Yeasts

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 (delta)ire1 cells was also suppressed by extra expression of KAR2, suggesting that BiP plays a principal role in protecting cell growth against misfolded proteins accumulated in the ER.

Genome Res, 1999 Nov, 9(11), 1128 - 34
A genetic strategy to eliminate self-activator baits prior to high-throughput yeast two-hybrid screens; Walhout AJ et al.; Large-scale sequencing projects have predicted high numbers of gene products for which no functional information is yet available . Hence, large-scale projects, such as gene knockouts, gene expression profiles, and protein-interaction mapping, are currently under way to initiate the understanding of the function of these gene products . The high-throughput strategies that are currently being developed to generate protein-interaction maps include automated versions of the yeast two-hybrid system . These strategies rely on the large-scale construction of DNA-binding domain/protein-of-interest hybrid constructs (DB-X baits) . An inherent problem of large-scale two-hybrid systems is that a high percentage of cloned sequences encode polypeptides that, when fused to DB, can activate transcription in the absence of any two-hybrid-interacting partner protein . Here, we describe and validate a genetic strategy that efficiently eliminates such self-activator baits prior to screening procedures . The strategy is based on a negative-growth selection and is compatible with high-throughput settings.

Genome Res, 1999 Nov, 9(11), 1059 - 73
A high-resolution physical map of human chromosome 21p using yeast artificial chromosomes; Wang SY et al.; The short arm of human chromosome 21 (21p) contains many different types of repetitive sequences and is highly homologous to the short arms of other acrocentric chromosomes . Owing to its repetitive nature and the lack of chromosome 21p-specific molecular markers, most physical maps of chromosome 21 exclude this region . We constructed a physical map of chromosome 21p using sequence tagged site (STS) content mapping of yeast artificial chromosomes (YACs) . To this end, 39 STSs located on the short arm or near the centromere of chromosome 21 were constructed, including four polymorphic simple tandem repeats (STRs) and two expressed sequence tags (ESTs) . Thirty YACs were selected from the St . Louis YAC library, the chromosome 21-enriched ICRF YAC library, and the CEPH YAC and megaYAC libraries . These were assembled in a YAC contig map ranging from the centromere to the rDNA gene cluster at 21p12 . The total size of the region covered by YACs is estimated between 2.9 and 5 Mb . The integrity of the YAC contig was confirmed by restriction enzyme fingerprinting and fluorescence in situ hybridization (FISH) . One gap with an estimated size of 400 kb remained near the telomeric end of the contig . This YAC contig map of the short arm of human chromosome 21 constitutes a basic framework for further structural and functional studies of chromosome 21p.

Genome Res, 1999 Nov, 9(11), 1040 - 7
Identification of target sites of the alpha2-Mcm1 repressor complex in the yeast genome; Zhong H et al.; The alpha2 and Mcm1 proteins bind DNA as a heterotetramer to repress transcription of cell-type-specific genes in the yeast Saccharomyces cerevisiae . Based on the DNA sequence requirements for binding by the alpha2-Mcm1 complex, we have searched the yeast genome for all potential alpha2-Mcm1 binding sites . Genes adjacent to the sites were examined for expression in the different cell mating types . These sites were further analyzed by cloning the sequences into a heterologous promoter and assaying for alpha2-Mcm1-dependent repression in vivo and DNA-binding affinity in vitro . Fifty-nine potential binding sites were identified in the search . Thirty-seven sites are located within or downstream of coding region of the gene . None of the sites assayed from this group are functional repressor sites in vivo or bound by the alpha2-Mcm1 complex in vitro . Among the remaining 22 sites, six are in the promoters of known alpha-specific genes and two other sites have an alpha2-Mcm1-dependent role in determining the direction of mating type switching . Among the remaining sequences, we have identified a functional site located in the promoter region of a previously uncharacterized gene, SCYJL170C . This site functions to repress transcription of a heterologous promoter and the alpha2-Mcm1 complex binds to the site in vitro . SCYJL170C is repressed by alpha2-Mcm1 in vivo and therefore using this method we have identified a new a-specific gene, which we call ASG7.

Int J Cancer, 1999 Dec 22, 84(6), 587 - 93
Prognostic significance of p53 mutation in breast cancer: frequent detection of non-missense mutations by yeast functional assay; Chappuis PO et al.; p53 status was tested in 180 patients with primary breast cancer using a yeast functional assay . Mutations were identified in 32% of cases . Only half were point missense mutations; the remainder were nonsense, insertion, deletion and splice site mutations . Twenty-two percent of mutations were located outside exons 5-8 . For a median follow-up of 88 months, survival analysis showed that p53 mutation conferred a worse prognosis in the whole population and the node-positive subgroup but not in node-negative patients . p53 status, tumour size >2 cm, axillary lymph node metastasis and high histological grade were major adverse risk factors in univariate analysis . Multivariate analysis of 153 patients for whom full data were available showed that p53 status contributed prognostic information when tumour size and lymph node status were taken into account but not when histological grade was included . p53 status thus contributes only limited new prognostic information in breast cancer when established prognostic factors are taken into account . Int . J . Cancer (Pred . Oncol.) 84:587-593, 1999 .

Mol Cell Biol, 1999 Dec, 19(12), 8559 - 69
Transcription factor UAF, expansion and contraction of ribosomal DNA (rDNA) repeats, and RNA polymerase switch in transcription of yeast rDNA; Oakes M et al.; Strains of the yeast Saccharomyces cerevisiae defective in transcription factor UAF give rise to variants able to grow by transcribing endogenous ribosomal DNA (rDNA) by RNA polymerase II (Pol II) . We have demonstrated that the switch to growth using the Pol II system consists of two steps: a mutational alteration in UAF and an expansion of chromosomal rDNA repeats . The first step, a single mutation in UAF, is sufficient to allow Pol II transcription of rDNA . In contrast to UAF mutations, mutations in Pol I or other Pol I transcription factors can not independently lead to Pol II transcription of rDNA, suggesting a specific role of UAF in preventing polymerase switch . The second step, expansion of chromosomal rDNA repeats to levels severalfold higher than the wild type, is required for efficient cell growth . Mutations in genes that affect recombination within the rDNA repeats, fob1 and sir2, decrease and increase, respectively, the frequency of switching to growth using Pol II, indicating that increased rDNA copy number is a cause rather than a consequence of the switch . Finally, we show that the switch to the Pol II system is accompanied by a striking alteration in the localization and morphology of the nucleolus . The altered state that uses Pol II for rDNA transcription is semistable and heritable through mitosis and meiosis . We discuss the significance of these observations in relation to the plasticity of rDNA tandem repeats and nucleolar structures as well as evolution of the Pol I machinery.

Mol Cell Biol, 1999 Dec, 19(12), 8344 - 52
PDK1 homologs activate the Pkc1-mitogen-activated protein kinase pathway in yeast; Inagaki M et al.; PDK1 (phosphoinositide-dependent kinase 1) is a mammalian growth factor-regulated serine/threonine kinase . Using a genetic selection based on a mutant form of the yeast MAP kinase kinase Ste7, we isolated a gene, PKH2, encoding a structurally and functionally conserved yeast homolog of PDK1 . Yeast cells lacking both PKH2 and PKH1, encoding another PDK1 homolog, were nonviable, indicating that Pkh1 and Pkh2 share an essential function . A temperature-sensitive mutant, pkh1(D398G) pkh2, was phenotypically similar to mutants defective in the Pkc1-mitogen-activated protein kinase (MAPK) pathway . Genetic epistasis analyses, the phosphorylation of Pkc1 by Pkh2 in vitro, and reduced Pkc1 activity in the pkh1(D398G) pkh2 mutant indicate that Pkh functions upstream of Pkc1 . The Pkh2 phosphorylation site in Pkc1 (Thr-983) is part of a conserved PDK1 target motif and essential for Pkc1 function . Thus, the yeast PDK1 homologs activate Pkc1 and the Pkc1-effector MAPK pathway.

Mol Cell Biol, 1999 Dec, 19(12), 8254 - 62
Ubiquitin metabolism affects cellular response to volatile anesthetics in yeast; Wolfe D et al.; To investigate the mechanism of action of volatile anesthetics, we are studying mutants of the yeast Saccharomyces cerevisiae that have altered sensitivity to isoflurane, a widely used clinical anesthetic . Several lines of evidence from these studies implicate a role for ubiquitin metabolism in cellular response to volatile anesthetics: (i) mutations in the ZZZ1 gene render cells resistant to isoflurane, and the ZZZ1 gene is identical to BUL1 (binds ubiquitin ligase), which appears to be involved in the ubiquitination pathway; (ii) ZZZ4, which we previously found is involved in anesthetic response, is identical to the DOA1/UFD3 gene, which was identified based on altered degradation of ubiquitinated proteins; (iii) analysis of zzz1Delta zzz4Delta double mutants suggests that these genes encode products involved in the same pathway for anesthetic response since the double mutant is no more resistant to anesthetic than either of the single mutant parents; (iv) ubiquitin ligase (MDP1/RSP5) mutants are altered in their response to isoflurane; and (v) mutants with decreased proteasome activity are resistant to isoflurane . The ZZZ1 and MDP1/RSP5 gene products appear to play important roles in determining effective anesthetic dose in yeast since increased levels of either gene increases isoflurane sensitivity whereas decreased activity decreases sensitivity . Like zzz4 strains, zzz1 mutants are resistant to all five volatile anesthetics tested, suggesting there are similarities in the mechanisms of action of a variety of volatile anesthetics in yeast and that ubiquitin metabolism affects response to all the agents examined.

Mol Cell Biol, 1999 Dec, 19(12), 8201 - 10
Dual role of the mitochondrial chaperone Mdj1p in inheritance of mitochondrial DNA in yeast; Duchniewicz M et al.; Mdj1p, a homolog of the bacterial DnaJ chaperone protein, plays an essential role in the biogenesis of functional mitochondria in the yeast Saccharomyces cerevisiae . We analyzed the role of Mdj1p in the inheritance of mitochondrial DNA (mtDNA) . Mitochondrial genomes were rapidly lost in a temperature-sensitive mdj1 mutant under nonpermissive conditions . The activity of mtDNA polymerase was severely reduced in the absence of functional Mdj1p at a nonpermissive temperature, demonstrating the dependence of the enzyme on Mdj1p . At a permissive temperature, the activity of mtDNA polymerase was not affected by the absence of Mdj1p . However, under these conditions, intact {rho(+)} genomes were rapidly converted to nonfunctional {rho(-)} genomes which were stably propagated in an mdj1 deletion strain . We propose that mtDNA polymerase depends on Mdj1p as a chaperone in order to acquire and/or maintain an active conformation at an elevated temperature . In addition, Mdj1p is required for the inheritance of intact mitochondrial genomes at a temperature supporting optimal growth; this second function appears to be unrelated to the function of Mdj1p in maintaining mtDNA polymerase activity.

Mol Cell Biol, 1999 Dec, 19(12), 8103 - 12
Evidence for a protein mutator in yeast: role of the Hsp70-related chaperone ssb in formation, stability, and toxicity of the {PSI} prion; Chernoff YO et al.; Propagation of the yeast protein-based non-Mendelian element {PSI}, a prion-like form of the release factor Sup35, was shown to be regulated by the interplay between chaperone proteins Hsp104 and Hsp70 . While overproduction of Hsp104 protein cures cells of {PSI}, overproduction of the Ssa1 protein of the Hsp70 family protects {PSI} from the curing effect of Hsp104 . Here we demonstrate that another protein of the Hsp70 family, Ssb, previously implicated in nascent polypeptide folding and protein turnover, exhibits effects on {PSI} which are opposite those of Ssa . Ssb overproduction increases, while Ssb depletion decreases, {PSI} curing by the overproduced Hsp104 . Both spontaneous {PSI} formation and {PSI} induction by overproduction of the homologous or heterologous Sup35 protein are increased significantly in the strain lacking Ssb . This is the first example when inactivation of an unrelated cellular protein facilitates prion formation . Ssb is therefore playing a role in protein-based inheritance, which is analogous to the role played by the products of mutator genes in nucleic acid-based inheritance . Ssb depletion also decreases toxicity of the overproduced Sup35 and causes extreme sensitivity to the {PSI}-curing chemical agent guanidine hydrochloride . Our data demonstrate that various members of the yeast Hsp70 family have diverged from each other in regard to their roles in prion propagation and suggest that Ssb could serve as a proofreading component of the enzymatic system, which prevents formation of prion aggregates.

Mol Cell Biol, 1999 Dec, 19(12), 8042 - 51
A subunit of yeast TFIIIC participates in the recruitment of TATA-binding protein; Deprez E et al.; TFIIIC plays a key role in nucleating the assembly of the initiation factor TFIIIB on class III genes . We have characterized an essential gene, TFC8, encoding the 60-kDa polypeptide, tau60, present in affinity-purified TFIIIC . Hemagglutinin-tagged variants of tau60 were found to be part of TFIIIC-tDNA complexes and to reside at least in part in the downstream DNA-binding domain tauB . Unexpectedly, the thermosensitive phenotype of N-terminally tagged tau60 was suppressed by overexpression of tau95, which belongs to the tauA domain, and by two TFIIIB components, TATA-binding protein (TBP) and B"/TFIIIB90 (but not by TFIIIB70) . Mutant TFIIIC was deficient in the activation of certain tRNA genes in vitro, and the transcription defect was selectively alleviated by increasing TBP concentration . Coimmunoprecipitation experiments support a direct interaction between TBP and tau60 . It is suggested that tau60 links tauA and tauB domains and participates in TFIIIB assembly via its interaction with TBP.

Mol Cell Biol, 1999 Dec, 19(12), 7983 - 94
The elm1 kinase functions in a mitotic signaling network in budding yeast; Sreenivasan A et al.; In budding yeast, the Clb2 mitotic cyclin initiates a signaling network that negatively regulates polar bud growth during mitosis . This signaling network appears to require the function of a Clb2-binding protein called Nap1, the Cdc42 GTPase, and two protein kinases called Gin4 and Cla4 . In this study, we demonstrate that the Elm1 kinase also plays a role in the control of bud growth during mitosis . Cells carrying a deletion of the ELM1 gene undergo a prolonged mitotic delay, fail to negatively regulate polar bud growth during mitosis, and show defects in septin organization . In addition, Elm1 is required in vivo for the proper regulation of both the Cla4 and Gin4 kinases and interacts genetically with Cla4, Gin4, and the mitotic cyclins . Previous studies have suggested that Elm1 may function to negatively regulate the Swe1 kinase . To further understand the functional relationship between Elm1 and Swe1, we have characterized the phenotype of Deltaelm1 Deltaswe1 cells . We found that Deltaelm1 Deltaswe1 cells are inviable at 37 degrees C and that a large proportion of Deltaelm1 Deltaswe1 cells grown at 30 degrees C contain multiple nuclei, suggesting severe defects in cytokinesis . In addition, we found that Elm1 is required for the normal hyperphosphorylation of Swe1 during mitosis . We propose a model in which the Elm1 kinase functions in a mitotic signaling network that controls events required for normal bud growth and cytokinesis, while the Swe1 kinase functions in a checkpoint pathway that delays nuclear division in response to defects in these events.

J Biol Chem, 1999 Nov 26, 274(48), 34396 - 402
Mutations in the yeast Hsp40 chaperone protein Ydj1 cause defects in Axl1 biogenesis and pro-a-factor processing; Meacham GC et al.; The heat shock protein (Hsp) 70/Hsp40 chaperone system plays an essential role in cell physiology, but few of its in vivo functions are known . We report that biogenesis of Axl1p, an insulinase-like endoprotease from yeast, is dependent upon the cytosolic Hsp40 protein Ydj1p . Axl1 is responsible for cleavage of the P2 processing intermediate of pro-a-factor, a mating pheromone, to its mature form . Mutant ydj1 strains exhibited a severe mating defect, which correlated with a 90% reduction in a-factor secretion . Reduced levels of a-factor export were caused by defects in the endoproteolytic processing of P2, which led to its intracellular accumulation . Defective P2 processing correlated with the reduction in the steady state level of active Axl1p . Two mechanisms were uncovered to explain why Axl1p activity was diminished in ydj1 strains . First, AXL1 mRNA levels were reduced ydj1 strains . Second, the half-life of newly synthesized Axl1p was greatly diminished in ydj1 strains . Collectively, these data indicate Ydj1p functions to promote AXL1 mRNA accumulation and in addition appears to facilitate the proper folding of nascent Axl1p . This study is the first to suggest a role for Ydj1p in RNA metabolism and identifies Axl1p as an in vivo substrate of the Hsp70/Ydj1p chaperone system.

J Biol Chem, 1999 Nov 26, 274(48), 33859 - 62
Mouse cyclin-dependent kinase (Cdk) 5 is a functional homologue of a yeast Cdk, pho85 kinase; Nishizawa M et al.; Mouse cyclin-dependent kinase (Cdk) 5 and yeast Pho85 kinase share similarities in structure as well as in the regulation of their activity . We found that mouse Cdk5 kinase produced in pho85Delta mutant cells could suppress some of pho85Delta mutant phenotypes including failure to grow on nonfermentable carbon sources, morphological defects, and growth defect caused by Pho4 or Clb2 overproduction . We also demonstrated that Cdk5 coimmunoprecipitated with Pho85-cyclins including Pcl1, Pcl2, Pcl6, Pcl9, and Pho80, and that the immunocomplex could phosphorylate Pho4, a native substrate of Pho85 kinase . Thus mouse Cdk5 is a functional homologue of yeast Pho85 kinase.

J Biol Chem, 1999 Nov 26, 274(48), 33839 - 42
Single strand DNA binding and annealing activities in the yeast recombination factor Rad59; Petukhova G et al.; Saccharomyces cerevisiae RAD59 gene is required for homologous recombination processes and normal level of resistance to ionizing radiation . To study the biochemical functions of Rad59, it was overproduced in yeast and purified to near homogeneity . Rad59 binds DNA, showing much higher affinity for ssDNA than dsDNA . Rad59 also anneals complementary DNA strands, and order of addition experiments indicate that maximal annealing efficiency is achieved when both complementary DNA strands are present upon addition of Rad59 . Thus, Rad59 resembles its homolog Rad52 in being able to bind ssDNA and anneal complementary DNA strands . However, unlike Rad52, DNA annealing by Rad59 is not accelerated by the ssDNA binding factor RPA . DNA binding and strand annealing are likely to be important for the biological functions of Rad59 in general recombination and in the single-strand annealing pathway of recombination.

Am J Physiol, 1999 Nov, 277(5 Pt 2), F734 - 41
Functional analysis of aquaporin-2 mutants associated with nephrogenic diabetes insipidus by yeast expression; Shinbo I et al.; Mutations of aquaporin-2 (AQP2) vasopressin water channel cause nephrogenic diabetes insipidus (NDI) . It has been suggested that impaired routing of AQP2 mutants to the plasma membrane causes the disease; however, no determinations have been made of mutation-induced alterations of AQP2 channel water permeability . To address this issue, a series of AQP2 mutants were expressed in yeast, and the osmotic water permeability (P(f)) of the isolated vesicles was measured . Wild-type and mutant AQP2 were expressed equally well in vesicles . P(f) of the vesicles containing wild-type AQP2 was 22 times greater than that of the control, which was sensitive to mercury and weakly dependent on the temperature . P(f) measurements and mercury inhibition examinations suggested that mutants L22V and P262L are fully functional, whereas mutants N68S, R187C, and S216P are partially functional . In contrast, mutants N123D, T125M, T126M, A147T, and C181W had very low water permeability . Our results suggest that the structure between the third and fifth hydrophilic loops is critical for the functional integrity of the AQP2 water channel and that disruption of AQP2 water permeability by mutations may cause NDI.

Methods Enzymol, 2000, 311, 223 - 32
Yeast sphingosine-1-phosphate phosphatases: assay, expression, deletion, purification, and cellular localization by GFP tagging; Mao C et al.; DHS-1-P phosphatases cloned from yeast represent novel lipid phosphatases, which were not thought to exist in yeast . Identification and characterization of YSR2 and YSR3 have demonstrated that the DHS-1-P phosphatase is an important mediator in the biosynthesis of sphingolipids and in the maintenance of the balance of signaling lipid molecules ceramide, sphingosine, and sphingosine-1-P . Methods introduced here for purification, activity assay, in vivo labeling, and cellular localization using GFP tagging are expected to facilitate our understanding of this enzyme.

J Cell Biol, 1999 Nov 15, 147(4), 791 - 808
The COOH-terminal domain of Myo2p, a yeast myosin V, has a direct role in secretory vesicle targeting; Schott D et al.; MYO2 encodes a type V myosin heavy chain needed for the targeting of vacuoles and secretory vesicles to the growing bud of yeast . Here we describe new myo2 alleles containing conditional lethal mutations in the COOH-terminal tail domain . Within 5 min of shifting to the restrictive temperature, the polarized distribution of secretory vesicles is abolished without affecting the distribution of actin or the mutant Myo2p, showing that the tail has a direct role in vesicle targeting . We also show that the actin cable-dependent translocation of Myo2p to growth sites does not require secretory vesicle cargo . Although a fusion protein containing the Myo2p tail also concentrates at growth sites, this accumulation depends on the polarized delivery of secretory vesicles, implying that the Myo2p tail binds to secretory vesicles . Most of the new mutations alter a region of the Myo2p tail conserved with vertebrate myosin Vs but divergent from Myo4p, the myosin V involved in mRNA transport, and genetic data suggest that the tail interacts with Smy1p, a kinesin homologue, and Sec4p, a vesicle-associated Rab protein . The data support a model in which the Myo2p tail tethers secretory vesicles, and the motor transports them down polarized actin cables to the site of exocytosis.

J Cell Biol, 1999 Nov 15, 147(4), 729 - 42
Sec34p, a protein required for vesicle tethering to the yeast Golgi apparatus, is in a complex with Sec35p; VanRheenen SM et al.; A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R . Duden, A . Eun, S . Hamamoto, P . Korn, R . Ram, and R . Schekman . 1996 . Genetics . 142:393-406) . We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes . The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)-associated protein Sly1p, Sly1-20p . Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p . This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering . sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis . Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an approximately 750-kD protein complex . Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well.

Plant Mol Biol, 1999 Sep, 41(1), 33 - 44
A constitutively expressed Myc-like gene involved in anthocyanin biosynthesis from Perilla frutescens: molecular characterization, heterologous expression in transgenic plants and transactivation in yeast cells; Gong ZZ et al.; The coordinate expression of anthocyanin biosynthetic genes in leaves and stems of a red forma of Perilla frutescens is presumably controlled by regulatory gene(s) . A Myc-like gene (Myc-rp) was isolated from a cDNA library prepared from the leaves of red P . frutescens, and its deduced amino acid sequence shows 64% identity with that of delila from snapdragon . The Myc-rp gene was expressed in leaves and roots of both red and green P . frutescens equally . Comparison of deduced amino acid sequence of Myc-rp with that of Myc-gp, the second allele isolated from a green forma of P . frutescens, indicates that the 132nd amino acid, alanine, existing in MYC-RP was changed to serine in MYC-GP . The heterologous expression of these two alleles of Myc-like gene in tobacco and tomato resulted in an increase of the anthocyanin contents in flowers of tobacco and vegetative tissues and flowers of tomato . However, the flowers of transgenic tobacco expressing the fragment with a partial deletion (encoding 1-115 amino acids deleted) of Myc(-gp gave no change in anthocyanin accumulation, but some morphological changes of the flower were observed . In yeast, the MYC-RP/GP and Delila protein exhibited transactivation activity on the GAL-1 promoter from yeast and the promoter of dihydroflavonol 4-reductase (DFR) gene from P . frutescens . A transactivation domain of MYC-RP/GP and Delila could be located in the region between the 193rd and the 420th amino acid of MYC-RP/GP proteins . Our data indicate that this Myc-like gene presumably functions in the regulation of anthocyanin biosynthesis similarly in different tissues of dicot plants.

