Curr Opin Microbiol, 1999 Dec, 2(6), 610 - 7 Altered states: programmed proteolysis and the budding yeast cell cycle; Jorgensen P et al.; The recent identification of an essential RING-H2 finger protein in the SCF E3 ubiquitin ligase complex of budding yeast has uncovered a family of related E3 enzymes, including the other main cell cycle E3 complex, the anaphase promoting complex (APC) . Recent insights into APC-dependent proteolysis include a novel protease activity that dissolves cohesion between sister chromatids at anaphase, and a crucial phosphatase, Cdc14, whose release from the nucleolus eliminates cyclin-dependent kinase activity and thereby drives exit from mitosis. Eur J Biochem, 2000 Jan, 267(1), 85 - 96 Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase; Bouvier-Nave P et al.; During the course of a search for cDNAs encoding plant sterol acyltransferases, an expressed sequence tag clone presenting substantial identity with yeast and animal acyl CoA:cholesterol acyltransferases was used to screen cDNA libraries from Arabidopsis and tobacco . This resulted in the isolation of two full-length cDNAs encoding proteins of 520 and 532 amino acids, respectively . Attempts to complement the yeast double-mutant are1 are2 defective in acyl CoA:cholesterol acyltransferase were unsuccessful, showing that neither gene encodes acyl CoA:cholesterol acyltransferase . Their deduced amino acid sequences were then shown to have 40 and 38% identity, respectively, with a murine acyl CoA:diacylglycerol acyltransferase and their expression in are1 are2 or wild-type yeast resulted in a strong increase in the incorporation of oleyl CoA into triacylglycerols . Incorporation was 2-3 times higher in microsomes from yeast transformed with these plant cDNAs than in yeast transformed with the void vector, clearly showing that these cDNAs encode acyl CoA:diacylglycerol acyltransferases . Moreover, during the preparation of microsomes from the Arabidopsis DGAT-transformed yeast, a floating layer was observed on top of the 100 000 g supernatant . This fraction was enriched in triacylglycerols and exhibited strong acyl CoA:diacylglycerol acyltransferase activity, whereas almost no activity was detected in the corresponding clear fraction from the control yeast . Thanks to the use of this active fraction and dihexanoylglycerol as a substrate, the de novo synthesis of 1,2-dihexanoyl 3-oleyl glycerol by AtDGAT could be demonstrated . Transformation of tobacco with AtDGAT was also performed . Analysis of 19 primary transformants allowed detection, in several individuals, of a marked increase (up to seven times) of triacylglycerol content which correlated with the AtDGAT mRNA expression . Furthermore, light-microscopy observations of leaf epidermis cells, stained with a lipid-specific dye, showed the presence of lipid droplets in the cells of triacylglycerol-overproducer plants, thus illustrating the potential application of acyl CoA:diacylglycerol acyltransferase-transformed plants. Genes Dev, 1999 Dec 1, 13(23), 3052 - 8 Yeast autonomously replicating sequence binding factor is involved in nucleotide excision repair; Reed SH et al.; Nucleotide excision repair (NER) in yeast is effected by the concerted action of a large complex of proteins . Recently, we identified a stable subcomplex containing the yeast Rad7 and Rad16 proteins . Here, we report the identification of autonomously replicating sequence binding factor 1 (ABF1) as a component of the Rad7/Rad16 NER subcomplex . Yeast ABF1 protein is encoded by an essential gene required for DNA replication, transcriptional regulation, and gene silencing . We show that ABF1 plays a direct role in NER in vitro . Additionally, consistent with a role of ABF1 protein in NER in vivo, we show that certain temperature-sensitive abf1 mutant strains that are defective in DNA replication are specifically defective in the removal of photoproducts by NER and are sensitive to killing by ultraviolet (UV) radiation . These studies define a novel and unexpected role for ABF1 protein during NER in yeast. J Exp Biol, 2000 Jan, 203(Pt 1), 155 - 60 Biogenesis and function of the yeast plasma-membrane H(+)-ATPase; Ambesi A et al.; One of the most abundant proteins in the yeast plasma membrane is the P-type H(+)-ATPase that pumps protons out of the cell, supplying the driving force for a wide array of H(+)-dependent cotransporters . The ATPase is a 100 kDa polypeptide, anchored in the lipid bilayer by 10 transmembrane alpha-helices . It is structurally and functionally related to the P-type Na(+),K(+)-, H(+),K(+)- and Ca(2+)-ATPases of animal cells and the H(+)-ATPases of plant cells, and it shares with them a characteristic reaction mechanism in which ATP is split to ADP and inorganic phosphate (P(i)) via a covalent beta-aspartyl phosphate intermediate . Cryoelectron microscopic images of the H(+)-ATPase of Neurospora crassa and the sarcoplasmic reticulum Ca(2+)-ATPase of animal cells have recently been obtained at 8 nm resolution . The membrane-embedded portion of the molecule, which presumably houses the cation translocation pathway, is seen to be connected via a narrow stalk to a large, multidomained cytoplasmic portion, known to contain the ATP-binding and phosphorylation sites . In parallel with the structural studies, efforts are being made to dissect structure/function relationships in several P-type ATPases by means of site-directed mutagenesis . This paper reviews three phenotypically distinct classes of mutant that have resulted from work on the yeast PMA1 H(+)-ATPase: (1) mutant ATPases that are poorly folded and retained in the endoplasmic reticulum; (2) mutants in which the conformational equilibrium has been shifted from the E(2) state, characterized by high affinity for vanadate, to the E(1) state, characterized by high affinity for ATP; and (3) mutants with altered coupling between ATP hydrolysis and proton pumping . Although much remains to be learned before the transport mechanism can be fully understood, these mutants serve to identify critical parts of the polypeptide that are required for protein folding, conformational change and H(+):ATP coupling. J Exp Biol, 2000 Jan, 203(Pt 1), 71 - 80 Structure, mechanism and regulation of the clathrin-coated vesicle and yeast vacuolar H(+)-ATPases; Forgac M; The vacuolar H(+)-ATPases (or V-ATPases) are a family of ATP-dependent proton pumps that carry out acidification of intracellular compartments in eukaryotic cells . This review is focused on our work on the V-ATPases of clathrin-coated vesicles and yeast vacuoles . The coated-vesicle V-ATPase undergoes trafficking to endosomes and synaptic vesicles, where it functions in receptor recycling and neurotransmitter uptake, respectively . The yeast V-ATPase functions to acidify the central vacuole and is necessary both for protein degradation and for coupled transport processes across the vacuolar membrane . The V-ATPases are multisubunit complexes composed of two functional domains . The V(1) domain is a 570 kDa peripheral complex composed of eight subunits of molecular mass 73-14 kDa (subunits A-H) that is responsible for ATP hydrolysis . The V(o) domain is a 260 kDa integral complex composed of five subunits of molecular mass 100-17 kDa (subunits a, d, c, c' and c") that is responsible for proton translocation . To explore the function of individual subunits in the V-ATPase complex as well as to identify residues important in proton transport and ATP hydrolysis, we have employed a combination of chemical modification, site-directed mutagenesis and in vitro reassembly . A central question concerns the mechanism by which vacuolar acidification is controlled in eukaryotic cells . We have proposed that disulfide bond formation between conserved cysteine residues at the catalytic site of the V-ATPase plays an important role in regulating V-ATPase activity in vivo . Other regulatory mechanisms that are discussed include reversible dissociation and reassembly of the V-ATPase complex, changes in the tightness of coupling between proton transport and ATP hydrolysis, differential targeting of V-ATPases within the cell and control of the Cl(-) conductance that is necessary for vacuolar acidification. J Exp Biol, 2000 Jan, 203(Pt 1), 61 - 70 Composition and assembly of the yeast vacuolar H(+)-ATPase complex; Graham LA et al.; The proton-translocating ATPase (H(+)-ATPase) found on the membrane of the yeast vacuole is the best characterized member of the V-type ATPase family . Biochemical and genetic screens have led to the identification of 14 genes, the majority designated VMA (for vacuolar membrane ATPase) encoding subunits of the enzyme complex . At least eight genes encode for proteins comprising the peripherally associated catalytic V(1) subcomplex, and six genes code for proteins forming the proton-translocating membrane V(o) subcomplex . Several additional genes have been identified that encode proteins that are not part of the final V-ATPase complex yet are required for its assembly . These non-subunit Vma proteins function as dedicated V-ATPase assembly factors since their absence appears to inhibit assembly of the V-ATPase only . The assembly factors designated Vma12p, Vma21p and Vma22p have been localized to the membrane of the endoplasmic reticulum and aid the association of newly synthesized V-ATPase subunits translocated into the endoplasmic reticulum membrane . Two of these proteins, Vma12p and Vma22p, function together in an assembly complex that interacts directly with nascent V-ATPase subunits. Genet Anal, 1999 Nov, 15(3-5), 175 - 8 The use of yeast artificial chromosomes in transgenic animals: expression studies of the tyrosinase gene in transgenic mice; Giraldo P et al.; Variegation and inherited somatic mosaicism has been observed in transgenic mice carrying yeast artificial chromosomes (YACs) in which a DNAse I hypersensitive site (HS) located -12 kb upstream of the mouse tyrosinase gene had been deleted . At present, we are generating new transgenic animals with minor deletions of the HS. Nucleic Acids Res . 2000 Jan 15;28(2):e2. Transcript quantitation in total yeast cellular RNA using kinetic PCR; Kang JJ et al.; Kinetically monitored, reverse transcriptase-initiated PCR (kinetic RT-PCR, kRT-PCR) is a novel application of kinetic PCR for high throughput transcript quantitation in total cellular RNA . The assay offers the simplicity and flexibility of an enzyme assay with distinct advantages over DNA microarray hybridization and SAGE technologies for certain applications . The reproducibility, sensitivity and accuracy of the kRT-PCR were assessed for yeast transcripts previously quantitated by a variety of methods including SAGE analysis . Changes in transcript levels between different genetic or physiological cell states were reproducibly quantitated with an accuracy of +/-20% . The assay was sufficiently sensitive to quantitate yeast transcripts over a range of more than five orders of magnitude, including low abundance transcripts encoding cell cycle and transcriptional regulators. Nucleic Acids Res, 2000 Jan 15, 28(2), 424 - 9 APOBEC-1 dependent cytidine to uridine editing of apolipoprotein B RNA in yeast; Dance GS et al.; Cytidine to uridine editing of apolipoprotein B (apoB) mRNA requires the cytidine deaminase APOBEC-1 as well as a tripartite sequence motif flanking a target cytidine in apoB mRNA and an undefined number of auxiliary proteins that mediate RNA recognition and determine site-specific editing . Yeast engineered to express APOBEC-1 and apoB mRNA supported editing under conditions of late log phase growth and stationary phase . The cis -acting sequence requirements and the intracellular distribution of APOBEC-1 in yeast were similar to those described in mammalian cells . These findings suggest that auxiliary protein functions necessary for the assembly of editing complexes, or 'editosomes', are expressed in yeast and that the distribution of editing activity is to the cell nucleus. RNA, 1999 Dec, 5(12), 1586 - 96 Reassembly and protection of small nuclear ribonucleoprotein particles by heat shock proteins in yeast cells; Bracken AP et al.; The process of mRNA splicing is sensitive to in vivo thermal inactivation, but can be protected by pretreatment of cells under conditions that induce heat-shock proteins (Hsps) . This latter phenomenon is known as "splicing thermotolerance" . In this article we demonstrate that the small nuclear ribonucleoprotein particles (snRNPs) are in vivo targets of thermal damage within the splicing apparatus in heat-shocked yeast cells . Following a heat shock, levels of the tri-snRNP (U4/U6.U5), free U6 snRNP, and a pre-U6 snRNP complex are dramatically reduced . In addition, we observe multiple alterations in U1, U2, U5, and U4/U6 snRNP profiles and the accumulation of precursor forms of U4- and U6-containing snRNPs . Reassembly of snRNPs following a heat shock is correlated with the recovery of mRNA splicing and requires both Hsp104 and the Ssa Hsp70 family of proteins . Furthermore, we correlate splicing thermotolerance with the protection of a subset of snRNPs by Ssa proteins but not Hsp104, and show that Hsp70 directly associates with U4- and U6-containing snRNPs in splicing thermotolerant cells . In addition, our results show that Hsp70 plays a role in snRNP assembly under normal physiological conditions. RNA, 1999 Dec, 5(12), 1526 - 34 Crystallographic structure of the amino terminal domain of yeast initiation factor 4A, a