast are evolutionarily dynamic in terms of subtelomeric repeat structure and variability.

Curr Genet, 1994 Mar, 25(3), 239 - 44
Suppression of a yeast mitochondrial RNA processing defect by nuclear mutations; Smooker PM et al.; The S . cerevisiae strain h56 is a temperature-sensitive mit- mutant containing a single nucleotide substitution in the region 5' to the reading frame of the mitochondrial var1 gene . The mutation decreases the efficiency of processing of a precursor RNA such that little var1 mRNA is produced at the restrictive temperature, 36 degrees C . This communication reports the isolation and characterization of several strains carrying nuclear mutations which suppress the temperature-sensitivity of h56 . Both dominant and recessive suppressor mutations were isolated . One dominant suppressor strain (h56-S4) was characterized biochemically, and the mechanism of suppression shown to involve a restoration of precursor RNA processing at the restrictive temperature, with a concomitant increase in the synthesis of the var1 protein . It appears likely that the suppressing allele encodes a component of an RNA processing endoribonuclease active on var1 transcripts . A genomic library was constructed from the h56-S4 strain, and several plasmids showing suppressed activity were isolated . A preliminary analysis of these plasmids is presented.

Curr Genet, 1994 Mar, 25(3), 196 - 201
Cloning and analysis of a FLO5 flocculation gene from S . cerevisiae; Bidard F et al.; A yeast flocculation gene was isolated from a genomic library of an FLO5 strain of S . cerevisiae on the basis of its ability to trigger flocculation in a non-flocculent strain . Characterization of the cloned gene by restriction mapping, Southern analysis, and chromosome mapping have shown that it corresponds to a FLO5 gene previously located on chromosome I and that this gene is related to the already described FLO1 gene . A study of gene expression in different yeast strains has indicated that, while this gene is dominant, its expression can be suppressed in some genetic backgrounds . A Northern-blot analysis has demonstrated that the same 5000-nt transcript was present in an FLO5 and an FLO1 strain . A gene disruption experiment has led to the conclusion that another flocculation gene is present and can be active in the FLO5 strain we used.

J Biotechnol, 1994 Feb 28, 32(3), 249 - 59
Production of recombinant hirudin by high cell density fed-batch cultivations of a Saccharomyces cerevisiae strain: physiological considerations during the bioprocess design; Mendoza-Vega O et al.; The conditions for the high cell density fed-batch culture of a Saccharomyces cerevisiae strain producing recombinant hirudin (rHV2-Lys47) have been established . A Leu+ derivative of S . cerevisiae c13ABYS86 was used as the host strain transformed with an expression plasmid containing the gene encoding rHV2-Lys47 and driven by the MF alpha 1 promoter . In order to develop the fed-batch culture protocol, the recombinant strains' physiology was first of all investigated in chemostat culture . The maximum respiratory capacity of the recombinant strain was observed to be between dilution rates of 0.2 and 0.26 h-1, which is typical for laboratory strains as compared to values published for baker's yeasts . Furthermore, maximum biomass yield and product secretion were observed at a dilution rate of approx . 0.15 h-1 . The plasmid segregational stability of the recombinant strain showed that the expression plasmid was stable, irrespective of the dilution rates used, for more than 80 generations of growth between dilution rates of 0.043 h-1 and 0.3 h-1 . The chemostat data was used to define a fed-batch process . The fed-batch results demonstrated a biomass production of 60 g l-1 CDW and a high production level of recombinant hirudin of 500 mg l-1 . Stability of the expression of the gene coding for rHV2-Lys47 was maintained during all the studied fed-batch conditions . The plasmid copy number in the fed-batch remained constant at approx . 43 at a specific growth rate of 0.12 h-1, whereas it increased by 60-95% at a lower dilution rate (mu = 0.06 h-1) . Although a variation of the plasmid copy number could be expected, it was postulated from the experimental data that the observed amplification could have been influenced by an environmental effect due to an accumulation of medium components in the supernatant . The results presented here illustrate the importance of a well-balanced medium when considering the production of a recombinant protein in a high cell density cultivation process with high production levels.

J Biol Chem, 1994 Feb 25, 269(8), 6117 - 23
Initiation factor eIF-4E of Saccharomyces cerevisiae . Distribution within the cell, binding to mRNA, and consequences of its overproduction; Lang V et al.; The eukaryotic translational initiation factor 4E (eIF-4E) is an essential protein that binds the 5' cap structure with high specificity and affinity . Yeast eIF-4E is homologous to eIF-4E of higher eukaryotes, but interacts with a different set of cap-binding complex proteins . In the present study the distribution of yeast eIF-4E in Saccharomyces cerevisiae was found to be similar to that observed in higher cells, whereby the yeast factor was more concentrated in the nucleus than in the cytoplasm . Overexpression of yeast eIF-4E in S . cerevisiae exerted at most a minimal effect on growth in liquid minimal medium and was not found to influence the translation of reporter gene mRNAs bearing secondary structure in their leader regions . In a new method to study mRNA-protein interactions, biotinylated mRNAs were synthesized in vitro for use in studies of the binding of eIF-4E in yeast extracts . Streptavidin was used to adsorb the biotinylated mRNAs plus bound initiation factors . Stem-loop structures in the leader region did not influence the binding of eIF-4E or, in comparative experiments, of eIF-4A . Thus yeast eIF-4E shows both similarities and differences with respect to the distribution and function of its counterparts in higher eukaryotes.

Gene, 1994 Feb 11, 139(1), 27 - 33
Two novel deduced serine/threonine protein kinases from Saccharomyces cerevisiae; Dahlkvist A et al.; We have cloned genes for two closely related protein kinase (PK) homologues from Saccharomyces cerevisiae . Sequence alignment of the kinase domain with previously characterized PK reveals the highest degree of similarity with second messenger-regulated kinases . RCK1 encodes a 58-kDa protein, and is weakly expressed . RCK2 encodes a 65-kDa protein, and transcripts from this gene are readily detected . RCK1 has been mapped to the L arm of chromosome VII, and RCK2 to chromosome XII . Disruption of these genes in the S . cerevisiae genome demonstrates that both genes are non-essential.

Gene, 1994 Feb 11, 139(1), 1 - 7
Two homologous introns from related Saccharomyces species differ in their mobility; Szczepanek T et al.; We have studied gene conversion initiated by the ai3 intron of the Saccharomyces cerevisiae mitochondrial (mt) COXI gene and its homologous intron (S.cap.ai1) from Saccharomyces capensis . The approach used involved the measurement of intron transmission amongst the progeny of crosses between constructed recipient and donor strains . We found that the S . cerevisiae ai3 intron is extremely active as a donor in gene conversion, whereas its homologous S . capensis intron is not . We have established the sequence of S.cap.ai1 and compared its open reading frame (ORF) with that of I-SceIII encoded by the homologous S . cerevisiae intron . The two protein-coding intron sequences are almost identical, except that the S . capensis ORF contains an in-frame stop codon . This finding provides a strong indication that the 3' part of the S . cerevisiae intron ORF encoding I-SceIII (which should not be translated in the S . capensis intron) must be critical for function of mtDNA endonucleases to mediate intron mobility.