Atherosclerosis, 1999 Dec, 147(2), 339 - 47
Macrophage specific overexpression of the human macrophage scavenger receptor in transgenic mice, using a 180-kb yeast artificial chromosome, leads to enhanced foam cell formation of isolated peritoneal macrophages; de Winther MP et al.; Macrophage scavenger receptors class A (MSR) are thought to play an important role in atherogenesis by mediating the unrestricted uptake of modified lipoproteins by macrophages in the vessel wall leading to foam cell formation . To investigate the in vivo role of the MSR in this process, a transgenic mouse model expressing both isoforms of the human MSR was generated . A 180-kb yeast artificial chromosome (YAC) containing the human MSR gene (MSR1) with 60- and 40-kb flanking sequence at the 5' and 3' end, respectively, was obtained by reducing the size of a 1050-kb YAC by homologous recombination . This 180-kb YAC was microinjected into mouse oocytes . In the resulting transgenic mice, high levels of mRNA for both type I and type II human MSR1 were detected in peritoneal macrophages and trace levels in other organs, known to contain macrophage-derived cells . Using an antibody against the human MSR, the Kupffer cells in the liver were shown to contain the MSR protein . In vivo clearance of acetyl-LDL was not changed in the MSR1-transgenic mice . However, in vitro studies using peritoneal macrophages from the transgenic mice showed a two-fold increased degradation of acetyl-LDL and cholesterolester accumulation concomitant with a four-fold increase in foam cell formation, as compared to wild-type macrophages . Thus, macrophage specific overexpression of the MSR may lead to increased foam cell formation, which is one of the initial and crucial steps in atherogenesis.

J Biol Chem, 1999 Nov 19, 274(47), 33462 - 8
Interaction between yeast RNA polymerase III and transcription factor TFIIIC via ABC10alpha and tau131 subunits; Dumay H et al.; Yeast TFIIIC mediates transcription of class III genes by promoting the assembly of a stable TFIIIB-DNA complex that is sufficient for RNA polymerase III recruitment and function . Unexpectedly, we found an interaction in vivo and in vitro between the TFIIIB-recruiting subunit of TFIIIC, tau131, and ABC10alpha, a small essential subunit common to the three forms of nuclear RNA polymerases . This interaction was mapped to the C-terminal region of ABC10alpha . A thermosensitive mutation in the C terminus region of ABC10alpha (rpc10-30) was found to be selectively suppressed by overexpression of a mutant form of tau131 (tau131-DeltaTPR2) that lacks the second TPR repeat . Remarkably, the rpc10-30 mutation weakened the ABC10alpha-tau131 interaction, and the suppressive mutation, tau131-DeltaTPR2 increased the interaction between the two proteins in the two-hybrid assay . These results point to the potential importance of a functional contact between TFIIIC and RNA polymerase III.

Mol Biol Cell, 1999 Nov, 10(11), 3971 - 8
Recognition of yeast mRNAs as "nonsense containing" leads to both inhibition of mRNA translation and mRNA degradation: implications for the control of mRNA decapping; Muhlrad D et al.; A critical step in the degradation of many eukaryotic mRNAs is a decapping reaction that exposes the transcript to 5' to 3' exonucleolytic degradation . The dual role of the cap structure as a target of mRNA degradation and as the site of assembly of translation initiation factors has led to the hypothesis that the rate of decapping would be specified by the status of the cap binding complex . This model makes the prediction that signals that promote mRNA decapping should also alter translation . To test this hypothesis, we examined the decapping triggered by premature termination codons to determine whether there is a down-regulation of translation when mRNAs were recognized as "nonsense containing." We constructed an mRNA containing a premature stop codon in which we could measure the levels of both the mRNA and the polypeptide encoded upstream of the premature stop codon . Using this system, we analyzed the effects of premature stop codons on the levels of protein being produced per mRNA . In addition, by using alterations either in cis or in trans that inactivate different steps in the recognition and degradation of nonsense-containing mRNAs, we demonstrated that the recognition of a nonsense codon led to a decrease in the translational efficiency of the mRNA . These observations argue that the signal from a premature termination codon impinges on the translation machinery and suggest that decapping is a consequence of the change in translational status of the mRNA.

Mol Biol Cell, 1999 Nov, 10(11), 3943 - 57
Specific sterols required for the internalization step of endocytosis in yeast; Munn AL et al.; Sterols are major components of the plasma membrane, but their functions in this membrane are not well understood . We isolated a mutant defective in the internalization step of endocytosis in a gene (ERG2) encoding a C-8 sterol isomerase that acts in the late part of the ergosterol biosynthetic pathway . In the absence of Erg2p, yeast cells accumulate sterols structurally different from ergosterol, which is the major sterol in wild-type yeast . To investigate the structural requirements of ergosterol for endocytosis in more detail, several erg mutants (erg2Delta, erg6Delta, and erg2Deltaerg6Delta) were made . Analysis of fluid phase and receptor-mediated endocytosis indicates that changes in the sterol composition lead to a defect in the internalization step . Vesicle formation and fusion along the secretory pathway were not strongly affected in the ergDelta mutants . The severity of the endocytic defect correlates with changes in sterol structure and with the abundance of specific sterols in the ergDelta mutants . Desaturation of the B ring of the sterol molecules is important for the internalization step . A single desaturation at C-8,9 was not sufficient to support internalization at 37 degrees C whereas two double bonds, either at C-5,6 and C-7,8 or at C-5,6 and C-8,9, allowed internalization.

Mol Biol Cell, 1999 Nov, 10(11), 3849 - 62
Two yeast La motif-containing proteins are RNA-binding proteins that associate with polyribosomes; Sobel SG et al.; We have characterized two Saccharomyces cerevisiae proteins, Sro9p and Slf1p, which contain a highly conserved motif found in all known La proteins . Originally described as an autoantigen in patients with rheumatic disease, the La protein binds to newly synthesized RNA polymerase III transcripts . In yeast, the La protein homologue Lhp1p is required for the normal pathway of tRNA maturation and also stabilizes newly synthesized U6 RNA . We show that deletions in both SRO9 and SLF1 are not synthetically lethal with a deletion in LHP1, indicating that the three proteins do not function in a single essential process . Indirect immunofluorescence microscopy reveals that although Lhp1p is primarily localized to the nucleus, Sro9p is cytoplasmic . We demonstrate that Sro9p and Slf1p are RNA-binding proteins that associate preferentially with translating ribosomes . Consistent with a role in translation, strains lacking either Sro9p or Slf1p are less sensitive than wild-type strains to certain protein synthesis inhibitors . Thus, Sro9p and Slf1p define a new and possibly evolutionarily conserved class of La motif-containing proteins that may function in the cytoplasm to modulate mRNA translation.

Mol Biol Cell, 1999 Nov, 10(11), 3689 - 703
Genetic and biochemical characterization of the yeast spo12 protein; Grether ME et al.; We have performed a genetic and biochemical analysis of the SPO12 gene, which regulates meiotic nuclear divisions in budding yeast . When sporulated, spo12 mutants undergo a single meiotic nuclear division most closely resembling meiosis II . We observed that Spo12 protein is localized to the nucleus during both meiotic divisions and that Clb1-Cdc28, Clb3-Cdc28, Clb4-Cdc28, and Clb5-Cdc28 kinase activities during meiosis were not affected by a spo12 mutation . Using two-hybrid analysis, we identified several genes, three of which are meiotically induced, that may code for proteins that interact with Spo12p . We also observed that two genes, BNS1 (Bypasses Need for Spo12p), which has homology to SPO12, and SPO13, whose mutant phenotype is like that of spo12, can partially suppress the meiotic defect of spo12 mutants when overexpressed . We found that Spo12p is also localized to the nucleus in vegetative cells and that its level peaks during G2/M . We observed that a spo12 mutation is synthetically lethal in vegetative cells with a mutation in HCT1, a gene necessary for cells to exit mitosis, suggesting that Spo12p may have a role in exit from mitosis.

Mol Biol Cell, 1999 Nov, 10(11), 3623 - 32
The cytoplasmic chaperone hsp104 is required for conformational repair of heat-denatured proteins in the yeast endoplasmic reticulum; Hanninen AL et al.; Severe heat stress causes protein denaturation in various cellular compartments . If Saccharomyces cerevisiae cells grown at 24 degrees C are preconditioned at 37 degrees C, proteins denatured by subsequent exposure to 48-50 degrees C can be renatured when the cells are allowed to recover at 24 degrees C . Conformational repair of vital proteins is essential for survival, because gene expression is transiently blocked after the thermal insult . Refolding of cytoplasmic proteins requires the Hsp104 chaperone, and refolding of lumenal endoplasmic reticulum (ER) proteins requires the Hsp70 homologue Lhs1p . We show here that conformational repair of heat-damaged glycoproteins in the ER of living yeast cells required functional Hsp104 . A heterologous enzyme and a number of natural yeast proteins, previously translocated and folded in the ER and thereafter denatured by severe heat stress, failed to be refolded to active and secretion-competent structures in the absence of Hsp104 or when an ATP-binding site of Hsp104 was mutated . During recovery at 24 degrees C, the misfolded proteins persisted in the ER, although the secretory apparatus was fully functional . Hsp104 appears to control conformational repair of heat-damaged proteins even beyond the ER membrane.

Nat Cell Biol, 1999 Sep, 1(5), 298 - 304
The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast; Bleazard W et al.; The dynamin-related GTPase Dnm1 controls mitochondrial morphology in yeast . Here we show that dnm1 mutations convert the mitochondrial compartment into a planar 'net' of interconnected tubules . We propose that this net morphology results from a defect in mitochondrial fission . Immunogold labelling localizes Dnm1 to the cytoplasmic face of constricted mitochondrial tubules that appear to be dividing and to the ends of mitochondrial tubules that appear to have recently completed division . The activity of Dnm1 is epistatic to that of Fzo1, a GTPase in the outer mitochondrial membrane that regulates mitochondrial fusion . dnm1 mutations prevent mitochondrial fragmentation in fzo1 mutant strains . These findings indicate that Dnm1 regulates mitochondrial fission, assembling on the cytoplasmic face of mitochondrial tubules at sites at which division will occur.

Nat Cell Biol, 1999 Aug, 1(4), 234 - 41
Yeast homologue of neuronal frequenin is a regulator of phosphatidylinositol-4-OH kinase; Hendricks KB et al.; In metazoans, certain calmodulin-related calcium-binding proteins (recoverins, neurocalcins and frequenins) are found at highest levels in excitable cells, but their physiological roles are largely uncharacterized . Here we show that Saccharomyces cerevisiae contains a frequenin homologue, Frq1, and that its target is Pik1, a phosphatidylinositol-4-OH kinase . Frq1 binds to a conserved sequence motif in Pik1 outside Pik1's catalytic domain and stimulates its activity in vitro . N-myristoylated Frq1 may also assist in Pik1 localization.

J Virol, 1999 Dec, 73(12), 10303 - 9
Brome mosaic virus RNA replication proteins 1a and 2a colocalize and 1a independently localizes on the yeast endoplasmic reticulum; Restrepo-Hartwig M et al.; The universal membrane association of positive-strand RNA virus RNA replication complexes is implicated in their function, but the intracellular membranes used vary among viruses . Brome mosaic virus (BMV) encodes two mutually interacting RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and the polymerase-like 2a protein . In cells from the natural plant hosts of BMV, 1a and 2a colocalize on the endoplasmic reticulum (ER) . 1a and 2a also direct BMV RNA replication and subgenomic mRNA synthesis in the yeast Saccharomyces cerevisiae, but whether the distribution of 1a, 2a, and active replication complexes in yeast duplicates that in plant cells has not been determined . For yeast expressing 1a and 2a and replicating BMV genomic RNA3, we used double-label confocal immunofluorescence to define the localization of 1a, 2a, and viral RNA and to explore the determinants of replication complex targeting . As in plant cells, 1a and 2a colocalized on and were retained on the yeast ER, with no detectable accumulation in the Golgi apparatus . 1a and 2a were distributed over most of the ER surface, with strongest accumulation on the perinuclear ER . In vivo labeling with bromo-UTP showed that the sites of 1a and 2a accumulation were the sites of nascent viral RNA synthesis . In situ hybridization showed that completed viral RNA products accumulated predominantly in the immediate vicinity of replication complexes but that some, possibly more mature cells also accumulated substantial viral RNA in the surrounding cytoplasm distal to replication complexes . Additionally, we find that 1a localizes to the ER when expressed in the absence of other viral factors . These results show that BMV RNA replication in yeast duplicates the normal localization of replication complexes, reveal the intracellular distribution of RNA replication products, and show that 1a is at least partly responsible for the ER localization and retention of the RNA replication complex.

Proc Natl Acad Sci U S A, 1999 Nov 9, 96(23), 13208 - 13
Participation of Bir1p, a member of the inhibitor of apoptosis family, in yeast chromosome segregation events; Yoon HJ et al.; Yeast two-hybrid and genetic interaction screens indicate that Bir1p, a yeast protein containing phylogenetically conserved antiapoptotic repeat domains called baculovirus inhibitor of apoptosis repeats (BIRs), is involved in chromosome segregation events . In the two-hybrid screen, Bir1p specifically interacts with Ndc10p, an essential component of the yeast kinetochore . Although Bir1p carries two BIR motifs in the N-terminal region, the C-terminal third of the protein is sufficient to provide strong interaction with Ndc10p and moderate interaction with Skp1p, another essential component of the yeast kinetochore . In addition, deletion of BIR1 is synthetically lethal with deletion of CBF1 or CTF19, genes specifying two other components of the yeast kinetochore . Yeast cells deleted of BIR1 have a chromosome-loss phenotype, which can be completely rescued by elevating NDC10 dosage . Furthermore, overexpression of either full-length or the C-terminal region of Bir1p can efficiently suppress the chromosome-loss phenotype of both bir1Delta null and skp1-4 mutants . Our data suggest that Bir1p participates in chromosome segregation events, either directly or via interaction with kinetochore proteins, and these effects are apparently not mediated by the BIR domains of Bir1p.

Genes Dev, 1999 Nov 1, 13(21), 2863 - 74
Essential functions of amino-terminal domains in the yeast telomerase catalytic subunit revealed by selection for viable mutants; Friedman KL et al.; Telomerase is a ribonucleoprotein complex that adds telomeric DNA repeats to the ends of most eukaryotic chromosomes . The reverse transcriptase subunit of telomerase (TERT) differs from retroviral reverse transcriptases in having a long basic amino-terminal extension . We made a large library containing random mutations in the amino terminus of the EST2 gene, which encodes the Saccharomyces cerevisiae TERT, and selected functional alleles by their ability to rescue senescence of telomerase-negative cells . Through analysis of 265 mutations, the amino terminus of Est2p was found to contain at least four essential regions . This domain structure was verified by a combination of deletion and alanine-block mutations . Mutations within two essential domains of the protein reduced RNA binding, suggesting that the amino terminus of Est2p makes important contacts with the intrinsic RNA component of telomerase . A mutant close to the amino terminus retained RNA binding and in vitro enzymatic activity but was defective in vivo, suggesting a role in interaction with other macromolecular components of telomerase.

FEBS Lett, 1999 Nov 5, 460(3), 491 - 4
The outer envelope protein OEP24 from pea chloroplasts can functionally replace the mitochondrial VDAC in yeast; Rohl T et al.; The chloroplastic outer envelope protein OEP24 from pea forms a high-conductance low specificity solute channel as shown by in vitro studies . In order to establish its function also in an in vivo-like system, the gene encoding OEP24 was transformed into a yeast strain which lacks the general mitochondria solute channel porin, also known as voltage-dependent anion channel (VDAC) . Transformation of the yeast VDAC(-) strain with the OEP24 gene resulted in the recovery of a phenotype indistinguishable from the wild-type . The OEP24 polypeptide is targeted to the mitochondrial outer membrane in this heterologous system . We conclude that OEP24 forms a solute channel in pea chloroplasts in planta.

Biochim Biophys Acta, 1999 Nov 9, 1461(1), 83 - 95
N-terminal chimeric constructs improve the expression of sarcoplasmic reticulum Ca(2+)-ATPase in yeast; Reis EM et al.; Wild-type and chimeric constructs comprising rabbit sarcoplasmic reticulum (SR) Ca(2+)-ATPase and the N-terminal cytoplasmic portion of yeast plasma membrane H(+)-ATPase were expressed in yeast under control of a heat-shock regulated promoter . The wild-type ATPase was found predominantly in endoplasmic reticulum (ER) membranes . Addition of the first 88 residues of H(+)-ATPase to the Ca(2+)-ATPase N-terminal end promoted a marked shift in the localization of chimeric H(+)/Ca(2+)-ATPase which accumulated in a light membrane fraction associated with yeast smooth ER . Furthermore, there was a three-fold increase in the overall level of expression of chimeric H(+)/Ca(2+)-ATPase . Similar results were obtained for a chimeric Ca(2+)-ATPase containing a hexahistidine sequence added to its N-terminal end . Both H(+)/Ca(2+)-ATPase and 6xHis-Ca(2+)-ATPase were functional as demonstrated by their ability to form a phosphorylated intermediate and undergo fast turnover . Conversely, a replacement chimera in which the N-terminal end of SR Ca(2+)-ATPase was replaced by the corresponding segment of H(+)-ATPase was not stably expressed in yeast membranes . These results indicate that the N-terminal segment of Ca(2+)-ATPase plays an important role in enzyme assembly and contains structural determinants necessary for ER retention of the ATPase.

Biochemistry, 1999 Nov 9, 38(45), 15017 - 24
The second stalk of the yeast ATP synthase complex: identification of subunits showing cross-links with known positions of subunit 4 (subunit b); Soubannier V et al.; A component of the stator of the yeast ATP synthase (subunit 4 or b) showed many cross-linked products with the homobifunctional reagent dithiobis{succinimidyl propionate}, which reacts with the amino group of lysine residues . The positions in subunit 4 that were involved in the cross-linkings were determined by using cysteine-generated mutants constructed by site-directed mutagenesis of ATP4 . Cross-linking experiments with the heterobifunctional reagent p-azidophenacyl bromide, which has a spacer arm of 9 A, were performed with mitochondria and crude Triton X-100 extracts containing the solubilized enzyme . Substitution of lysine residues by cysteine residues in the hydrophilic C-terminal part of subunit 4 allowed cross-links with subunit h from C98 and with subunit d from C141, C143, and C151 . OSCP was cross-linked from C174 and C209 . A cross-linked product, 4+beta, was also obtained from C174 . It is concluded that the C-terminus of subunit 4 is distant from the membrane surface and close to F(1) and OSCP . The N-terminal part of subunit 4 is close to subunit g, as demonstrated by the identification of a cross-linked product involving subunit g and the cysteine residues 7 or 14 of subunit 4.

Biochemistry, 1999 Nov 9, 38(45), 14860 - 7
Differences in structural dynamics of muscle and yeast actin accompany differences in functional interactions with myosin; Prochniewicz E et al.; We have used spectroscopic probes ErIA and IAEDANS attached to Cys374 to compare the structural dynamics of yeast actin filaments with that of muscle actin, to understand the structural basis of the less productive interaction of yeast actin with myosin . Time-resolved phosphorescence anisotropy (TPA) of ErIA and steady-state fluorescence of IAEDANS were measured . TPA indicated more rapid rotational motion and more restricted angular amplitude in yeast actin . The fluorescence spectrum was less intense and more red-shifted in yeast actin, suggesting more exposure of the probe to solvent . These results indicate that the two actins differ substantially in the conformational dynamics of the C-terminal region . Binding of myosin S1 induced significantly different spectroscopic changes in TPA and fluorescence of muscle and yeast actin . As a result, the spectroscopic differences between the two actins were decreased by the addition of S1 . These results suggest that yeast actin is less effective at activating myosin because of larger changes required in the structure of actin upon strong myosin binding . These results provide insight into the relationship between actomyosin dynamics and function, and they provide a useful framework for structure-function analysis of mutant yeast actin.

Br J Cancer, 1999 Nov, 81(5), 800 - 7
Differential expression of topoisomerase I and RAD52 protein in yeast reveals new facets of the mechanism of action of bisdioxopiperazine compounds; van Hille B et al.; A screening procedure which permits identification of compounds based on their activities against specific biological targets directly in a living organism, Saccharomyces cerevisiae, has been established as part of our new drug discovery programme . Use of this assay has provided the first direct evidence that TOP1 and RAD52 proteins are involved in the mode of action of bisdioxopiperazine ICRF compounds, which thus express a mode of action quite distinctive from the other known TOP2 inhibitors evaluated . The functional assay is based on a comparison of pairs of yeast differing in their phenotypes by specific traits: the expression or lack of expression of ectopic human DNA topoisomerase I, with or without that of the RAD52 gene . Amongst a series of anticancer agents, inhibitors of topoisomerase I (camptothecin) were identified as such in yeast expressing human topoisomerase I, whilst the presence or absence of RAD52 protein permitted the discrimination of compounds generating double-stranded DNA breaks, either directly (bleomycin) or involving DNA adduct formation (cisplatin), or indirectly with DNA damage mediated via inhibition of the topoisomerase II enzyme (etoposide) . Notably, however, both the RAD52 protein and the lack of TOP1 enzyme appeared implicated in the cytotoxic activities of the series of bisdioxopiperazine ICRF compounds tested . This functional assay in a living organism therefore appears to provide a valuable tool for probing distinctive and specific mode(s) of action of diverse anticancer agents.

Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1933 - 8
A natural chimeric yeast containing genetic material from three species; Groth C et al.; The Saccharomyces sp . CID1 isolate (CBS 8614) and several other Saccharomyces sensu stricto yeasts were analysed for their mitochondrial and nuclear genes . The data show that Saccharomyces sp . CID1, found so far only in one location in Europe, is a natural hybrid between three different Saccharomyces yeast species . Two of them, Saccharomyces cerevisiae-like and Saccharomyces bayanus-like, are ubiquitous and contributed parts of the nuclear genome; the third, Saccharomyces sp . IFO 1802-like, which has been found only in Japan, contributed the mitochondrial DNA molecule . These data suggest that the yeast cell is able to accommodate, express and propagate genetic material that originates from different species, and the very existence of the resulting natural hybrids indicates that such hybrids are well adapted to their habitats.

J Biol Chem, 1999 Nov 12, 274(46), 33025 - 34
Yeast mitochondrial protein, Nfs1p, coordinately regulates iron-sulfur cluster proteins, cellular iron uptake, and iron distribution; Li J et al.; Nfs1p is the yeast homolog of the bacterial proteins NifS and IscS, enzymes that release sulfur from cysteine for iron-sulfur cluster assembly . Here we show that the yeast mitochondrial protein Nfs1p regulates cellular and mitochondrial iron homeostasis . A strain of Saccharomyces cerevisiae, MA14, with a missense NFS1 allele (I191S) was isolated in a screen for altered iron-dependent gene regulation . This mutant exhibited constitutive up-regulation of the genes of the cellular iron uptake system, mediated through effects on the Aft1p iron-regulatory protein . Iron accumulating in the mutant cells was retained in the mitochondrial matrix while, at the same time, iron-sulfur proteins were deficient . In this work, the yeast protein was localized to mitochondria, and the gene was shown to be essential for viability . Furthermore, Nfs1p in the MA14 mutant was found to be markedly decreased, suggesting that this low protein level produced the observed regulatory effects . This hypothesis was confirmed by experiments in which expression of wild-type Nfs1p from a regulated galactose-induced promoter was turned off, leading to recapitulation of the iron regulatory phenotypes characteristic of the MA14 mutant . These phenotypes include decreases in iron-sulfur protein activities coordinated with increases in cellular iron uptake and iron distribution to mitochondria.