representative DEAD-box RNA helicase; Johnson ER et al.; The eukaryotic translation initiation factor 4A (elF4A) is a representative of the DEAD-box RNA helicase protein family . We have solved the crystallographic structure of the amino-terminal domain (residues 1-223) of yeast elF4A . The domain is built around a core scaffold, a parallel alpha-beta motif with five beta strands, that is found in other RNA and DNA helicases, as well as in the RecA protein . The amino acid sequence motifs that are conserved within the helicase family are localized to the beta strand-->alpha helix junctions within the core . The core of the amino terminal domain of elF4A is amplified with additional structural elements that differ from those of other helicases . The phosphate binding loop (the Walker A motif) is in an unusual closed conformation . The crystallographic structure reveals specific interactions between amino acid residues of the phosphate binding loop, the DEAD motif, and the SAT motif, whose alteration is known to impair coupling between the ATPase cycle and the RNA unwinding activity of elF4A. Trends Microbiol, 1999 Dec, 7(12), 500 - 5 Pipes and wiring: the regulation of copper uptake and distribution in yeast; Labbe S et al.; Copper is required for processes as conserved as respiration and as specialized as protein modification . Recent exciting findings from studies in yeast cells have revealed the presence of specific pathways for copper transport, trafficking and signal transduction that maintain the delicate balance of this essential yet toxic metal ion. Cell Motil Cytoskeleton, 1999 Dec, 44(4), 284 - 95 Lethal level overexpression of gamma-tubulin in fission yeast causes mitotic arrest; Horio T et al.; gamma-Tubulin is a member of the tubulin superfamily and plays essential roles in microtubule nucleation . While the level of other tubulins, alpha- and beta-tubulin, is strictly regulated in higher eukaryotes and overexpression of beta-tubulin is toxic in yeasts, gamma-tubulin can be overexpressed by fivefold in fission yeast without any obvious defect in growth . Extreme overexpression of gamma-tubulin in mammalian cells caused growth arrest; however, the exact level of gamma-tubulin and the critical level of gamma-tubulin necessary for growth defect were undetermined . We have constructed strains that over- or underexpress gamma-tubulin by placing the gamma-tubulin gene under the control of the inducible nmt1 promoter and its variants . Among these, the weakest promoter was able to produce enough gamma-tubulin to support normal growth when its expression was induced . A strain in which the gamma-tubulin gene was placed under the control of the strongest inducible promoter achieved 160-fold overexpression of gamma-tubulin and its growth was suppressed . Normal cytoplasmic microtubules were mostly lost in gamma-tubulin overexpressing cells and gamma-tubulin was accumulated around the periphery of nuclei . Many of the cells were arrested in mitosis . A small fraction of cells did proceed to undergo nuclear division; however, its process looked either significantly deterred or abnormal . Our results presented here suggest that excess gamma-tubulin disrupts the microtubule array and significantly deters the formation of the mitotic spindle, most likely because of random nucleation of microtubules from excess gamma-tubulin in the cytoplasm . J Cell Biol, 1999 Dec 13, 147(6), 1223 - 36 Role for Drs2p, a P-type ATPase and potential aminophospholipid translocase, in yeast late Golgi function; Chen CY et al.; ADP-ribosylation factor appears to regulate the budding of both COPI and clathrin-coated transport vesicles from Golgi membranes . An arf1Delta synthetic lethal screen identified SWA3/DRS2, which encodes an integral membrane P-type ATPase and potential aminophospholipid translocase (or flippase) . The drs2 null allele is also synthetically lethal with clathrin heavy chain (chc1) temperature-sensitive alleles, but not with mutations in COPI subunits or other SEC genes tested . Consistent with these genetic analyses, we found that the drs2Delta mutant exhibits late Golgi defects that may result from a loss of clathrin function at this compartment . These include a defect in the Kex2-dependent processing of pro-alpha-factor and the accumulation of abnormal Golgi cisternae . Moreover, we observed a marked reduction in clathrin-coated vesicles that can be isolated from the drs2Delta cells . Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p . These observations indicate a novel role for a P-type ATPase in late Golgi function and suggest a possible link between membrane asymmetry and clathrin function at the Golgi complex. J Biol Chem, 1999 Dec 24, 274(52), 37139 - 46 Casein kinase I-dependent phosphorylation and stability of the yeast multidrug transporter Pdr5p; Decottignies A et al.; The pleiotropic drug resistance protein, Pdr5p, is an ATP-binding cassette transporter of the plasma membrane of Saccharomyces cerevisiae . Overexpression of Pdr5p results in increased cell resistance to a variety of cytotoxic compounds, a phenotype reminiscent of the multiple drug resistance seen in tumor cells . Pdr5p and two other yeast ATP-binding cassette transporters, Snq2p and Yor1p, were found to be phosphorylated on serine residues in vitro . Mutations in the plasma membrane-bound casein kinase I isoforms, Yck1p and Yck2p, abolished Pdr5p phosphorylation and modified the multiple drug resistance profile . We showed Pdr5p to be ubiquitylated when overexpressed . However, instability of Pdr5p was only seen in Yck1p- and Yck2p-deficient strains, in which it was degraded in the vacuole via a Pep4p-dependent mechanism . Our results suggest that casein kinase I activity is required for membrane trafficking of Pdr5p to the cell surface . In the absence of functional Yck1p and Yck2p, Pdr5p is transported to the vacuole for degradation. J Biol Chem, 1999 Dec 24, 274(52), 37035 - 40 The action of N-terminal acetyltransferases on yeast ribosomal proteins; Arnold RJ et al.; Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to determine the state of N-terminal acetylation of 68 ribosomal proteins from a normal strain of Saccharomyces cerevisiae and from the ard1-Delta, nat3-Delta, and mak3-Delta mutants (), each lacking a catalytic subunit of three different N-terminal acetyltransferases . A total 30 of the of 68 ribosomal proteins were N-terminal-acetylated, and 24 of these (80%) were NatA substrates, unacetylated in solely the ard1-Delta mutant and having mainly Ac-Ser- termini and a few with Ac-Ala- or Ac-Thr- termini . Only 4 (13%) were NatB substrates, unacetylated in solely the nat3-Delta mutant, and having Ac-Met-Asp- or Ac-Met-Glu- termini . No NatC substrates were uncovered, e.g . unacetylated in solely mak3-Delta mutants, consistent with finding that none of the ribosomal proteins had Ac-Met-Ile-, Ac-Met-Leu-, or Ac-Met-Phe- termini . Interestingly, two new types of the unusual NatD substrates were uncovered, having either Ac-Ser-Asp-Phe- or Ac-Ser-Asp-Ala- termini that were unacetylated in the ard1-Delta mutant, and only partially acetylated in the mak3-Delta mutant and, for one case, also only partially in the nat3-Delta mutant . We suggest that the acetylation of NatD substrates requires not only Ard1p and Nat1p, but also auxiliary factors that are acetylated by the Mak3p and Nat3p N-terminal acetyltransferases. EMBO J, 1999 Dec 15, 18(24), 7041 - 55 SIR repression of a yeast heat shock gene: UAS and TATA footprints persist within heterochromatin; Sekinger EA et al.; Previous work has suggested that products of the Saccharomyces cerevisiae Silent Information Regulator (SIR) genes form a complex with histones, nucleated by cis-acting silencers or telomeres, which represses transcription in a position-dependent but sequence-independent fashion . While it is generally thought that this Sir complex works through the establishment of heterochromatin, it is unclear how this structure blocks transcription while remaining fully permissive to other genetic processes such as recombination or integration . Here we examine the molecular determinants underlying the silencing of HSP82, a transcriptionally potent, stress-inducible gene . We find that HSP82 is efficiently silenced in a SIR-dependent fashion, but only when HMRE mating-type silencers are configured both 5' and 3' of the gene . Accompanying dominant repression are novel wrapped chromatin structures within both core and upstream promoter regions . Strikingly, DNase I footprints mapping to the binding sites for heat shock factor (HSF) and TATA-binding protein (TBP) are strengthened and broadened, while groove-specific interactions, as detected by dimethyl sulfate, are diminished . Our data are consistent with a model for SIR repression whereby transcriptional activators gain access to their cognate sites but are rendered unproductive by a co-existing heterochromatic complex. Science, 1999 Dec 17, 286(5448), 2339 - 42 Requirement of yeast SGS1 and SRS2 genes for replication and transcription; Lee SK et al.; The SGS1 gene of the yeast Saccharomyces cerevisiae encodes a DNA helicase with homology to the human Bloom's syndrome gene BLM and the Werner's syndrome gene WRN . The SRS2 gene of yeast also encodes a DNA helicase . Simultaneous deletion of SGS1 and SRS2 is lethal in yeast . Here, using a conditional mutation of SGS1, it is shown that DNA replication and RNA polymerase I transcription are drastically inhibited in the srs2Delta sgs1-ts strain at the restrictive temperature . Thus, SGS1 and SRS2 function in DNA replication and RNA polymerase I transcription . These functions may contribute to the various defects observed in Werner's and Bloom's syndromes. Plant Mol Biol, 1999 Oct, 41(3), 339 - 49 The maize EmBP-1 orthologue differentially regulates opaque2-dependent gene expression in yeast and cultured maize endosperm cells; Carlini LE et al.; In addition to the bZIP protein Opaque2 (O2), there are other maize endosperm nuclear proteins that recognize the O2 box in 22 kDa zein gene promoters . In an effort to understand the effect of these factors on 22 kDa zein expression, we have cloned one of these and identified it as the putative maize (Zea mays L.) orthologue of the wheat bZIP protein EmBP-1 (mEmBP-1) . The mEmBP-1 protein exhibits 52% sequence identity and 68% similarity with the wheat protein and recognizes a similar spectrum of DNA sequences, albeit with slightly altered specificity . The mEmBP-1 gene exists as duplicate loci in maize on chromosomes 7S (mEmBP-1a) and 2L (mEmBP-1b) . The mEmBP-1 genes are expressed in endosperm, embryo, immature ears, tassel, roots, and seedling shoots at low levels . Although mEmBP-1 binds to the O2 box from the 22 kDa zein gene promoter as a homodimer, it is unable to heterodimerize with O2 . The mEmBP-1 protein can activate transcription from a truncated promoter containing a pentamer of the O2 site in yeast cells; however, it inhibited regulated transcription of a 22 kDa zein promoter in a transient expression assay using cultured maize endosperm cells. J Agric Food Chem, 1999 Mar, 47(3), 1139 - 44 Yeast influence on volatile composition of wines; Antonelli A et al.; Nine Saccharomyces cerevisiae and four Saccharomyces bayanus strains used in the production of Trebbiano wine were examined . The aim of this study is to evaluate the different abilities of various yeast strains in wine-making . The possibility of yeast discrimination on the basis of their volatile production is another possibility . Wine chemical analyses showed statistically significant differences depending on the yeast strain used . Some compounds such as 2-phenylethanol, 2-phenethyl acetate, ethyl lactate, 3-ethoxypropanol, and, to a lesser extent, diethyl succinate and propionic acid characterized examined Saccharomyces bayanus yeasts . Moreover, these strains did not produce any undesirable compounds, such as acetic acid and sulfur anhydride . For these reasons and because they synthesized malic acid, they could be more suitable for white wine production . The other yeasts showed great differences, which are difficult to correlate with the strain . However, some strains had peculiar characteristics, such as an uncommonly high concentration of n-propanol and 3-ethoxypropanol. Cancer Res, 1999 Nov 1, 59(21), 5625 - 8 B-cell tumorigenesis in mice carrying a yeast artificial chromosome-based immunoglobulin heavy/c-myc translocus is independent of the heavy chain intron enhancer (Emu); Palomo C et al.; We have used YAC (yeast artificial chromosome) technology to create large translocation regions where the c-myc proto-oncogene is coupled to the core region of the human immunoglobulin heavy chain (IgH) locus (from VH2-5 through to Cdelta) . Chimeric mice were obtained from embryonic stem cells carrying a single copy of the 240-kb IgH/c-myc translocation region . B-cell tumorigenesis occurs in the translocus mice, even when the entire Emu intron enhancer region between the joining segments and switch mu is deleted . This demonstrates that as yet unidentified regulatory elements in the IgH locus, independent from the known enhancers, are sufficient to cause B-cell specific activation of c-myc after translocation . The phenotype of tumors from IgH/c-myc YAC transgenic mice with or without Emu (B220+, IgM+/IgD+) is reminiscent of Burkitt's lymphoma . A rapidly expanding abnormal B-cell population is present at birth and accumulates in bone marrow, periphery, and