EMBO J, 1994 Feb 1, 13(3), 655 - 64
YAP1 dependent activation of TRX2 is essential for the response of Saccharomyces cerevisiae to oxidative stress by hydroperoxides; Kuge S et al.; The role of the YAP1 transcription factor in the response of Saccharomyces cerevisiae cells to a variety of conditions that induce oxidative stress has been investigated . Cells deficient in YAP1 were found to be hypersensitive to hydroperoxides and thioloxidants, whereas overexpression of YAP1 conferred hyper-resistance to the same conditions . These treatments resulted in an increase in YAP1-specific binding to DNA together with an increase in YAP1 dependent transcription . Our results indicate that this increase is not due to an increase in synthesis of YAP1 protein, but rather results from modification of pre-existing protein . Using a specific genetic screen, the TRX2 gene, one of two genes of S . cerevisiae that encode thioredoxin protein, was identified as being essential for YAP1 dependent resistance to hydroperoxides . Furthermore, efficient expression of TRX2 was dependent on YAP1 and enhanced under conditions of oxidative stress.

Mol Cell Biol, 1994 Feb, 14(2), 1278 - 92
Homologous, homeologous, and illegitimate repair of double-strand breaks during transformation of a wild-type strain and a rad52 mutant strain of Saccharomyces cerevisiae; Mezard C et al.; Different modes of in vivo repair of double-strand breaks (DSBs) have been described for various organisms: the recombinational DSB repair (DSBR) mode, the single-strand annealing (SSA) mode, and end-to-end joining . To investigate these modes of DSB repair in Saccharomyces cerevisiae, we have examined the fate of in vitro linearized replicative plasmids during transformation with respect to several parameters . We found that (i) the efficiencies of both intramolecular and intermolecular linear plasmid DSB repair are homology dependent (according to the amount of DNA used during transformation {100 ng or less}, recombination between similar but not identical {homeologous} P450s sequences sharing 73% identity is 2- to 18-fold lower than recombination between identical sequences); (ii) the RAD52 gene product is not essential for intramolecular recombination between homologous and homeologous direct repeats (as in the wild-type strain, recombination occurs with respect to the overall alignment of the parental sequences); (iii) in contrast, the RAD52 gene product is required for intermolecular interactions (the rare transformants which are obtained contain plasmids resulting from deletion-forming intramolecular events involving little or no sequence homology); (iv) similarly, sequencing data revealed examples of intramolecular joining within the few terminal nucleotides of the transforming DNA upon transformation with a linear plasmid with no repeat in the wild-type strain . The recombinant junctions of the rare illegitimate events obtained with S . cerevisiae are very similar to those observed in the repair of DSB in mammalian cells . Together, these and previous results suggest the existence of alternative modes for DSB repair during transformation which differ in their efficiencies and in the structure of their products . We discuss the implications of these results with respect to the existence of alternative pathways and the role of the RAD52 gene product.

Mol Cell Biol, 1994 Feb, 14(2), 1104 - 12
Identification of residues of the H-ras protein critical for functional interaction with guanine nucleotide exchange factors; Mosteller RD et al.; Ras proteins are activated in vivo by guanine nucleotide exchange factors encoded by genes homologous to the CDC25 gene of Saccharomyces cerevisiae . We have taken a combined genetic and biochemical approach to probe the sites on Ras proteins important for interaction with such exchange factors and to further probe the mechanism of CDC25-catalyzed GDP-GTP exchange . Random mutagenesis coupled with genetic selection in S . cerevisiae was used to generate second-site mutations within human H-ras-ala15 which could suppress the ability of the Ala-15 substitution to block CDC25 function . We transferred these second-site suppressor mutations to normal H-ras and oncogenic H-rasVal-12 to test whether they induced a general loss of function or whether they selectively affected CDC25 interaction . Four highly selective mutations were discovered, and they affected the surface-located amino acid residues 62, 63, 67, and 69 . Two lines of evidence suggested that these residues may be involved in binding to CDC25: (i) using the yeast two-hybrid system, we demonstrated that these mutants cannot bind CDC25 under conditions where the wild-type H-Ras protein can; (ii) we demonstrated that the binding to H-Ras of monoclonal antibody Y13-259, whose epitope has been mapped to residues 63, 65, 66, 67, 70, and 73, is blocked by the mouse sos1 and yeast CDC25 gene products . We also present evidence that the mechanism by which CDC25 catalyzes exchange is more involved than simply catalyzing the release of bound nucleotide and passively allowing nucleotides to rebind . Most critically, a complex of Ras and CDC25 protein, unlike free Fas protein, possesses significantly greater affinity for GTP than for GDP . Furthermore, the Ras CDC25 complex is more readily dissociated into free subunits by GTP than it is by GDP . Both of these results suggest a function for CDC25 in promoting the selective exchange of GTP for GDP.

J Cell Sci, 1994 Feb, 107 ( Pt 2), 581 - 8
Alternative splicing of human cyclin E; Sewing A et al.; Cyclin E is a regulatory subunit of the cdc2-related protein kinase cdk2, which is activated shortly before S-phase entry, thus defining it as a G1 cyclin . We report here the existence of a 43 kDa splice variant of human cyclin E, termed cyclin Es, which lacks 49 amino acids within the cyclin box compared to the known 48 kDa cyclin E . Cyclin Es is expressed at approximately 1/10 of the level of full-length cyclin E in several cell lines analysed . The two cyclin E forms differ functionally in that cyclin E, but not cyclin Es, is able to complex with cdk2, to activate the histone H1, pRb and p107 in vitro kinase activity of cdk2 and to rescue a triple CLN mutation in S . cerevisiae . Cyclin Es is the first splice variant of a cell cycle regulatory protein to be described . Our findings also indicate that the cyclin box in cyclin E mediates the interaction with cdk2.

Yeast, 1994 Feb, 10(2), 199 - 209
Analysis of PFK3--a gene involved in particulate phosphofructokinase synthesis reveals additional functions of TPS2 in Saccharomyces cerevisiae; Sur IP et al.; The pfk3 mutation of Saccharomyces cerevisiae causes glucose-negativity in a pfk1 genetic background, the mutant is temperature-sensitive for growth and homozygous diploids do not sporulate . It fails to accumulate trehalose, and has an altered glycogen accumulation profile under glucose-starvation conditions . pfk3-6, one of the alleles of pfk3, has an altered morphology, forming long chain-like structures at 36 degrees C . The PFK3 gene was cloned by complementation of the mutant phenotypes . Integrative transformation demonstrated that the complementing fragment encoded the authentic PFK3 gene . The disruption of the gene does not affect viability . Like the EMS-induced pfk3 mutant, the disruptants are temperature-sensitive and in a pfk1 genetic background are also glucose-negative . The PFK3 transcript is induced by heat-shock . Partial DNA sequence shows that PFK3 is identical to TPS2 (De Virgilio et al., 1993) . We demonstrate that, apart from being a structural determinant of trehalose 6-phosphate phosphatase, PFK3 (TPS2) is required for PFKII synthesis and normal regulation of S . cerevisiae response to nutrient and thermal stresses.

J Membr Biol, 1994 Feb, 138(1), 29 - 35
Dependence of the kinetics of secondary active transports in yeast on H(+)-ATPase acidification; Kotyk A; Acidification of the external medium of the yeast Saccharomyces cerevisiae, mainly caused by proton extrusion by plasma membrane H(+)-ATPase, was inhibited to different degrees by D2O, diethylstilbestrol, suloctidil, vanadate, erythrosin B, cupric sulfate and dicyclohexylcarbodiimide . The same pattern of inhibition was found with the uptake of amino acids, adenine, uracil, and phosphate and sulfate anions . An increase of the acidification rate by dioctanoylglycerol also increased the rates of uptake of adenine and of glutamic acid . In contrast, a decrease of the membrane potential at pH 4.5 from a mean of -40 to -20 mV caused by 20 mM KCl had no effect on the transport rates . The ATPase-deficient mutant S . cerevisiae pmal-105 showed a markedly lower uptake of all the above solutes as compared with the wild type, while its membrane potential and delta pH were unchanged . Other types of acidification (spontaneous upon suspension; K+ stimulated) did not affect the secondary uptake systems . A partially competitive inhibition between some individual transport systems was observed, most pronouncedly with adenine as the most avidly transported solute . These observations, together with the earlier results that inhibition of H(+)-ATPase activity affects more the acidic than the basic amino acids and that it is more pronounced at higher pH values and at greater solute concentrations, support the view that it is the protons in or at the membrane, as they are extruded by the ATPase, that govern the rates of uptake by secondary active transport systems in yeast.