J Biol Chem, 1999 Nov 12, 274(46), 32869 - 74
Photoaffinity labeling of wild-type and mutant forms of the yeast V-ATPase A subunit by 2-azido-{(32)P}ADP; MacLeod KJ et al.; Molecular modeling studies have previously suggested the possible presence of four aromatic residues (Phe(452), Tyr(532), Tyr(535), and Phe(538)) near the adenine binding pocket of the catalytic site on the yeast V-ATPase A subunit (MacLeod, K . J., Vasilyeva, E., Baleja, J . D., and Forgac, M . (1998) J . Biol . Chem . 273, 150-156) . To test the proximity of these aromatic residues to the adenine ring, the yeast V-ATPase containing wild-type and mutant forms of the A subunit was reacted with 2-azido-{(32)P}ADP, a photoaffinity analog that stably modifies tyrosine but not phenylalanine residues . Mutant forms of the A subunit were constructed in which the two endogenous tyrosine residues were replaced with phenylalanine and in which a single tyrosine was introduced at each of the four positions . Strong ATP-protectable labeling of the A subunit was observed for the wild-type and the mutant containing tyrosine at 532, significant ATP-protectable labeling was observed for the mutants containing tyrosine at positions 452 and 538, and only very weak labeling was observed for the mutants containing tyrosine at 535 or in which all four residues were phenylalanine . These results suggest that Tyr(532) and possibly Phe(452) and Tyr(538) are in close proximity to the adenine ring of ATP bound to the A subunit . In addition, the effects of mutations at Phe(452), Tyr(532), Tyr(535), and Glu(286) on dissociation of the peripheral V(1) and integral V(0) domains both in vivo and in vitro were examined . The results suggest that in vivo dissociation requires catalytic activity while in vitro dissociation requires nucleotide binding to the catalytic site.

J Biol Chem, 1999 Nov 12, 274(46), 32704 - 11
A yeast Golgi E-type ATPase with an unusual membrane topology; Zhong X et al.; E-type ATPases are involved in many biological processes such as modulation of neural cell activity, prevention of intravascular thrombosis, and protein glycosylation . In this study, we show that a gene of Saccharomyces cerevisiae, identified by similarity to that of animal ectoapyrase CD39, codes for a new member of the E-type ATPase family (Apy1p) . Overexpression of Apy1p in yeast cells causes an increase in intracellular membrane-bound nucleoside di- and triphosphate hydrolase activity . The activity is highest with ADP as substrate and is stimulated similarly by Ca (2+), Mg(2+), and Mn(2+) . The results also indicate that Apy1p is an integral membrane protein located predominantly in the Golgi compartment . Sequence analysis reveals that Apy1p contains one large NH(2)-terminal hydrophilic apyrase domain, one COOH-terminal hydrophilic domain, and two hydrophobic stretches in the central region of the polypeptide . Although no signal sequence is found at the NH(2)-terminal portion of the protein and no NH(2)-terminal cleavage of the protein is observed, demonstrated by the detection of NH(2)-terminal tagged Apy1p, the NH(2)-terminal domain of Apylp is on the luminal side of the Golgi apparatus, and the COOH-terminal hydrophilic domain binds to the cytoplasmic face of the Golgi membrane . The second hydrophobic stretch of Apy1p is the transmembrane domain . These results indicate that Apylp is a type III transmembrane protein; however, the size of the Apy1p extracytoplasmic NH(2) terminus is much larger than those of other type III transmembrane proteins, suggesting that a novel translocation mechanism is utilized.

Biotechnol Bioeng, 1999 Dec 20, 65(6), 649 - 58
Protein transmission during Dean vortex microfiltration of yeast suspensions; Kluge T et al.; Substantially higher rates of protein and fluid volume transport for microfiltration of yeast suspensions were possible with improved hydrodynamics using centrifugal fluid instabilities called Dean vortices . Under constant permeate flux operation with suspended yeast cells, a helical module exhibited 19 times the filtration capacity of a linear module . For feed containing both BSA and beer yeast under constant transmembrane pressure with diafiltration, about twice as much protein (BSA and other proteins from cell lysis) was transported out of the feed by the helical module as compared with the linear module . The volumetric permeation flux improvements for the helical over the linear module ranged from 18 to 43% for yeast concentrations up to 4.5 dry wt % .

Nat Genet, 1999 Nov, 23(3), 367 - 71
Meiotic instability of human minisatellite CEB1 in yeast requires DNA double-strand breaks; Debrauwere H et al.; Minisatellites are tandemly repeated DNA sequences of 10-100-bp units . Some minisatellite loci are highly unstable in the human germ line, and structural analysis of mutant alleles has suggested that repeat instability results from a recombination-based process . To provide insights into the molecular mechanism of human minisatellite instability, we developed Saccharomyces cerevisiae strains carrying alleles of the most unstable human minisatellite locus, CEB1 (ref . 2) . We observed that CEB1 is destabilized in meiosis, resulting in a variety of intra- and inter-allelic gains or losses of repeat units, similar to rearrangements described in humans . Using mutations affecting the initiation of recombination (spo11) or mismatch repair (msh2 pms1 ), we demonstrate that meiotic destabilization depends on the initiation of homologous recombination at nearby DNA double-strand break (DSBs) sites and involves a 'rearranged heteroduplex' intermediate . Most of the human and yeast data can be explained and unified in the context of DSB repair models.

J Cell Sci, 1999 Nov, 112 ( Pt 22), 4135 - 42
Yeast ER-Golgi v-SNAREs Bos1p and Bet1p differ in steady-state localization and targeting; Ossipov D et al.; Vesicle specific SNAP receptors (v-SNAREs) Bos1p and Bet1p are involved in targeting of anterograde vesicles between the endoplasmic reticulum (ER) and early Golgi of Saccharomyces cerevisiae . To analyze factors that influence the targeting of these proteins, alpha-factor tagged versions of Bos1p and Bet1p were employed . The alpha-factor can be cleaved off by the Kex2p protease as soon as the hybrid proteins reach the late Golgi compartment . The data obtained by monitoring of Kex2p cleavage, by immunofluorescence microscopy and cell fractionation showed that Bos1-alpha and Bet1-alpha have different cellular localization and dynamics . Bos1-alpha is an ER protein, which recycles between the Golgi and the ER in COPI-dependent manner . Bet1-alpha is an early Golgi protein and it does not change its localization under conditions when other recycling Golgi proteins can be trapped in the ER.

Cell Stress Chaperones, 1999 Sep, 4(3), 171 - 6
Medicinal yeast extracts; Schlemm DJ et al.; Alcoholic extracts of bakers' yeast (Saccharomyces cerevisiae) have been used for over 60 years in over-the-counter medications for the treatment of hemorrhoids, burns, and wounds . Although previous studies suggested that small peptides were responsible for the medical observations, the peptides were never resolved into separate fractions and identified . In the present report, a protein fraction was prepared by RPC18 chromatography of the extract which enhances wound closure in both diabetic and non-diabetic littermates . The peptides are active in nanomolar amounts and are 600 times more active than the initial extract . SDS-PAGE and N-terminal amino acid sequencing identified 4 polypeptides in the extract . Three of the proteins were small molecular weight stress-associated proteins: copper, zinc superoxide-dismutase, ubiquitin, and glucose lipid regulated protein (HSP 12) . The fourth protein, acyl-CoA binding protein II, has not been previously associated with stress proteins.

Acta Biochim Pol, 1999, 46(2), 249 - 53
Deficiency in superoxide dismutases shortens life span of yeast cells; Wawryn J et al.; Deficiencies in superoxide dismutases (Cu,ZnSOD or Mn-SOD) strongly shorten the life span of yeast cells . The effects of these deficiencies are additive . In contrast, deficiencies in catalases do not influence life span . Our results confirm that free radical processes may be involved in aging.

Biochem Cell Biol, 1999, 77(4), 375 - 82
An allele of the yeast RPB7 gene, encoding an essential subunit of RNA polymerase II, reduces cellular resistance to the antitumor drug bleomycin; He CH et al.; Bleomycin is an antitumor drug that kills cells by introducing lesions in DNA . Thus, normal cells exposed to bleomycin must rely on efficient DNA repair mechanisms to survive . In the yeast Saccharomyces cerevisiae, the transcriptional activator Imp2 is required to fend off the toxic effects of bleomycin . However, it remains unclear whether Imp2 controls the expression of a protein that either repairs bleomycin-induced DNA lesions, or detoxifies the drug, and or both . To gain further insight into the mechanisms by which yeast cells mount a response towards bleomycin, we began to sequentially characterize the genetic defect in a collection of bleomycin-sensitive mutants that were previously isolated by mini-Tn3 transposon mutagenesis . A rescue plasmid designed to integrate at the site of the mini-Tn3 insertion was used to identify the defective gene in one of the mutant strains, HCY53, which was not allelic to IMP2 . We showed that in strain HCY53, the mini-Tn3 was inserted at the distal end of an essential gene RPB7, which encodes one of the two subunits, Rpb4-Rbp7, that forms a subcomplex with RNA polymerase II . Since rpb7 null mutants are nonviable, it would appear that the rpb7::mini-Tn3 allele produces a protein that retains partial biological function thus permitting cell viability, but which is unable to provide bleomycin resistance to strain HCY53 . The defective phenotype of strain HCY53 could be corrected by a plasmid bearing the entire RPB7 gene . Two dimensional gel analysis revealed that the expression of several proteins were diminished or absent in the rpb7::mini-Tn3 mutant when challenged with bleomycin . These results are in accord with our previous report that bleomycin resistance in yeast is controlled at the transcriptional level.

Hum Mol Genet, 1999 Nov, 8(12), 2285 - 92
Homologous DNA exchanges in humans can be explained by the yeast double-strand break repair model: a study of 17p11.2 rearrangements associated with CMT1A and HNPP; Lopes J et al.; Rearrangements in 17p11.2, responsible for the 1.5 Mb duplications and deletions associated, respectively, with autosomal dominant Charcot-Marie-Tooth type 1A disease (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are a suitable model for studying human recombination . Rearrangements in 17p11.2 are caused by unequal crossing-over between two homologous 24 kb sequences, the CMT1A-REPs, that flank the disease locus and occur in most cases within a 1.7 kb hotspot . We sequenced this hotspot in 28 de novo patients (25 CMT1A and three HNPP), in order to localize precisely, at the DNA sequence level, the crossing-overs . We show that some chimeric CMT1A-REPs in de novo patients (10/28) present conversion of DNA segments associated with the crossing-over . These rearrangements can be explained by the double-strand break (DSB) repair model described in yeast . Fine mapping of the de novo rearrangements provided evidence that the successive steps of this model, heteroduplex DNA formation, mismatch correction and gene conversion, occurred in patients . Furthermore, the model explains 17p11.2 recombinations between chromosome homologues as well as between sister chromatids . In addition, defective mismatch repair of the heteroduplex DNA, observed in two patients, resulted in two heterozygous chimeric CMT1A-REPs which can be explained, as in yeast, by post-meiotic segregation . This work supports the hypothesis that the DSB repair model of DNA exchange may apply universally from yeasts to humans.

Genetics, 1999 Nov, 153(3), 1271 - 83
Crossing over during Caenorhabditis elegans meiosis requires a conserved MutS-based pathway that is partially dispensable in budding yeast; Zalevsky J et al.; Formation of crossovers between homologous chromosomes during Caenorhabditis elegans meiosis requires the him-14 gene . Loss of him-14 function severely reduces crossing over, resulting in lack of chiasmata between homologs and consequent missegregation . Cytological analysis showing that homologs are paired and aligned in him-14 pachytene nuclei, together with temperature-shift experiments showing that him-14 functions during the pachytene stage, indicate that him-14 is not needed to establish pairing or synapsis and likely has a more direct role in crossover formation . him-14 encodes a germline-specific member of the MutS family of DNA mismatch repair (MMR) proteins . him-14 has no apparent role in MMR, but like its Saccharomyces cerevisiae ortholog MSH4, has a specialized role in promoting crossing over during meiosis . Despite this conservation, worms and yeast differ significantly in their reliance on this pathway: whereas worms use this pathway to generate most, if not all, crossovers, yeast still form 30-50% of their normal number of crossovers when this pathway is absent . This differential reliance may reflect differential stability of crossover-competent recombination intermediates, or alternatively, the presence of two different pathways for crossover formation in yeast, only one of which predominates during nematode meiosis . We discuss a model in which HIM-14 promotes crossing over by interfering with Holliday junction branch migration.

Genetics, 1999 Nov, 153(3), 1145 - 52
Catalytic roles of yeast GSK3beta/shaggy homolog Rim11p in meiotic activation; Malathi K et al.; In Saccharomyces cerevisiae, many meiotic genes are activated by a heteromeric transcription factor composed of Ime1p and Ume6p . Ime1p-Ume6p complex formation depends upon the protein kinase Rim11p, which interacts with and phosphorylates both Ime1p and Ume6p in vitro . Rim11p may promote complex formation through its phosphorylation of Ime1p and Ume6p or simply through its interaction with both proteins . Here, we characterize mutant Ime1p derivatives that interact with Rim11p but are not phosphorylated in vitro . These mutant proteins are also defective in interaction with Ume6p . These results argue that Ime1p must be phosphorylated to interact with Ume6p . Our genetic observations suggest that Ime1p tyrosine residues are among the Rim11p phosphoacceptors, and we find that Ime1p reacts with an anti-phosphotyrosine antibody . Ime1p and Rim11p have been thought to act only through Ume6p, but we find that Ime1p and Rim11p promote meiosis at a very low level in the absence of Ume6p . A nonphosphorylatable mutant Ime1p derivative promotes sporulation through this Ume6p-independent pathway, as does a mutant Rim11p derivative that fails to interact with Ime1p . Therefore, Ime1p and Rim11p have two genetically separable functions in the sporulation program . However, catalytic activity of Rim11p is required for sporulation in the presence or absence of Ume6p.

Biochemistry, 1999 Nov 2, 38(44), 14534 - 41
An N-terminal EF hand-like motif modulates ion transport by Pmr1, the yeast Golgi Ca(2+)/Mn(2+)-ATPase; Wei Y et al.; Pmr1, a novel member of the family of P-type ATPases, localizes to the Golgi compartment in yeast where it provides Ca(2+) and Mn(2+) for a variety of normal secretory processes . We have previously characterized Ca(2+) transport in isolated Golgi vesicles, and described an expression system for the analysis of Pmr1 mutants in a yeast strain devoid of background Ca(2+) pump activity {Sorin, A., Rosas, G., and Rao, R . (1997) J . Biol . Chem . 272, 9895-9901} . Here we show, using recombinant bacterial fusions, that an N-terminal EF hand-like motif in Pmr1 binds Ca(2+) . Increasing disruptions of this motif led to progressive loss of pump function; thus, the single point mutations D51A and D53A retained pump activity but with drastic reductions in the affinity for Ca(2+) transport, while the double mutant was largely unable to exit the endoplasmic reticulum . In-frame deletions of the Ca(2+)-binding motif resulted in complete loss of function . Interestingly, the single point mutations conferred differential affinities for transport of Ca(2+) and Mn(2+) ions . Further, the proteolytic stability of the catalytic ATP-binding domain is altered by the N-terminal mutations, suggesting an interaction between these two regions of polypeptide . These studies implicate the N-terminal domain of Pmr1 in the modulation of ion transport, and may help elucidate the role of N-terminal metal-binding sites of Cu(2+)-ATPases, defective in Wilson and Menkes disease.

Trends Biochem Sci, 1999 Nov, 24(11), 437 - 40
The economics of ribosome biosynthesis in yeast; Warner JR; In a rapidly growing yeast cell, 60% of total transcription is devoted to ribosomal RNA, and 50% of RNA polymerase II transcription and 90% of mRNA splicing are devoted to ribosomal proteins (RPs) . Coordinate regulation of the approximately 150 rRNA genes and 137 RP genes that make such prodigious use of resources is essential for the economy of the cell . This is entrusted to a number of signal transduction pathways that can abruptly induce or silence the ribosomal genes, leading to major implications for the expression of other genes as well.

J Biol Chem, 1999 Nov 5, 274(45), 32360 - 7
The yeast nucleoporin Nup2p is involved in nuclear export of importin alpha/Srp1p; Booth JW et al.; The importin alpha.beta heterodimer mediates nuclear import of proteins containing classical nuclear localization signals . After carrying its cargo into the nucleus, the importin dimer dissociates, and Srp1p (the yeast importin alpha subunit) is recycled to the cytoplasm in a complex with Cse1p and RanGTP . Nup2p is a yeast FXFG nucleoporin that contains a Ran-binding domain . We find that export of Srp1p from the nucleus is impaired in Deltanup2 mutants . Also, Srp1p fusion proteins accumulate at the nuclear rim in wild-type cells but accumulate in the nuclear interior in Deltanup2 cells . A deletion of NUP2 shows genetic interactions with mutants in SRP1 and PRP20, which encodes the Ran nucleotide exchange factor . Srp1p binds directly to an N-terminal domain of Nup2p . This region of Nup2p is sufficient to allow accumulation of an Srp1p fusion protein at the nuclear rim, but the C-terminal Ran-binding domain of Nup2p is required for efficient Srp1p export . Formation of the Srp1p.Cse1p . RanGTP export complex releases Srp1p from its binding site in Nup2p . We propose that Nup2p may act as a scaffold that facilitates formation of the Srp1p export complex.

Toxicol Appl Pharmacol, 1999 Nov 1, 160(3), 297 - 303
A human aryl hydrocarbon receptor signaling pathway constructed in yeast displays additive responses to ligand mixtures; Miller CA 3rd; An optimized signal transduction pathway that reproduces the response of human aryl hydrocarbon (Ah) receptor to ligands has been established in Saccharomyces cerevisiae . Ligand treatment induced a 50-fold increase in beta-galactosidase activity from a reporter plasmid in yeast engineered to express human Ah receptor and Ah nuclear translocator (Arnt) proteins . The archetypal Ah receptor ligand, 2,3,7,8-tetrachlorodibenzo(p)dioxin, activated Ah receptor and induced lacZ reporter activity at concentrations of >/=0.3 nM . Mixtures of halogenated and nonhalogenated Ah receptor ligands produced additive signaling responses in this yeast bioassay . These results were consistent with the existence of a common binding site and mechanism of ligand-mediated Ah receptor activation . Although yeast have no natural counterpart to the Ah receptor pathway, expression of human Ah receptor and Arnt under the appropriate conditions provides a functional model system for studying Ah receptor activation and signal transduction .

EMBO J, 1999 Nov 1, 18(21), 6134 - 45
An internal open reading frame triggers nonsense-mediated decay of the yeast SPT10 mRNA; Welch EM et al.; Yeast cells containing a temperature-sensitive mutation in the PRT1 gene were found to selectively stabilize mRNAs harboring early nonsense codons . The similarities between the mRNA decay phenotypes of prt1-1 cells and those lacking the nonsense-mediated mRNA decay (NMD) factor Upf1p led us to determine whether both types of mutations cause the accumulation of the same mRNAs . Differential display analysis and mRNA half-life measurements demonstrated that the HHF2 mRNA increased in abundance in prt1-1 and upf1Delta cells, but did not manifest a change in decay rate . In both mutant strains this increase was attributable to stabilization of the SPT10 transcript, an mRNA encoding a transcriptional regulator of HHF2 . Analyses of chimeric mRNAs used to identify the cis-acting basis for NMD of the SPT10 mRNA indicated that ribosomes scan beyond its initiator AUG and initiate at the next downstream AUG, resulting in premature translation termination . By searching a yeast database for transcripts with sequence features similar to those of the SPT10 mRNA, other transcripts that decay by the NMD pathway were identified . Our results demonstrate that mRNAs undergoing leaky scanning are a new class of endogenous NMD substrate, and suggest the existence of a novel cellular regulatory circuit.

EMBO J, 1999 Nov 1, 18(21), 5972 - 82
Eps1, a novel PDI-related protein involved in ER quality control in yeast; Wang Q et al.; PMA1 is an essential gene encoding the yeast plasma membrane {H(+)}ATPase . A pma1-D378N mutant has a dominant-negative effect on cell growth because both newly synthesized mutant and wild-type Pma1 molecules are retained and degraded in the endoplasmic reticulum (ER) . Like other substrates for ER-associated degradation, Pma1-D378N is stabilized in mutants defective in components of the ubiquitination machinery . A genetic selection was performed for eps (ER-retained pma1 suppressing) mutants in which the growth defect caused by the D378N allele is suppressed . In an eps1 mutant, both mutant and wild-type Pma1 molecules are allowed to travel to the plasma membrane; however, normal retention of resident ER proteins Shr3 and Kar2 is not perturbed . Eps1 is a novel membrane protein belonging to the protein disulfide isomerase (PDI) family, and Eps1 co-localizes with Pma1-D378N in the ER . In the absence of Pma1-D378N, ER export of wild-type Pma1 is not affected by eps1 deletion, but export of the plasma membrane protein Gas1 is delayed . Because Eps1 is required for retention and degradation of Pma1-D378N, we propose a model in which Eps1 acts as a novel membrane-bound chaperone in ER quality control.

EMBO J, 1999 Nov 1, 18(21), 5911 - 21
Identification of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes; Sciorra VA et al.; Phosphoinositides are both substrates for second messenger-generating enzymes and spatially localized membrane signals that mediate vital steps in signal transduction, cytoskeletal regulation and membrane trafficking . Phosphatidylcholine-specific phospholipase D (PLD) activity is stimulated by phosphoinositides, but the mechanism and physiological requirement for such stimulation to promote PLD-dependent cellular processes is not known . To address these issues, we have identified a site at which phosphoinositides interact with PLD and have assessed the role of this region in PLD function . This interacting motif contains critical basic amino acid residues that are required for stimulation of PLD activity by phosphoinositides . Although PLD alleles mutated at this site fail to bind to phosphoinositides in vitro, they are membrane-associated and properly localized within the cell but are inactive against cellular lipid substrates . Analogous mutations of this site in yeast PLD, Spo14p, result in enzymes that localize normally, but with catalytic activity that has dramatically reduced responsiveness to phosphoinositides . The level of responsiveness to phosphoinositides in vitro correlated with the ability of PLD to function in vivo . Taken together, these results provide the first evidence that phosphoinositide regulation of PLD activity observed in vitro is physiologically important in cellular processes in vivo including membrane trafficking and secretion.

FEBS Lett, 1999 Oct 29, 460(2), 275 - 9
Yeast ascospore wall assembly requires two chitin deacetylase isozymes; Christodoulidou A et al.; Chitin deacetylases are required for spore wall rigidity in Saccharomyces cerevisiae . Two chitin deacetylase genes (CDA1 and CDA2) have been identified in yeast . In this report we studied the biochemical properties of the chitin deacetylases encoded by CDA1 and CDA2 and we show how their elimination directly affects the ascospore wall assembly.

Environ Health Perspect, 1999 Nov, 107(11), 855 - 60
A yeast screen system for aromatase inhibitors and ligands for androgen receptor: yeast cells transformed with aromatase and androgen receptor; Mak P et al.; Endocrine disruptors are hormone mimics that modify hormonal action in humans and animals . It is thought that some endocrine disruptors modify estrogen and androgen action in humans and animals by suppressing aromatase activity . Aromatase cytochrome P450 is the key enzyme that converts C19 androgens to aromatic C18 estrogenic steroids . We have developed a novel aromatase inhibitor screening method that allows us to identify antiaromatase activity of various environmental chemicals . The screen was developed by coexpressing the human aromatase and the mouse androgen receptor in yeast cells, which carry the androgen-responsive ss-galactosidase reporter plasmid . Functional expression of aromatase in yeast has been demonstrated using the {3H}-water release assay with intact cells as well as with yeast microsomes . The aromatase activity could be blocked by known aromatase inhibitors such as aminoglutethimide (AG) . Yeast-produced androgen receptors were able to transactivate a yeast basal promoter linked to an androgen-responsive element in response to androgens . The resultant triple yeast transformant responded to the treatment of testosterone, androstenedione, or 5 alpha-dihydrotestosterone (5 alpha-DHT) . In the absence of the aromatase inhibitor AG, transcriptional activation was observed only for the nonaromatizable androgen 5 alpha-DHT . However, the two aromatizable androgens (testosterone and androstenedione) induced the reporter activity in the presence of AG . Using this yeast-based assay, we confirmed that two flavones, chrysin and alpha-naphtholflavone, are inhibitors of aromatase . Thus, this yeast system allows us to develop a high-throughput screening method, without using radioactive substrate, to identify aromatase inhibitors as well as new ligands (nonaromatizable androgen mimics) for the androgen receptors . In addition, this screening method also allows us to distinguish nonandrogenic aromatase inhibitors from inhibitors with androgenic activity . This yeast screening method will be useful to screen environmental chemicals for their antiaromatase activity and for their interaction with androgen receptor.