spleen, well before discrete tumor establishment . Molecular analysis identified a clonal origin, with rearrangement of one mouse heavy chain allele retained in tumor cells from different sites, whereas subsequent rearrangements of heavy or light chain loci can be diverse . These mice routinely develop mature B-cell tumors early in life and may provide an invaluable resource of a B-cell lymphoma model. Syst Appl Microbiol, 1999 Sep, 22(3), 486 - 91 Effect of growth phase on the content and composition of ceramides of the hydrocarbon-assimilating yeast Candida lipolytica; Rupcic J et al.; Candida lipolytica yeast was grown batchwise on n-hexadecane as the carbon and energy source . Ceramides were quantitatively isolated from total lipids of exponential and stationary phase cells by a combination of column chromatography and preparative high-performance thin-layer chromatography . After acid methanolysis their composition was analyzed by gas-liquid chromatography . The ceramide content of the exponential phase cells was two times higher than the one of the stationary phase cells . The composition of long-chain base moiety of ceramides did not change significantly during the growth . In both growth phases 19-phytosphingosine was the major long-chain base . However, the fatty acid composition of ceramides changed greatly during the growth . In the exponential growth phase, ceramides contained predominantly fatty acids greater than 20 carbon atoms, while fatty acids shorter than 20 atoms predominated in ceramides of the stationary phase, 16:0 being the main one . In the exponential growth phase fatty acid moiety of ceramides was characterized by unusually high degree of unsaturation and relatively high proportion of odd-numbered fatty acids . However, the proportion of both, unsaturated and odd-numbered fatty acid decreased significantly in ceramides of the stationary phase . The unexpected finding was the absence of fatty acid hydroxylation of ceramides in the exponential phase cells and unusually low degree of hydroxylation in the stationary phase. Syst Appl Microbiol, 1999 Sep, 22(3), 445 - 53 Restriction enzyme analysis of PCR amplified rDNA as a taxonomic tool in yeast identification; Dlauchy D et al.; A method has been developed to simplify the identification of yeast strains . We used the restriction fragment patterns of PCR-amplified 18S rRNA-coding DNA with the neighbouring ITS1 region for differentiation and identification of 169 yeast strains representing 128 species associated mainly with food, wine, beer, and soft drinks . The amplicons were digested with four different four-base-cutting restriction enzymes . To construct a database of restriction fragment patterns, the gels have been scanned and analyzed using the Molecular Analyst Fingerprint 2.0 software . The use of four enzymes proved to be sufficient for strain identification. Mol Microbiol, 1999 Dec, 34(5), 1049 - 57 Genome-wide analysis of gene expression regulated by the yeast cell wall integrity signalling pathway; Jung US et al.; The cell integrity pathway of Saccharomyces cerevisiae monitors cell wall remodelling during growth and differentiation . Additionally, this pathway responds to environmental stresses that challenge the integrity of the cell wall . We conducted a genome-wide survey of genes whose expression was altered in response to activation of Mpk1/Slt2, the MAP kinase, under the control of cell integrity signalling . We identified 25 genes whose regulation was altered by Mpk1 activity . Among these, 20 were positively regulated by Mpk1, and five were negatively regulated . Most of the genes identified encode either known or suspected cell wall proteins or enzymes involved in cell wall biogenesis . These include glycosyl-phosphatidylinositol (GPI) proteins, members of the Pir family of cell wall proteins, Mpk1 itself and others . All of the regulation detected was mediated by the Rlm1 transcription factor, a MADS-box protein that is phosphorylated and activated by Mpk1, but for which no transcriptional targets had been identified . A similar pattern of regulation was observed when cell integrity signalling was induced by environmental stress (i.e . temperature upshift). Protein Sci, 1999 Nov, 8(11), 2465 - 73 Structures of yeast vesicle trafficking proteins; Tishgarten T et al.; In protein transport between organelles, interactions of v- and t-SNARE proteins are required for fusion of protein-containing vesicles with appropriate target compartments . Mammalian SNARE proteins have been observed to interact with NSF and SNAP, and yeast SNAREs with yeast homologues of NSF and SNAP proteins . This observation led to the hypothesis that, despite low sequence homology, SNARE proteins are structurally similar among eukaryotes . SNARE proteins can be classified into two groups depending on whether they interact with SNARE binding partners via conserved glutamine (Q-SNAREs) or arginine (R-SNAREs) . Much of the published structural data available is for SNAREs involved in exocytosis (either in yeast or synaptic vesicles) . This paper describes circular dichroism, Fourier transform infrared spectroscopy, and dynamic light scattering data for a set of yeast v- and t-SNARE proteins, Vti1p and Pep12p, that are Q-SNAREs involved in intracellular trafficking . Our results suggest that the secondary structure of Vti1p is highly alpha-helical and that Vti1p forms multimers under a variety of solution conditions . In these respects, Vti1p appears to be distinct from R-SNARE proteins characterized previously . The alpha-helicity of Vti1p is similar to that of Q-SNARE proteins characterized previously . Pep12p, a Q-SNARE, is highly alpha-helical . It is distinct from other Q-SNAREs in that it forms dimers under many of the solution conditions tested in our experiments . The results presented in this paper are among the first to suggest heterogeneity in the functioning of SNARE complexes. Mol Cell Biol, 2000 Jan, 20(1), 26 - 33 Association of yeast adenylyl cyclase with cyclase-associated protein CAP forms a second Ras-binding site which mediates its Ras-dependent activation; Shima F et al.; Posttranslational modification, in particular farnesylation, of Ras is crucial for activation of Saccharomyces cerevisiae adenylyl cyclase (CYR1) . Based on the previous observation that association of CYR1 with cyclase-associated protein (CAP) is essential for its activation by posttranslationally modified Ras, we postulated that the associated CAP might contribute to the formation of a Ras-binding site of CYR1, which mediates CYR1 activation, other than the primary Ras-binding site, the leucine-rich repeat domain . Here, we observed a posttranslational modification-dependent association of Ras with a complex between CAP and CYR1 C-terminal region . When CAP mutants defective in Ras signaling but retaining the CYR1-binding activity were isolated by screening of a pool of randomly mutagenized CAP, CYR1 complexed with two of the obtained three mutants failed to be activated efficiently by modified Ras and exhibited a severely impaired ability to bind Ras, providing a genetic evidence for the importance of the physical association with Ras at the second Ras-binding site . On the other hand, CYR1, complexed with the other CAP mutant, failed to be activated by Ras but exhibited a greatly enhanced binding to Ras . Conversely, a Ras mutant E31K, which exhibits a greatly enhanced binding to the CYR1-CAP complex, failed to activate CYR1 efficiently . Thus, the strength of interaction at the second Ras-binding site appears to be a critical determinant of CYR1 regulation by Ras: too-weak and too-strong interactions are both detrimental to CYR1 activation . These results, taken together with those obtained with mammalian Raf, suggest the importance of the second Ras-binding site in effector regulation. Mol Cell Biol, 2000 Jan, 20(1), 12 - 25 Pan1p, End3p, and S1a1p, three yeast proteins required for normal cortical actin cytoskeleton organization, associate with each other and play essential roles in cell wall morphogenesis; Tang HY et al.; The EH domain proteins Pan1p and End3p of budding yeast have been known to form a complex in vivo and play important roles in organization of the actin cytoskeleton and endocytosis . In this report, we describe new findings concerning the function of the Pan1p-End3p complex . First, we found that the Pan1p-End3p complex associates with Sla1p, another protein known to be required for the assembly of cortical actin structures . Sla1p interacts with the first long repeat region of Pan1p and the N-terminal EH domain of End3p, thus leaving the Pan1p-End3p interaction, which requires the second long repeat of Pan1p and the C-terminal repeat region of End3p, undisturbed . Second, Pan1p, End3p, and Sla1p are also required for normal cell wall morphogenesis . Each of the Pan1-4, sla1Delta, and end3Delta mutants displays the abnormal cell wall morphology previously reported for the act1-1 mutant . These cell wall defects are also exhibited by wild-type cells overproducing the C-terminal region of Sla1p that is responsible for interactions with Pan1p and End3p . These results indicate that the functions of Pan1p, End3p, and Sla1p in cell wall morphogenesis may depend on the formation of a heterotrimeric complex . Interestingly, the cell wall abnormalities exhibited by these cells are independent of the actin cytoskeleton organization on the cell cortex, as they manifest despite the presence of apparently normal cortical actin cytoskeleton . Examination of several act1 mutants also supports this conclusion . These observations suggest that the Pan1p-End3p-Sla1p complex is required not only for normal actin cytoskeleton organization but also for normal cell wall morphogenesis in yeast. J Biol Chem, 1999 Dec 17, 274(51), 36679 - 83 Yeast Sml1, a protein inhibitor of ribonucleotide reductase; Chabes A et al.; Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides; this step is rate-limiting in DNA precursor synthesis . A number of regulatory mechanisms ensure optimal deoxyribonucleotide pools, which are essential for cell viability . The best studied mechanisms are transcriptional regulation of the RNR genes during the cell cycle and in the response to DNA damage, and the allosteric regulation of ribonucleotide reductase by nucleoside triphosphates . Recently, another mode of RNR regulation has been hypothesized in yeast . A novel protein, Sml1, was shown to bind to the Rnr1 protein of the yeast ribonucleotide reductase; this interaction was proposed to inhibit ribonucleotide reductase activity when DNA synthesis is not required (Zhao, X., Muller, E.G.D., and Rothstein, R . (1998) Mol . Cell 2, 329-340) . Here, we use highly purified recombinant proteins to directly demonstrate that the Sml1 protein is a strong inhibitor of yeast RNR . The Sml1p specifically binds to the yeast Rnr1p in a 1:1 ratio with a dissociation constant of 0.4 microM . Interestingly, Sml1p also specifically binds to the mouse ribonucleotide reductase R1 protein . However, the inhibition observed in an in vitro mouse ribonucleotide reductase assay is less pronounced than the inhibition in yeast and probably occurs via a different mechanism. J Biol Chem, 1999 Dec 17, 274(51), 36643 - 8 Characterization of active reverse transcriptase and nucleoprotein complexes of the yeast retrotransposon Ty3 in vitro; Cristofari G et al.; Human immunodeficiency virus (HIV) and the distantly related yeast Ty3 retrotransposon encode reverse transcriptase (RT) and a nucleic acid-binding protein designated nucleocapsid protein (NCp) with either one or two zinc fingers, required for HIV-1 replication and Ty3 transposition, respectively . In vitro binding of HIV-1 NCp7 to viral 5' RNA and primer tRNA(3)(Lys) catalyzes formation of nucleoprotein complexes resembling the virion nucleocapsid . Nucleocapsid complex formation functions in viral RNA dimerization and tRNA annealing to the primer binding site (PBS) . RT is recruited in these nucleoprotein complexes and synthesizes minus-strand cDNA initiated at the PBS . Recent results on yeast Ty3 have shown that the homologous NCp9 promotes annealing of primer tRNA(i)(Met) to a 5'-3' bipartite PBS, allowing RNA:tRNA dimer formation and initiation of cDNA synthesis at the 5' PBS () . To compare specific cDNA synthesis in a retrotransposon and HIV-1, we have established a Ty3 model system comprising Ty3 RNA with the 5'-3' PBS, primer tRNA(i)(Met), NCp9, and for the first time, highly purified Ty3 RT . Here we report that Ty3 RT is as active as retroviral HIV-1 or murine leukemia virus RT using a synthetic template-primer system . Moreover, and in contrast to what was found with retroviral RTs, retrotransposon Ty3 RT was unable to direct cDNA synthesis by self-priming . We also show that Ty3 nucleoprotein complexes were formed in vitro and that the N terminus of NCp9, but not the zinc finger, is required for complex formation, tRNA annealing to the PBS, RNA dimerization, and primer tRNA-directed cDNA synthesis by Ty3 RT . These results indicate that NCp9 chaperones bona fide cDNA synthesis by RT in the yeast Ty3 retrotransposon, as illustrated for NCp7 in HIV-1, reinforcing the notion that Ty3 NCp9 is an ancestor of HIV-1 NCp7. J Biol Chem, 1999 Dec 17, 274(51), 36387 - 91 Feedback phosphorylation of an RGS protein by MAP kinase in yeast; Garrison TR et al.; Regulators of G protein signaling (RGS proteins) are well known to accelerate G protein GTPase