Protein Eng, 1994 Feb, 7(2), 243 - 53
Mutational analysis supports a structural model for the cell cycle protein kinase p34; Endicott JA et al.; Structural models for the eukaryotic cell cycle control protein p34 from human, S . pombe and S . cerevisiae have been derived from the crystallographic coordinates of the cAMP-dependent protein kinase (cAPK) catalytic subunit (active conformation) and compared with the structure of inactive CDK2 apoenzyme . Differences between the p34 and cAPK catalytic sites provide a possible explanation for their different substrate specificities . The p34 models localize Tyr15 and Thr14 close to the sites of catalysis and substrate recognition where their phosphorylation could inhibit p34 kinase activity either by blocking MgATP or substrate binding . The conserved sequences PSTAIRE and LYLIFEFL are both close to the catalytic site and accessible on the protein surface available to mediate interactions with other proteins . It is predicted that p34 has an active-site cleft composed almost entirely of sequences common to all protein kinases and sequences unique to the p34 protein family . Genetic and biochemical analyses of p34 have shown that it interacts extensively with a number of other proteins . The model allows the relative disposition of these sites of mutation to each other and to the sites of catalysis and substrate recognition to be appreciated . Surface regions on p34 that are important for function have been identified . These sites identify residues that may interact with p13suc1, cyclin, p107wee1 and p80cdc25.

Mol Gen Genet, 1994 Feb, 242(3), 263 - 71
Sequence and promoter analysis of the highly expressed TEF gene of the filamentous fungus Ashbya gossypii; Steiner S et al.; Ashbya gossypii carries only a single gene (TEF) coding for the abundant translation elongation factor 1 alpha . Cloning and sequencing of this gene and deletion analysis of the promoter region revealed an extremely high degree of similarity with the well studied TEF genes of the yeast Saccharomyces cerevisiae including promoter upstream activation sequence (UAS) elements . The open reading frames in both species are 458 codons long and show 88.6% identity at the DNA level and 93.7% identity at the protein level . A short DNA segment in the promoter, between nucleotides -268 and -213 upstream of the ATG start codon, is essential for high-level expression of the A . gossypii TEF gene . It carries two sequences, GCCCATACAT and ATCCATACAT, with high homology to the UASrpg sequence of S . cerevisiae, which is an essential promoter element in genes coding for highly expressed components of the translational apparatus . UASrpg sequences are binding sites for the S . cerevisiae protein TUF, also called RAP1 or GRF1 . In gel retardation with A . gossypii protein extracts we demonstrated specific protein binding to the short TEF promoter segment carrying the UASrpg homologous sequences.

J Cell Biol, 1994 Feb, 124(4), 507 - 19
Localization of the Kar3 kinesin heavy chain-related protein requires the Cik1 interacting protein; Page BD et al.; The Kar3 protein (Kar3p), a protein related to kinesin heavy chain, and the Cik1 protein (Cik1p) appear to participate in the same cellular processes in S . cerevisiae . Phenotypic analysis of mutants indicates that both CIK1 and KAR3 participate in spindle formation and karyogamy . In addition, the expression of both genes is induced by pheromone treatment . In vegetatively growing cells, both Cik1::beta-gal and Kar3::beta-gal fusions localize to the spindle pole body (SPB), and after pheromone treatment both fusion proteins localize to the spindle pole body and cytoplasmic microtubules . The dependence of Cik1p and Kar3p localization upon one another was investigated by indirect immunofluorescence of fusion proteins in pheromone-treated cells . The Cik1p::beta-gal fusion does not localize to the SPB or microtubules in a kar3 delta strain, and the Kar3p::beta-gal fusion protein does not localize to microtubule-associated structures in a cik1 delta strain . Thus, these proteins appear to be interdependent for localization to the SPB and microtubules . Analysis by both the two-hybrid system and co-immunoprecipitation experiments indicates that Cik1p and kar3p interact, suggesting that they are part of the same protein complex . These data indicate that interaction between a putative kinesin heavy chain-related protein and another protein can determine the localization of motor activity and thereby affect the functional specificity of the motor complex.

J Bacteriol, 1994 Feb, 176(4), 953 - 8
Affinity of glucose transport in Saccharomyces cerevisiae is modulated during growth on glucose; Walsh MC et al.; By using a modified technique to measure glucose uptake in Saccharomyces cerevisiae, potential uncertainties have been identified in previous determinations . These previous determinations had led to the proposal that S . cerevisiae contained a constitutive low-affinity glucose transporter and a glucose-repressible high-affinity transporter . We show that, upon transition from glucose-repressed to -derepressed conditions, the maximum rate of glucose transport is constant and only the affinity for glucose changes . We conclude that the transporter or group of transporters is constitutive and that regulation of glucose transport occurs via a factor that modifies the affinity of the transporters and not via the synthesis of different kinetically independent transporters . Such a mechanism could, for instance, be accommodated by the binding of kinases causing a change in affinity for glucose.

Curr Genet, 1994 Feb, 25(2), 178 - 9
Yeast ts secretory mutation rgs1 is suppressed by the SEC4 gene of Saccharomyces cerevisiae; Gerassimenko OG; Yeast rgs1 cells accumulate secretory vesicles in the cytoplasm and stop the secretion of proteins at the restrictive temperature . The ts mutation rgs1 may be suppressed by several different genes; the S . cerevisiae SEC4 gene, encoding the small G-protein involved in the late secretory stage, is one of them . Synthetic lethality of the double rgs1 sec4 mutant is demonstrated.

Braz J Med Biol Res, 1994 Feb, 27(2), 145 - 50
Isolation of temperature-sensitive yeast GPI-anchoring mutants; Orlean P et al.; We are using a genetic approach to explore the synthesis and function of glycosylphosphatidylinositol (GPI) . We have developed a novel strategy to isolate Saccharomyces cerevisiae mutants blocked in GPI anchoring by screening colonies of mutagenized yeast cells for those that fail to incorporate {3H}inositol into protein . Among our isolates are strains blocked in mannosylation of the GPI-anchorprecursor, and strains defective in the synthesis of N-acetylglucosaminyl phosphatidylinositol (GlcNAc-PI) . We have characterized one mutant, gpi1, further . This strain is defective in GlcNAc-PI synthesis and is temperature-sensitive for growth . Completion of the first step in GPI assembly is therefore required for the growth of the unicellular eukaryote S . cerevisiae . We have isolated plasmids that complement the gpi1 mutation from S . cerevisiae genomic DNA-and fission yeast cDNA libraries.

Mol Biol Cell, 1994 Feb, 5(2), 173 - 81
Raf-1 interferes with Ras and Rap1A effector functions in yeast; Ruggieri R et al.; Raf-1 is a serine/threonine kinase that acts downstream of Ras in mitogenic signal transduction pathways, but the mechanism by which Ras transmits signals to Raf-1 is not known . We have examined the interaction between Raf-1 and human H-ras in three different systems that utilize H-ras-induced phenotypes in Saccharomyces cerevisiae . In each system, the effects of H-ras depend on guanosine triphosphate and appear to be mediated through the H-ras effector binding region . H-ras effector function was blocked in each case by expression of the N-terminal regulatory domain of Raf-1 . These inhibitory effects did not require the Raf-1 kinase domain . Raf-1 also blocked Rap1A effector function in S . cerevisiae . Raf-1, therefore, appears to interact with H-Ras and Rap1A in these in vivo systems with properties that suggest it is an immediate downstream effector.