Nucleic Acids Res, 1999 Nov 15, 27(22), 4451 - 6
Pyrophosphate mediates the effect of certain tRNA mutations on aminoacylation of yeast tRNA(Phe); Khvorova A et al.; The influence of pyrophosphate hydrolysis by inorganic pyrophosphatase on homologous aminoacylation of different yeast tRNA(Phe) mutants was studied . The addition of pyrophosphatase significantly improved the aminoacylation efficiency of tRNA(Phe) structural mutants as well as the mutant with substitution at position 20, while having no effect on the charge of wild-type tRNA(Phe) . Aminoacylation of tRNA(Phe) anticodon and discriminator base (N(73)) mutants was not affected by pyrophosphatase . Activation of wild-type tRNA(Phe) transcript aminoacylation by inorganic pyrophosphatase was observed only at low Mg(2+) concentrations due to distortion of the tRNA(Phe) structure under these conditions . Our results demonstrate that pyrophosphate dissociation becomes a rate-limiting step of the reaction in yeast phenylalanyl-tRNA synthetase catalyzed aminoacylation of tRNA(Phe) variants with altered tertiary structure . A possible mechanism of pyrophosphate-mediated inhibition of tRNA mutants aminoacylation is discussed.

Curr Biol, 1999 Oct 7, 9(19), 1123 - 6
Yeast Ku protein plays a direct role in telomeric silencing and counteracts inhibition by rif proteins; Mishra K et al.; Yku70p/Yku80p, the yeast Ku protein homologue, is a DNA end-binding heterodimer involved in non-homologous end joining . It also binds to telomeres, where it plays an important role in the maintenance of telomeric DNA structure {1} {2} {3} {4} {5} . Ku protein, together with Rap1p, a telomeric DNA (TG(1-3) repeat)-binding protein, is also required to initiate transcriptional silencing, or telomere-position effect (TPE) . Here, we provide evidence for a direct role of Ku in TPE, which is most likely to be in either the recruitment or activation of Sir4 protein at the telomere . Surprisingly, however, the essential role of Ku in TPE is to overcome the inhibitory effect of two Rap1p-interacting proteins, Rif1p and Rif2p, both of which also play an important role in telomere length regulation {6} {7} . Previous studies showed that Rif and Sir proteins compete for binding to the carboxyl terminus of Rap1p {7} {8} {9} . In the absence of this competition, for example, when RIF genes are mutated, Ku is no longer necessary for TPE, whereas the Rap1p carboxyl terminus is still absolutely required . We show that Rif1p is localized to telomeres, indicating that its inhibitory effect on TPE is direct . Our data implicate a role for Ku in the competition between Sir and Rif proteins for access to the telomeric array of Rap1p molecules, which results in a balance between telomeric silencing and telomere length control.

Curr Biol, 1999 Oct 7, 9(19), 1111 - 4
A retention mechanism for distribution of mitochondria during cell division in budding yeast; Yang HC et al.; Mitochondria are indispensable for normal eukaryotic cell function . As they cannot be synthesized de novo and are self-replicating, mitochondria must be transferred from mother to daughter cells . Studies in the budding yeast Saccharomyces cerevisiae indicate that mitochondria enter the bud immediately after bud emergence, interact with the actin cytoskeleton for linear, polarized movement of mitochondria from mother to bud, but are equally distributed among mother and daughter cells {1} {2} {3} . It is not clear how the mother cell maintains its own supply of mitochondria . Here, we found that mother cells retain mitochondria by immobilization of some mitochondria in the 'retention zone', the base of the mother cell distal to the bud . Retention requires the actin cytoskeleton as mitochondria colocalized with actin cables in the retention zone, and mutations that perturb actin dynamics or actin-mitochondrial interactions produced retention defects . Our results support the model that equal distribution of mitochondria during cell division is a consequence of two actin-dependent processes: movement of some mitochondria into the daughter bud and immobilization of others in the mother cell.

Curr Biol, 1999 Oct 7, 9(19), 1085 - 94
The yeast CDC9 gene encodes both a nuclear and a mitochondrial form of DNA ligase I; Willer M et al.; BACKGROUND: The yeast CDC9 gene encodes a DNA ligase I activity required during nuclear DNA replication to ligate the Okazaki fragments formed when the lagging DNA strand is synthesised . The only other DNA ligase predicted from the yeast genome sequence, DNL4/LIG4, is specifically involved in a non-homologous DNA end-joining reaction . What then is the source of the DNA ligase activity required for replication of the yeast mitochondrial genome? RESULTS: We report that CDC9 encodes two distinct polypeptides expressed from consecutive in-frame AUG codons . Translational initiation at these two sites gives rise to polypeptides differing by a 23 residue amino-terminal extension, which corresponds to a functional mitochondrial pre-sequence sufficient to direct import into yeast mitochondria . Initiation at the first AUG codon results in a 755 amino-acid polypeptide that is imported into mitochondria, whereupon the pre-sequence is proteolytically removed to yield the mature mitochondrial form of Cdc9p . Initiation at the second AUG codon produces a 732 amino-acid polypeptide, which is localised to the nucleus . Cells expressing only the nuclear isoform were found to be specifically defective in the maintenance of the mitochondrial genome . CONCLUSIONS: CDC9 encodes two distinct forms of DNA ligase I . The first is targeted to the mitochondrion and is required for propagation and maintenance of mitochondrial DNA, the second localises to the nucleus and is sufficient for the essential cell-division function associated with this gene.

Cell Struct Funct, 1999 Aug, 24(4), 197 - 208
Mutation of the yeast epsilon-COP gene ANU2 causes abnormal nuclear morphology and defects in intracellular vesicular transport; Kimata Y et al.; Previously we reported an original method of visualizing the shape of yeast nuclei by the expression of green fluorescent protein (GFP)-tagged Xenopus nucleoplasmin in Saccharomyces cerevisiae . To identify components that determine nuclear structure, we searched for mutants exhibiting abnormal nuclear morphology from a collection of temperature-sensitive yeast strains expressing GFP-tagged nucleoplasmin . Four anu mutant strains (anu1-1, 2-1, 3-1 and 4-1; ANU=abnormal nuclear morphology) that exhibited strikingly different nuclear morphologies at the restrictive temperature as compared to the wild-type were isolated . The nuclei of these mutants were irregularly shaped and often consisted of multiple lobes . ANU1, 3 and 4 were found to encode known factors Sec24p, Sec13p and Sec18p, respectively, all of which are involved in the formation or fusion of intracellular membrane vesicles of protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus . On the other hand, ANU2 was not well characterized . Disruption of ANU2 (delta anu2) was not lethal but conferred temperature-sensitivity for growth . Electron microscopic analysis of anu2-1 cells revealed not only the abnormal nuclear morphology but also excessive accumulation of ER membranes . In addition, both anu2-1 and delta anu2 cells were defective in protein transport between the ER and the Golgi, suggesting that Anu2p has an important role in vesicular transport in the early secretory pathway . Here we show that ANU2 encodes a 34 kDa polypeptide, which shares a 20% sequence identity with the mammalian epsilon-COP . Our results suggest that Anu2p is the yeast homologue of mammalian epsilon-COP and the abrupt accumulation of the ER membrane caused by a blockage of the early protein transport pathway leads to alteration of nuclear morphology of the budding yeast cells.

Appl Microbiol Biotechnol, 1999 Sep, 52(3), 311 - 20
Yeast cells as tools for target-oriented screening; Munder T et al.; Information about biomolecular interaction networks is crucial for understanding cellular functions and the development of disease processes . Many diseases are known to be based on aberrations of DNA sequences encoding proteins with key functions in the cellular metabolism . Alterations in the respective proteins often lead to disturbances in biomolecular interactions caused by unbalanced stoichiometries, and thus result in alterations of molecule fluxes, cell architecture and signalling pathways . Drug discovery programmes have been designed to find promising chemical lead structures with the help of target-oriented bioassay systems . These are, in most cases, based upon the interaction of small molecules to specific macromolecular targets in vivo or in vitro, as exemplified by enzyme assays or small-ligand-based receptor systems . In addition, interactions between large biomolecules, such as proteins or nucleic acids, offer a huge arsenal of potential drug targets that can be addressed by small chemical compounds . This latter approach is gaining considerable attention because many potential target structures are becoming available through genomic research . Funnelling these new targets into high-throughput screening programs represents a major challenge for today's pharmaceutical research . An important outcome of the ongoing genome projects is the fact that the basic cellular structures, pathways and signalling principles show a high degree of conservation . Model organisms that are easily approachable by genetic, biochemical and physiological means can thus play an important role in the design of target-oriented screening systems . They offer the possibility to express individual proteins, nucleic acids or even more complex aggregates of biomolecules such as protein-interaction networks or transcription-initiation complexes, which can be addressed by small effector molecules in vivo . Combining these targets with biological signalling systems is an attractive way of creating robust cellular assay systems.

J Biol Chem, 1999 Oct 29, 274(44), 31531 - 42
The human homologue of the yeast proteins Skb1 and Hsl7p interacts with Jak kinases and contains protein methyltransferase activity; Pollack BP et al.; To expand our understanding of the role of Jak2 in cellular signaling, we used the yeast two-hybrid system to identify Jak2-interacting proteins . One of the clones identified represents a human homologue of the Schizosaccaromyces pombe Shk1 kinase-binding protein 1, Skb1, and the protein encoded by the Saccharomyces cerevisiae HSL7 (histone synthetic lethal 7) gene . Since no functional motifs or biochemical activities for this protein or its homologues had been reported, we sought to determine a biochemical function for this human protein . We demonstrate that this protein is a protein methyltransferase . This protein, designated JBP1 (Jak-binding protein 1), and its homologues contain motifs conserved among protein methyltransferases . JBP1 can be cross-linked to radiolabeled S-adenosylmethionine (AdoMet) and methylates histones (H2A and H4) and myelin basic protein . Mutants containing substitutions within a conserved region likely to be involved in AdoMet binding exhibit little or no activity . We mapped the JBP1 gene to chromosome 14q11.2-21 . In addition, JBP1 co-immunoprecipitates with several other proteins, which serve as methyl group acceptors and which may represent physiological targets of this methyltransferase . Messenger RNA for JBP1 is widely expressed in human tissues . We have also identified and sequenced a homologue of JBP1 in Drosophila melanogaster . This report provides a clue to the biochemical function for this conserved protein and suggests that protein methyltransferases may have a role in cellular signaling.

Biochemistry, 1999 Oct 5, 38(40), 13138 - 46
Yeast protein farnesyltransferase . pKas of peptide substrates bound as zinc thiolates; Rozema DB et al.; Protein farnesyltransferase (PFTase) is a zinc metalloenzyme that catalyzes the posttranslational alkylation of the cysteine in C-terminal -Ca(1)a(2)X sequences by a 15-carbon farnesyl residue, where C is cysteine, a(1) and a(2) are normally aliphatic amino acids, and X is an amino acid that specifies selectivity for the farnesyl moiety . Formation of a Zn(2+) thiolate in the PFTase . peptide complex was detected by the appearance of an absorbance at 236 nm (epsilon = 15 000 M(-1) cm(-1)), which was dependent on the concentration of peptide, in a UV difference spectrum in a solution of PFTase and the peptide substrate RTRCVIA . We developed a fluorescence anisotropy binding assay to measure the dissociation constants as a function of pH for peptide analogues by appending a 2',7'-difluorofluorescein to their N-terminus . The electron-withdrawing fluorine atoms allowed us to measure peptide binding down to pH 5.5 without having to correct for the changes in fluorescence intensity that accompany protonation of the fluorophore . Measurements of the pK(a)s for thiol groups in free and bound peptide indicate that peptide binding is accompanied by formation of a zinc thiolate and that binding to PFTase lowers the pK of the peptide thiol by 3 units . In similar studies with the betaY310F mutant, the pK(a) of the thiol moiety was lowered by 2 units upon binding, indicating that the hydroxyl group in the conserved tyrosine helps stabilize the bound thiolate.

Biochemistry, 1999 Oct 5, 38(40), 13042 - 54
The single amino acid changes in the yeast mitochondrial S4 ribosomal protein cause temperature-sensitive defect in the accumulation of mitochondrial 15S rRNA; Biswas TK et al.; Four different mutant alleles of a nuclear gene (MNA6), which lose mt 15S rRNA at nonpermissive temperature (36 degrees C), were previously generated by EMS mutagenesis of Saccharomyces cerevisiae . To understand the biochemical basis for the loss of 15S rRNA in these mutants, the wild-type and mutant alleles of the MNA6 gene were isolated and characterized . The DNA sequencing of the cloned MNA6 gene revealed that it has an open reading frame specifying a 486 amino acid polypeptide, which appears to be a yeast mt homologue of the S4 r-protein family . The large size of this yeast S4 homologue is due to a nonhomologous long C-terminal extension . The MNA6 gene also appeared to be identical to the previously isolated yeast NAM9 gene . The in vitro expression under coupled transcription-translation reaction conditions followed by mt import demonstrated that MNA6 indeed encodes a approximately 56 kDa protein targeted to the mitochondria . We have also demonstrated by Western blot analysis using anti-Mna6p antibody that Mna6p is associated with the small subunit of mitoribosomes . The sequence analysis of the four mutant mna6 alleles revealed that Leu(109) --> Phe, Arg(111) --> Lys, Pro(424) --> Leu, or Pro(438) --> Leu amino acid substitution in Mna6p causes temperature-dependent loss of the 15S rRNA . These mutations do not affect the mitochondrial import or accumulation of Mna6p . Rather the evidence points to an inability of mutant Mna6p to be assembled into the mitoribosomes of cells grown at 36 degrees C.

Can J Microbiol, 1999 Aug, 45(8), 686 - 90
Trichosporon guehoae sp.nov., an anamorphic basidiomycetous yeast; Middelhoven WJ et al.; A morphological and physiological description of an anamorphic basidiomycetous yeast species, named Trichosporon guehoae (CBS 8521T), is presented . The ability to assimilate several aliphatic and aromatic compounds as sole source of carbon and energy is reported . The phylogenetic position within the genus, based on nuclear base sequencing of the D1/D2 region of the large subunit of rDNA is discussed.

Methods, 1999 Oct, 19(2), 330 - 7
Applications of the yeast two-hybrid system; McAlister-Henn L et al.; In recent years, the yeast two-hybrid system has become the method of choice for detection and analysis of protein-protein interactions in an in vivo context . This system, which capitalizes on the significant genetic history and ease of protocols for manipulation of Saccharomyces cerevisiae, is accessible to most laboratories and is applicable to the pursuit of a large variety of experimental goals . To date, the two-hybrid system has seen widespread application for identification of interaction partners by screening methods using a particular protein of interest as a "bait." Large-scale ventures are also in progress, for example, a cataloging of interactions among the cellular proteins in yeast . However, this method also has tremendous potential for more focused analyses of specific proteins and should become more routine as an alternative or adjunct approach for many structure-function investigations .

Genes Cells, 1999 Sep, 4(9), 517 - 27
Overproduction of elongation factor 1alpha, an essential translational component, causes aberrant cell morphology by affecting the control of growth polarity in fission yeast; Suda M et al.; BACKGROUND: Elongation factor 1alpha (EF1alpha), an essential component of the eukaryotic translational machinery, has been shown to possess various biochemical and biological activities, including F-actin-binding and -bundling, microtubule- severing, and the activity of making fibroblasts highly susceptible to transformation . However, our understanding of the biological significance of EF1alpha with respect to these various biochemical or biological activities remains limited . Here we report the identification of EF1alpha-encoding genes as genes whose over-expression causes aberrant cell morphology in fission yeast . RESULTS: Overproduction of EF1alpha caused aberrant cell morphology-elliptic, curved or branched-and growth defects in yeast cells at high temperatures . EF1alpha-overproducing cells showed a supersensitivity to the actin inhibitor cytochalasin D and to the tubulin inhibitor thiabendazole . Genetic analyses using cdc mutants suggested that excess EF1alpha disturbed the establishment and the maintenance of growth polarity in the G1 phase by pre- venting the localization of F-actin to the polarized growing site and the organization of microtubules . Results from DNase I column chromatography indicated that EF1alpha was bound to G-actin . Indeed, the fission yeast actin was immunoprecipitated along with EF1alpha . Moreover, the temperature sensitivity caused by the overproduction of EF1alpha was restored by co-overproduction of actin . CONCLUSIONS: Fission yeast EF1alpha has the ability to alter the cell morphology of yeast by affecting the control of actin and microtubule cytoskeletons.

Genes Cells, 1999 Aug, 4(8), 445 - 63
Fission yeast APC/cyclosome subunits, Cut20/Apc4 and Cut23/Apc8, in regulating metaphase-anaphase progression and cellular stress responses; Yamashita YM et al.; BACKGROUND: The 20S cyclosome/APC complex promotes metaphase-anaphase transition by ubiquitinating its specific substrates such as mitotic cyclins and anaphase inhibitor Cut2/Pds1/securin . The complex has been shown to contain more than 10 proteins in budding yeast and frog . In fission yeast, however, only five (Cut4, Cut9, Nuc2, Apc10, Hcn1) have been identified . RESULTS: More than five hundred temperature-sensitive mutants were screened for identifying those defective in mitotic anaphase . Fifty-five showed the cut (cell untimely torn) phenotype or metaphase-arrest phenotypes, 27 of them locating at new loci . Their extracts were run in sucrose gradient centrifugation, and four showed alterations in the sedimentation profiles . The gene products of cut20+ and cut23+ were thus identified . Phenotypes of cut20-100 mutant highly resemble cut4-533 in many ways: they are hypersensitive to canavanine and CdCl2, and suppressed by PKA-inactivating regulators, cAMP-dependent phosphodiesterase and PKA regulatory subunits . Cut20 interacts closely with Cut4 in the assembly process of cyclosome . But cut20 mutant differs from cut4, as a novel gene stw1+ suppresses cut20 mutant but not cut4 . cut23-194 mutant cells are sterile and blocked at metaphase, but does not show sensitivity to the stress and cAMP . TPR repeat-containing Cut23 may not be the stable component of APC/cyclosome, and its level significantly fluctuates during cell cycle . Cut23 may be ubiquitinated and degraded in a cell cycle dependent fashion . CONCLUSIONS: We identified two new subunits of fission yeast cyclosome/APC complex . Our observations indicate that cyclosome components are divided into several subgroups with distinctly different roles.

FEBS Lett, 1999 Oct 15, 459(3), 458 - 62
The yeast Rgd1p is a GTPase activating protein of the Rho3 and rho4 proteins; Doignon F et al.; The RGD1 gene, identified during sequencing of the Saccharomyces cerevisiae genome, encodes a protein with a Rho-GTPase activating protein (GAP) domain at the carboxy-terminal end . The Rgd1 protein showed two-hybrid interactions with the activated forms of Rho2p, Rho3p and Rho4p . Using in vitro assays, we demonstrated that Rgd1p stimulated the GTPase activity of both Rho3p and Rho4p; no stimulation was observed on Rho2p . In addition, the rho3Deltargd1Delta double mutant exhibited a dramatic growth defect compared to the single mutants, suggesting that Rgd1p has a GAP activity in vivo . The present study allowed the identification of the first GAP of Rho3p and Rho4p.

Chromosoma, 1999 Sep, 108(5), 278 - 90
Tel2p, a regulator of yeast telomeric length in vivo, binds to single-stranded telomeric DNA in vitro; Kota RS et al.; The telomeres of the yeast Saccharomyces cerevisiae consist of a duplex region of TG(1-3) repeats that acquire a single-stranded 3' extension of the TG(1-3) strand at the end of S-phase . The length of these repeats is kept within a defined range by regulators such as the TEL2-encoded protein (Tel2p) . Here we show that Tel2p can specifically bind to single-stranded TG(1-3) . Tel2p binding produced several shifted bands; however, only the slowest migrating band contained Tel2p . Methylation protection and interference experiments as well as gel shift experiments using inosine-containing probes indicated that the faster migrating bands resulted from Tel2p-mediated formation of DNA secondary structures held together by G-G interactions . Tel2p bound to single-stranded substrates that were at least 19 bases in length and contained 14 bases of TG(1-3), and also to double-stranded/single-stranded hybrid substrates with a 3' TG(1-3) overhang . Tel2p binding to a hybrid substrate with a 24 base single-stranded TG(1-3) extension also produced a band characteristic of G-G-mediated secondary structures . These data suggest that Tel2p could regulate telomeric length by binding to the 3' single-stranded TG(1-3) extension present at yeast telomeres.

Curr Microbiol, 1999 Dec, 39(6), 369 - 0373
Transcriptional and structural study of a region of two convergent overlapping yeast genes; Puig S et al.; The exceptionally close packing of many yeast genes and other chromosomal elements raises the question of how those elements are functionally insulated . All published work shows that natural insulators are very effective, but transcriptional interference (TI) occurs if they are mutated or if their natural context is altered . Mechanisms to avoid TI are poorly understood, but are thought to involve an interplay of cis sequences and trans factors in a chromatin context . We have studied the case of two convergent closely packed ORFs (56 bp of separation) in chromosome IX of Saccharomyces cerevisiae . mRNAs from POT1 and YIL161w overlap by up to 115 nt . Convergent transcription causes a small but noticeable negative effect on the level of POT1 mRNA and nucleosome displacement in the intergenic region . This suggests for the first time that some TI could occur in convergently transcribed yeast genes, even in a natural chromosomal context.

J Cell Biol, 1999 Oct 18, 147(2), 435 - 46
Formation process of autophagosome is traced with Apg8/Aut7p in yeast; Kirisako T et al.; We characterized Apg8/Aut7p essential for autophagy in yeast . Apg8p was transcriptionally upregulated in response to starvation and mostly existed as a protein bound to membrane under both growing and starvation conditions . Immunofluorescence microscopy revealed that the intracellular localization of Apg8p changed drastically after shift to starvation . Apg8p resided on unidentified tiny dot structures dispersed in the cytoplasm at growing phase . During starvation, it was localized on large punctate structures, some of which were confirmed to be autophagosomes and autophagic bodies by immuno-EM . Besides these structures, we found that Apg8p was enriched on isolation membranes and in electron less-dense regions, which should contain Apg8p-localized membrane- or lipid-containing structures . These structures would represent intermediate structures during autophagosome formation . Here, we also showed that microtubule does not play an essential role in the autophagy in yeast . The result does not match with the previously proposed role of Apg8/Aut7p, delivery of autophagosome to the vacuole along microtubule . Moreover, it is revealed that autophagosome formation is severely impaired in the apg8 null mutant . Apg8p would play an important role in the autophagosome formation.

J Mol Biol, 1999 Oct 1, 292(4), 827 - 36
Molecular modeling of the three-dimensional architecture of the RNA component of yeast RNase MRP; Schmitt ME; RNase mitochondrial RNA processing (MRP) is a ribonucleoprotein endoribonuclease that is involved in RNA processing events in both the nucleus and the mitochondria . The MRP RNA is both structurally and evolutionarily related to RNase P, the ribonucleoprotein endoribonuclease that processes the 5'-end of tRNAs . Previous analysis of the RNase MRP RNA by phylogenetic analysis and chemical modification has revealed strikingly conserved secondary structural elements in all characterized RNase MRP RNAs . Utilizing successive constraint modeling and energy minimization I derived a three-dimensional model of the yeast RNase MRP RNA . The final model predicts several notable features . First, the enzyme appears to contain two separate structural domains, one that is highly conserved among all MRP and P RNAs and a second that is only conserved in MRP RNAs . Second, nearly all of the highly conserved nucleotides cluster in the first domain around a long-range interaction (LRI-I) . This LRI-I is characterized by a ubiquitous uridine base, which points into a cleft between these two structural domains generating a potential active site for RNA cleavage . Third, helices III and IV (the yeast equivalent of the To-binding site) model as a long extended helix . This region is believed to be the binding site of shared proteins between RNase P and RNase MRP and would provide a necessary platform for binding these seven proteins . Indeed, several residues conserved between the yeast MRP and P RNAs cluster in the central region of these helixes . Lastly, characterized mutations in the MRP RNA localize in the model based on their severity . Those mutations with little or no effect on the activity of the enzyme localize to the periphery of the model, while the most severe mutations localize to the central portion of the molecule where they would be predicted to cause large structural defects . Press.