activity in vitro and to promote G protein desensitization in vivo . Less is known about how RGS proteins are themselves regulated . To address this question we purified the RGS in yeast, Sst2, and used electrospray ionization mass spectrometry to identify post-translational modifications . This analysis revealed that Sst2 is phosphorylated at Ser-539 and that phosphorylation occurs in response to pheromone stimulation . Ser-539 lies within a consensus mitogen-activated protein (MAP) kinase phosphorylation site, Pro-X-Ser-Pro . Phosphorylation is blocked by mutations in the MAP kinase genes (FUS3, KSS1), as well as by mutations in components needed for MAP kinase activation (STE11, STE7, STE4, STE18) . Phosphorylation is also blocked by replacing Ser-539 with Ala, Asp, or Glu (but not Thr) . These point mutations do not alter pheromone sensitivity, as determined by growth arrest and reporter transcription assays . However, phosphorylation appears to slow the rate of Sst2 degradation . These findings indicate that the G protein-regulated MAP kinase in yeast can act as a feedback regulator of Sst2, itself a regulator of G protein signaling. Biosci Biotechnol Biochem, 1999 Sep, 63(9), 1618 - 20 Induction of isoflavonoid and retrochalcone branches of the flavonoid pathway in cultured Glycyrrhiza echinata cells treated with yeast extract; Nakamura K et al.; Yeast extract-treated suspension cultures of a new cell line, AK-1, of Glycyrrhiza echinata were induced to produce an isoflavonoid phytoalexin (medicarpin) and metabolites of retrochalcone/flavone pathway (echinatin, licodione, and 7,4'-dihydroxyflavone) . From these cells, putative full-length cDNAs encoding cytochrome P450s, (2S)-flavanone 2-hydroxylase and isoflavone 2'-hydroxylase, were cloned. Nucleic Acids Res, 2000 Jan 1, 28(1), 85 - 6 YIDB: the Yeast Intron DataBase; Lopez PJ et al.; The Yeast Intron DataBase (YIDB) contains currently available information about all introns encoded in the nuclear and mitochondrial genomes of the yeast Saccharomyces cerevisiae . Introns are divided according to their mechanism of excision: group I and group II introns, pre-mRNA introns, tRNA introns and the HAC1 intron . Information about the host genome, the type of RNA in which they are inserted and their primary structure are provided together with references . For nuclear pre-mRNA introns, transcription frequencies, as determined by microarray experiments, have also been included . This updated database is accessible at: de/ExternalInfo/seraphin/yidb.html Nucleic Acids Res, 2000 Jan 1, 28(1), 73 - 6 The yeast proteome database (YPD) and Caenorhabditis elegans proteome database (WormPD): comprehensive resources for the organization and comparison of model organism protein information; Costanzo MC et al.; The Yeast Proteome Database (YPDtrade mark) has been for several years a resource for organized and accessible information about the proteins of Saccharomyces cerevisiae . We have now extended the YPD format to create a database containing complete proteome information about the model organism Caenorhabditis elegans (WormPDtrade mark) . YPD and WormPD are designed for use not only by their respective research communities but also by the broader scientific community . In both databases, information gleaned from the literature is presented in a consistent, user-friendly Protein Report format: a single Web page presenting all available knowledge about a particular protein . Each Protein Report begins with a Title Line, a concise description of the function of that protein that is continually updated as curators review new literature . Properties and functions of the protein are presented in tabular form in the upper part of the Report, and free-text annotations organized by topic are presented in the lower part . Each Protein Report ends with a comprehensive reference list whose entries are linked to their MEDLINE s . YPD and WormPD are seamlessly integrated, with extensive links between the species . They are freely accessible to academic users on the WWW at proteome.com/databases/index.html, and are available by subscription to corporate users. Cell Biochem Biophys, 1999, 31(2), 165 - 74 Cloning, sequencing, and characterization of five genes coding for acyl-CoA oxidase isozymes in the yeast Yarrowia lipolytica; Wang H et al.; The Acyl-CoA oxidase (AOX) isozymes catalyze the first steps of peroxisomal beta-oxidation, which is important for the degradation of fatty acids . Using conserved blocks in previously identified yeast POX genes encoding AOXs, the authors have shown that five POX genes are present in the yeast Yarrowia lipolytica . These genes show approx 63% identity among themselves, and 42% identity with the POX genes from other yeasts . Mono-disrupted Y . lipolytica strains were constructed using a variation of the sticky-end polymerase chain reaction method . AOX activity in the mono-disrupted strains revealed that a long-chain oxidase is encoded by the POX2 gene and a short-chain oxidase by the POX3 gene. J Bioenerg Biomembr, 1999 Jun, 31(3), 215 - 24 The role of various domains of the iron-sulfur protein in the assembly and activity of the cytochrome bc1 complex of yeast mitochondria; Beattie DS et al.; Assembly studies in vitro of deletion mutants of the iron-sulfur protein into the cytochrome bc1 complex revealed that mutants localized in the extramembranous regions of the protein were not assembled into the complex in contrast to the efficient assembly of mutants in the membrane-spanning region . Charged amino acids located in the extramembranous alpha1-beta4 loop and the alpha1 helix were mutated and expressed in yeast cells lacking the gene for the iron-sulfur protein . Mutating the charged amino acid residues H124, E125, R146, K148, and D149 as well as V132 and W152 resulted in loss of enzymatic activity due to the loss of iron-sulfur protein suggesting that these amino acids are required to maintain protein stability . By contrast, no loss of iron-sulfur protein accompanied the 30-50% loss of bc1 complex activity in mutants of three conserved alanine residues, A86, A90, and A92, suggesting that these residues may be involved in the proposed movement of the flexible tether of the iron-sulfur protein during catalysis. Microb Pathog, 1999 Dec, 27(6), 395 - 405 The effect of canine macrophages on the adherence and growth of Blastomyces dermatitidis yeast: evidence of a soluble factor that enhances the growth of B . dermatitidis yeast; Giles S et al.; Blastomycosis is a medically important systemic fungal infection of dogs and humans . Phagocytic cells are the first line of cellular defence against B . dermatitidis, and are a prominent feature in the lesions and exudate of canine blastomycosis . The adherence of B . dermatitidis yeast to canine phagocytes, and the effects of such adherence on the growth of B . dermatitidis yeast, has not been previously reported . The results of this study demonstrate that canine complement enhances the adherence of B . dermatitidis yeast to canine macrophages . Initiation of the canine complement cascade by B . dermatitidis yeast appeared to occur predominantly by the classical pathway . Adherence of B . dermatitidis yeast to canine macrophages enhanced the growth of the yeast . In the absence of macrophages, this effect could be duplicated by incubating yeast in conditioned medium from co-cultures of macrophages and yeast . This observation suggests that a soluble factor is involved in the growth enhancement of the yeast, These findings provide new insights into the adherence of B . dermatitidis yeast to canine macrophages, and how adherence influences the proliferation of B . dermatitidis yeast . Mol Biol Cell, 1999 Dec, 10(12), 4263 - 81 The yeast GRD20 gene is required for protein sorting in the trans-Golgi network/endosomal system and for polarization of the actin cytoskeleton; Spelbrink RG et al.; The proper localization of resident membrane proteins to the trans-Golgi network (TGN) involves mechanisms for both TGN retention and retrieval from post-TGN compartments . In this study we report identification of a new gene, GRD20, involved in protein sorting in the TGN/endosomal system of Saccharomyces cerevisiae . A strain carrying a transposon insertion allele of GRD20 exhibited rapid vacuolar degradation of the resident TGN endoprotease Kex2p and aberrantly secreted approximately 50% of the soluble vacuolar hydrolase carboxypeptidase Y . The Kex2p mislocalization and carboxypeptidase Y missorting phenotypes were exhibited rapidly after loss of Grd20p function in grd20 temperature-sensitive mutant strains, indicating that Grd20p plays a direct role in these processes . Surprisingly, little if any vacuolar degradation was observed for the TGN membrane proteins A-ALP and Vps10p, underscoring a difference in trafficking patterns for these proteins compared with that of Kex2p . A grd20 null mutant strain exhibited extremely slow growth and a defect in polarization of the actin cytoskeleton, and these two phenotypes were invariably linked in a collection of randomly mutagenized grd20 alleles . GRD20 encodes a hydrophilic protein that partially associates with the TGN . The discovery of GRD20 suggests a link between the cytoskeleton and function of the yeast TGN. Nature, 1999 Nov 25, 402(6760), 418 - 21 Chromosomal landscape of nucleosome-dependent gene expression and silencing in yeast; Wyrick JJ et al.; Eukaryotic genomes are packaged into nucleosomes, which are thought to repress gene expression generally . Repression is particularly evident at yeast telomeres, where genes within the telomeric heterochromatin appear to be silenced by the histone-binding silent information regulator (SIR) complex (Sir2, Sir3, Sir4) and Rap1 (refs 4-10) . Here, to investigate how nucleosomes and silencing factors influence global gene expression, we use high-density arrays to study the effects of depleting nucleosomal histones and silencing factors in yeast . Reducing nucleosome content by depleting histone H4 caused increased expression of 15% of genes and reduced expression of 10% of genes, but it had little effect on expression of the majority (75%) of yeast genes . Telomere-proximal genes were found to be de-repressed over regions extending 20 kilobases from the telomeres, well beyond the extent of Sir protein binding and the effects of loss of Sir function . These results indicate that histones make Sir-independent contributions to telomeric silencing, and that the role of histones located elsewhere in chromosomes is gene specific rather than generally repressive. Biophys J, 1999 Dec, 77(6), 3060 - 70 K(+)-dependent composite gating of the yeast K(+) channel, Tok1; Loukin SH et al.; TOK1 encodes an outwardly rectifying K(+) channel in the plasma membrane of the budding yeast Saccharomyces cerevisiae . It is capable of dwelling in two kinetically distinct impermeable states, a near-instantaneously activating R state and a set of related delayed activating C states (formerly called C(2) and C(1), respectively) . Dwell in the R state is dependent on membrane potential and both internal and external K(+) in a manner consistent with the K(+) electrochemical potential being its determinant, where dwell in the C states is dependent on voltage and only external K(+) . Whereas activation from the C states showed high temperature dependencies, typical of gating transitions in other Shaker-like channels, activation from the R state had a temperature dependence nearly as low as that of simple ionic diffusion . These findings lead us to conclude that although the C states reflect the activity of an internally oriented channel gate, the R state results from an intrinsic gating property of the channel filter region. Mol Gen Genet, 1999 Oct, 262(3), 508 - 14 Mechanism of transcription termination: PTRF interacts with the largest subunit of RNA polymerase I and dissociates paused transcription complexes from yeast and mouse; Jansa P et al.; Transcription termination by RNA polymerase I (Pol I) is a stepwise process . First the elongating RNA polymerase is forced to pause by DNA-bound transcription termination factor (TTF-I) . Then the ternary transcription complex is dissociated by PTRF, a novel factor that promotes release of both nascent transcripts and Pol I from the template . In this study we have investigated the ability of PTRF to liberate transcripts from ternary transcription complexes isolated from yeast and mouse . Using immobilized, tailed templates that contain terminator sequences from Saccharomyces cerevisiae and mouse, respectively, we demonstrate that PTRF promotes release of terminated transcripts, irrespective of whether mouse Pol I has interacted with the murine termination factor TTF-I or its yeast homolog Reb1p . In contrast, mouse Pol I paused by the lac repressor remains bound to the template both in the presence and absence of PTRF . We demonstrate that PTRF interacts with the largest subunit of murine Pol I, with TTF-I and Reb1p, but not the lac repressor . The results imply that Pol I transcription termination in yeast and mouse is mediated by conserved interactions between Pol I, Reb1p/TTF-I and PTRF. Microbiology, 1999 Nov, 145 ( Pt 11), 3221 - 8 The yeast endosomal Na+/H+ exchanger, Nhx1, confers osmotolerance following acute hypertonic shock; Nass R et al.; Osmotolerance in yeast is regulated by at least two distinct mechanisms . The acquired response occurs following long-term exposure to hypertonic medium and