Eur J Cell Biol, 1994 Feb, 63(1), 130 - 9
Activity of Kex2 dibasic endoprotease is localized throughout the secretory pathway in Saccharomyces cerevisiae . An ultracytochemical study; Vorisek J; The sites of calcium-dependent dibasic endoprotease (Kex2; yscF) activity have been ultracytochemically localized in exponential cultures of haploid (alpha) wild-type strain of Saccharomyces cerevisiae, in its pep4-3 mutant and in the kex2 mutant . The gently prefixed cells were thoroughly washed and incubated in a buffered mixture containing Ca2+, benzyloxycarbonyl-L-tyrosyl-L-lysyl-arginin-4-methoxy-2-naphthylam ide (Z-Tyr-Lys-Arg-MNA) as substrate and hexazotized p-rosaniline (HPR) as coupler of the liberated MNA . The precipitated azo-dye was osmicated, and the cells were embedded for ultrathin sectioning . In the pep4-3 mutant the reaction product labeled the periphery of lipoprotein condensates, the lumen of nuclear envelope plus endoplasmic membrane cisternae, the matrix of juvenile vacuoles and the lumen of microvesicles--the membrane vesicles and the smaller coated or uncoated globules . These were dispersed in the cytoplasm or in the senescent vacuoles . The reaction product labeled also both faces of the plasmalemma-restricted cells . In the presence of EDTA the reaction product appeared only in the lipoprotein condensates . In the absence of substrate and in the presence of HgCl2, no reaction product was formed . In the wild-type strain the enzyme activity was detectable in the cytoplasmic microvesicles and in the tonoplast of vacuoles . No reaction product formed in the kex2 mutant cells . Enzymic assay of total activity of dibasic endoprotease in investigated strains confirmed the substrate was hydrolyzed principally by calcium-dependent protease . The study was supplemented by ultracytochemical localization of glycoproteins in cells of secretory mutants cultivated under restrictive conditions . The results of both topochemical studies give further arguments against the established model of a polar compartmentalized Golgi apparatus in S . cerevisiae.

Protein Sci, 1994 Feb, 3(2), 303 - 13
The 2.6-A refined structure of the Escherichia coli recombinant Saccharomyces cerevisiae flavocytochrome b2-sulfite complex; Tegoni M et al.; Flavocytochrome b2 from Saccharomyces cerevisiae catalyzes the oxidation of L-lactate to pyruvate and the electron transfer to cytochrome c in the mitochondrial intermembrane space . It is a homotetramer with a molecular weight of 4 x 58 kDa, each monomer of which is composed of 2 distinct domains, the one carrying FMN and the other, a "b5-like" heme . The native structure has been described at a resolution of 2.4 A (Xia ZX, Mathews FS, 1990, J Mol Biol 212:837-863) . The heme domains protrude from the central body of the tetramer consisting of the 4 FMN binding domains . Because only 2 heme domains are visible in the electron density map, the other 2 are probably disordered . We crystallized the Escherichia coli recombinant flavocytochrome b2 from S . cerevisiae inhibited by sulfite . Although the crystals were obtained under very different conditions from those of the pyruvate-containing native enzyme, they were found to be isostructural (P 3(2) 2 1, a = b = 164.5 A, c = 114.0 A) . The 2.6-A X-ray structure was extensively refined with X-PLOR (R = 17.3%), which made it possible to describe in detail the recombinant flavocytochrome b2 molecular structure . There exist few differences between the native and recombinant structures, in line with the fact that they show similar kinetic behavior, and they further confirm the intrinsic mobility of the heme domain (Labeyrie F, Beloil JC, Thomas MA, 1988, Biochim Biophys Acta 953:134-141) . This structure will be used as a starting model in the structural resolution of flavocytochrome b2 point mutants.

J Mol Biol, 1994 Jan 28, 235(4), 1173 - 92
Analysis of RNA chain elongation and termination by Saccharomyces cerevisiae RNA polymerase III; Matsuzaki H et al.; When Saccharomyces cerevisiae RNA polymerase (Pol) III transcribes the S . cerevisiae SUP4 tRNA(Tyr) gene, it is obliged to navigate past a large, multi-subunit DNA-bound complex of proteins . We have analyzed individual steps of RNA chain elongation on this gene . Slow steps of transcriptional initiation were by-passed by forming 5'-end-labeled, arrested and precisely positioned transcription complexes . Synchronous resumption of chain elongation by these complexes allowed a single round of RNA synthesis and termination to be analyzed in detail . Results for synthesis at 20 degrees C and 0 degrees C, in the presence of 100 microM and 1 mM ribonucleoside triphosphates (NTPs) are presented . RNA chain elongation through assembled transcription complexes was uneven but relatively rapid: at 20 degrees C with 1 mM NTPs, the fastest RNA chains elongated at an average rate of 29 nucleotides (nt)/second, and the median RNA chains elongated at 21 to 22 nt/second on average . These rates are comparable with a recent measurement of the average rate of chain elongation in vivo by Drosophila RNA polymerase II at 25 degrees C . At 0 degree C, RNA chain elongation rates were, on average, approximately 30-fold slower . Quantitative analysis of the individual steps of RNA chain elongation showed that steps of adding U and A to U-terminated RNA chains tended to be relatively slow, and to be more strongly influenced by nucleotide concentration . Termination of transcription occurred in the sequence T7GT6 (in the non-template DNA strand) and was progressive . Transcripts with five, six and seven U residues were formed, and there was even slow readthrough of the T7 stretch, with GU3 adding rapidly, suggesting that incorporation of a single G into the RNA chain served to reset elongation rates substantially or entirely . Stripping transcription factor (TF) IIIC from transcription complexes did not substantially increase overall RNA chain growth rate, but did diminish pausing at a single site upstream of the boxB binding site of TFIIIC . The TFIIIC-generated delay at this single site was estimated to be only approximately 0.15 to 0.2 seconds at 20 degrees C . Quantitative analysis of RNA chain elongation yielded kinetic parameters for the individual steps of nucleotide addition that were used in computer simulations of RNA chain growth . Elongation modeled as a simple sequence of pseudo-first-order reactions yielded computed RNA chain length distributions that remained relatively synchronous during elongation, while observed chain growth quickly became desynchronized.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell, 1994 Jan 28, 76(2), 403 - 10
The FET3 gene of S . cerevisiae encodes a multicopper oxidase required for ferrous iron uptake; Askwith C et al.; S . cerevisiae accumulate iron by a process requiring a ferrireductase and a ferrous transporter . We have isolated a mutant, fet3, defective for high affinity Fe(II) uptake . The wild-type FET3 gene was isolated by complementation of the mutant defect . Sequence analysis of the gene revealed the presence of an open reading frame coding for a protein with strong similarity to the family of blue multicopper oxidoreductases . Consistent with the role of copper in iron transport, growth of wild-type cells in copper-deficient media resulted in decreased ferrous iron transport . Addition of copper, but not other transition metals (manganese or zinc), to the assay media resulted in the recovery of Fe(II) transporter activity . We suggest that the catalytic activity of the Fet3 protein is required for cellular iron accumulation.