FEMS Microbiol Rev, 1999 Oct, 23(5), 537 - 50
Phosphorylation of the yeast ribosomal stalk . Functional effects and enzymes involved in the process; Ballesta JP et al.; The ribosomal stalk is directly involved 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 the P0 protein . All these proteins are found phosphorylated in eukaryotic organisms, and previous in vitro data suggested this modification was involved in the activity of this structure . Results from mutational studies have shown that phosphorylation takes place at a serine residue close to the carboxy end of the P proteins . Modification of this serine residue does not affect the formation of the stalk and the activity of the ribosome in standard conditions but induces an osmoregulation-related phenotype at 37 degrees C . The phosphorylatable serine is part of a consensus casein kinase II phosphorylation site . However, although CKII seems to be responsible for part of the stalk phosphorylation in vivo, it is probably not the only enzyme in the cell able to perform this modification . Five protein kinases, RAPI, RAPII and RAPIII, in addition to the previously reported CKII and PK60 kinases, are able to phosphorylate the stalk proteins . A comparison of the five enzymes shows differences among them that suggest some specificity regarding the phosphorylation of the four yeast acidic proteins . It has been found that some typical effectors of the PKC kinase stimulate the in vitro phosphorylation of the stalk proteins . All the data suggest that although phosphorylation is not involved in the interaction of the acidic P proteins with the ribosome, it can affect the ribosome activity and might participate in a possible ribosome regulatory mechanism.

J Protein Chem, 1999 Jul, 18(5), 523 - 32
Antibody immunodiversity: a study on the marked specificity difference between two anti-yeast iso-1 cytochrome c monoclonal antibodies whose epitopes are closely related; Rizzo P et al.; Anti-yeast iso-1 cytochrome c (cyt . c) monoclonal antibodies 2-96-12 and 4-74-6 have closely related epitopes (antigenic determinants) . However, while the specificity of 4-74-6 is stringent, 2-96-12 cross-reacts with many evolutionarily related cytochromes c . Such a marked difference in specificity of antibodies with overlapping epitopes may represent unique antibody immunodiversity . Thus, we constructed Fv fragment models consisting of the variable domains of the heavy and light chains of 2-96-12 and 4-74-6 and that of another anti-iso-1 cyt . c as a control to gain insight into the origin of this difference in specificity . Our models show that 4-74-6 and 2-96-12 contain five and two aromatic side chains, respectively, in or near the central area of the antigen-combining site . The side chains of Arg95H (heavy chain) in 2-96-12 and Arg91L (light chain) in 4-74-6 project toward the central area of the combining site in our model . Antigen docking to our Fv models, combined with previous immunological studies, suggests that iso-1 cyt . c Asp60 may interact with Arg95H in 2-96-12 and Arg91L in 4-74-6 and that both epitopes of 2-96-12 and 4-74-7 may include iso-1 cyt . c Leu58, Asp60, Asn62, and Asn63 . The effect of the Arg95H to Lys mutation on the antigen binding is also in accord with our model . The difference in specificity may be partly explained by a greater degree of conformational flexibility in and around the central area of the combining site in 2-96-12 compared to 4-74-6 due to differences in aromatic side chain packing.

Biotechniques, 1999 Oct, 27(4), 772 - 4, 776-7
Budding yeast as a screening tool for discovery of nucleoside analogs for use in HSV-1 TK suicide-gene therapy; Wera S et al.; We present a fast, convenient and inexpensive method that allows the automated, large-scale screening of chemical libraries for compounds that are converted by the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) into inhibitors of cell growth . The method is based on the use of budding yeast (Saccharomyces cerevisiae) transformed with the HSV-1 TK gene on a multicopy plasmid . Eight nucleoside analogs (acyclovir, ganciclovir, penciclovir, lobucavir, brivudin, sorivudine, IVDU and ara-T), for which the cytostatic action against mammalian cells expressing the HSV-1 TK gene has been well documented, were studied for their inhibitory effect on the growth of yeast expressing the viral TK . These nucleoside analogs had little or no inhibitory effect on the growth of yeasts transformed with the empty vector, but inhibited to a significant extent the growth of yeast expressing the viral TK . Use of HSV-1 TK-expressing yeast allows quick screening in multi-well plate format for compounds with potential use in HSV-1 TK suicide gene therapy . The method may also be used as a tool to selectively suppress or arrest the growth of one population of yeast out of mixed yeast cell cultures.

Oncol Rep, 1999 Nov-Dec, 6(6), 1267 - 71
Yeast functional assay of the p53 gene status in 11 cell lines and 26 surgical specimens of human hepatocellular carcinoma; Gao C et al.; The structural abnormalities of the p53 gene have frequently been detected in hepatocellular carcinomas (HCCs) . To ascertain whether or not functional inactivation of this gene also occurs in HCCs, the transactivation of p53 gene products in 11 HCC cell lines maintained in our laboratory and 26 HCC surgical specimens was examined by yeast functional assay (functional analysis of separated alleles in yeast: FASAY), which determines the functional status of the individual p53 alleles . The p53 gene product was inactivated in 8 of 11 (72.7%) HCC cell lines and in 12 of 26 (46.2%) HCC surgical specimens . The inactivation frequency of the gene was 37.5%, 36.4%, and 71.4% in well, moderately, and poorly differentiated HCCs, respectively . In HCC surgical specimens larger than 5 cm in diameter, the inactivation frequency was 83.3% while in those smaller than 2 . 5 cm, it was 14.3% . These results show that functional inactivation of p53 gene products often occurs in HCCs and that the inactivation frequency of the gene in HCCs is well correlated with differentiation degree and tumor size, suggesting that the inactivation of p53 gene products plays a role in progression from well to poorly differentiated HCC.

Mol Cell Biol, 1999 Nov, 19(11), 7857 - 69
Mutagenesis of SNM1, which encodes a protein component of the yeast RNase MRP, reveals a role for this ribonucleoprotein endoribonuclease in plasmid segregation; Cai T et al.; RNase MRP is a ribonucleoprotein endoribonuclease that has been shown to have roles in both mitochondrial DNA replication and nuclear 5.8S rRNA processing . SNM1 encodes an essential 22.5-kDa protein that is a component of yeast RNase MRP . It is an RNA binding protein that binds the MRP RNA specifically . This 198-amino-acid protein can be divided into three structural regions: a potential leucine zipper near the amino terminus, a binuclear zinc cluster in the middle region, and a serine- and lysine-rich region near the carboxy terminus . We have performed PCR mutagenesis of the SNM1 gene to produce 17 mutants that have a conditional phenotype for growth at different temperatures . Yeast strains carrying any of these mutations as the only copy of snm1 display an rRNA processing defect identical to that in MRP RNA mutants . We have characterized these mutant proteins for RNase MRP function by examining 5.8S rRNA processing, MRP RNA binding in vivo, and the stability of the RNase MRP RNA . The results indicate two separate functional domains of the protein, one responsible for binding the MRP RNA and a second that promotes substrate cleavage . The Snm1 protein appears not to be required for the stability of the MRP RNA, but very low levels of the protein are required for processing of the 5.8S rRNA . Surprisingly, a large number of conditional mutations that resulted from nonsense and frameshift mutations throughout the coding regions were identified . The most severe of these was a frameshift at amino acid 7 . These mutations were found to be undergoing translational suppression, resulting in a small amount of full-length Snm1 protein . This small amount of Snm1 protein was sufficient to maintain enough RNase MRP activity to support viability . Translational suppression was accomplished in two ways . First, CEN plasmid missegregation leads to plasmid amplification, which in turn leads to SNM1 mRNA overexpression . Translational suppression of a small amount of the superabundant SNM1 mRNA results in sufficient Snm1 protein to support viability . CEN plasmid missegregation is believed to be the result of a prolonged telophase arrest that has been recently identified in RNase MRP mutants . Either the SNM1 gene is inherently susceptible to translational suppression or extremely small amounts of Snm1 protein are sufficient to maintain essential levels of MRP activity.

Mol Cell Biol, 1999 Nov, 19(11), 7816 - 27
HDAC4, a human histone deacetylase related to yeast HDA1, is a transcriptional corepressor; Wang AH et al.; Histone acetylation plays an important role in regulating chromatin structure and thus gene expression . Here we describe the functional characterization of HDAC4, a human histone deacetylase whose C-terminal part displays significant sequence similarity to the deacetylase domain of yeast HDA1 . HDAC4 is expressed in various adult human tissues, and its gene is located at chromosome band 2q37 . HDAC4 possesses histone deacetylase activity intrinsic to its C-terminal domain . When tethered to a promoter, HDAC4 represses transcription through two independent repression domains, with repression domain 1 consisting of the N-terminal 208 residues and repression domain 2 containing the deacetylase domain . Through a small region located at its N-terminal domain, HDAC4 interacts with the MADS-box transcription factor MEF2C . Furthermore, HDAC4 and MEF2C individually upregulate but together downmodulate c-jun promoter activity . These results suggest that HDAC4 interacts with transcription factors such as MEF2C to negatively regulate gene expression.

Mol Cell Biol, 1999 Nov, 19(11), 7751 - 8
The glycine-phenylalanine-rich region determines the specificity of the yeast Hsp40 Sis1; Yan W et al.; Hsp40s are ubiquitous, conserved proteins which function with molecular chaperones of the Hsp70 class . Sis1 is an essential Hsp40 of the cytosol of Saccharomyces cerevisiae, thought to be required for initiation of translation . We carried out a genetic analysis to determine the regions of Sis1 required to perform its key function(s) . A C-terminal truncation of Sis1, removing 231 amino acids but retaining the N-terminal 121 amino acids encompassing the J domain and the glycine-phenylalanine-rich (G-F) region, was able to rescue the inviability of a Deltasis1 strain . The yeast cytosol contains other Hsp40s, including Ydj1 . To determine which regions carried the critical determinants of Sis1 function, we constructed chimeric genes containing portions of SIS1 and YDJ1 . A chimera containing the J domain of Sis1 and the G-F region of Ydj1 could not rescue the lethality of the Deltasis1 strain . However, a chimera with the J domain of Ydj1 and the G/F region of Sis1 could rescue the strain's lethality, indicating that the G-F region is a unique region required for the essential function of Sis1 . However, a J domain is also required, as mutants expected to cause a disruption of the interaction of the J domain with Hsp70 are inviable . We conclude that the G-F region, previously thought only to be a linker or spacer region between the J domain and C-terminal regions of Hsp40s, is a critical determinant of Sis1 function.

Mol Cell Biol, 1999 Nov, 19(11), 7733 - 40
Pta1, a component of yeast CF II, is required for both cleavage and poly(A) addition of mRNA precursor; Zhao J et al.; CF II, a factor required for cleavage of the 3' ends of mRNA precursor in Saccharomyces cerevisiae, has been shown to contain four polypeptides . The three largest subunits, Cft1/Yhh1, Cft2/Ydh1, and Brr5/Ysh1, are homologs of the three largest subunits of mammalian cleavage-polyadenylation specificity factor (CPSF), an activity needed for both cleavage and poly(A) addition . In this report, we show by protein sequencing and immunoreactivity that the fourth subunit of CF II is Pta1, an essential 90-kDa protein originally implicated in tRNA splicing . Yth1, the yeast homolog of the CPSF 30-kDa subunit, is not detected in this complex . Extracts prepared from pta1 mutant strains are impaired in the cleavage and the poly(A) addition of both GAL7 and CYC1 substrates and exhibit little processing activity even after prolonged incubation . However, activity is efficiently rescued by the addition of purified CF II to the defective extracts . Extract from a strain with a mutation in the CF IA subunit Rna14 also restored processing, but extract from a brr5-1 strain did not . The amounts of Pta1 and other CF II subunits are reduced in pta1 strains, suggesting that levels of the subunits may be coordinately regulated . Coimmunoprecipitation experiments indicate that the CF II in extract can be found in a stable complex containing Pap1, CF II, and the Fip1 and Yth1 subunits of polyadenylation factor I . While purified CF II does not appear to retain the association with these other factors, this larger complex may be the form recruited onto pre-mRNA in vivo . The involvement of Pta1 in both steps of mRNA 3'-end formation supports the conclusion that CF II is the functional homolog of CPSF.

Mol Cell Biol, 1999 Nov, 19(11), 7705 - 11
Dual lipid modification of the yeast ggamma subunit Ste18p determines membrane localization of Gbetagamma; Hirschman JE et al.; The pheromone response in the yeast Saccharomyces cerevisiae is mediated by a heterotrimeric G protein . The Gbetagamma subunit (a complex of Ste4p and Ste18p) is associated with both internal and plasma membranes, and a portion is not stably associated with either membrane fraction . Like Ras, Ste18p contains a farnesyl-directing CaaX box motif (C-terminal residues 107 to 110) and a cysteine residue (Cys 106) that is a potential site for palmitoylation . Mutant Ste18p containing serine at position 106 (mutation ste18-C106S) migrated more rapidly than wild-type Ste18p during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . The electrophoretic mobility of wild-type Ste18p (but not the mutant Ste18p) was sensitive to hydroxylamine treatment, consistent with palmitoyl modification at Cys 106 . Furthermore, immunoprecipitation of the Gbetagamma complex from cells cultured in the presence of {(3)H}palmitic acid resulted in two radioactive species on nonreducing SDS-PAGE gels, with molecular weights corresponding to Ggamma and Gbetagamma . Substitution of serine for either Cys 107 or Cys 106 resulted in the failure of Gbetagamma to associate with membranes . The Cys 107 substitution also resulted in reduced steady-state accumulation of Ste18p, suggesting that the stability of Ste18p requires modification at Cys 107 . All of the mutant forms of Ste18p formed complexes with Ste4p, as assessed by coimmunoprecipitation . We conclude that tight membrane attachment of the wild-type Gbetagamma depends on palmitoylation at Cys 106 and prenylation at Cys 107 of Ste18p.

Mol Cell Biol, 1999 Nov, 19(11), 7661 - 71
Control of meiotic recombination and gene expression in yeast by a simple repetitive DNA sequence that excludes nucleosomes; Kirkpatrick DT et al.; Tandem repeats of the pentanucleotide 5'-CCGNN (where N indicates any base) were previously shown to exclude nucleosomes in vitro (Y . -H . Wang and J . D . Griffith, Proc . Natl . Acad . Sci . USA 93:8863-8867, 1996) . To determine the in vivo effects of these sequences, we replaced the upstream regulatory sequences of the HIS4 gene of Saccharomyces cerevisiae with either 12 or 48 tandem copies of CCGNN . Both tracts activated HIS4 transcription . We found that (CCGNN)(12) tracts elevated meiotic recombination (hot spot activity), whereas the (CCGNN)(48) tract repressed recombination (cold spot activity) . In addition, a "pure" tract of (CCGAT)(12) activated both transcription and meiotic recombination . We suggest that the cold spot activity of the (CCGNN)(48) tract is related to the phenomenon of the suppressive interactions of adjacent hot spots previously described in yeast (Q.-Q . Fan, F . Xu, and T . D . Petes, Mol . Cell . Biol . 15:1679-1688, 1995; Q.-Q . Fan, F . Xu, M . A . White, and T . D . Petes, Genetics 145:661-670, 1997; T.-C . Wu and M . Lichten, Genetics 140:55-66, 1995; L . Xu and N . Kleckner, EMBO J . 16:5115-5128, 1995).

Mol Cell Biol, 1999 Nov, 19(11), 7651 - 60
Differential regulation of the cell wall integrity mitogen-activated protein kinase pathway in budding yeast by the protein tyrosine phosphatases Ptp2 and Ptp3; Mattison CP et al.; Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity and protein tyrosine phosphatases (PTPs) in yeasts . In Saccharomyces cerevisiae, two PTPs, Ptp2 and Ptp3, inactivate the MAPKs, Hog1 and Fus3, with different specificities . To further examine the functions and substrate specificities of Ptp2 and Ptp3, we tested whether they could inactivate a third MAPK, Mpk1, in the cell wall integrity pathway . In vivo and in vitro evidence indicates that both PTPs inactivate Mpk1, but Ptp2 is the more effective negative regulator . Multicopy expression of PTP2, but not PTP3, suppressed growth defects due to the MEK kinase mutation, BCK1-20, and the MEK mutation, MKK1-386, that hyperactivate this pathway . In addition, deletion of PTP2, but not PTP3, exacerbated growth defects due to MKK1-386 . Other evidence supported a role for Ptp3 in this pathway . Expression of MKK1-386 was lethal in the ptp2Delta ptp3Delta strain but not in either single PTP deletion strain . In addition, the ptp2Delta ptp3Delta strain showed higher levels of heat stress-induced Mpk1-phosphotyrosine than the wild-type strain or strains lacking either PTP . The PTPs also showed differences in vitro . Ptp2 was more efficient than Ptp3 at binding and dephosphorylating Mpk1 . Another factor that may contribute to the greater effectiveness of Ptp2 is its subcellular localization . Ptp2 is predominantly nuclear whereas Ptp3 is cytoplasmic, suggesting that active Mpk1 is present in the nucleus . Last, PTP2 but not PTP3 transcript increased in response to heat shock in a Mpk1-dependent manner, suggesting that Ptp2 acts in a negative feedback loop to inactivate Mpk1.

Mol Cell Biol, 1999 Nov, 19(11), 7529 - 38
The yeast ras/cyclic AMP pathway induces invasive growth by suppressing the cellular stress response; Stanhill A et al.; Haploid yeast cells are capable of invading agar when grown on rich media . Cells of the Sigma1278b genetic background manifest this property, whereas other laboratory strains are incapable of invasive growth . We show that disruption of the RAS2 gene in the Sigma1278b background significantly reduces invasive growth but that expression of a constitutively active Ras2p (Ras2(Val19)p) in this strain has a minimal effect on its invasiveness . On the other hand, expression of Ras2(Val19)p in another laboratory strain, SP1, rendered it invasive . These results suggest that a hyperactive Ras2 pathway induces invasive growth and that this pathway might be overactive in the Sigma1278b genetic background . Indeed, cells of the Sigma1278b are defective in the induction of stress-responsive genes, while their Gcn4 target genes are constitutively transcribed . This pattern of gene expression was previously shown to be associated with an active Ras/cyclic AMP (cAMP) pathway . We show that suppression of stress-related genes in Sigma1278b cells is a result of their inability to activate transcription through the stress response element (STRE) . Disruption of RAS2, which abolished invasiveness, induced an increase in STRE activity . Further, in the SP1 genetic background, disruption of either the MSN2/4 genes (encoding activators of STRE) or the yAP-1 gene was sufficient to restore invasive growth in ras2Delta cells . We conclude that Ras2-mediated suppression of the stress response is sufficient to induce invasiveness . Accordingly, the fact that the stress response is suppressed in Sigma1278b background explains its invasiveness . It seems that invasiveness is a phenotype related to unregulated growth and is therefore manifested by cells harboring an overactive Ras/cAMP cascade . In this respect, invasiveness in yeast is reminiscent of the property of ras-transformed fibroblasts to invade soft agar.

Mol Cell Biol, 1999 Nov, 19(11), 7481 - 90
In vivo analysis of functional regions within yeast Rap1p; Graham IR et al.; We have analyzed the in vivo importance of different regions of Rap1p, a yeast transcriptional regulator and telomere binding protein . A yeast strain (SCR101) containing a regulatable RAP1 gene was used to test functional complementation by a range of Rap1p derivatives . These experiments demonstrated that the C terminus of the protein, containing the putative transcriptional activation domain and the regions involved in silencing and telomere function, is not absolutely essential for cell growth, a result confirmed by sporulation of a diploid strain containing a C terminal deletion derivative of RAP1 . Northern analysis with cells that expressed Rap1p lacking the transcriptional activation domain revealed that this region is important for the expression of only a subset of Rap1p-activated genes . The one essential region within Rap1p is the DNA binding domain . We have investigated the possibility that this region has additional functions . It contains two Myb-like subdomains separated by a linker region . Individual point mutations in the linker region had no effect on Rap1p function, although deletion of the region abolished cell growth . The second Myb-like subdomain contains a large unstructured loop of unknown function . Domain swap experiments with combinations of elements from DNA binding domains of Rap1p homologues from different yeasts revealed that major changes can be made to the amino acid composition of this region without affecting Rap1p function.

Mol Cell Biol, 1999 Nov, 19(11), 7410 - 9
DNA damage and replication checkpoints in fission yeast require nuclear exclusion of the Cdc25 phosphatase via 14-3-3 binding; Zeng Y et al.; In fission yeast as well as in higher eukaryotic organisms, entry into mitosis is delayed in cells containing damaged or unreplicated DNA . This is accomplished in part by maintaining the Cdc25 phosphatase in a phosphorylated form that binds 14-3-3 proteins . In this study, we generated a mutant of fission yeast Cdc25 that is severely impaired in its ability to bind 14-3-3 proteins . Loss of both the DNA damage and replication checkpoints was observed in fission yeast cells expressing the 14-3-3 binding mutant . These findings indicate that 14-3-3 binding to Cdc25 is required for fission yeast cells to arrest their cell cycle in response to DNA damage and replication blocks . Furthermore, the 14-3-3 binding mutant localized almost exclusively to the nucleus, unlike wild-type Cdc25, which localized to both the cytoplasm and the nucleus . Nuclear accumulation of wild-type Cdc25 was observed when fission yeast cells were treated with leptomycin B, indicating that Cdc25 is actively exported from the nucleus . Nuclear exclusion of wild-type Cdc25 was observed upon overproduction of Rad 24, one of the two fission yeast 14-3-3 proteins, indicating that one function of Rad 24 is to keep Cdc25 out of the nucleus . In support of this conclusion, Rad 24 overproduction did not alter the nuclear location of the 14-3-3 binding mutant . These results indicate that 14-3-3 binding contributes to the nuclear exclusion of Cdc25 and that the nuclear exclusion of Cdc25 is required for a normal checkpoint response to both damaged and unreplicated DNA.

Mol Cell Biol, 1999 Nov, 19(11), 7357 - 68
A sampling of the yeast proteome; Futcher B et al.; In this study, we examined yeast proteins by two-dimensional (2D) gel electrophoresis and gathered quantitative information from about 1,400 spots . We found that there is an enormous range of protein abundance and, for identified spots, a good correlation between protein abundance, mRNA abundance, and codon bias . For each molecule of well-translated mRNA, there were about 4,000 molecules of protein . The relative abundance of proteins was measured in glucose and ethanol media . Protein turnover was examined and found to be insignificant for abundant proteins . Some phosphoproteins were identified . The behavior of proteins in differential centrifugation experiments was examined . Such experiments with 2D gels can give a global view of the yeast proteome.

J Cell Sci, 1999 Nov, 112 ( Pt 21), 3703 - 12
The fission yeast origin recognition complex is constitutively associated with chromatin and is differentially modified through the cell cycle; Lygerou Z et al.; The origin recognition complex (ORC) binds to the well defined origins of DNA replication in budding yeast . Homologous proteins in other eukaryotes have been identified but are less well characterised . We report here the characterisation of a fission yeast ORC complex (SpORC) . Database searches identified a fission yeast Orc5 homologue . SpOrc5 is essential for cell viability and its deletion phenotype is identical to that of two previously identified ORC subunit homologues, SpOrc1 (Orp1/Cdc30) and SpOrc2 (Orp2) . Co-immunoprecipitation experiments demonstrate that SpOrc1 forms a complex with SpOrc2 and SpOrc5 and gel filtration chromatography shows that SpOrc1 and SpOrc5 fractionate as high molecular mass complexes . SpORC subunits localise to the nucleus in a punctate distribution which persists throughout interphase and mitosis . We developed a chromatin isolation protocol and show that SpOrc1, 2 and 5 are associated with chromatin at all phases of the cell cycle . While the levels, nuclear localisation and chromatin association of SpORC remain constant through the cell cycle, one of its subunits, SpOrc2, is differentially modified . We show that SpOrc2 is a phosphoprotein which is hypermodified in mitosis and is rapidly converted to a faster migrating isoform as cells proceed into G(1) in preparation for S-phase.