requires the induction of the HOG-MAP (high-osmolarity glycerol mitogen-activated protein) kinase cascade to increase levels of the osmolyte glycerol . The acute response occurs following sudden exposure to high osmotica and appears to be dependent on normal vacuole function . In this study it is reported that the yeast endosomal/prevacuolar Na+/H+ exchanger Nhx1 contributes to osmotolerance following sudden exposure to hyperosmotic media . Vacuolar shrinkage and recovery in response to osmotic shock was altered in the (delta)nhx1 null mutant . Our results also show that the osmotolerance conferred by Nhx1 contributes to the postdiauxic/stationary-phase resistance to osmotic stress and allows for the continued growth of cells until the acquired osmotolerance response can occur. Cell, 1999 Nov 24, 99(5), 533 - 43 Structure and mechanism of yeast RNA triphosphatase: an essential component of the mRNA capping apparatus; Lima CD et al.; RNA triphosphatase is an essential mRNA processing enzyme that catalyzes the first step in cap formation . The 2.05 A crystal structure of yeast RNA triphosphatase Cet1p reveals a novel active site fold whereby an eight-stranded beta barrel forms a topologically closed triphosphate tunnel . Interactions of a sulfate in the center of the tunnel with a divalent cation and basic amino acids projecting into the tunnel suggest a catalytic mechanism that is supported by mutational data . Discrete surface domains mediate Cet1p homodimerization and Cet1p binding to the guanylyltransferase component of the capping apparatus . The structure and mechanism of fungal RNA triphosphatases are completely different from those of mammalian mRNA capping enzymes . Hence, RNA triphosphatase presents an ideal target for structure-based antifungal drug discovery. Proc Natl Acad Sci U S A, 1999 Dec 7, 96(25), 14445 - 50 Mammalian Cdk5 is a functional homologue of the budding yeast Pho85 cyclin-dependent protein kinase; Huang D et al.; Mammalian Cdk5 is a member of the cyclin-dependent kinase family that is activated by a neuron-specific regulator, p35, to regulate neuronal migration and neurite outgrowth . p35/Cdk5 kinase colocalizes with and regulates the activity of the Pak1 kinase in neuronal growth cones and likely impacts on actin cytoskeletal dynamics through Pak1 . Here, we describe a functional homologue of Cdk5 in budding yeast, Pho85 . Like Cdk5, Pho85 has been implicated in actin cytoskeleton regulation through phosphorylation of an actin-regulatory protein . Overexpression of CDK5 in yeast cells complemented most phenotypes associated with pho85Delta, including defects in the repression of acid phosphatase expression, sensitivity to salt, and a G(1) progression defect . Consistent with the functional complementation, Cdk5 associated with and was activated by the Pho85 cyclins Pho80 and Pcl2 in yeast cells . In a reciprocal series of experiments, we found that Pho85 associated with the Cdk5 activators p35 and p25 to form an active kinase complex in mammalian and insect cells, supporting our hypothesis that Pho85 and Cdk5 are functionally related . Our results suggest the existence of a functionally conserved pathway involving Cdks and actin-regulatory proteins that promotes reorganization of the actin cytoskeleton in response to regulatory signals. Folia Microbiol (Praha), 1999, 44(2), 153 - 63 Structure and function of the stalk, a putative regulatory element of the yeast ribosome . Role of stalk protein phosphorylation; Rodriguez-Gabriel MA et al.; The ribosomal stalk is involved directly in the interaction of the elongation factors with the ribosome during protein synthesis . The stalk is formed by a complex of five proteins, four small acidic polypeptides and a larger protein which directly interacts with the rRNA at the GTPase center . In eukaryotes, the acidic components correspond to the 12 kDa P1 and P2 proteins, and the RNA binding component is protein P0 . All these proteins are found to be phosphorylated in eukaryotic organisms . Previous in vitro data suggested this modification was involved in the activity of this structure . To confirm this possibility a mutational study has shown that phosphorylation takes place at a serine residue close to the carboxyl end of proteins P1, P2 and P0 . This serine is part of a consensus casein kinase II phosphorylation site . However, by using a yeast strain carrying a temperature sensitive mutant, it has been shown that CKII is probably not the only enzyme responsible for this modification . Three new protein kinases, RAPI, RAPII and RAPIII, have been purified and compared with CKII and PK60, a previously reported enzyme that phosphorylates the stalk proteins . Differences among the five enzymes have been studied . It has also been found that some typical effectors of the PKC kinase stimulate the in vitro phosphorylation of the stalk proteins . All the data available suggest that phosphorylation, although it is not involved in the interaction of the acidic proteins with the ribosome, affects ribosome activity and might participate in some ribosome regulatory mechanism. Can J Microbiol, 1999 Nov, 45(11), 949 - 58 Sequencing as a tool in yeast molecular taxonomy; Valente P et al.; The literature on sequencing as a tool for yeast molecular taxonomy is reviewed . Ribosomal DNA has been preferred for sequencing over other molecules such as mitochondrial DNA, and a large database is now available . rDNA consists of regions that evolve at different rates, allowing comparison of different levels of relationship among yeasts . Sequences of the 18S rDNA and the 25S rDNA have been largely used for yeast systematics and phylogeny, but the search for regions with increased resolving power has led to the study of the spacer regions of the rDNA . Few studies are concerned with signature sequences. J Immunol, 1999 Dec 15, 163(12), 6898 - 906 Antibody repertoires of four- and five-feature translocus mice carrying human immunoglobulin heavy chain and kappa and lambda light chain yeast artificial chromosomes; Nicholson IC et al.; We have produced mice that carry the human Ig heavy (IgH) and both kappa and lambda light chain transloci in a background in which the endogenous IgH and kappa loci have been inactivated . The B lymphocyte population in these translocus mice is restored to about one-third of normal levels, with preferential (3:1) expression of human lambda over human kappa . Human IgM is found in the serum at levels between 50 and 400 microg/ml and is elevated following immunization . This primary human Ab repertoire is sufficient to yield diverse Ag-specific responses as judged by analysis of mAbs . The use of DH and J segments is similar to that seen in human B cells, with an analogous pattern of N nucleotide insertion . Maturation of the response is accompanied by somatic hypermutation, which is particularly effective in the light chain transloci . These mice therefore allow the production of Ag-specific repertoires of both IgM,kappa and IgM,lambda Abs and should prove useful for the production of human mAbs for clinical use. Protein Eng, 1999 Nov, 12(11), 895 - 7 Homology modeling of the multicopper oxidase Fet3 gives new insights in the mechanism of iron transport in yeast; di Patti MC et al.; Fet3, the multicopper oxidase of yeast, oxidizes extracellular ferrous iron which is then transported into the cell through the permease Ftr1 . A three-dimensional model structure of Fet3 has been derived by homology modeling . Fet3 consists of three cupredoxin domains joined by a trinuclear copper cluster which is connected to the blue copper site located in the third domain . Close to this site, which is the primary electron acceptor from the substrate, residues for a potential iron binding site could be identified . The surface disposition of negatively charged residues suggests that Fet3 can translocate Fe(3+) to the permease Ftr1 through a pathway under electrostatic guidance. J Biol Chem, 1999 Dec 10, 274(50), 35873 - 80 Interaction in vivo and in vitro between the yeast fimbrin, SAC6P, and a polymerization-defective yeast actin (V266G and L267G); Cheng D et al.; A mutant yeast actin (GG) has decreased hydrophobicity in a subdomain 3/4 hydrophobic plug believed to be involved in a hydrophobic cross-strand "plug-pocket" interaction necessary for actin filament stability . This actin will not polymerize in vitro but is compatible with cell viability . We have assessed the ability of Sac6p, the yeast homologue of the actin filament stabilizing and bundling protein fimbrin, to restore polymerization in vitro and to facilitate GG-actin function in vivo . Sac6p rescues GG-actin polymerization at 25 degrees C but not at 4 degrees C . The actin polymerizes into bundles at room temperature with a fimbrin:actin molar ratio of 1:4 . At this ratio, every actin monomer contacts a Sac6p actin binding domain . Following cold-induced depolymerization, actin/Sac6p mixtures repolymerize beginning at 15 degrees C instead of the 25 degrees C required for de novo assembly, because of the presence of residual actin-Sac6p nuclei . Generation of haploid Deltasac6/GG-actin cells from either diploid or haploid cells was unsuccessful . The facile isolation of cells with either mutation alone indicates a synthetic lethal relationship between this actin allele and the SAC6 gene . Sac6p may allow GG-actin function in vivo by stabilizing the actin in bundles thereby helping maintain sufficient levels of an otherwise destabilized actin monomer within the cell. Philos Trans R Soc Lond B Biol Sci, 1999 Sep 29, 354(1389), 1551 - 7 Two distinct ubiquitin-proteolysis pathways in the fission yeast cell cycle; Toda T et al.; The SCF complex (Skp1-Cullin-1-F-box) and the APC/cyclosome (anaphase-promoting complex) are two ubiquitin ligases that play a crucial role in eukaryotic cell cycle control . In fission yeast F-box/WD-repeat proteins Pop1 and Pop2, components of SCF are required for cell-cycle-dependent degradation of the cyclin-dependent kinase (CDK) inhibitor Rum1 and the S-phase regulator Cdc18 . Accumulation of these proteins in pop1 and pop2 mutants leads to re-replication and defects in sexual differentiation . Despite structural and functional similarities, Pop1 and Pop2 are not redundant homologues . Instead, these two proteins form heterodimers as well as homodimers, such that three distinct complexes, namely SCFPop1/Pop1, SCFPop1/Pop2 and SCFPop2/Pop2, appear to exist in the cell . The APC/cyclosome is responsible for inactivation of CDK/cyclins through the degradation of B-type cyclins . We have identified two novel components or regulators of this complex, called Apc10 and Ste9, which are evolutionarily highly conserved . Apc10 (and Ste9), together with Rum1, are required for the establishment of and progression through the G1 phase in fission yeast . We propose that dual downregulation of CDK, one via the APC/cyclosome and the other via the CDK inhibitor, is a universal mechanism that is used to arrest the cell cycle at G1. Nat Struct Biol, 1999 Dec, 6(12), 1139 - 47 A novel loop-loop recognition motif in the yeast ribosomal protein L30 autoregulatory RNA complex; Mao H et al.; The yeast Saccharomyces cerevisiae ribosomal protein L30 negatively autoregulates its production by binding to a helix-loop-helix structure formed in its pre-mRNA and its mRNA . A three-dimensional solution structure of the L30 protein in complex with its regulatory RNA has been solved using NMR spectroscopy . In the complex, the helix-loop-helix RNA adopts a sharply bent conformation at the internal loop region . Unusual RNA features include a purine stack, a reverse Hoogsteen base pair (G11anti-G56syn) and highly distorted backbones . The L30 protein is folded in a three-layer alpha/beta/alpha sandwich topology, and three loops at one end of the sandwich make base-specific contacts with the RNA internal loop . The protein-RNA binding interface is divided into two clusters, including hydrophobic and aromatic stacking interactions centering around G56, and base-specific hydrogen-bonding contacts to A57, G58 and G10-U60 wobble base pair . Both the protein and the RNA exhibit a partially induced fit for binding, where loops in the protein and the internal loop in the RNA become more ordered upon complex formation . The specific interactions formed between loops on L30 and the internal loop on the mRNA constitute a novel loop-loop recognition motif where an intimate RNA-protein interface is formed between regions on both molecules that lack regular secondary structure. Biochim Biophys Acta, 1999 Dec 6, 1461(2), 217 - 36 Inventory and function of yeast ABC proteins: about sex, stress, pleiotropic drug and heavy metal resistance; Bauer BE et al.; Saccharomyces cerevisiae was the first eukaryotic organism whose complete genome sequence has been determined, uncovering the existence of numerous genes encoding proteins of the ATP-binding cassette (ABC) family . Fungal ABC proteins are implicated in a variety of cellular functions, ranging from clinical drug resistance development, pheromone secretion, mitochondrial function, peroxisome biogenesis, translation elongation, stress response to cellular detoxification . Moreover, some yeast ABC proteins are orthologues of human disease genes, which makes yeast an excellent model system to study the molecular mechanisms of ABC protein-mediated disease . This review provides a comprehensive discussion and update on the function and transcriptional regulation of all known