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