EMBO J, 1999 Oct 15, 18(20), 5577 - 91
A novel regulator of G protein signalling in yeast, Rgs2, downregulates glucose-activation of the cAMP pathway through direct inhibition of Gpa2; Versele M et al.; We have characterized a novel member of the recently identified family of regulators of heterotrimeric G protein signalling (RGS) in the yeast Saccharomyces cerevisiae . The YOR107w/RGS2 gene was isolated as a multi-copy suppressor of glucose-induced loss of heat resistance in stationary phase cells . The N-terminal half of the Rgs2 protein consists of a typical RGS domain . Deletion and overexpression of Rgs2, respectively, enhances and reduces glucose-induced accumulation of cAMP . Overexpression of RGS2 generates phenotypes consistent with low activity of cAMP-dependent protein kinase A (PKA), such as enhanced accumulation of trehalose and glycogen, enhanced heat resistance and elevated expression of STRE-controlled genes . Deletion of RGS2 causes opposite phenotypes . We demonstrate that Rgs2 functions as a negative regulator of glucose-induced cAMP signalling through direct GTPase activation of the Gs-alpha protein Gpa2 . Rgs2 and Gpa2 constitute the second cognate RGS-G-alpha protein pair identified in yeast, in addition to the mating pheromone pathway regulators Sst2 and Gpa1 . Moreover, Rgs2 and Sst2 exert specific, non-overlapping functions, and deletion mutants in Rgs2 and Sst2 are complemented to some extent by different mammalian RGS proteins.

Biochemistry (Mosc), 1999 Sep, 64(9), 1061 - 7
Adaptation to salt stress in a salt-tolerant strain of the yeast yarrowia lipolytica
Andreishcheva EN, Isakova EP, Sidorov NN, Abramova NB.
We have studied the cellular mechanisms underlying adaptation to salt stress in a newly isolated osmo- and salt-tolerant strain of the yeast Yarrowia lipolytica . When cells are incubated in the presence of 9% NaCl, a rapid change in their size and shape is observed . Salt stress is accompanied by an increase in the intracellular level of glycerol, free amino acids (notably proline and aliphatic amino acids), and Na+, as well as by changes in lipid and fatty acid composition.

J Biol Chem, 1999 Oct 22, 274(43), 31068 - 75
Characterization of a mammalian gene related to the yeast CCR4 general transcription factor and revealed by transposon insertion; Dupressoir A et al.; Murine intracisternal A-particles (IAPs) are reiterated retrovirus-like transposable elements that can act as insertional mutagens . Accordingly, we previously identified a chimeric transcript initiated at an IAP promoter and extending through a 3'-located open reading frame with significant similarity to the C-terminal domain of the yeast CCR4 general transcription factor . In this report, we characterize the corresponding murine gene, mCCR4, and its human homologue, thus providing the first description of CCR4-like factors in mammals . cDNA cloning revealed two mCCR4 mRNAs of 2.7 and 3.1 kilobases, differing by their transcription start sites within the native mCCR4 gene promoter, and encoding a putative 430-amino acid protein . The mCCR4 gene contains three exons and two introns spanning almost 27 kilobases . The IAP insertion, detected only in some laboratory mouse strains, is recent and lies within the first intron . The 5'-region of the gene has features of housekeeping gene promoters . It lacks a TATA box but contains a CpG island and Sp1 sites . This region discloses strong promoter activity in transient transfection assays and also stimulates transcription in the reverse orientation, a feature common to other CpG island-containing promoters . Transcripts were detected in all the organs tested, although at a variable level, and displayed no strain-dependent differences relative to the IAP insertion, suggesting the existence of mechanisms preserving mCCR4 transcription from the usually deleterious effects of intronic transposition . The strong amino acid conservation between the human, murine, and the previously identified Xenopus CCR4-like proteins, is consistent with an important and conserved role for this protein in vertebrates.

J Biol Chem, 1999 Oct 22, 274(43), 30690 - 6
Yeast topoisomerase II is inhibited by etoposide after hydrolyzing the first ATP and before releasing the second ADP; Morris SK et al.; Topoisomerase II-catalyzed DNA transport requires coordination between two distinct reactions: ATP hydrolysis and DNA cleavage/religation . To further understand how these reactions are coupled, inhibition by the clinically used anticancer drug etoposide was studied . The IC(50) for perturbing the DNA cleavage/religation equilibrium is nucleotide-dependent; its value is 6 microM in the presence of ATP, 25 microM in the presence of a nonhydrolyzable ATP analog, and 45 microM in the presence of ADP or no nucleotide . This inhibition was further characterized using steady-state and pre-steady-state ATPase and decatenation assays . Etoposide is a hyperbolic noncompetitive inhibitor of the ATPase activity with a K(i)(app) of 5.6 microM no inhibition of ATP hydrolysis is seen in the absence of DNA cleavage . In order to determine which steps of the ATPase mechanism etoposide inhibits, pre-steady-state analysis was performed . These results showed that etoposide does not reduce the rate of binding two ATP, hydrolyzing the first ATP, or releasing the second ADP . Inhibition is therefore associated with the first product release step or hydrolysis of the second ATP, suggesting that DNA religation normally occurs at one of these two steps . Multiple turnover decatenation is inhibited when etoposide is present; however, single turnover decatenation occurs normally . The implications of these results are discussed in terms of their contribution to our current model for the topoisomerase II mechanism.

J Biol Chem, 1999 Oct 22, 274(43), 30402 - 9
Conservation and divergence of the yeast and mammalian unfolded protein response . Activation of specific mammalian endoplasmic reticulum stress element of the grp78/BiP promoter by yeast Hac1; Foti DM et al.; Yeast Hac1 (yHac1), the transcription factor that binds and activates the unfolded protein response element of endoplasmic reticulum (ER)-chaperone gene promoters, only accumulates in stressed cells after an unconventional splicesosome-free mRNA processing step and escape from translation block . In determining whether the novel regulatory mechanisms for yHac1 are conserved in mammalian cells, we discovered a unique unfolded protein response element-like sequence within the endoplasmic reticulum stress element 163, one of the three ER stress elements recently identified in the rat grp78 promoter . The unspliced form of yHac1 is stably expressed in nonstressed mammalian cells and is as active as the spliced form in stimulating the promoter activities of grp genes . Further, the yHac1 mRNA is not processed in response to ER stress in mammalian cells . We identified a CCAGC motif as the yHac1 binding site, which is contained within a YY1 binding site previously shown to be important for mammalian UPR . Dissection of the yHac1 and the YY1 binding sites uncovered specific contact points for an activator protein predicted to be the mammalian homolog of yHac1, the activity of which can be stimulated by YY1 . A model of the conserved and unique features of the yeast and mammalian unfolded protein response transcription machinery is proposed.

Genes Dev, 1999 Oct 1, 13(19), 2581 - 93
Meiotic DNA replication checkpoint control in fission yeast; Murakami H et al.; In eukaryotes, the DNA replication checkpoint prevents entry into mitosis when DNA replication is incomplete and is crucial for maintaining genomic integrity . Much less is known about equivalent controls that operate during meiosis . Here, we show that a DNA replication checkpoint control operates during meiosis in fission yeast . The mitotic checkpoint Rad genes and the Cds1 protein kinase are required for the DNA replication checkpoint during meiosis, with Cds1 playing a more prominent role than it does during mitosis . When DNA replication is blocked, the checkpoint maintains Cdc2 tyrosine 15 phosphorylation keeping Cdc2 protein kinase activity low and preventing onset of meiosis I . Additionally, there is a second checkpoint acting during meiosis that is revealed if cells are prevented from maintaining Cdc2 tyrosine 15 phosphorylation when DNA replication is blocked . Such cells arrest with high Cdc2 protein kinase activity and separated spindle pole bodies, an arrest state similar to that observed in mitotic budding yeast cells when DNA replication is incomplete . This second checkpoint is meiosis specific and may reflect processes occurring only during meiosis such as increased recombination rates, an extended duration of nuclear division, or homolog chromosome pairing.

Genes Dev, 1999 Oct 1, 13(19), 2484 - 9
TFIID-specific yeast TAF40 is essential for the majority of RNA polymerase II-mediated transcription in vivo; Komarnitsky PB et al.; Many questions remain concerning the role of TFIID TBP-associated factors (TAFs) in transcription, including whether TAFs are required at most or only a small subset of promoters . It was shown previously that three histone-like TAFs are broadly required for transcription, but interpretation of this observation is complicated because these proteins are components of both TFIID and the SAGA histone acetyltransferase complex . Here we show that mutations in the yeast TFIID-specific protein Taf40 lead to a general cessation of transcription, even in the presence of excess TBP, suggesting that the TFIID complex is required at most promoters in vivo.

Science, 1999 Oct 15, 286(5439), 552 - 5
Yeast gene for a Tyr-DNA phosphodiesterase that repairs topoisomerase I complexes; Pouliot JJ et al.; Covalent intermediates between topoisomerase I and DNA can become dead-end complexes that lead to cell death . Here, the isolation of the gene for an enzyme that can hydrolyze the bond between this protein and DNA is described . Enzyme-defective mutants of yeast are hypersensitive to treatments that increase the amount of covalent complexes, indicative of enzyme involvement in repair . The gene is conserved in eukaryotes and identifies a family of enzymes that has not been previously recognized . The presence of this gene in humans may have implications for the effectiveness of topoisomerase I poisons, such as the camptothecins, in chemotherapy.

Biol Cell, 1999 Jul, 91(6), 439 - 50
Mechanisms of genetic instability revealed by analysis of yeast spindle pole body duplication; Chial HJ et al.; Aneuploidy and polyploidy are commonly observed in transformed cells . These states arise from failures during mitotic chromosome segregation, some of which can be traced to defects in the function or duplication of the centrosome . The centrosome is the organizing center for the mitotic spindle, and the equivalent organelle in the budding yeast, Saccharomyces cerevisiae, is the spindle pole body . We review how defects in spindle pole body duplication or function lead to genetic instability in yeast . There are several well documented instances of genetic instability in yeast that can be traced to the spindle pole body, all of which serve as models for genetic instability in transformed cells.

Biol Cell, 1999 May-Jun, 91(4-5), 305 - 12
Life cycles of yeast spindle pole bodies: getting microtubules into a closed nucleus; McIntosh JR et al.; The spindle pole body (SPB) is the principal microtubule organizing center of budding and fission yeast . We have examined SPBs and their associated microtubules from both organisms, using electron microscopy and three-dimensional reconstruction techniques, to identify the structural changes that accompany progression through the cell cycle . In this report, we compare these changes in the two kinds of yeasts and present a model for how microtubules get into a closed nucleus.

Biol Cell, 1999 May-Jun, 91(4-5), 291 - 304
Microtubule organization by the budding yeast spindle pole body; Knop M et al.; In budding yeast microtubule organizing functions are provided by the spindle pole body (SPB), a multi-layered structure that is embedded in the nuclear envelope throughout the cell cycle . The SPB organizes the nuclear and cytoplasmic microtubules which are spatially and functionally distinct . Microtubule formation in yeast requires the Tub4p-complex, containing the gamma-tubulin Tub4p, and two additional proteins, the SPB components Spc97p and Spc98p . The Tub4p complex assembles in the cytoplasm and is then anchored to the sides of the SPB which organize microtubules . This is achieved by the binding of Spc97p and Spc98p to so-called gamma-tubulin complex binding proteins (GTBPs) at the SPB . Spc72p is the yeast GTBP at the cytoplasmic side of the SPB, while Spc110p is the nuclear GTBP . Both GTBPs control the number of Tub4p complexes associated with the SPB and thereby the number of microtubules formed . In addition, the GTBPs may regulate the activity of the Tub4p complex . Homologues of Spc97p and Spc98p have been identified from yeast to mammalian cells and these are also part of gamma-tubulin complexes, suggesting that these related proteins may also interact with GTBPs at the centrosome . Candidates for GTBPs have been identified in mammalian and insect cells.

FEBS Lett, 1999 Sep 10, 458(1), 51 - 4
The nascent polypeptide-associated complex (NAC) of yeast functions in the targeting process of ribosomes to the ER membrane; Wiedmann B et al.; We study here the binding of ribosomes to the endoplasmic reticulum (ER) membrane and its dependence on nascent polypeptide-associated complex (NAC) . For this, we use an in vitro translation system in combination with isolated microsomes . Importantly, all components in the system are derived from a single source, Saccharomyces cerevisiae . Ribosome nascent chains (RNCs) of the two naturally occurring invertase species (secreted or cytosolic) were prepared in wild-type, delta alpha NAC or delta alpha beta 1 beta 3 NAC translation lysates and tested for binding to the corresponding microsomal membranes . We provide evidence that NAC prevents binding of RNCs without a signal sequence to yeast membranes . In the absence of NAC, signal-less RNCs are able to bind to ER membranes . However, following puromycin treatment, only very few nascent chains translocate into the lumen, as detected by glycosylation.

Nucleic Acids Res, 1999 Nov 1, 27(21), 4276 - 81
Construction of a recombinant adenovirus for efficient delivery of the I-SceI yeast endonuclease to human cells and its application in the in vivo cleavage of chromosomes to expose new potential telomeres; Anglana M et al.; We have constructed a replication-defective adenovirus vector encoding the yeast I- Sce I endonuclease under the control of the murine cytomegalovirus immediate-early gene promoter (AdM Sce I) for efficient delivery of this enzyme to mammalian cells . We present evidence of AdM Sce I-mediated I- Sce I protein expression and cleavage activity in replication-permissive 293 cells, and of cleavage of chromosomes in vivo in both 293 cells and in non-permissive human cells . We have exploited this system for the generation of chromosomes capped by artificial telomeric sequences in cells with integrated plasmids containing telomeric DNA arrays adjacent to an I- Sce I recognition site . The properties of the AdM Sce I virus described here make it a useful tool for studying biological processes involving induction of DNA breaks, recombination and gene targeting in cells grown in culture and in vivo.

Proc Natl Acad Sci U S A, 1999 Oct 12, 96(21), 11934 - 9
The yeast HML I silencer defines a heterochromatin domain boundary by directional establishment of silencing; Bi X et al.; The eukaryotic genome is divided into functional domains defined in part by local differences in chromatin structure and delimited in many cases by boundary elements . The HML and HMR loci in the yeast Saccharomyces cerevisiae are transcriptionally silent chromosome domains . Each locus is bracketed by two cis-acting sequences, designated E and I, that serve to establish and maintain repression of genes within each locus . We show that repression at HML is uniformly high between E and I but decreases sharply beyond I . The region of repression at HML generally correlates with the domain of histone hypoacetylation . Despite the sharp definition of the boundaries of HML, no sequence capable of blocking the spread of heterochromatin resides in the sequences flanking HML . We find, though, that inverting the orientation of I increases silencing outside of HML while weakening silencing within HML . These results indicate that the HML I silencer establishes a boundary between active and inactive chromatin at HML, but does so by organizing inactive chromatin in only one direction . This represents a different mechanism for delimiting the boundaries of a eukaryotic chromosome domain.

Nature, 1999 Sep 23, 401(6751), 394 - 7
Evidence that a free-running oscillator drives G1 events in the budding yeast cell cycle; Haase SB et al.; In yeast and somatic cells, mechanisms ensure cell-cycle events are initiated only when preceding events have been completed . In contrast, interruption of specific cell-cycle processes in early embryonic cells of many organisms does not affect the timing of subsequent events, indicating that cell-cycle events are triggered by a free-running cell-cycle oscillator . Here we present evidence for an independent cell-cycle oscillator in the budding yeast Saccharomyces cerevisiae . We observed periodic activation of events normally restricted to the G1 phase of the cell cycle, in cells lacking mitotic cyclin-dependent kinase activities that are essential for cell-cycle progression . As in embryonic cells, G1 events cycled on schedule, in the absence of S phase or mitosis, with a period similar to the cell-cycle time of wild-type cells . Oscillations of similar periodicity were observed in cells responding to mating pheromone in the absence of G1 cyclin (Cln)- and mitotic cyclin (Clb)-associated kinase activity, indicating that the oscillator may function independently of cyclin-dependent kinase dynamics . We also show that Clb-associated kinase activity is essential for ensuring dependencies by preventing the initiation of new G1 events when cell-cycle progression is delayed.

Plant Physiol, 1999 Oct, 121(2), 609 - 17
The Arabidopsis homolog of yeast TAP42 and mammalian alpha4 binds to the catalytic subunit of protein phosphatase 2A and is induced by chilling; Harris DM et al.; Type 2A serine/threonine protein phosphatases (PP2A) have been implicated as important mediators of a number of plant growth and developmental processes . In an effort to identify plant PP2A substrates and/or regulators, we performed a yeast two-hybrid screen using an Arabidopsis PP2A catalytic subunit cDNA as bait . All true positives identified by this screen were derived from the same gene, which we have named TAP46 (2A phosphatase associated protein of 46 kD) . The TAP46 gene appears to be a single-copy gene and is expressed in all Arabidopsis organs . Transcripts derived from this gene are induced by chilling treatment but not by heat or anaerobic stress . Immunoprecipitation assays using antibodies generated to a peptide spanning amino acids 356 to 366 of TAP46 indicate that TAP46 is associated with a type 2A protein phosphatase in vivo . A search of the database identified TAP46 as a homolog of Saccharomyces cerevisiae TAP42 and mammalian alpha4 . These two proteins are known to bind to the catalytic subunit of PP2A and to function in the target-of-rapamycin signaling pathway . Our results identify TAP46 as a plant PP2A-associated protein, with a possible function in the chilling response, and suggest that a target-of-rapamycin-like signaling pathway may exist in plants.

Mol Gen Genet, 1999 Sep, 262(2), 367 - 77
Imbalance in dosage of the genes for the heterochromatin components Sir3p and histone H4 results in changes in the length and sequence organization of yeast telomeres; Venditti S et al.; Telomeric heterochromatin plays an essential role in telomere function, including the regulation of telomere length . We observe that in Saccharomyces cerevisiae an imbalance in the dosage of genes for two protein components of heterochromatin (namely Sir3p and histone H4) causes modifications in telomere length and telomere sequence organization . The effects of Sir3p/H4 imbalance were analyzed in yeast strains in which the wild-type SIR3 gene (normally a single-copy gene) was either absent or present in 20-30 copies, and both histone H4 genes (HHF1 and HHF2) were present or HHF1 was deleted, thus covering a wide range of viable gene-dosage combinations . Modifications of telomeres and of subtelomeric regions were identified by analyzing both the overall telomere population and by focusing on two single telomeric regions: the left telomere of chromosome III (LIII) and the right telomere of chromosome XI (RXI) . The modifications induced by alteration of the Sir3p/H4 ratio consist of a reduction in the length and an increase in the instability of the terminal block of (C(1-3)A)n repeats and in susceptibility to insertion of Y' elements into this repeat element . Restoration of the wild-type gene ratio (by removal of the extra copies of SIR3 or by complementation with the missing second copy of HHF) restored the original telomere organization, both with respect to the length of the (C(1-3)A)n repeat stretch and the absence of Y' elements . This behavior shows that the stability of the wild-type sequence organization requires maintenance of the normal structure of telomeric heterochromatin.

Biotechnol Bioeng, 1999 Dec 5, 65(5), 572 - 8
Preservation of frozen yeast cells by trehalose; Diniz-Mendes L et al.; Two different methods commonly used to preserve intact yeast cells-freezing and freeze-drying-were compared . Different yeast cells submitted to these treatments were stored for 28 days and cell viability assessed during this period . Intact yeast cells showed to be less tolerant to freeze-drying than to freezing . The rate of survival for both treatments could be enhanced by exogenous trehalose (10%) added during freezing and freeze-drying treatments or by a combination of two procedures: a pre-exposure of cells to 40 degrees C for 60 min and addition of trehalose . A maximum survival level of 71.5 +/- 6.3% after freezing could be achieved at the end of a storage period of 28 days, whereas only 25.0 +/- 1.4% showed the ability to tolerate freeze-drying treatment, if both low-temperature treatments were preceded by a heat exposure and addition of trehalose to yeast cells . Increased survival ability was also obtained when the pre-exposure treatment of yeast cells was performed at 10 degrees C for 3 h and trehalose was added: these treatments enhanced cell survival following freezing from 20.5 +/- 7 . 7% to 60.0 +/- 3.5% . Although both mild cold and heat shock treatments could enhance cell tolerance to low temperature, only the heat treatment was able to increase the accumulation of intracellular trehalose whereas, during cold shock exposure, the intracellular amount of trehalose remained unaltered . Intracellular trehalose levels seemed not to be the only factor contributing to cell tolerance against freezing and freeze-drying treatments; however, the protection that this sugar confers to cells can be exerted only if it is to be found on both sides of the plasma membrane .

J Bacteriol, 1999 Oct, 181(20), 6456 - 62
Biochemical and genetic analyses of the role of yeast casein kinase 2 in salt tolerance; de Nadal E et al.; Saccharomyces cerevisiae cells lacking the regulatory subunit of casein kinase 2 (CK-2), encoded by the gene CKB1, display a phenotype of hypersensitivity to Na(+) and Li(+) cations . The sensitivity of a strain lacking ckb1 is higher than that of a calcineurin mutant and similar to that of a strain lacking HAL3, the regulatory subunit of the Ppz1 protein phosphatase . Genetic analysis indicated that Ckb1 participates in regulatory pathways different from that of Ppz1 or calcineurin . Deletion of CKB1 increased the salt sensitivity of a strain lacking Ena1 ATPase, the major determinant for sodium efflux, suggesting that the function of the kinase is not mediated by Ena1 . Consistently, ckb1 mutants did not show an altered cation efflux . The function of Ckb1 was independent of the TRK system, which is responsible for discrimination of potassium and sodium entry, and in the absence of the kinase regulatory subunit, the influx of sodium was essentially normal . Therefore, the salt sensitivity of a ckb1 mutant cannot be attributed to defects in the fluxes of sodium . In fact, in these cells, both the intracellular content and the cytoplasm/vacuole ratio for sodium were similar to those features of wild-type cells . The possible causes for the salt sensitivity phenotype of casein kinase mutants are discussed in the light of these findings.

Yeast, 1999 Oct, 15(14), 1485 - 501
A novel vacuolar protein encoded by SSU21 / MCD4 is involved in cell wall integrity in yeast; Packeiser AN et al.; Using a screening procedure for obtaining yeast strains with enhanced ability to secrete heterologous protein, we have isolated a mutant with alteration of the cell wall structure . This mutant displayed strong decrease in cell wall mannoprotein content, which was not accompanied by decreased glycosylation of secreted proteins . The mutation defines a gene, designated SSU21(identical to previously characterized MCD4), which encodes a novel vacuolar protein . SSU21 is probably connected to the cell integrity protein kinase C-mediated pathway, since ssu21 and pkc1Delta double mutant is synthetic lethal . To our knowledge, this is the first example of a yeast vacuolar protein whose alteration results in a cell wall defect .

Mol Biol Cell, 1999 Oct, 10(10), 3389 - 400
Association of the cell cycle transcription factor Mbp1 with the Skn7 response regulator in budding yeast; Bouquin N et al.; We previously isolated the SKN7 gene in a screen designed to isolate new components of the G1-S cell cycle transcription machinery in budding yeast . We have now found that Skn7 associates with Mbp1, the DNA-binding component of the G1-S transcription factor DSC1/MBF . SKN7 and MBP1 show several genetic interactions . Skn7 overexpression is lethal and is suppressed by a mutation in MBP1 . Similarly, high overexpression of Mbp1 is lethal and can be suppressed by skn7 mutations . SKN7 is also required for MBP1 function in a mutant compromised for G1-specific transcription . Gel-retardation assays indicate that Skn7 is not an integral part of MBF . However, a physical interaction between Skn7 and Mbp1 was detected using two-hybrid assays and GST pulldowns . Thus, Skn7 and Mbp1 seem to form a transcription factor independent of MBF . Genetic data suggest that this new transcription factor could be involved in the bud-emergence process.