ABC genes from yeasts, including those discovered in fungal pathogens. Genetics, 1999 Dec, 153(4), 1617 - 28 Dominant mutations in three different subunits of replication factor C suppress replication defects in yeast PCNA mutants; Amin NS et al.; To identify proteins that interact with the yeast proliferating cell nuclear antigen (PCNA), we used a genetic approach to isolate mutations that compensate for the defects in cold-sensitive (Cs(-)) mutants of yeast PCNA (POL30) . Because the cocrystal structure of human PCNA and a p21(WAF1/CIP1) peptide shows that the interdomain region of PCNA is a site of p21 interaction, we specifically looked for new mutations that suppress mutations in the equivalent region of yeast PCNA . In independent screens using three different Cs(-) mutants, we identified spontaneously arising dominant suppressor mutations in the RFC3 gene . In addition, dominant suppressor mutations were identified in the RFC1 and RFC2 genes using a single pol30 mutant . An intimate association between PCNA and RFC1p, RFC2p, and RFC3p is suggested by the allele-restricted suppression of 10 different pol30 alleles by the RFC suppressors . RFC1, RFC2, and RFC3 encode three of the five subunits of the replication factor C complex, which is required to load PCNA onto DNA in reconstituted DNA replication reactions . Genomic sequencing reveals a common region in RFC1p, RFC2p, and RFC3p that is important for the functional interaction with PCNA . Biochemical analysis of the wild type and mutant PCNA and RFC3 proteins shows that mutant RFC3p enhances the production of long DNA products in pol delta-dependent DNA synthesis, which is consistent with an increase in processivity. Genetics, 1999 Dec, 153(4), 1591 - 600 Analysis of the seven-member AAD gene set demonstrates that genetic redundancy in yeast may be more apparent than real; Delneri D et al.; Saccharomyces cerevisiae has seven genes encoding proteins with a high degree (>85%) of amino-acid sequence identity to the aryl-alcohol dehydrogenase of the lignin-degrading, filamentous fungus, Phanerochaete chrysosporium . All but one member of this gene set are telomere associated . Moreover, all contain a sequence similar to the DNA-binding site of the Yap1p transcriptional activator either upstream of or within their coding sequences . The expression of the AAD genes was found to be induced by chemicals, such as diamide and diethyl maleic acid ester (DEME), that cause an oxidative shock by inactivating the glutathione (GSH) reservoir of the cells . In contrast, the oxidizing agent hydrogen peroxide has no effect on the expression of these genes . We found that the response to anti-GSH agents was Yap1p dependent . The very high level of nucleotide sequence similarity between the AAD genes makes it difficult to determine if they are all involved in the oxidative-stress response . The use of single and multiple aad deletants demonstrated that only AAD4 (YDL243c) and AAD6 (YFL056/57c) respond to the oxidative stress . Of these two genes, only AAD4 is likely to be functional since the YFL056/57c open reading frame is interrupted by a stop codon . Thus, in terms of the function in response to oxidative stress, the sevenfold redundancy of the AAD gene set is more apparent than real. Genetics, 1999 Dec, 153(4), 1573 - 81 Transcriptional activation in yeast cells lacking transcription factor IIA; Chou S et al.; The general transcription factor IIA (TFIIA) forms a complex with TFIID at the TATA promoter element, and it inhibits the function of several negative regulators of the TATA-binding protein (TBP) subunit of TFIID . Biochemical experiments suggest that TFIIA is important in the response to transcriptional activators because activation domains can interact with TFIIA, increase recruitment of TFIID and TFIIA to the promoter, and promote isomerization of the TFIID-TFIIA-TATA complex . Here, we describe a double-shut-off approach to deplete yeast cells of Toa1, the large subunit of TFIIA, to <1% of the wild-type level . Interestingly, such TFIIA-depleted cells are essentially unaffected for activation by heat shock factor, Ace1, and Gal4-VP16 . However, depletion of TFIIA causes a general two- to threefold decrease of transcription from most yeast promoters and a specific cell-cycle arrest at the G2-M boundary . These results indicate that transcriptional activation in vivo can occur in the absence of TFIIA. Plant Physiol Biochem, 1999 Nov, 37(11), 821 - 829 Transcriptional activation of the parsley chalcone synthase promoter in heterologous pea and yeast systems; Kalbin G et al.; Introduction by electroporation of different parsley (Petroselinum crispum) CHS-promoter/beta-glucuronidase(GUS)-reporter constructs into pea (Pisum sativum L.) protoplasts leads to a high constitutive GUS-expression and to the loss of the light-inducibility seen in the homologous parsley protoplast system . These results indicate that Unit 1 of the parsley CHS-promoter is only partly responsible for the GUS-expression detected . Instead, additional cis-elements, which are located downstream within 100 bp from the transcriptional start site, mediate the de-repression in pea protoplasts . In contrast, in yeast (Saccharomyces cerevisiae) cells, the GUS expression from the heterologous CHS/GUS construct is controlled by elements between Unit 1 and -100 bp . In both pea and yeast cells, transcription factors different from those regulating UV-responsiveness in parsley, are probably mediating the constitutive expression from the heterologous construct . The results with pea protoplasts imply that protoplastation of pea leaf cells itself induces de-repression as a result of stress to the protoplasts . This notion was strengthened by the finding that mRNA levels of the endogenous chalcone synthase were drastically increased as the result of the protoplastation procedure. Mol Cell Endocrinol, 1999 Sep 10, 155(1-2), 135 - 42 TOR kinase homologs function in a signal transduction pathway that is conserved from yeast to mammals; Cutler NS et al.; Rapamycin is a natural product with potent antifungal and immunosuppressive activities . Rapamycin binds to the FKBP12 prolyl isomerase, and the resulting protein-drug complex inhibits the TOR kinase homologs . Both the FKBP12 and the TOR proteins are highly conserved from yeast to man, and genetic and biochemical studies reveal that these proteins are the targets of rapamycin in vivo . Treatment of yeast or mammalian cells with rapamycin inhibits translational initiation of a subset of mRNAs and dramatically represses ribosomal mRNA and tRNA transcription . Furthermore, rapamycin exposure blocks cell cycle progression in the early G1 phase of the cell cycle, driving cells into a G0 state and, ultimately, triggering autophagy . Recent findings reveal that the upstream factors regulating the TOR signaling cascade are involved in detecting amino acids, nutrients, or growth factors . These findings indicate that the TOR proteins function in a signal transduction pathway that coordinates nutritional and mitogenic signals to control protein biosynthesis and degradation. Jpn J Pharmacol, 1999 Sep, 81(1), 94 - 8 The profile of FR140423, a novel anti-inflammatory compound, in yeast-induced rat hyperalgesia; Ochi T et al.; The mechanism of action of FR140423 (3-(difluoromethyl)-1-(4-methoxyphenyl)-5-{4-(methylsulfinyl)phenyl}pyra zole), a novel anti-inflammatory compound, in a rat yeast-induced hyperalgesic model was investigated and compared with those of indomethacin and morphine . We tested the inhibitory effects of FR140423 on the formation of arachidonic acid metabolites, prostaglandin (PG) E2, thromboxane (TX) B2 and leukotriene (LT) B4, in yeast-injected inflamed paws and the effect of the opioid receptor antagonist naloxone on FR140423-induced anti-hyperalgesic effect and inhibition of the formation of arachidonic acid metabolites . Oral administration of FR140423 showed a dose-dependent anti-hyperalgesic effect . This effect was fourfold more potent than that of indomethacin but less potent than that of morphine . Unlike morphine, FR140423 suppressed the levels of PGE2 and TXB2 but not LTB4 in inflamed paws . FR140423 did not inhibit yeast-induced paw edema . The anti-hyperalgesic effect of FR140423 in yeast-injected rat paws was partially blocked by naloxone . However, the inhibitory effects of FR140423 on the formation of PGE2 and TXB2 in yeast-injected rat paws were not antagonized by naloxone . These results suggest that FR140423 shows a potent anti-hyperalgesic effect mediated by inhibition of PGs in inflamed tissue and by activation of opioid receptors. FEBS Lett, 1999 Nov 26, 462(1-2), 221 - 4 Synergy of importin alpha recognition and DNA binding by the yeast transcriptional activator GAL4; Chan CK et al.; The N-terminus of the yeast transcriptional activator GAL4 contains partially overlapping nuclear targeting and DNA binding functions . We have previously shown that GAL4 is recognised with high affinity by importin beta and not by the conventional nuclear localisation sequence binding importin alpha subunit of the importin alpha/beta heterodimer . The present study uses ELISA-based binding and electrophoretic mobility shift assays to show that recognition of GAL4 by importin alpha can occur, but only when GAL4 is bound to its specific DNA recognition sequence . Intriguingly, binding by importin alpha enhances DNA binding on the part of GAL4, implying a synergistic co-operation between these two functions . The results implicate a possible role for importin alpha in the nucleus additional to its established role in nuclear transport, as well as having implications for the use of GAL4 as a DNA carrier in gene therapy applications. FEBS Lett, 1999 Nov 26, 462(1-2), 108 - 12 Farnesol-induced generation of reactive oxygen species dependent on mitochondrial transmembrane potential hyperpolarization mediated by F(0)F(1)-ATPase in yeast; Machida K et al.; An isoprenoid farnesol (FOH) inhibited cellular oxygen consumption and induced mitochondrial generation of reactive oxygen species (ROS) in cells of Saccharomyces cerevisiae in correlation with hyperpolarization of the mitochondrial transmembrane potential (mtDeltaPsi) . The FOH-induced events were coordinately abolished with the F(1)-ATPase inhibitor sodium azide as well as the F(0)F(1)-ATPase inhibitor oligomycin, suggesting the dependence of ROS generation on mtDeltaPsi hyperpolarization mediated by the proton pumping function of F(0)F(1)-ATPase as a result of ATP hydrolysis . The role of F(1)-ATPase activity in mtDeltaPsi hyperpolarization was supported by the intracellular depletion of ATP in FOH-treated cells and its protection with sodium azide . An indirect mechanism was suggested to exist in the regulation of F(0)F(1)-ATPase by FOH to accelerate its ATP-hydrolyzing activity. J Cell Biol, 1999 Nov 29, 147(5), 981 - 94 Cell cycle-regulated attachment of the ubiquitin-related protein SUMO to the yeast septins; Johnson ES et al.; SUMO is a ubiquitin-related protein that functions as a posttranslational modification on other proteins . SUMO conjugation is essential for viability in Saccharomyces cerevisiae and is required for entry into mitosis . We have found that SUMO is attached to the septins Cdc3, Cdc11, and Shs1/Sep7 specifically during mitosis, with conjugates appearing shortly before anaphase onset and disappearing abruptly at cytokinesis . Septins are components of a belt of 10-nm filaments encircling the yeast bud neck . Intriguingly, only septins on the mother cell side of the bud neck are sumoylated . We have identified four major SUMO attachment-site lysine residues in Cdc3, one in Cdc11, and two in Shs1, all within the consensus sequence (IVL)KX(ED) . Mutating these sites eliminated the vast majority of bud neck-associated SUMO, as well as the bulk of total SUMO conjugates in G(2)/M-arrested cells, indicating that sumoylated septins are the most abundant SUMO conjugates at this point in the cell cycle . This mutant has a striking defect in disassembly of septin rings, resulting in accumulation of septin rings marking previous division sites . Thus, SUMO conjugation plays a role in regulating septin ring dynamics during the cell cycle. Carbohydr Res, 1999 May 31, 318(1-4), 110 - 5 Competitive inhibitors of yeast phosphoglucose isomerase: synthesis and evaluation of new types of phosphorylated sugars from the synthon D-arabinolactone-5-phosphate; Hardre R et al.; Designed as competitive inhibitors of the isomerization reaction catalyzed by the potential chemotherapeutic target phosphoglucose isomerases (PGI), D-arabinonamide-5-phosphate and D-arabinohydrazine-5-phosphate were synthesized and fully characterized . These new types of phosphorylated sugar derivatives were easily and efficiently obtained in a one-step procedure from the promising synthon D-arabinono-1,4-lactone 5-phosphate . These two compounds proved to be new good competitive inhibitors of yeast PGI with the substrate D-fructose-6-phosphate, though not as strong as D-arabinohydroxamic acid-5-phosphate . Overall, our results are in accord with the postulated 1,2-cis-enediolate species as a probable high-energy intermediate of the PGI-catalyzed reaction. N Z Med J, 1999 Sep 10, 112(1095), 331 - 3 Immunity to hepatitis B in two birth cohorts given plasma-derived or yeast-derived vaccine; Salmond CE et al.; AIM: To determine the antibody response to either yeast-derived or low-dose, plasma-derived hepatitis B vaccine, in