Cell, 1994 Jan 28, 76(2), 393 - 402
Molecular characterization of a copper transport protein in S . cerevisiae: an unexpected role for copper in iron transport; Dancis A et al.; We report the identification and characterization of CTR1, a gene in the yeast S . cerevisiae that encodes a multispanning plasma membrane protein specifically required for high affinity copper transport into the cell . The predicted protein contains a methionine- and serine-rich domain that includes 11 examples of the sequence Met-X2-Met, a motif noted in proteins involved in bacterial copper metabolism . CTR1 mutants and deletion strains have profound deficiency in ferrous iron uptake, thus revealing a requirement for copper in mediating ferrous transport into the cell . Genetic evidence suggests that the target for this requirement is the FET3 gene (detailed in a companion study), predicted to encode a copper-containing protein that acts as a cytosolic ferro-oxidase . These findings provide an unexpected mechanistic link between the uptake of copper and iron.

Gene, 1994 Jan 28, 138(1-2), 213 - 7
Cloning of a rat cDNA encoding dihydroxypolyprenylbenzoate methyltransferase by functional complementation of a Saccharomyces cerevisiae mutant deficient in ubiquinone biosynthesis; Marbois BN et al.; 3,4-Dihydroxy-5-hexaprenylbenzoate methyltransferase (DHHB-MTase) is the product of the COQ3 gene in Saccharomyces cerevisiae and catalyses the fourth step in the biosynthesis of ubiquinone (coenzyme Q) from p-hydroxybenzoic acid . A full-length cDNA encoding a mammalian homologue of DHHB-MTase was isolated from a newly constructed rat testis cDNA library by functional complementation of a coq3 deletion mutant of S . cerevisiae . The complementing clone contained a 1.1-kb poly(A)(+)-tailed insert with a 858-bp open reading frame and presumably encodes 3,4-dihydroxy-5-polyprenylbenzoate-MTase . The deduced rat amino acid (aa) sequence has a 39% identity over 138 aa with the yeast DHHB-MTase and a 37% identity over this same region with an Escherichia coli protein encoded by the ubiG gene, a MTase that catalyses the terminal step of ubiquinone biosynthesis . The rescue of the yeast coq3 mutant by the rat homologue suggests that yeast and rat synthesize ubiquinone via the same early steps in this pathway.

J Biol Chem, 1994 Jan 14, 269(2), 968 - 73
Noncontiguous domains of the alpha-factor receptor of yeasts confer ligand specificity; Sen M et al.; The Saccharomyces cerevisiae alpha-factor receptor has a 3400-fold higher affinity for the S . cerevisiae alpha-factor peptide (c-alpha-f) than for the Saccharomyces kluyveri alpha-factor peptide (k-alpha-f) as determined by competition for {3H} c-alpha-f binding . The S . kluyveri alpha-factor receptor has an approximately 2-fold higher affinity for k-alpha-f than for c-alpha-f . The S . kluyveri receptor gene (k-STE2) is incompletely regulated by S . cerevisiae mating type and poorly expressed on the surface of an S . cerevisiae mating type a strain . A chimeric receptor (c/k1) with amino acid residues 1-45 derived from S . cerevisiae and amino acid residues 46-427 from S . kluyveri exhibits the binding specificity of the S . kluyveri receptor . However, chimeric receptors containing residues 1-168 (c/k2) or 1-250 (c/k3) from S . cerevisiae and the remainder from the S . kluyveri receptor exhibit specificities similar to one another, but intermediate between the parent S . cerevisiae and S . kluyveri receptors . The relative ability of c-alpha-f and k-alpha-f to induce growth arrest in strains expressing chimeric receptors parallels relative affinity . Thus, two noncontiguous domains that include putative extracellular loops 1 and 3 and associated transmembrane segments, but exclude the extracellular NH2 terminus and loop 2, appear to contribute to alpha-factor receptor ligand specificity . COOH-terminal regions of the S . kluyveri receptor appear to confer a desensitization defect when expressed in S . cerevisiae . The S . cerevisiae receptor truncated at residue 296 retains ligand specificity for growth arrest.

Cell, 1994 Jan 14, 76(1), 65 - 75
Meiosis-specific formation of joint DNA molecules containing sequences from homologous chromosomes; Collins I et al.; All recombination models postulate one or more recombination intermediates that are joint molecules containing two homologous parental molecules . A spike of branched DNA molecules not seen in DNA from mitotic cells was found in the two-dimensional gel analysis of meiotic DNA from S . cerevisiae . The mass of molecules in the spike, the timing of its appearance and disappearance, and its absence from a recombination-defective spo11 mutant are consistent with the hypothesis that it contains recombination intermediates . The spike changes in mass as predicted for joint molecules containing DNA from homologous chromosomes rather than sister chromatids in a strain heterozygous for an RFLP . Finally, joint molecules containing DNA from homologous chromosomes were not found, suggesting that the block to recombination between homologous sequences occurs prior to the formation of joint molecules.

Cell, 1994 Jan 14, 76(1), 51 - 63
Identification of joint molecules that form frequently between homologs but rarely between sister chromatids during yeast meiosis; Schwacha A et al.; We have investigated DNA interactions between homologs and between sister chromatids during meiosis in S . cerevisiae . We have detected a DNA species containing information from both parental chromosomes at a specific hotspot for meiotic recombination and double strand breaks (DSBs) . These joint molecules are a prominent feature of meiotic prophase . They appear to be a major intermediate stage in DSB-promoted recombination, because they occur with appropriate timing and require known recombination functions . Other possibilities cannot be completely dismissed, however . Most or all joint molecules contain two full-length nonrecombinant strands from each parental duplex and thus do not consist of single Holliday junctions . Joint molecules form between sister chromatids at approximately 10% the interhomolog level . Also, joint molecule formation is aberrant in a mutant defective in the HOP1 gene, which encodes a meiotic chromosome structure component . General models for discrimination between homologs and sisters during meiosis are discussed.

J Mol Biol, 1994 Jan 7, 235(1), 396 - 401
SAV, an archaebacterial gene with extensive homology to a family of highly conserved eukaryotic ATPases; Confalonieri F et al.; Nucleotide sequencing of a region of the hyperthermophilic archaebacterium Sulfolobus acidocaldarius allowed us to identify an open reading frame of 780 amino acids strikingly similar to a family of eukaryotic ATPases, involved in a variety of biological functions . Sequence analysis of the predicted polypeptide revealed 63 to 66% similarity with S . cerevisiae CDC 48p and its related genes in amphibians (p97ATPase) and mammals (Valosin Containing Protein, VCP), all possibly involved in the regulation of the cell cycle . The finding of an archaebacterial equivalent of these proteins with a high degree of similarity suggests that it represents the same gene in these various species . The new archaebacterial ORF, called SAV (S . acidocaldarius VCP-like) exhibited the usual signature of all members of the family, a highly conserved domain of about 200 amino acids, which is duplicated . Thus, apart from the VCP-like proteins, SAV also appeared similar, although less clearly, to other ATPases, members of the family, involved in vesicle-mediated transport (NSF, Sec18p), peroxysome assembly (PAS1p), and gene expression in yeast (SUG1p) and in human immunodeficiency virus (TBP-1) . Finally, the discovery of the archaebacterial gene could enlighten not only the evolutionary relationships between the members of this complex ATPase family, but also the cellular function of these proteins, that is presently obscure.