Mol Biol Cell, 1999 Oct, 10(10), 3331 - 43
A double-strand break repair component is essential for S phase completion in fission yeast cell cycling; Suto K et al.; Fission yeast rad22(+), a homologue of budding yeast RAD52, encodes a double-strand break repair component, which is dispensable for proliferation . We, however, have recently obtained a cell division cycle mutant with a temperature-sensitive allele of rad22(+), designated rad22-H6, which resulted from a point mutation in the conserved coding sequence leading to one amino acid alteration . We have subsequently isolated rad22(+) and its novel homologue rti1(+) as multicopy suppressors of this mutant . rti1(+) suppresses all the defects of cells lacking rad22(+) . Mating type switch-inactive heterothallic cells lacking either rad22(+) or rti1(+) are viable, but those lacking both genes are inviable and arrest proliferation with a cell division cycle phenotype . At the nonpermissive temperature, a synchronous culture of rad22-H6 cells performs DNA synthesis without delay and arrests with chromosomes seemingly intact and replication completed and with a high level of tyrosine-phosphorylated Cdc2 . However, rad22-H6 cells show a typical S phase arrest phenotype if combined with the rad1-1 checkpoint mutation . rad22(+) genetically interacts with rad11(+), which encodes the large subunit of replication protein A . Deletion of rad22(+)/rti1(+) or the presence of rad22-H6 mutation decreases the restriction temperature of rad11-A1 cells by 4-6 degrees C and leads to cell cycle arrest with chromosomes incompletely replicated . Thus, in fission yeast a double-strand break repair component is required for a certain step of chromosome replication unlinked to repair, partly via interacting with replication protein A.

Mol Biol Cell, 1999 Oct, 10(10), 3301 - 16
Regulation of G2/M progression by the STE mitogen-activated protein kinase pathway in budding yeast filamentous growth; Ahn SH et al.; Inoculation of diploid budding yeast onto nitrogen-poor agar media stimulates a MAPK pathway to promote filamentous growth . Characteristics of filamentous cells include a specific pattern of gene expression, elongated cell shape, polar budding pattern, persistent attachment to the mother cell, and a distinct cell cycle characterized by cell size control at G2/M . Although a requirement for MAPK signaling in filamentous gene expression is well established, the role of this pathway in the regulation of morphogenesis and the cell cycle remains obscure . We find that ectopic activation of the MAPK signal pathway induces a cell cycle shift to G2/M coordinately with other changes characteristic of filamentous growth . These effects are abrogated by overexpression of the yeast mitotic cyclins Clb1 and Clb2 . In turn, yeast deficient for Clb2 or carrying cdc28-1N, an allele of CDK defective for mitotic functions, display enhanced filamentous differentiation and supersensitivity to the MAPK signal . Importantly, activation of Swe1-mediated inhibitory phosphorylation of Thr-18 and/or Tyr-19 of Cdc28 is not required for the MAPK pathway to affect the G2/M delay . Mutants expressing a nonphosphorylatable mutant Cdc28 or deficient for Swe1 exhibit low-nitrogen-dependent filamentous growth and are further induced by an ectopic MAPK signal . We infer that the MAPK pathway promotes filamentous growth by a novel mechanism that inhibits mitotic cyclin/CDK complexes and thereby modulates cell shape, budding pattern, and cell-cell connections.

Mol Biol Cell, 1999 Oct, 10(10), 3289 - 99
Nascent polypeptide-associated complex stimulates protein import into yeast mitochondria; Funfschilling U et al.; To identify yeast cytosolic proteins that mediate targeting of precursor proteins to mitochondria, we developed an in vitro import system consisting of purified yeast mitochondria and a radiolabeled mitochondrial precursor protein whose C terminus was still attached to the ribosome . In this system, the N terminus of the nascent chain was translocated across both mitochondrial membranes, generating a translocation intermediate spanning both membranes . The nascent chain could then be completely chased into the mitochondrial matrix after release from the ribosome . Generation of this import intermediate was dependent on a mitochondrial membrane potential, mitochondrial surface proteins, and was stimulated by proteins that could be released from the ribosomes by high salt . The major salt-released stimulatory factor was yeast nascent polypeptide-associated complex (NAC) . Purified NAC fully restored import of salt-washed ribosome-bound nascent chains by enhancing productive binding of the chains to mitochondria . We propose that ribosome-associated NAC facilitates recognition of nascent precursor chains by the mitochondrial import machinery.

Mol Biol Cell, 1999 Oct, 10(10), 3223 - 38
Requirement of sequences outside the conserved kinase domain of fission yeast Rad3p for checkpoint control; Chapman CR et al.; The fission yeast Rad3p checkpoint protein is a member of the phosphatidylinositol 3-kinase-related family of protein kinases, which includes human ATMp . Mutation of the ATM gene is responsible for the disease ataxia-telangiectasia . The kinase domain of Rad3p has previously been shown to be essential for function . Here, we show that although this domain is necessary, it is not sufficient, because the isolated kinase domain does not have kinase activity in vitro and cannot complement a rad3 deletion strain . Using dominant negative alleles of rad3, we have identified two sites N-terminal to the conserved kinase domain that are essential for Rad3p function . One of these sites is the putative leucine zipper, which is conserved in other phosphatidylinositol 3-kinase-related family members . The other is a novel motif, which may also mediate Rad3p protein-protein interactions.

Biochemistry, 1999 Sep 7, 38(36), 11634 - 42
An NMR analysis of ubiquitin recognition by yeast ubiquitin hydrolase: evidence for novel substrate recognition by a cysteine protease; Sakamoto T et al.; Yeast ubiquitin hydrolase 1 (YUH1), a cysteine protease that catalyzes the removal of ubiquitin C-terminal adducts, is important for the generation of monomeric ubiquitin . Heteronuclear NMR spectroscopy has been utilized to map the YUH1 binding surface on ubiquitin . When YUH1 was titrated into a sample of ubiquitin, approximately 50% of the (1)H-(15)N correlation peaks of ubiquitin were affected to some degree, as a result of binding to YUH1 . It is noteworthy that the amide resonances of the basic residues (Arg42, Lys48, Arg72, and Lys74) were highly perturbed . These positively charged basic residues may be involved in direct interactions with the negatively charged acidic residues on YUH1 . In addition to the electrostatic surface, the hydrophobic surfaces on ubiquitin (Leu8, Ile44, Phe45, Val70, Leu71, and Leu73) and YUH1 are also likely to contribute to the binding interaction . Furthermore, the amide resonances of Ile13, Leu43, Leu50, and Leu69, the side chains of which are not on the surface, were also highly perturbed, indicating substrate-induced changes in the environments of these residues as well . These large changes, observed from residues located throughout the five-stranded beta-sheet surface and the C-terminus, suggest that substrate recognition by YUH1 involves a wider area on ubiquitin.

Biospectroscopy, 1999, 5(5 Suppl), S42 - 52
Influence of protein environment on magnetic circular dichroism spectral properties of ferric and ferrous ligand complexes of yeast cytochrome c peroxidase; Pond AE et al.; The addition of exogenous ligands to the ferric and ferrous states of yeast cytochrome c peroxidase (CCP) is investigated with magnetic circular dichroism (MCD) at 4 degrees C to determine the effect the protein environment may exercise on spectral properties . The MCD spectrum of each derivative is directly compared to those of analogous forms of horseradish peroxidase (HRP) and myoglobin (Mb), two well-characterized histidine-ligated heme proteins . The ferric azide adduct of CCP is a hexacoordinate, largely low-spin species with an MCD spectrum very similar to that of ferric azide HRP . This complex displays an MCD spectrum dissimilar from that of the Mb derivative, possibly because of the stabilizing interaction between the azide ligand and the distal arginine of CCP (Arg 48) . For the ferric fluoride derivative all three proteins display varied MCD data, indicating that the differences in the distal pocket of each protein influences their respective MCD characteristics . The MCD data for the cyanoferric complexes are similar for all three proteins, demonstrating that a strong field ligand bound in the sixth axial position dominates the MCD characteristics of the derivative . Similarly, the ferric NO complexes of the three proteins show MCD spectra similar in feature position and shape, but vary somewhat in intensity . Reduction of CCP at neutral pH yields a typical pentacoordinate high-spin complex with an MCD spectrum similar to that of deoxyferrous HRP . Formation of the NO and cyanide complexes of ferrous CCP gives derivatives with MCD spectra similar to the analogous forms of HRP and Mb in both feature position and shape . Addition of CO to deoxyferrous CCP results in a ferrous-CO complex with MCD spectral similarity to that of ferrous-CO HRP but not Mb, indicating that interactions between the ligand and the distal residues affects the MCD characteristics . Examination of alkaline (pH 9.7) deoxyferrous CCP indicates that a pH dependent conformational change has occurred, leading to a coordination structure similar to that of ferrous cytochrome b5, a known bis-histidine complex . Exposure of this complex to CO further confirms that a conformational change has taken place in that the MCD spectral characteristics of the resulting complex are similar to those of ferrous-CO Mb but not ferrous-CO HRP.

Genetics, 1999 Oct, 153(2), 681 - 92
A core activity associated with the N terminus of the yeast RAD52 protein is revealed by RAD51 overexpression suppression of C-terminal rad52 truncation alleles; Asleson EN et al.; C-terminal rad52 truncation and internal deletion mutants were characterized for their ability to repair MMS-induced double-strand breaks and to produce viable spores during meiosis . The rad52-Delta251 allele, encoding the N-terminal 251 amino acids of the predicted 504-amino-acid polypeptide, supports partial activity for both functions . Furthermore, RAD51 overexpression completely suppresses the MMS sensitivity of a rad52-Delta251 mutant . The absence of the C terminus in the truncated protein makes it likely that suppression occurs by bypassing the C-terminal functions of Rad52p . RAD51 overexpression does not suppress the low level of spore viability that the rad52-Delta251 allele causes and only partially suppresses the defect in rad52 alleles encoding the N-terminal 292 or 327 amino acids . The results of this study also show that intragenic complementation between rad52 alleles is governed by a complex relationship that depends heavily on the two alleles involved and their relative dosage . In heteroallelic rad52 diploids, the rad52-Delta251 allele does not complement rad52 missense mutations altering residues 61 or 64 in the N terminus . However, complementation is achieved with each of these missense alleles when the rad52-Delta251 allele is overexpressed . Complementation also occurs between rad52-Delta327 and an internal deletion allele missing residues 210 through 327 . We suggest that the first 251 amino acids of Rad52p constitute a core domain that provides critical RAD52 activities.

Genetics, 1999 Oct, 153(2), 665 - 79
Multiple heterologies increase mitotic double-strand break-induced allelic gene conversion tract lengths in yeast; Nickoloff JA et al.; Spontaneous and double-strand break (DSB)-induced allelic recombination in yeast was investigated in crosses between ura3 heteroalleles inactivated by an HO site and a +1 frameshift mutation, with flanking markers defining a 3.4-kbp interval . In some crosses, nine additional phenotypically silent RFLP mutations were present at approximately 100-bp intervals . Increasing heterology from 0.2 to 1% in this interval reduced spontaneous, but not DSB-induced, recombination . For DSB-induced events, 75% were continuous tract gene conversions without a crossover in this interval; discontinuous tracts and conversions associated with a crossover each comprised approximately 7% of events, and 10% also converted markers in unbroken alleles . Loss of heterozygosity was seen for all markers centromere distal to the HO site in 50% of products; such loss could reflect gene conversion, break-induced replication, chromosome loss, or G2 crossovers . Using telomere-marked strains we determined that nearly all allelic DSB repair occurs by gene conversion . We further show that most allelic conversion results from mismatch repair of heteroduplex DNA . Interestingly, markers shared between the sparsely and densely marked interval converted at higher rates in the densely marked interval . Thus, the extra markers increased gene conversion tract lengths, which may reflect mismatch repair-induced recombination, or a shift from restoration- to conversion-type repair.

Genetics, 1999 Oct, 153(2), 621 - 41
Genetic control of recombination partner preference in yeast meiosis . Isolation and characterization of mutants elevated for meiotic unequal sister-chromatid recombination; Thompson DA et al.; Meiotic exchange occurs preferentially between homologous chromatids, in contrast to mitotic recombination, which occurs primarily between sister chromatids . To identify functions that direct meiotic recombination events to homologues, we screened for mutants exhibiting an increase in meiotic unequal sister-chromatid recombination (SCR) . The msc (meiotic sister-chromatid recombination) mutants were quantified in spo13 meiosis with respect to meiotic unequal SCR frequency, disome segregation pattern, sporulation frequency, and spore viability . Analysis of the msc mutants according to these criteria defines three classes . Mutants with a class I phenotype identified new alleles of the meiosis-specific genes RED1 and MEK1, the DNA damage checkpoint genes RAD24 and MEC3, and a previously unknown gene, MSC6 . The genes RED1, MEK1, RAD24, RAD17, and MEC1 are required for meiotic prophase arrest induced by a dmc1 mutation, which defines a meiotic recombination checkpoint . Meiotic unequal SCR was also elevated in a rad17 mutant . Our observation that meiotic unequal SCR is elevated in meiotic recombination checkpoint mutants suggests that, in addition to their proposed monitoring function, these checkpoint genes function to direct meiotic recombination events to homologues . The mutants in class II, including a dmc1 mutant, confer a dominant meiotic lethal phenotype in diploid SPO13 meiosis in our strain background, and they identify alleles of UBR1, INP52, BUD3, PET122, ELA1, and MSC1-MSC3 . These results suggest that DMC1 functions to bias the repair of meiosis-specific double-strand breaks to homologues . We hypothesize that the genes identified by the class II mutants function in or are regulators of the DMC1-promoted interhomologue recombination pathway . Class III mutants may be elevated for rates of both SCR and homologue exchange.

Yeast, 1999 Sep 30, 15(13), 1377 - 91
Signalling between mitochondria and the nucleus regulates the expression of a new D-lactate dehydrogenase activity in yeast; Chelstowska A et al.; We have adapted a LacZ promoter trap screen developed by Burns et al . (1994) to search for genes whose expression is dependent on Rtg2p, a protein with an N-terminal hsp70/actin/sugar kinase ATP binding domain . Rtg2p acts upstream of the basic helix-loop-helix/leucine zipper transcription factors, Rtg1p and Rtg3p . All three proteins are known to be required for the expression of the CIT2 gene, which encodes a peroxisomal isoform of citrate synthase whose expression is also dependent on the functional state of mitochondria . Using this screen, we have identified a previously uncharacterized gene, YEL071w, predicted to encode a protein of 496 amino acids that shares 80% homology and 60% sequence identity with actin interacting protein 2, encoded by the AIP2 gene; both proteins also share sequence similarity to aD-lactate dehydrogenase encoded by the DLD1 gene . Expression of YEL071w is dependent on the functional state of mitochondria and on all three of the Rtg proteins, whereas AIP2 expression is independent of the Rtg proteins and the functional state of mitochondria . Like CIT2, the 5' flanking region of YEL071w contains two R box binding sites for the Rtg1p/Rtg3p heterodimeric transcription complex . Both R boxes are necessary for full YEL071w expression . We show that YEL071w and AIP2 encode proteins withD-lactate dehydrogenase activity, the former located in the cytoplasm and the latter in the mitochondrial matrix . Our data thus provide gene assignments for two previously unrecognized D-lactate dehydrogenase activities in yeast .

Yeast, 1999 Sep 30, 15(13), 1357 - 64
Mouse CAF1, a mouse homologue of the yeast POP2 gene, complements the yeast pop2 null mutation; Shimizu-Yoshida Y et al.; The yeast POP2 protein (Pop2p) is a component of a global transcription regulatory complex and is required for gene expression of many genes in Saccharomyces cerevisiae . We constructed POP2 deletion plasmids encoding various Pop2p regions under the native POP2 promoter and found that the minimum functional region was located in two-thirds of the carboxyl terminal region . A mouse homologue of the POP2 gene (mCAF1), which corresponds to the Pop2p minimum region, partially rescued the growth defect of pop2 null mutant cells . Addition of the Pop2p amino terminal region to mCAF1 strengthened the suppression . mCAF1 also weakly suppressed the relatively high expression of the SUC2 gene of pop2 cells under glucose-repressing conditions; however, it failed to suppress the defect of full expression of the SUC2 gene under glucose-derepressing conditions . Our findings clearly demonstrate that a mammalian homologue can substitute for the yeast POP2 gene in some aspect .

Yeast, 1999 Sep 30, 15(13), 1307 - 21
A selection system for human apoptosis inhibitors using yeast; Greenhalf W et al.; Apoptosis is a regulated series of events which leads to the death and elimination of mammalian cells during development or in disease control . It is regulated in part by members of the Bcl-2 family of genes . Some of these stimulate cell death, while others prevent it . Expression of one of these death inducers, Bax-alpha (Bax), in the yeast Saccharomyces cerevisiae induces growth arrest and subsequently can cause cell death . Proteins of the Bcl-2 family that are known to inhibit apoptosis in mammalian cells overcome Bax-induced growth arrest in yeast . We describe here a system for isolation of human genes that are able to overcome Bax sensitivity in yeast . Two novel proteins, identified with this system, have been named 'Bax antagonists selected in saccharomyces' (BASS) . These proteins not only overcome toxicity of Bax in yeast but also protect mammalian cells from apoptosis that is induced by staurosporine or Bax overexpression . We find that BASS2 is the more effective of the two genes .

FEBS Lett, 1999 Oct 1, 459(1), 80 - 4
Ricin A chain utilises the endoplasmic reticulum-associated protein degradation pathway to enter the cytosol of yeast; Simpson JC et al.; Cytotoxic proteins such as ricin A chain (RTA) have target substrates in the cytosol and therefore have to reach this cellular compartment in order to act . RTA is thought to translocate into the cytosol from the lumen of the endoplasmic reticulum (ER), although how it traverses the ER membrane has not been established . Using yeast mutants defective in various aspects of the ER-associated protein degradation (ERAD) pathway, we show that RTA introduced into the yeast ER subverts this pathway to enter the cytosol via the Sec61p translocon . A significant proportion of the exported RTA avoided proteasomal degradation . These data are consistent with the contention that the RTA component from ricin endocytosed by mammalian cells may likewise exploit ERAD to translocate into the cytosol.

Curr Opin Microbiol, 1999 Oct, 2(5), 548 - 54
Yeast genome evolution in the post-genome era; Seoighe C et al.; The Saccharomyces cerevisiae genome sequence, augmented by new data on gene expression and function, continues to yield new findings about eukaryote genome evolution . Analysis of the duplicate gene pairs formed by whole-genome duplication indicates that selection for increased levels of gene expression was a significant factor determining which genes were retained as duplicates and which were returned to a single-copy state, possibly in addition to selection for novel gene functions . Proteome comparisons between worm and yeast show that genes for core metabolic processes are shared among eukaryotes and unchanging in function, while comparisons between different yeast species identify 'orphan' genes as the most rapidly evolving fraction of the proteome . Natural hybridisation among yeast species is frequent, but its long-term evolutionary significance is unknown.

Curr Opin Biotechnol, 1999 Oct, 10(5), 422 - 7
Applications of yeast in biotechnology: protein production and genetic analysis; Cereghino GP et al.; Improvements in yeast expression systems, coupled with the development of yeast surface display and refinements in two-hybrid methodology, are expanding the role of yeasts in the process of understanding and engineering eukaryotic proteins.

Biochemistry, 1999 Sep 14, 38(37), 11926 - 32
Mimics of yeast tRNAAsp and their recognition by aspartyl-tRNA synthetase; Wolfson AD et al.; Assuming that the L-shaped three-dimensional structure of tRNA is an architectural framework allowing the proper presentation of identity nucleotides to aminoacyl-tRNA synthetases implies that altered and/or simplified RNA architectures can fulfill this role and be functional substrates of these enzymes, provided they contain correctly located identity elements . In this work, this paradigm was submitted to new experimental verification . Yeast aspartyl-tRNA synthetase was the model synthetase, and the extent to which the canonical structural framework of cognate tRNAAsp can be altered without losing its ability to be aminoacylated was investigated . Three novel architectures recognized by the synthetase were found . The first resembles that of metazoan mitochondrial tRNASer lacking the D-arm . The second lacks both the D- and T-arms, and the 5'-strand of the amino acid acceptor arm . The third structure is a construct in which the acceptor and anticodon helices are joined by two connectors . Aspartylation specificity of these RNAs is verified by the loss of aminoacylation activity upon mutation of the putative identity residues . Kinetic data indicate that the first two architectures are mimics of the whole tRNAAsp molecule, while the third one behaves as an aspartate minihelix mimic . Results confirm the primordial role of the discriminator nucleotide G73 in aspartylation and demonstrate that neither a helical structure in the acceptor domain nor the presence of a D- or T-arm is mandatory for specific aspartylation, but that activity relies on the presence of the cognate aspartate GUC sequence in the anticodon loop.

EMBO J, 1999 Oct 1, 18(19), 5234 - 41
Apg10p, a novel protein-conjugating enzyme essential for autophagy in yeast; Shintani T et al.; Autophagy is a cellular process for bulk degradation of cytoplasmic components . The attachment of Apg12p, a modifier with no significant similarity to ubiquitin, to Apg5p is crucial for autophagy in yeast . This reaction proceeds in a ubiquitination-like manner, and requires Apg7p and Apg10p . Apg7p exhibits a considerable similarity to ubiquitin-activating enzyme (E1) and is found to activate Apg12p with ATP hydrolysis . Apg10p, on the other hand, shows no significant similarity to other proteins whose functions are known . Here, we show that after activation by Apg7p, Apg12p is transferred to the Cys-133 residue of Apg10p to form an Apg12p-Apg10p thioester . Cells expressing Apg10p(C133S) do not generate the Apg12p-Apg5p conjugate, which leads to defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I . These findings indicate that Apg10p is a new type of protein-conjugating enzyme that functions in the Apg12p-Apg5p conjugation pathway.

EMBO J, 1999 Oct 1, 18(19), 5216 - 25
Identification of the catalytic domains and their functionally critical arginine residues of two yeast GTPase-activating proteins specific for Ypt/Rab transport GTPases; Albert S et al.; Ypt/Rab proteins constitute the largest subfamily of the Ras superfamily of monomeric GTPases and are regulators of vesicular protein transport . Their slow intrinsic GTPase activity (10(-4)-10(-3) min(-1) at 30 degrees C) has to be accelerated to switch the active to the inactive conformation . We have identified the catalytic domain within the C-terminal halves of two yeast GTPase-activating proteins (GAPs), Gyp1p and Gyp7p, with specificity for Ypt/Rab GTPases . The catalytically active fragments of Gyp1p and Gyp7p were more active than the full-length proteins and accelerated the intrinsic GTP hydrolysis rates of their preferred substrates by factors of 4.5 x 10(4) and 7.8 x 10(5), respectively . The K(m) values for the Gyp1p and Gyp7p active fragments (143 and 42 microM, respectively) indicate that the affinities of those GAPs for their substrates are very low . The catalytic domains of Gyp1p and Gyp7p contain five invariant arginine residues; substitutions of only one of them (R343 in Gyp1p and R458 in the analogous position of Gyp7p) rendered the GAPs almost completely inactive . We suggest that Ypt/Rab-GAPs, like Ras- and Rho-GAPs, follow the same mode of action and provide a catalytic arginine ('arginine finger') in trans to accelerate the GTP hydrolysis rate of the transport GTPases.

J Biol Chem, 1999 Oct 8, 274(41), 29564 - 7
The TFB4 subunit of yeast TFIIH is required for both nucleotide excision repair and RNA polymerase II transcription; Feaver WJ et al.; The N-degron strategy has been used to generate a yeast strain harboring a temperature-sensitive allele of TFB4 (tfb4(td)), the gene that encodes the 37-kDa subunit of the transcription/repair factor TFIIH . The tfb4(td) strain was sensitive to UV radiation and is defective in nucleotide excision repair in vitro . The mutant strain was also found to be an inositol auxotroph due at least in part to an inability to properly induce expression of the INO1 gene . These results indicate that like other subunits of TFIIH, Tfb4 is required for both RNA polymerase II transcription and DNA repair.