two cohorts of infants monitored by an immunisation coordinator and immunised by general practitioners . METHODS: Infants born to two cohorts of non-carrier mothers in Northland were followed up, the first receiving a low-dose, plasma-derived vaccine, the second a yeast-derived vaccine . An immunisation coordinator enrolled the mothers into the programme during pregnancy, promoted full immunisation against hepatitis B and later obtained blood samples from their babies . In each cohort, four subsamples of babies, randomly assigned, were bled for estimation of antibody levels to hepatitis B at ages 18, 30, 42 and 54 months (1 1/2, 2 1/2, 3 1/2, 4 1/2 years) . No infant was bled more than once . RESULTS: In both cohorts, antibody levels declined significantly with age . By age 4 1/2 years, 5.1% of children (95% confidence interval (CI): 3.5-7.1) immunised with yeast-derived vaccine were estimated to have antibody levels to hepatitis B below the acceptable level for protection of 10 IU/L . The proportion for those immunised with plasma-derived vaccine was 14.3% (95% CI: 7.4-24.1) . CONCLUSIONS: Children receiving yeast-derived vaccine do not require a second booster dose at school entry, although this might be considered at age 11 . There are grounds to suggest that those who received low-dose, plasma-derived vaccine (prior to 1990) should be offered a booster before age 11. Curr Biol, 1999 Nov 18, 9(22), R845 - 7 Yeast prions: bungee cord domains' balancing act; Lansbury PT Jr; The yeast prion-like protein Sup35 has repeats responsible for the reversible induction of an altered, but advantageous phenotype . The expansion of similar repeat domains in several mammalian proteins is associated with neurodegenerative disease - so why are these 'bungee cord' domains conserved? Ital J Gastroenterol Hepatol, 1999 Aug-Sep, 31(6), 494 - 500 From yeast to man--from mitochondria to liver regeneration: a new essential gene family; Polimeno L et al.; The purpose of this review is to bring to the attention of the reader the latest developments in research on an important new emerging gene family . The respective genes are found in eukaryotes from yeast to man and even on the genome of some doubled-stranded DNA viruses . They have essential functions in the biogenesis of mitochondria, the cell division cycle and, in higher eukaryotes, in the development of organs like liver and testis . The most important medical implication is their probable role in liver regeneration that will, therefore, be addressed in detail . Aspects of molecular biology, medical implications and problems of developmental biology reflect the complexity of the functions of these proteins and the subjects of the respective research . This is just the beginning of an interdisciplinary effort directed towards the elucidation of the precise function of these essential factors inside the eukaryotic cell . In the general part of this review, we will concentrate on the history of the discovery of these genes and on a summary of their characteristic features . In the more specialized section, the specific role as augmenter of liver regeneration will be addressed in detail. Cytogenet Cell Genet, 1999, 86(3-4), 307 - 16 Identification and characterization of a mouse homolog to yeast Cdc6p; Berger C et al.; Periodic expression of the Cdc6 protein is essential for the entry of budding yeast cells into S phase, and also for participating in checkpoint controls that ensure that DNA replication is completed before mitosis is initiated . We have identified a mouse protein closely related to Cdc6p (MmCdc6p) as well as to its human and Xenopus homologs . The gene coding for MmCdc6p (Cdc6) is located at band D on murine chromosome 11 . Analysis of its genomic region revealed that the 13-kb Cdc6 gene is divided into 12 exons by 11 introns . MmCdc6p has putative cyclin-dependent phosphorylation sites, a destruction box, nuclear localization signals, a nucleotide triphosphate-binding motif, and a potential leucine zipper . None of these consensus motifs except the leucine-zipper and the destruction box overlaps an intron . Expression of MmCdc6 mRNA and protein is suppressed in mouse NIH3T3 fibroblasts made quiescent by serum starvation . Upon replenishment of the medium, transcript and protein levels increase during progression through G(1), peaking as cells enter S phase . MmCdc6p is phosphorylated in vitro by cdk1/cyclin B, cdk4/cyclin D, cdk2/cyclin E, and cdk2/cyclin A, respectively at serine-residues . In vivo however, phosphorylation of MmCdc6p is carried out by cdk2/cyclin A at serine-residues exclusively . Conservation of structures among members of the Cdc6-related proteins suggests that these proteins play a key role in the regulation of DNA replication during the cell cycle in all eukaryotes . These results strongly suggest, that Cdc6p plays an important role in cell cycle regulation and replication licensing. J Biol Chem, 1999 Dec 3, 274(49), 35099 - 106 Pig-n, a mammalian homologue of yeast Mcd4p, is involved in transferring phosphoethanolamine to the first mannose of the glycosylphosphatidylinositol; Hong Y et al.; Many cell surface proteins are anchored to the membrane via a glycosylphosphatidylinositol (GPI) moiety, which is attached to the C terminus of the proteins . The core of the GPI anchor is conserved in all eukaryotes but is modified by various side chains . We cloned a mouse phosphatidylinositol glycan-class N (Pig-n) gene that encodes a 931amino acid protein expressed in the endoplasmic reticulum, which is homologous to yeast Mcd4p . We disrupted the gene in F9 embryonal carcinoma cells . In the Pig-n knockout cells, the first mannose in the GPI precursors was not modified by phosphoethanolamine . Nevertheless, further biosynthetic steps continued with the addition of the third mannose and the terminal phosphoethanolamine . The surface expression of Thy-1 was only partially affected, indicating that modification of the first mannose by phosphoethanolamine is not essential for attachment of GPI anchors in mammalian cells . An inhibitor of GPI biosynthesis, YW3548/BE49385A, inhibited transfer of phosphoethanolamine to the first mannose in mammalian cells but only slightly affected the surface expression of GPI-anchored proteins . Biosynthesis of GPI in the Pig-n knockout cells was not affected by YW3548/BE49385A, and yeast overexpressing MCD4 was highly resistant to YW3548/BE49385A, suggesting that Pig-n and Mcd4p are targets of this drug. J Biol Chem, 1999 Dec 3, 274(49), 35089 - 94 Yeast SMF1 mediates H(+)-coupled iron uptake with concomitant uncoupled cation currents; Chen XZ et al.; Yeast membrane proteins SMF1, SMF2, and SMF3 are homologues of the DCT1 metal ion transporter family . Their functional characteristics and the implications of these characteristics in vivo have not yet been reported . Here we show that SMF1 expressed in Xenopus oocytes mediates H(+)-dependent Fe(2+) transport and uncoupled Na(+) flux . SMF1-mediated Fe(2+) transport exhibited saturation kinetics (K(m) = 2.2 microM), whereas the Na(+) flux did not, although both processes were electrogenic . SMF1 is also permeable to Li(+), Rb(+), K(+), and Ca(2+), which likely share the same uncoupled pathway . SMF2 (but not SMF3) mediated significant increases in both Fe(2+) and Na(+) transport compared with control oocytes . These data are consistent with the concept that uptake of divalent metal ions by SMF1 and SMF2 is essential to yeast cell growth . Na(+) inhibited metal ion uptake mediated by SMF1 and SMF2 expressed in oocytes . Consistent with this, we found that increased sensitivity of yeast to EGTA in the high Na(+) medium is due to inhibition of SMF1- and SMF2-mediated metal ion transport by uncoupled Na(+) pathway . Interestingly, DCT1 also mediates Fe(2+)-activated uncoupled currents . We propose that uncoupled ion permeabilities in metal ion transporters protect cells from metal ion overload. Nature, 1999 Nov 4, 402(6757), 96 - 100 Mitochondrial DNA repairs double-strand breaks in yeast chromosomes; Ricchetti M et al.; The endosymbiotic theory for the origin of eukaryotic cells proposes that genetic information can be transferred from mitochondria to the nucleus of a cell, and genes that are probably of mitochondrial origin have been found in nuclear chromosomes . Occasionally, short or rearranged sequences homologous to mitochondrial DNA are seen in the chromosomes of different organisms including yeast, plants and humans . Here we report a mechanism by which fragments of mitochondrial DNA, in single or tandem array, are transferred to yeast chromosomes under natural conditions during the repair of double-strand breaks in haploid mitotic cells . These repair insertions originate from noncontiguous regions of the mitochondrial genome . Our analysis of the Saccharomyces cerevisiae mitochondrial genome indicates that the yeast nuclear genome does indeed contain several short sequences of mitochondrial origin which are similar in size and composition to those that repair double-strand breaks . These sequences are located predominantly in non-coding regions of the chromosomes, frequently in the vicinity of retrotransposon long terminal repeats, and appear as recent integration events . Thus, colonization of the yeast genome by mitochondrial DNA is an ongoing process. FEBS Lett, 1999 Sep 24, 458(3), 285 - 91 Mutations in the yeast two pore K+ channel YKC1 identify functional differences between the pore domains; Vergani P et al.; The K+ channel of Saccharomyces cerevisiae encoded by the YKC1 gene includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore . Gating of the channel is promoted by membrane depolarisation and is regulated by the extracellular K+ concentration ({K+}o) both in the yeast and when expressed in Xenopus oocytes . Our previous work showed that substitutions of equivalent residues L293 and A428 within the pore-loops had qualitatively similar effects on both the {K+}o-sensitivity of channel gating and its voltage-dependence . Here, we report that mutations of equivalent residues N275 and N410, N-terminal from the K+ channel signature sequences of the two pores, have very different actions on channel gating and, in this case, are without effect on its voltage-sensitivity . The mutation N410D slowed current activation in a {K+}o-dependent manner and it accelerated deactivation, but without significant effect on the apparent affinity for K+ . The N275D mutant, by contrast, had little effect on the {K+}o-sensitivity for activation and it greatly altered the . {K+}o-dependence of current deactivation . Neither mutant affected the voltage-dependence of the steady-state current nor the ability for other alkali cations to substitute for K+ in regulating gating . The double mutant N410D-N275D showed characteristics of N410D in the {K+}o-sensitivity of current activation and of N275D in the {K+}o-sensitivity of deactivation, suggesting that little interaction occurs between pore domains with mutations at these sites . The results indicate that the two pore domains are not functionally equivalent and they suggest that the regulation of gating by external K+ is mediated by K+ binding at two physically distinct sites with different actions. Glycobiology, 2000 Jan, 10(1), 51 - 8 In yeast the export of small glycopeptides from the endoplasmic reticulum into the cytosol is not affected by the structure of their oligosaccharide chains; Suzuki T et al.; A "quality control" system associated with the endoplasmic reticulum (ER) that discriminates between misfolded proteins and correctly folded proteins is present in a variety of eukaryotic cells, including yeast . Recently, it has been shown that misfolded proteins that are N -glycosylated in the lumen of the ER are transported out of the ER, de-N-glycosylated by a soluble peptide: N -glycanase (PNGase) and degraded by action of the proteasome . It also has been shown that small N -glycosylatable peptides follow a fate similar to that of misfolded proteins, i.e., glycosylation in the lumen of the ER, transport out of the ER, and de- N -glycosylation in the cytosol . These processes of retrograde glycopeptide transport and de- N -glycosylation have been observed in mammalian cells, as well as in yeast cells . However, little is known about the mechanism involved in the movement of glycopeptides from the ER to the cytosol . Here we report a simple method for assaying N -glycosylation/de- N -glycosylation by simple paper chromatographic and electrophoretic techniques using an N -glycosylatable(3)H-labeled tripeptide as a substrate . With this method, we confirmed the cytosolic localization of the de- N -glycosylated peptide, which supports the idea that de- N -glycosylation occurs after the export of the glycopeptide from the lumen of the ER to the cytosol . Further, we found that the variations in the structure of the oligosaccharide chain on the glycopeptide did not cause differences in the export of the glycopeptide . This finding suggests that the mechanism for the export of small glycopeptides may differ from that of misfolded (glyco)proteins. RNA, 1999 Oct, 5(10), 1333 - 47 The Alu domain homolog of the yeast signal recognition particle consists of an Srp14p homodimer and a yeast-specific RNA structure; Strub K et al.; The mammalian Alu domain of the signal recognition particle (SRP) consists of a heterodimeric protein SRP9/14 and the Alu portion of 7SL RNA and comprises the elongation arrest function of the particle . To define the domain in Saccharomyces cerevisiae SRP that is homologous to the mammalian Alu domain {Alu domain homolog in yeast (Adhy)}, we examined the assembly of a yeast protein homologous to mammalian SRP14 (Srp14p) and scR1 RNA . Srp14p binds as a homodimeric complex to the 5' sequences of scR1 RNA . Its minimal binding site consists of 99 nt . (Adhy RNA), comprising a short hairpin structure followed by an extended stem . As in mammalian SRP9/14, the motif UGUAAU present in most SRP RNAs is part of the Srp14p binding sites as shown by footprint and mutagenesis studies . In addition, certain basic amino acid residues conserved between mammalian SRP14 and Srp14p are essential for RNA binding in both proteins . These findings confirm the common ancestry of the yeast and the mammalian components and indicate that Srp14p together with Adhy RNA represents the Alu domain homolog in yeast SRP that may comprise its elongation arrest function . Despite the similarities, Srp14p selectively recognizes only scR1 RNA, revealing substantial changes in RNA-protein recognition as well as in the overall structure of the complex . The alignment of the three yeast SRP RNAs known to date suggests a common structure for the putative elongation arrest domain of all three organisms. Yeast, 1999 Nov, 15(15), 1645 - 56 trans-dominant mutations in the GPR1 gene cause high sensitivity to acetic acid and ethanol in the yeast Yarrowia lipolytica; Tzschoppe K et al.; Acetate non-utilizing strains harbouring trans-dominant mutations in the GPR1 gene (GPR1(d)) of the dimorphic yeast Yarrowia lipolytica have been selected and characterized . These mutants are highly sensitive to low concentrations of acetic acid and ethanol, even in presence of glucose . The toxic effect of acetic acid is pH-dependent and has the strongest effect at low pH . In contrast, the action of ethanol is pH-independent . One GPR1(d) mutant has been detected that was highly sensitive to acetic acid but could still grow on ethanol, which indicates putative differences in the function of the GPR1 gene product in the sensitivity to acetic acid and ethanol . The GPR1(d) mutants exhibit a complex pleiotropic phenotype . The mutations cause changed colony morphology as well as dimorphism of cells, and induce early cell death during growth on glucose, even without the presence of dicarbon compounds . Composition of intracellular membranes, as well as morphology of vacuole and mitochondria, were strongly changed . Back-crosses with wild-type strains and analysis of recombinant strains have shown that the expression of the pleiotropic phenotype depends on the site of mutation in the GPR1 gene, as well as on the genetic background of the strain harbouring the responsive mutation . Our data suggest that Gpr1p is involved in a general response of cells to the toxic action of dicarbon compounds like acetic acid and ethanol . Yeast, 1999 Nov, 15(15), 1585 - 93 An alternative model for the transmembrane segments of the yeast H+-ATPase; Pardo JP et al.; An alternative topological model for the yeast plasma membrane H(+)-ATPase from K . lactis was deduced by joint prediction, using 11 algorithms for the prediction of transmembrane segments complemented with hydrophobic moment analysis . Similarly to the model currently used in the literature, this alternative model contains 10 transmembrane segments, four in the N-half and six in the C-half of the protein . However, the distribution of the membrane-associated segments on the C-half of the enzyme differs in both models . Nine of the 10 transmembrane segments are highly hydrophobic with low hydrophobic moments, and are probably involved in structural roles . The fifth transmembrane segment is, on the other hand, less hydrophobic, with the highest hydrophobic moment, suggesting that this segment might have a dynamic role in the coupling of the hydrolysis of ATP with the translocation of protons across the membrane . The alignment of the Ca(2+)-ATPase, the Na(+)/K(+)-ATPase and the H(+)-ATPase sequences showed that these proteins have the same topology in the N-half, but important differences were found at the C-half of the enzymes . In contrast with the mammalian ATPases, the fifth transmembrane segment in the H(+)-ATPase appears early in the sequence, giving rise to a shorter cytoplasmic central loop . This alternative model will be useful in the designing of site-directed mutagenesis experiments and contains information for the fitting of the amino acid sequence into the transmembrane region of the three-dimensional model of the ATPase . Int J Radiat Oncol Biol Phys, 1999 Nov 1, 45(4), 975 - 80 A point mutation of human p53, which was not detected as a mutation by a yeast functional assay, led to apoptosis but not p21Waf1/Cip1/Sdi1 expression in response to ionizing radiation in a human osteosarcoma cell line, Saos-2; Okaichi K et al.; PURPOSE: The 123A point mutation of p53 showed increased radiosensitivity, whereas other mutations (143A, 175H, and 273H) were not affected . To determine the reason for increased radiosensitivity of the 123A mutation, the response of the transformant of 123A mutation to ionizing radiation (IR) was examined and compared to those of transformants with the wild type p53 or other point mutations (143A, 175H, and 273H) . METHODS AND MATERIALS: Stable transformants with a mutant or wild type p53 made by introducing cDNA into the human osteosarcoma cell line, Saos-2, which lacks an endogenous p53 were used . The transcriptional activity of mutant p53 was examined using a yeast functional assay . The transformants were examined for the accumulation of p53, the induction of p21Waf1/Cip1/Sdi1 (hereafter referred to as p21), and the other response of p53-responsive genes (MDM2, Bax, and Bcl-2) by Western blotting . Apoptosis was analyzed by detection of DNA fragmentation . RESULTS: The 123A point mutation of p53 was detected as a wild type in the yeast functional assay . The 123A mutant accumulated p53 in response to IR . The 123A mutant did not induce p21, but normally responded to MDM2, Bax, and Bcl-2 . The 123A mutant entered apoptosis earlier than the wild type p53 transformant, and induced Fas at earlier in response to IR . CONCLUSION: The 123A mutant led to apoptosis, but not p21 expression in response to IR . The occurrence of apoptosis, but not induction of p21, corresponded to the radiosensitivity in the transformant . The early occurrence of apoptosis in 123A transformants may depend on the early induction of Fas. Gene, 1999 Oct 18, 239(1), 29 - 38 Meiotic interallelic conversion at the human minisatellite MS32 in yeast triggers recombination in several chromatids; Appelgren H et al.; Tandem repetitive DNA sequences such as minisatellites include the most polymorphic loci yet identified in the human genome . The high mutation rates at many of these loci are driven by incompletely understood recombination-based mechanisms that operate in the germline . To analyse aspects of minisatellite mutation processes and general eukaryotic recombination in meiosis that cannot be studied in humans or other mammals, including crosstalk and interplay between all four chromatids, we have previously constructed a eukaryotic model system, enabling the analysis of all four products of meiosis . In this system we have integrated alleles of the human minisatellite MS32, flanked by synthetic markers, in the vicinity of a meiotic recombination hot spot in chromosome III of Saccharomyces cerevisiae . In the present study, tetrad analysis showed that gene conversion is the predominant and possibly the universal pathway leading to interallelic transfer of repeats, with or without exchange of flanking regions . The data also suggest a hyper-recombinogenic state, triggered by interallelic mutation processes which generate a cascade of mutant alleles in the same meiosis . A number of tetrads contained identical mutant alleles of meiotic origin . Several tetrads could not be explained by the current models for minisatellite mutation . Accordingly, we here present a modified model based on the successive repair of multiple double-strand breaks. Gene, 1999 Sep 30, 238(1), 253 - 61 Updated map of duplicated regions in the yeast genome; Seoighe C et al.; We have updated the map of duplicated chromosomal segments in the Saccharomyces cerevisiae genome originally published by Wolfe and Shields in 1997 (Nature 387, 708-713) . The new analysis is based on the more sensitive Smith Waterman search method instead of BLAST . The parameters used to identify duplicated chromosomal regions were optimized such as to maximize the amount of the genome placed into paired regions, under the assumption that the hypothesis that the entire genome was duplicated in a single event is correct . The core of the new map, with 52 pairs of regions containing three or more duplicated genes, is largely unchanged from our original map . 39 tRNA gene pairs and one snRNA pair have been added . To find additional pairs of genes that may have been formed by whole genome duplication, we searched through the parts of the genome that are not covered by this core map, looking for putative duplicated chromosomal regions containing only two duplicate genes instead of three, or having lower-scoring gene pairs . This approach identified a further 32 candidate paired regions, bringing the total number of protein-coding genes on the duplication map to 905 (16% of the proteome) . The updated map suggests that a second copy of the ribosomal DNA array has been deleted from chromosome IV. Biochemistry, 1999 Nov 23, 38(47), 15597 - 605 Structural characterization of manganese(II)-nucleotide complexes bound to yeast 3-phosphoglycerate kinase: 13C relaxation measurements using {U-13C}ATP and {U-13C}ADP; Raghunathan V et al.; A complete characterization of the conformations of Mn.ADP and Mn.ATP bound to the active site of yeast 3-P-glycerate kinase is presented . These conformations have been deduced on the basis of paramagnetic effects on 13C spin-lattice relaxation rates in {U-13C}nucleotides due to Mn(II), used as a substituent activating cation . The 13C relaxation measurements were performed on exclusively enzyme-bound complexes E.Mn.{U-13C}ATP and E.Mn.{U-13C}ADP at three distinct 13C NMR frequencies: 75.4, 125.7, and 181 MHz . The frequency dependence of the relaxation data has been analyzed in an effort to evaluate distances from the cation for all 10 13C nuclei in the adenosine moieties of E.Mn.ATP and E.Mn.ADP . These distance data, taken along with previously published cation-31P distances, have been used as constraints in the molecular modeling program Quanta, in which molecular dynamics simulations and energy minimization have been performed to determine the conformations that are compatible with the distance data . It was possible to model the distances on the basis of a single enzyme-bound conformation for each of the nucleotides . The details of the enzyme-bound Mn.ATP and Mn.ADP conformations are distinguishably different from each other, indicating that structural alterations occur in the enzyme-bound reaction complex as the enzyme turns over . For example, when the adenosine moieties in the bound structures of Mn.ATP and Mn.ADP are superposed, the cation is found to be displaced by approximately 2.4 A between the two conformations, suggesting that these structural changes may involve movements with significant amplitudes . Furthermore, the NMR-determined structures of enzyme-bound Mn.ATP and Mn.ADP are significantly different from those in published X-ray crystal structures of the enzyme-nucleotide complexes. Eur J Cell Biol, 1999 Oct, 78(10), 726 - 38 Unfolded protein response-induced BiP/Kar2p production protects cell growth against accumulation of misfolded protein aggregates in the yeast endoplasmic reticulum; Umebayashi K et al.; Overproduction of delta(pro), a mutated secretory proteinase derived from a filamentous fungus Rhizopus niveus, results in formation of gross aggregates (delta(pro) aggregates) in the yeast endoplasmic reticulum (ER) lumen, activation of the unfolded protein response (UPR) and ER membrane proliferation . To investigate the roles of the UPR against the delta(pro) aggregates, we constructed an IRE1-deleted ((delta)ire1) strain . In contrast to wild-type cells, (delta)ire1 cells ceased to grow several hours after the overproduction of (delta)pro . Two lines of evidence argued against the possibility that the growth defect was due to the inability to make extra ER membrane which accommodates the (delta)pro aggregates . First, by electron microscopy, ER membrane proliferation was observed in (delta)ire1 cells overproducing (delta)pro . Second, disruption of the OPI1 gene in the (delta)ire1 mutant, which is considered to derepress the activities of phospholipid-synthesizing enzymes, did not restore the growth upon the overproduction of (delta)pro . Instead, the growth was restored when an extra copy of the KAR2 gene, which encodes yeast BiP, was introduced, indicating that an increase in the amount of BiP is essential for cell growth when the (delta)pro aggregates accumulate in the ER . Since BiP is included in the insoluble (delta)pro aggregates, it is likely that the amount of free BiP in the ER lumen is insufficient without the UPR to fully exert its functions . Consistently, overproduction of (delta)pro impaired protein translocation and folding in (delta)ire1 cells but not in wild-type cells . The tunicamycin sensitivity of (de