J Biol Chem, 1994 Jan 7, 269(1), 207 - 11
The Golgi guanosine diphosphatase is required for transport of GDP-mannose into the lumen of Saccharomyces cerevisiae Golgi vesicles; Berninsone P et al.; The Saccharomyces cerevisiae Golgi lumenal guanosine diphosphatase is hypothesized to generate GMP which in turn allows entry of GDP-mannose into the lumen to serve as substrate for mannosylation of proteins and lipids . We have recently shown in studies in vivo that this GDPase is required for protein and sphingolipid mannosylation in the Golgi lumen of S . cerevisiae . We have now isolated Golgi-vesicles from wild type and gda1 null mutants (GDPase defective) and have found that the initial rate of GDP-mannose entry into mutant vesicles was 5-fold lower than into those of wild type . Because the concentration of GDP within vesicles is insufficient to inhibit Golgi lumenal mannosyltransferases and the null mutant vesicles are impaired in synthesis of Golgi mannoproteins, the above results demonstrate that the reduced availability of GDP-mannose in the null mutants is the cause for altered Golgi mannosylation of macromolecules.

Nature, 1994 Jan 6, 367(6458), 91 - 4
DNA repair gene RAD3 of S . cerevisiae is essential for transcription by RNA polymerase II; Guzder SN et al.; The RAD3 gene of Saccharomyces cerevisiae is required for excision repair of ultraviolet-damaged DNA and is essential for cell viability . The RAD3-encoded protein shares a high degree of homology with the human ERCC2(XPD) gene product . Mutations in XPD, besides causing the cancer-prone syndrome xeroderma pigmentosum, can also result in Cockayne's syndrome and trichothiodystrophy . To investigate the role of RAD3 in viability, we examined here the effect of a recessive, temperature-sensitive (ts) conditional lethal mutation of the gene on transcription by RNA polymerase II . Upon transfer to the restrictive temperature, the rad3-ts mutant rapidly ceases growth and poly(A)+ RNA synthesis is inhibited drastically . Messenger RNA levels of all the genes examined, HIS3, TRP3, STE2, MET19, RAD23, CDC7, CDC9 and ACT1, decline rapidly upon loss of RAD3 activity . The synthesis of heat-shock-inducible HSP26 mRNA and galactose-inducible GAL7 and GAL10 mRNAs is also drastically inhibited in the rad3-ts mutant at the restrictive temperature . The RNA polymerase II transcriptional activity in extract from the rad3-ts14 strain is thermolabile, and this in vitro transcriptional defect can be fully corrected by the addition of homogeneous RAD3 protein . These findings indicate that RAD3 protein has a direct and essential role in RNA polymerase II transcription.

Folia Microbiol (Praha), 1994, 39(6), 507 - 9
Inward and outward rectifying potassium currents in Saccharomyces cerevisiae mediated by endogenous and heterelogously expressed ion channels; Bertl A et al.; Disruption of genes encoding endogenous transport proteins in Saccharomyces cerevisiae has facilitated the recent cloning, by functional expression, of cDNAs encoding K+ channels and amino acid transporters from the plant Arabidopsis thaliana {1-4} . In the present study, we demonstrate in whole-cell patch clamp experiments that the inability of trk1deltatrk2delta mutants of S . cerevisiae to grow on submillimolar K+ correlates with the lack of K+ inward currents, which are present in wild-type cells, and that transformation of the trk1deltatrk2delta double-deletion mutant with KAT1 from Arabidopsis thaliana restores this phenotype by encoding a plasma membrane protein that allows large K+ inward currents . Similar K+ inward currents are induced by transformation of a trk1 mutant with AKT1 from A . thaliana.

DNA Res, 1994, 1(4), 191 - 9
Stability of YACs containing ribosomal or RCP/GCP locus DNA in wild-type S . cerevisiae and RAD mutant strains; Kohno K et al.; About 2% of human YAC clones, including tandemly repeated segments color vision pigment DNA, ribosomal DNA and alphoid DNA have been reported to be inherently unstable in yeast hosts, producing more stable deletion products . YACs containing color vision red pigment gene DNA or 1.5 rDNA tandem repeat units were transformed into hosts bearing lesions at the RAD1, RAD6, RAD51, or RAD52 loci . YACs susceptible to deletion during outgrowth of wild-type cells (or in preliminary experiments, in RAD6 transformants) were stable for up to 100 generations or more in the other strains . Thus both the RAD1 and RAD51/RAD52 epistatic pathways are apparently involved in the instability of YACs containing tandem repeat loci, presumably during recombination-based deletion formation; and a yeast host disarmed in these pathways will likely maintain YACs intact that are otherwise unstable.

EMBO J, 1994 Jan 1, 13(1), 61 - 70
MSG5, a novel protein phosphatase promotes adaptation to pheromone response in S . cerevisiae; Doi K et al.; Pheromone-stimulated yeast cells and haploid gpa1 deletion mutants arrest their cell cycle in G1 . Overexpression of a novel gene called MSG5 suppresses this inhibition of cell division . Loss of MSG5 function leads to a diminished adaptive response to pheromone . Genetic analysis indicates that MSG5 acts at a stage where the protein kinases STE7 and FUS3 function to transmit the pheromone-induced signal . Since loss of MSG5 function causes an increase in FUS3 enzyme activity but not STE7 activity, we propose that MSG5 impinges on the pathway at FUS3 . Sequence analysis suggests that MSG5 encodes a protein tyrosine phosphatase . This is supported by the finding that recombinant MSG5 has phosphatase activity in vitro and is able to inactivate autophosphorylated FUS3 . Thus MSG5 might stimulate recovery from pheromone by regulating the phosphorylation state of FUS3.

Mol Gen Genet, 1994 Jan, 242(1), 17 - 22
Cloning and characterisation of the cytochrome c gene of Aspergillus nidulans; Raitt DC et al.; The cytochrome c gene (cycA) of the filamentous fungus Aspergillus nidulans has been isolated and sequenced . The gene is present in a single copy per haploid genome and encodes a polypeptide of 112 amino acid residues . The nucleotide sequence of the A . nidulans cycA gene shows 87% identity to the DNA sequence of the Neurospora crassa cytochrome c gene, and approximately 72% identity to the sequence of the Saccharomyces cerevisiae iso-1-cytochrome c gene (CYC1) . The S . cerevisiae CYC1 gene was used as a heterologous probe to isolate the homologous gene in A . nidulans . The A . nidulans cytochrome c sequence contains two small introns . One of these is highly conserved in terms of position, but the other has not been reported in any of the cytochrome c genes so far sequenced . Expression of the cycA gene is not affected by glucose repression, but has been shown to be induced approximately tenfold in the presence of oxygen and three- to fourfold under heat-shock conditions.

Mol Cell Biol, 1994 Jan, 14(1), 744 - 58
The effector domain of Rab6, plus a highly hydrophobic C terminus, is required for Golgi apparatus localization; Beranger F et al.; C-terminal lipid modifications are essential for the interaction of Ras-related proteins with membranes . While all Ras proteins are farnesylated and some palmitoylated, the majority of other Ras-related proteins are geranylgeranylated . One such protein, Rab6, is associated with the Golgi apparatus and has a C-terminal CXC motif that is geranylgeranylated on both cysteines . We show here that farnesylation alone cannot substitute for geranylgeranylation in targeting Rab6 to the Golgi apparatus and that whereas Ras proteins that are farnesylated and palmitoylated are targeted to the plasma membrane, mutant Rab proteins that are both farnesylated and palmitoylated associate with the Golgi apparatus . Using chimeric Ras-Rab proteins, we find that there are sequences in the N-terminal 71 amino acids of Rab6 which are required for Golgi complex localization and show that these sequences comprise or include the effector domain . The C-terminal hypervariable domain is not essential for the Golgi complex targeting of Rab6 but is required to prevent prenylated and palmitoylated Rab6 from localizing to the plasma membrane . Functional analysis of these mutant Rab6 proteins in Saccharomyces cerevisiae shows that wild-type Rab6 and C-terminal mutant Rab6 proteins which localize to the Golgi apparatus in mammalian cells can complement the temperature-sensitive phenotype of ypt6 null mutants . Interestingly, therefore, the C-terminal hypervariable domain of Rab6 is not required for this protein to function in S . cerevisiae.

Mol Cell Biol, 1994 Jan, 14(1), 407 - 15
Dual requirement in yeast DNA mismatch repair for MLH1 and PMS1, two homologs of the bacterial mutL gene; Prolla TA et al.; We have identified a new Saccharomyces cerevisiae gene, MLH1 (mutL homolog), that encodes a predicted protein product with sequence similarity to DNA mismatch repair proteins of bacteria (MutL and HexB) and S . cerevisiae yeast (PMS1) . Disruption of the MLH1 gene results in elevated spontaneous mutation rates during vegetative growth as measured by forward mutation to canavanine resistance and reversion of the hom3-10 allele . Additionally, the mlh1 delta mutant displays a dramatic increase in the instability of simple sequence repeats, i.e., (GT)n (M . Strand, T . A . Prolla, R . M . Liskay, and T . D . Petes, Nature {London} 365:274-276, 1993) . Meiotic studies indicate that disruption of the MLH1 gene in diploid strains causes increased spore lethality, presumably due to the accumulation of recessive lethal mutations, and increased postmeiotic segregation at each of four loci, the latter being indicative of inefficient repair of heteroduplex DNA generated during genetic recombination . mlh1 delta mutants, which should represent the null phenotype, show the same mutator and meiotic phenotypes as isogenic pms1 delta mutants . Interestingly, mutator and meiotic phenotypes of the mlh1 delta pms1 delta double mutant are indistinguishable from those of the mlh1 delta and pms1 delta single mutants . On the basis of our data, we suggest that in contrast to Escherichia coli, there are two MutL/HexB-like proteins in S . cerevisiae and that each is a required component of the same DNA mismatch repair pathway.

Mol Cell Biol, 1994 Jan, 14(1), 327 - 38
Two upstream activation sequences control the expression of the XPR2 gene in the yeast Yarrowia lipolytica; Blanchin-Roland S et al.; We have initiated a study of the promoter region of the alkaline extracellular protease gene (XPR2) from Yarrowia lipolytica to identify upstream sequences possibly involved in carbon, nitrogen, and peptone control of XPR2 expression . Deletion analysis showed that the TATA box and two major upstream activation sequences (UASs) were essential for promoter activity under conditions of repression or full induction . Within the distal UAS (UAS1), in vivo footprinting studies with dimethyl sulfate (DMS) identified two sequences similar to Saccharomyces cerevisiae GCN4 (-800 to -792)- and TUF/RAP1 (-790 to -778)-binding sites and two sequences which partially overlap a repeated sequence (-778 to -771 and -720 to -713) similar to the CAR1 upstream repression sequence of S . cerevisiae . Oligonucleotides carrying the TUF/RAP1-like-binding site and adjacent downstream nucleotides restored full transcriptional activity of a UAS1-deleted promoter . Within the proximal UAS (UAS2), a directly repeated decameric sequence (-146 to -137 and -136 to -127) was protected against DMS in vivo . Sequences identical to the ABF1-binding site of S . cerevisiae (-121 to -109) or similar to the GCN4-binding site (-113 to -105) were not clearly protected from DMS in vivo . An oligomer (-150 to -106) carrying these three sequences, inserted into a UAS2-deleted promoter, increased the transcriptional activity . The results from footprints under different physiological conditions suggested that protein binding to both UASs was constitutive . Deletion of both UASs greatly reduced XPR2 expression without abolishing its regulation . Our results strongly suggest that these UASs are targets for transcriptional factors required for assisting specific regulatory proteins.

Mol Cell Biol, 1994 Jan, 14(1), 238 - 54
Functional analysis of mouse Hoxa-7 in Saccharomyces cerevisiae: sequences outside the homeodomain base contact zone influence binding and activation; Gross MK et al.; The murine developmental control gene product, Hoxa-7, was shown to function as a DNA-binding transactivator in Saccharomyces cerevisiae . The importance of the ATTA core, the preference for antp class flanking nucleotides, the importance of Asn-51 of the homeodomain (HD), and the synergism of multiple binding sites all reflect properties that have previously been described for HOM or Hox proteins in tissue culture systems . A comparison of contact positions among genes of paralog groups and classes of mammalian HDs points to a lack of diversity in positions that make base contact, suggesting that besides the combination of HD amino acid-base pair contacts, another means of recognizing differences between targets must exist if Hox genes select different targets . The HD of antennapedia is identical to the Hoxa-7 HD . The interaction of Hoxa-7 with the exact sequence used in the nuclear magnetic resonance three-dimensional structural analysis on the antennapedia HD was studied . Hoxa-7 binding and transactivation was influenced by sequences outside of the known base contact zone of this site . We conclude that Hoxa-7 protein has a second means to interact with DNA or/and that the sequences flanking the base contact zone influence HD interactions by distorting DNA within the contact zone (base or backbone) . This result is discussed in terms of DNA flexure and two modes of transcription used in S . cerevisiae.

Mol Cell Biol, 1994 Jan, 14(1), 226 - 37
The sua8 suppressors of Saccharomyces cerevisiae encode replacements of conserved residues within the largest subunit of RNA polymerase II and affect transcription start site selection similarly to sua7 (TFIIB) mutations; Berroteran RW et al.; Mutations in the Saccharomyces cerevisiae sua8 gene were found to be suppressors of an aberrant ATG translation initiation codon in the leader region of the cyc1 gene . Analysis of cyc1 transcripts from sua8 mutants revealed that suppression is a consequence of diminished transcription initiation at the normal start sites in favor of initiation at downstream sites, including a site between the aberrant and normal ATG start codons . This effect is not cyc1 gene specific since initiation at other genes, including ADH1, CYC7, and HIS4, was similarly affected, although initiation at HIS3 and SPT15 was unaffected . The SUA8 gene was cloned and partially sequenced, revealing identity to RPB1, which encodes the largest subunit of RNA polymerase II . The sua8 suppressors are the result of single amino acid replacements of highly conserved residues . Three replacements were found either within or immediately preceding homology block D, and a fourth was found adjacent to homology block H, indicating that these regions play a role in defining start sites in vivo . Nearly identical effects on start site selection were observed for sua7 suppressors, which encode altered forms of TFIIB . Synthetic lethality was associated with double sua7 sua8 suppressor mutations, and recessive sua7 mutants failed to fully complement recessive sua8 mutants in heterozygous diploids (nonallelic noncomplementation) . These data indicate that the largest subunit of RNA polymerase II and TFIIB are important determinants of transcription start site selection in S . cerevisiae and suggest that this function might be conferred by interaction between these two proteins.

Yeast, 1994 Jan, 10(1), 93 - 104
Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks; Larionov V et al.; Rearrangements within plasmid DNA are commonly observed during transformation of eukaryotic cells . One possible cause of rearrangements may be recombination between repeated sequences induced by some lesions in the plasmid . We have examined the mechanisms of transformation-associated recombination in the yeast Saccharomyces cerevisiae using a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied . The plasmids contain homologous or diverged (19%) repeats of the URA3 genes (from S . cerevisiae or S . carlsbergensis) separated by the genetically detectable ADE2 colour marker . Recombination during transformation for covalently closed circular plasmids was over 100-fold more frequent than during mitotic growth . The frequency of recombination is partly dependent on the method of transformation in that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation . When present in the repeats, unique single-strand breaks that are ligatable, as well as double-strand breaks, lead to high levels of recombination between diverged and identical repeats . The transformation-associated recombination between repeat DNAs is under the influence of the RAD52 and RAD1 genes.

Mol Biochem Parasitol, 1994 Jan, 63(1), 135 - 42
Isolation of the genes encoding the 51-kilodalton and 28-kilodalton subunits of Crithidia fasciculata replication protein A; Brown GW et al.; The genes encoding the 51-kilodalton subunit (p51) and the 28-kilodalton subunit (p28) of replication protein A (RP-A), designated CfaRPA1 and CfaRPA2 respectively, were cloned from the trypanosomatid Crithidia fasciculata by screening a genomic DNA library in the expression vector lambda gt11 with antibodies raised against purified C . fasciculata RP-A . CfaRPA1 has a single open reading frame encoding a polypeptide of 467 amino acids and a molecular mass of 52.0 kDa . The predicted p51 polypeptide has sequence similarity to the corresponding subunits from human, Xenopus laevis, and Saccharomyces cerevisiae, but is lacking a segment of approximately 20 kDa from its amino terminus, accounting for its smaller molecular weight when compared to the large subunits of RP-A from these other organisms . CfaRPA1 contains a zinc-finger motif that is also found in the RP-A large subunits from human, frog, and yeast . CfaRPA2 contains a single large open reading frame encoding a polypeptide of 258 amino acids and a molecular mass of 27.5 kDa . The predicted polypeptide has significant sequence similarity to the middle subunit of RP-A from human cells, mouse cells, and the budding yeast S . cerevisiae . Northern hybridization analysis of polyadenylated RNA from C . fasciculata indicates that both cloned genes are expressed as polyadenylated transcripts . CfaRPA1 hybridized with a 2.30-kb transcript and CfaRPA2 hybridized with a 1.44-kb transcript.

Hereditas, 1994, 120(2), 121 - 6
Identification of new chromosomes of Saccharomyces bayanus using gene probes from S . cerevisiae; Naumov GI et al.; The Saccharomyces cerevisiae genes HIS3 (chr . XV), LYS2 (chr . II), ARG4 (chr . VIII), MF alpha 1 (chr . XVI) and MEL1 (chr . II) and the S . pastorianus gene MELx were used for identification of the homeologous chromosomes in S . bayanus by using pulsed-field gel electrophoresis and Southern blot hybridization . The results showed differences in hybridization intensities and mobilities between the homeologous chromosomes of the two sibling yeasts S . cerevisiae and S . bayanus . The probes HIS3, LYS2, MEL1, and MF alpha 1 identified four new homeologous chromosomes of S . bayanus . The ARG4 probe confirmed the previously identified location of chr . VIII (NAUMOV et al . 1992b) . Using the MF alpha 1 gene, another chromosome besides chr . XVI was identified in some S . bayanus strains, suggesting translocation of this sequence to the S . bayanus chromosome that normally migrates to the position of chr . XI of S . cerevisiae.

Curr Genet, 1994 Jan, 25(1), 30 - 3
Basidiomycetous ras cDNA functionally replaces its homolog genes in yeast; Ishibashi O et al.; It was shown by a plasmid exchange procedure that the Ras-encoding cDNA of the basidiomycete Lentinus edodes (named Leras cDNA) can functionally replace its homolog genes (ScRAS1 and ScRAS2) in the yeast Saccharomyces cerevisiae to maintain the viability of an yeast strain containing genetic disruptions of both RAS genes . The strain replaced by a Leras-cDNA-carrying plasmid, however, grew slower than the strains replaced by a ScRAS1- or a ScRAS2-carrying plasmid . The intracellular level of cAMP in the strain harboring the Leras-cDNA-carrying plasmid was clearly higher than that of a parental strain which maintains a plasmid carrying the S . cerevisiae cAMP-dependent protein kinase catalytic subunit C1 gene, TPK1, but was lower than that in a strain harboring an ScRAS2-carrying plasmid . These results suggest that the Leras cDNA can complement the ras1- ras2- mutation of yeast by virture of the stimulation of adenylate cyclase activity, although the complementation is not as efficient as that obtained by expressing the ScRAS2 gene.

Microbios, 1994, 78(316), 155 - 61
Analysis of Sec 19-1 mutant of Saccharomyces cerevisiae; Sabanero M et al.; Saccharomyces cerevisiae Sec 19-1 cells are secretion mutants defective at 37 degrees C . The cells were analysed in order to ascertain the effect of mutation temperature on cell wall formation . At the restrictive temperature of 37 degrees C, the Sec 19-1 mutants had 37 micrograms/mg N-acetylglucosamine in the wall cells, while the wild type S . cerevisiae showed 84 micrograms/mg hexosamine . The mutants Sec 19-1 showed a maximum activity of chitin synthetase of 0.113 nmoles/min/ml, and the activity increased to 0.33 nmoles/min/ml in the wild type cells . On the other hand, variations of chitin distribution in the wall cells occurred at the restrictive temperature, but changes in actin organization were not evident . The results indicated that the mutation caused variations in the levels of N-acetylglucosamine and chitin synthetase, as well as in cell wall chitin distribution.

Arch Microbiol, 1994, 162(3), 211 - 4
Kinetic studies of killer toxin K1 binding to yeast cells indicate two receptor populations; Kurzweilova H et al.; A recently described new method for determination of killer toxin activity was used for kinetic measurements of K1 toxin binding . The cells of the killer sensitive strain Saccharomyces cerevisiae S6 were shown to carry two classes of toxin binding sites differing widely in their half-saturation constants and maximum binding rates . The low-affinity and high-velocity binding component (KT1 = 2.6 x 10(9) L.U./ml, Vmax1 = 0.19 s-1) probably reflects diffusion-limited binding to cell wall receptors; the high-affinity and low-velocity component (KT2 = 3.2 x 10(7) L.U./ml, Vmax2 = 0.03 s-1) presumably indicates the binding of the toxin to plasma membrane receptors . Adsorption of most of the killer toxin K1 to the surface of sensitive cells occurred within 1 min and was virtually complete within 5 min . The amount of toxin that saturated practically all cell receptors was about 600 lethal units (L.U.) per cell of S . cerevisiae S6.

Antisense Res Dev, 1994 Summer, 4(2), 109 - 17
Artificial ribozyme and antisense gene expression in Saccharomyces cerevisiae; Atkins D et al.; A sensitive, quantitative reporter gene-based experimental system for the in vivo analysis of hammerhead ribozyme and antisense gene function in Saccharomyces cerevisiae is described . The system was constructed to test the activity of ribozyme and antisense genes targeting the chloramphenicol acetyltransferase gene (CAT) in both a cis and trans configuration relative to the target . When both target and ribozyme or antisense genes were transcribed in the same mRNA from an expression vector, CAT expression was reduced by up to 90% . Although the cis-positioned ribozyme molecule cleaved the target RNA in vitro, the steady state RNA levels of these chimeric transcripts were increased several fold relative to control mRNAs . This observation indicates a mechanism of suppression of CAT gene expression other than duplex-dependent degradation of mRNA . When the ribozyme or antisense genes were transcribed in trans from a plasmid-based expression vector, expression of a CAT gene integrated into a chromosome was unaffected . The effect of the cis-located RNA molecules may be dependent on an interaction requiring co-localization of ribozyme or antisense and target mRNAs during or immediately after target gene transcription . The failure of such a co-localization of these RNAs when synthesized in trans may contribute to the lack of efficacy seen in the trans-acting ribozymes or antisense RNAs . These observations are consistent with other studies reporting inefficient trans-acting ribozyme and antisense activity in S . cerevisiae.

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