J Biol Chem, 1999 Oct 8, 274(41), 29453 - 62
Yeast Rad54 promotes Rad51-dependent homologous DNA pairing via ATP hydrolysis-driven change in DNA double helix conformation; Petukhova G et al.; Saccharomyces cerevisiae RAD54 gene functions in the formation of heteroduplex DNA, a key intermediate in recombination processes . Rad54 is monomeric in solution, but forms a dimer/oligomer on DNA . Rad54 dimer/oligomer alters the conformation of the DNA double helix in an ATP-dependent manner, as revealed by a change in the DNA linking number in a topoisomerase I-linked reaction . DNA conformational alteration does not occur in the presence of non-hydrolyzable ATP analogues, nor when mutant rad54 proteins defective in ATP hydrolysis replace Rad54 . Accordingly, the Rad54 ATPase activity is shown to be required for biological function in vivo and for promoting Rad51-mediated homologous DNA pairing in vitro . Taken together, the results are consistent with a model in which a Rad54 dimer/oligomer promotes nascent heteroduplex joint formation via a specific interaction with Rad51 protein and an ability to transiently unwind duplex DNA.

Mol Gen Genet, 1999 Aug, 262(1), 145 - 53
Genetic evidence for interaction between components of the yeast 26S proteasome: combination of a mutation in RPN12 (a lid component gene) with mutations in RPT1 (an ATPase gene) causes synthetic lethality; Takeuchi J et al.; The 19S regulatory particle of the yeast 26S proteasome consists of six related ATPases (Rpt proteins) and at least 11 non-ATPase proteins (Rpn proteins) . RPN12 (formerly NIN1) encodes an Rpn component of the 19S regulatory particle and is essential for growth . To determine which subunit(s) of the 26S proteasome interact(s) with Rpn12, we attempted to screen for mutations that cause synthetic lethality in the presence of the rpn12-1 (formerly nin1-1) mutation . Among the candidates recovered was a new allele of RPT1 (formerly CIM5) . This mutant allele was designated rpt1-2; on its own this mutation caused no phenotypic change, whereas the rpn12-1 rpt1-2 double mutant was lethal, suggesting a strong interaction between Rpn12 and Rpt1 . The site of the rpt1-2 mutation was determined by DNA sequencing of the RPT1 locus retrieved from the mutant, and a single nucleotide alteration was found . This changes amino acid 446 of the RPT1 product from alanine to valine . The alanine residue is conserved in all Rpt proteins, except Rpt5, but no function has yet been assigned to the region that contains it . We propose that this region is necessary for Rpt1 to interact with Rpn12 . The terminal phenotype of the rpn12-1 rpt1-2 double mutant was not cell cycle specific, suggesting that in the double mutant cells the function of the 26S proteasome is completely eliminated, thereby inducing multiple defects in cellular functions.

Mol Biotechnol, 1999 Jun, 11(3), 213 - 20
Reconstitution of fibroblast growth factor receptor interactions in the yeast two hybrid system; Aloni-Grinstein R et al.; Fibroblast growth factors (FGF) activate their receptors through the formation of trimolecular complexes, composed of a ligand, a receptor, and a heparan sulfate oligosaccharide, all of which are members of particularly large families capable of multiple interactions in a combinatorial fashion . Understanding this large network of interactions not only presents a great challenge, but is practically beyond the capacity of most classical techniques routinely used to study ligand receptor interactions . We have used the yeast two hybrid system to study protein-protein interactions in the FGF family . Both ligand and receptor ectodomains are properly folded and functional in the yeast . Basic FGF (bFGF) expressed in the yeast dimerizes spontaneously . This self-assembly occurs at low affinity, which can be greatly enhanced by the introduction of heparin, supporting a defined role for heparin in bFGF dimerization . Screening a rat embryo cDNA library with bFGF in the yeast two hybrid system identified a short variant of FGF receptor 1, found most frequently in embryonal and tumor cells and which possesses affinity toward bFGF that is significantly greater than that of the more abundant, full-length receptor . We find the yeast two hybrid system, a most suitable alternative method for the analysis of growth factor-receptor interactions as well as for screening for novel interacting proteins and modulators of FGF and its receptors.

Mamm Genome, 1999 Oct, 10(10), 1000 - 4
Conservation of the Caenorhabditis elegans timing gene clk-1 from yeast to human: a gene required for ubiquinone biosynthesis with potential implications for aging; Vajo Z et al.; Mutations in the Caenorhabditis elegans gene clk-1 have a major effect on slowing development and increasing life span . The Saccharomyces cerevisiae homolog COQ7 encodes a mitochondrial protein involved in ubiquinone biosynthesis and, hence, is required for respiration and gluconeogenesis . In this study, RT-PCR and 5' RACE were used to isolate both human and mouse clk-1/COQ7 homologs . Human CLK-1 was mapped to Chr 16(p12-13.1) by Radiation Hybrid (RH) and fluorescence in situ hybridization (FISH) methods . The number and location of human CLK1 introns were determined, and the location of introns II and IV are the same as in C . elegans . Northern blot analysis showed that three different isoforms of CLK-1 mRNA are present in several tissues and that the isoforms differ in the amount of expression . The functional equivalence of human CLK-1 to the yeast COQ7 homolog was tested by introducing either a single or multicopy plasmid containing human CLK-1 cDNA into yeast coq7 deletion strains and assaying for growth on a nonfermentable carbon source . The human CLK-1 gene was able to functionally complement yeast coq7 deletion mutants . The protein similarities and the conservation of function of the CLK-1/clk-1/COQ7 gene products suggest a potential link between the production of ubiquinone and aging.

Vaccine, 1999 Aug 20, 18(1-2), 57 - 67
Over a decade of experience with a yeast recombinant hepatitis B vaccine; Assad S et al.; Experience with the yeast recombinant hepatitis B vaccine Engerix-B now exceeds 10 years . We reviewed published studies on this vaccine . These show the vaccine to be safe, causing mostly only minor local symptoms and to be highly immunogenic both in monitored clinical trials and under field conditions . Engerix-B consistently elicits high geometric mean antibody titres and a high protective efficacy has been established in three groups at high-risk of hepatitis B infection, homosexual men, institutionalised mentally handicapped subjects and neonates of chronic carrier mothers . The profile of the recombinant hepatitis B vaccine in certain high-risk groups and immuno compromised people is discussed . Finally we present updated post marketing surveillance data based on 496 million distributed doses of vaccine.

Genes Dev, 1999 Sep 15, 13(18), 2425 - 38
Luc7p, a novel yeast U1 snRNP protein with a role in 5' splice site recognition; Fortes P et al.; The characterization of a novel yeast-splicing factor, Luc7p, is presented . The LUC7 gene was identified by a mutation that causes lethality in a yeast strain lacking the nuclear cap-binding complex (CBC) . Luc7p is similar in sequence to metazoan proteins that have arginine-serine and arginine-glutamic acid repeat sequences characteristic of a family of splicing factors . We show that Luc7p is a component of yeast U1 snRNP and is essential for vegetative growth . The composition of yeast U1 snRNP is altered in luc7 mutant strains . Extracts of these strains are unable to support any of the defined steps of splicing unless recombinant Luc7p is added . Although the in vivo defect in splicing wild-type reporter introns in a luc7 mutant strain is comparatively mild, splicing of introns with nonconsensus 5' splice site or branchpoint sequences is more defective in the mutant strain than in wild-type strains . By use of reporters that have two competing 5' splice sites, a loss of efficient splicing to the cap proximal splice site is observed in luc7 cells, analogous to the defect seen in strains lacking CBC . CBC can be coprecipitated with U1 snRNP from wild-type, but not from luc7, yeast strains . These data suggest that the loss of Luc7p disrupts U1 snRNP-CBC interaction, and that this interaction contributes to normal 5' splice site recognition.

Genes Dev, 1999 Sep 15, 13(18), 2360 - 4
Activation of dormant origins of DNA replication in budding yeast; Santocanale C et al.; Eukaryotic genomes often contain more potential replication origins than are actually used during S phase . The molecular mechanisms that prevent some origins from firing are unknown . Here we show that dormant replication origins on the left arm of budding yeast chromosome III become activated when both passive replication through them is prevented and the Mec1/Rad53 checkpoint that blocks late-origin firing is inactivated . Under these conditions, dormant origins fire very late relative to other active origins . These experiments show that some dormant replication origins are competent to fire during S phase and that passage of a replication fork through such origins can inactivate them.

Cell, 1999 Sep 17, 98(6), 799 - 810
Yeast RNA polymerase II at 5 A resolution; Fu J et al.; Appropriate treatment of X-ray diffraction from an unoriented 18-heavy atom cluster derivative of a yeast RNA polymerase II crystal gave significant phase information to 5 A resolution . The validity of the phases was shown by close similarity of a 6 A electron density map to a 16 A molecular envelope of the polymerase from electron crystallography . Comparison of the 6 A X-ray map with results of electron crystallography of a paused transcription elongation complex suggests functional roles for two mobile protein domains: the tip of a flexible arm forms a downstream DNA clamp; and a hinged domain may serve as an RNA clamp, enclosing the transcript from about 8-18 residues upstream of the 3'-end in a tunnel.

Lett Appl Microbiol, 1999 Aug, 29(2), 77 - 80
Molybdate induces thermotolerance in yeast; Tiligada E et al.; Application of a mild heat pretreatment, performed by shifting cells from 27 degrees C to 37 degrees C led to the protection of yeast cells from death due to a subsequent extreme heat shock at 53 degrees C . The presence of cycloheximide inhibited this induction of thermotolerance, indicating the involvement of de novo protein . The phosphatase inhibitor sodium molybdate induced thermotolerance to the non-pretreated yeast cells . This induction of thermotolerance did not seem to depend upon de novo protein synthesis . Thus, acquisition of thermotolerance in yeast may involve a number of cellular mechanisms depending on the conditions the organism encounters at any particular time.

Biochemistry (Mosc), 1999 Aug, 64(8), 945 - 51
Development and activation of cyanide-resistant respiration in the yeast Yarrowia lipolytica; Medentsev AG et al.; Changes in respiratory activity and in the contents of adenine nucleotides (ATP, ADP, AMP) were studied in cells of the yeast Yarrowia lipolytica during the development of cyanide-resistant respiration . The transition of the yeast from the logarithmic to the stationary growth phase due to exhaustion of glucose was associated with decreased endogenous respiration and with the activation of a cyanide-resistant oxidase . Cyanide activated cell respiration during the stationary growth phase . The cyanide-resistant respiration was inhibited by benzohydroxamic acid (BHA), an inhibitor of the alternative oxidase . In the absence of cyanide, BHA had no effect on the cells which had the cyanide-resistant oxidase . This indicates that the cells do not use the alternative pathway in vivo . The decreased endogenous respiration of the cells was accompanied by decreased contents of adenine nucleotides . Addition of cyanide resulted in a sharp decrease in the content of ATP, in a twofold increase in the content of ADP, and in a fivefold increase in the content of AMP . In the absence of cyanide, BHA had virtually no effect on the contents of adenine nucleotides . The decreased rate of oxygen consumption during the transition of the cells to the stationary growth phase was caused by the decreased activity of the main cytochrome-containing respiratory chain (2,4-dinitrophenol (DNP) stimulated respiration) . The alternative oxidase was synthesized in the cell but was inactive . Cyanide stimulated respiration due to activation of the alternative oxidase via the AMP produced . The decrease in the cell content of ATP is suggested to be a factor inducing the synthesis of the alternative oxidase.

Hokkaido Igaku Zasshi, 1999 Sep, 74(5), 377 - 86
{Development and establishment of a yeast-based stop codon assay for detection of NF2 gene premature-terminating mutations}; Kobayashi H; Neurofibromatosis type 2 (NF2) is an autosomally inherited disorder, caused by a mutation in NF2 tumor suppressor gene on chromosome 22q12, being characterized by multiple intracranial tumors including schwannomas, meningiomas and ependymomas . The protein encoded by the NF2 gene has a similarity to ezrin, radixin and moesin (ERM) proteins that link membrane proteins to the cytoskeleton . It has been reported that the majority of NF2 gene mutations are nonsense mutations that result in a premature termination of translation . We have developed and established a yeast-based stop codon assay for detection of NF2 gene premature terminating mutations . This assay utilizes an autonomously replicating yeast vector that expresses an NF2::ADE2 chimera protein, which gives a normal white colony when a sample NF2 cDNA is homologously recombinated, while it gives a red colony when the sample cDNA contains a nonsense mutation . The assay gave 8.0 +/- 3.5 (mean +/- SD) background red colonies when tested on clinical samples which did not contain an NF2 gene mutation . A total of 16 schwannomas (including three NF2 cases) were tested by the assay . NF2 gene mutations were detected as red colonies more than 10% in 13 of the 16 cases . Sequence analyses of plasmids recovered from the red colonies showed single base substitutions giving stop codons in five cases, and base deletions leading to frameshift and premature termination in four . Additionally, in-frame exon skippings were found in three cases . One case that gave 14% red colonies did not show a clonal mutation . This study demonstrates that the newly established assay is capable of an efficient detection of nonsense mutations of NF2 gene in clinical samples.

Biol Chem, 1999 Jul-Aug, 380(7-8), 729 - 33
Felix Hoppe-Seyler Lecture 1999 . Cyclin dependent kinases and regulation of the fission yeast cell cycle; Nurse P; The cyclin dependent kinases (CDKs), formed by complexes between Cdc2p and the B-cyclins Cig2p and Cdc13p, have a central role in regulating the fission yeast cell cycle and maintaining genomic stability . The CDK Cig2p/Cdc2p controls the onset of S-phase and the CDK Cdc13p/Cdc2p controls the onset of mitosis and ensures that there is only one S-phase in each cell . Cdc13p/Cdc2p can replace Cig2p/Cdc2p forthe onset of S-phase, suggesting that the increasing activity of a single CDK during the cell cycle is sufficient to drive a cell in an orderly fashion into S-phase and into mitosis . If S-phase is incomplete, then inhibition of Cdc13p/ Cdc2p prevents cells with unreplicated DNA from undergoing a catastrophic entry into mitosis . Control of CDK activity is also important to allow cells to exit the cell cycle and accumulate in G1 in response to nutritional deprivation and the presence of pheromone.

J Mol Biol, 1999 Sep 17, 292(2), 345 - 59
Local folding coupled to RNA binding in the yeast ribosomal protein L30; Mao H et al.; The ribosomal protein L30 from yeast Saccharomyces cerevisiae auto-regulates its own synthesis by binding to a structural element in both its pre-mRNA and its mRNA . The three-dimensional structures of L30 in the free (f L30) and the pre-mRNA bound (b L30) forms have been solved by nuclear magnetic resonance spectroscopy . Both protein structures contain four alternating alpha-helices and four beta-strands segments and adopt an overall topology that is an alphabetaalpha three-layer sandwich, representing a unique fold . Three loops on one end of the alphabetaalpha sandwich have been mapped as the RNA binding site on the basis of structural comparison, chemical shift perturbation and the inter-molecular nuclear Overhauser effects to the RNA . The structural and dynamic comparison of f L30 and b L30 reveals that local dynamics may play an important role in the RNA binding . The fourth helix in b L30 is longer than in f L30, and is stabilized by RNA binding . The exposed hydrophobic surface that is buried upon RNA binding may provide the energy necessary to drive secondary structure formation, and may account for the increased stability of b L30 .

J Mol Biol, 1999 Sep 10, 292(1), 65 - 73
Yeast Ty retrotransposons assemble into virus-like particles whose T-numbers depend on the C-terminal length of the capsid protein; AL-Khayat HA et al.; The virus-like particles (VLPs) produced by the yeast Ty retrotransposons are structurally and functionally related to retroviral cores . Using cryo-electron microscopy (cryo-EM) and three-dimensional (3D) reconstruction, we have examined the structures of VLPs assembled from full-length and truncated forms of the capsid structural protein . The VLPs are highly polydisperse in their radius distribution . We have found that the length of the C-terminal region of the capsid structural protein dictates the T -number, and thus the size, of the assembled particles . Each construct studied appears to assemble into at least two or three size classes, with shorter C termini giving rise to smaller particles . This assembly property provides a model for understanding the variable assembly of retroviral core proteins . The particles are assembled from trimer-clustered units and there are holes in the capsid shells .

Eur J Biochem, 1999 Sep, 264(3), 840 - 7
Comparison of the chemical mechanisms of action of yeast and equine liver alcohol dehydrogenase; Leskovac V et al.; The pH-dependence of the steady-state kinetic parameters and the ligand-binding parameters for competitive dead-end inhibitors for the yeast alcohol dehydrogenase (EC 1.1.1.1, constitutive, cytoplasmic) reaction was studied in the pH range 6-10 . These studies were designed in order to assign the appropriate pKa values to all dissociation forms of enzyme in the chemical mechanism of action for the yeast enzyme, previously proposed by Cook and Cleland {P . F . Cook & W . W . Cleland (1981) Biochemistry 20, 1796-1816} . In addition, the chemical mechanism of action for the yeast enzyme, proposed in this work, was compared with a similar mechanism of action for the horse liver enzyme, proposed by Cook and Cleland . Substantial differences were found, especially in the binding of coenzymes and in the structure of enzyme-coenzyme complexes.

Eur J Biochem, 1999 Sep, 264(2), 545 - 53
Association between the endoplasmic reticulum and mitochondria of yeast facilitates interorganelle transport of phospholipids through membrane contact; Achleitner G et al.; Membrane association between mitochondria and the endoplasmic reticulum of the yeast Saccharomyces cerevisiae is probably a prerequisite for phospholipid translocation between these two organelles . This association was visualized by fluorescence microscopy and computer-aided three-dimensional reconstruction of electron micrographs from serial ultrathin sections of yeast cells . A mitochondria-associated membrane (MAM), which is a subfraction of the endoplasmic reticulum, was isolated and re-associated with mitochondria in vitro . In the reconstituted system, phosphatidylserine synthesized in MAM was imported into mitochondria independently of cytosolic factors, bivalent cations, ATP, and ongoing synthesis of phosphatidylserine . Proteolysis of mitochondrial surface proteins by treatment with proteinase K reduced the capacity to import phosphatidylserine . Phosphatidylethanolamine formed in mitochondria by decarboxylation of phosphatidylserine is exported to the endoplasmic reticulum where part of it is converted into phosphatidylcholine . In contrast with previous observations with permeabilized yeast cells {Achleitner, G., Zweytick, D., Trotter, P., Voelker, D . & Daum, G . (1995) J . Biol . Chem . 270, 29836-29842}, export of phosphatidylethanolamine from mitochondria to the endoplasmic reticulum was shown to be energy-independent in the reconstituted yeast system.

Mol Cell Biol, 1999 Oct, 19(10), 7228 - 36
Association of fission yeast Orp1 and Mcm6 proteins with chromosomal replication origins; Ogawa Y et al.; We have previously shown that replication of fission yeast chromosomes is initiated in distinct regions . Analyses of autonomous replicating sequences have suggested that regions required for replication are very different from those in budding yeast . Here, we present evidence that fission yeast replication origins are specifically associated with proteins that participate in initiation of replication . Most Orp1p, a putative subunit of the fission yeast origin recognition complex (ORC), was found to be associated with chromatin-enriched insoluble components throughout the cell cycle . In contrast, the minichromosome maintenance (Mcm) proteins, SpMcm2p and SpMcm6p, encoded by the nda1(+)/cdc19(+) and mis5(+) genes, respectively, were associated with chromatin DNA only during the G(1) and S phases . Immunostaining of spread nuclei showed SpMcm6p to be localized at discrete foci on chromatin during the G(1) and S phases . A chromatin immunoprecipitation assay demonstrated that Orp1p was preferentially localized at the ars2004 and ars3002 origins of the chromosome throughout the cell cycle, while SpMcm6p was associated with these origins only in the G(1) and S phases . Both Orp1p and SpMcm6p were associated with a 1-kb region that contains elements required for autonomous replication of ars2004 . The results suggest that the fission yeast ORC specifically interacts with chromosomal replication origins and that Mcm proteins are loaded onto the origins to play a role in initiation of replication.

Mol Cell Biol, 1999 Oct, 19(10), 6872 - 90
Functional characterization of rpn3 uncovers a distinct 19S proteasomal subunit requirement for ubiquitin-dependent proteolysis of cell cycle regulatory proteins in budding yeast; Bailly E et al.; By selectively eliminating ubiquitin-conjugated proteins, the 26S proteasome plays a pivotal role in a large variety of cellular regulatory processes, particularly in the control of cell cycle transitions . Access of ubiquitinated substrates to the inner catalytic chamber within the 20S core particle is mediated by the 19S regulatory particle (RP), whose subunit composition in budding yeast has been recently elucidated . In this study, we have investigated the cell cycle defects resulting from conditional inactivation of one of these RP components, the essential non-ATPase Rpn3/Sun2 subunit . Using temperature-sensitive mutant alleles, we show that rpn3 mutations do not prevent the G(1)/S transition but cause a metaphase arrest, indicating that the essential Rpn3 function is limiting for mitosis . rpn3 mutants appear severely compromised in the ubiquitin-dependent proteolysis of several physiologically important proteasome substrates . Thus, RPN3 function is required for the degradation of the G(1)-phase cyclin Cln2 targeted by SCF; the S-phase cyclin Clb5, whose ubiquitination is likely to involve a combination of E3 (ubiquitin protein ligase) enzymes; and anaphase-promoting complex targets, such as the B-type cyclin Clb2 and the anaphase inhibitor Pds1 . Our results indicate that the Pds1 degradation defect of the rpn3 mutants most likely accounts for the metaphase arrest phenotype observed . Surprisingly, but consistent with the lack of a G(1) arrest phenotype in thermosensitive rpn3 strains, the Cdk inhibitor Sic1 exhibits a short half-life regardless of the RPN3 genotype . In striking contrast, Sic1 turnover is severely impaired by a temperature-sensitive mutation in RPN12/NIN1, encoding another essential RP subunit . While other interpretations are possible, these data strongly argue for the requirement of distinct RP subunits for efficient proteolysis of specific cell cycle regulators . The potential implications of these data are discussed in the context of possible Rpn3 function in multiubiquitin-protein conjugate recognition by the 19S proteasomal regulatory particle.

Mol Cell Biol, 1999 Oct, 19(10), 6720 - 8
A transcriptional switch in the expression of yeast tricarboxylic acid cycle genes in response to a reduction or loss of respiratory function; Liu Z et al.; The Hap2,3,4,5p transcription complex is required for expression of many mitochondrial proteins that function in electron transport and the tricarboxylic acid (TCA) cycle . We show that as the cells' respiratory function is reduced or eliminated, the expression of four TCA cycle genes, CIT1, ACO1, IDH1, and IDH2, switches from HAP control to control by three genes, RTG1, RTG2, and RTG3 . The expression of four additional TCA cycle genes downstream of IDH1 and IDH2 is independent of the RTG genes . We have previously shown that the RTG genes control the retrograde pathway, defined as a change in the expression of a subset of nuclear genes, e.g., the glyoxylate cycle CIT2 gene, in response to changes in the functional state of mitochondria . We show that the cis-acting sequence controlling RTG-dependent expression of CIT1 includes an R box element, GTCAC, located 70 bp upstream of the Hap2,3,4,5p binding site in the CIT1 upstream activation sequence . The R box is a binding site for Rtg1p-Rtg3p, a heterodimeric, basic helix-loop-helix/leucine zipper transcription factor complex . We propose that in cells with compromised mitochondrial function, the RTG genes take control of the expression of genes leading to the synthesis of alpha-ketoglutarate to ensure that sufficient glutamate is available for biosynthetic processes and that increased flux of the glyoxylate cycle, via elevated CIT2 expression, provides a supply of metabolites entering the TCA cycle sufficient to support anabolic pathways . Glutamate is a potent repressor of RTG-dependent expression of genes encoding both mitochondrial and nonmitochondrial proteins, suggesting that it is a specific feedback regulator of the RTG system.

Mol Cell Biol, 1999 Oct, 19(10), 6710 - 9
Yeast Upf proteins required for RNA surveillance affect global expression of the yeast transcriptome; Lelivelt MJ et al.; mRNAs are monitored for errors in gene expression by RNA surveillance, in which mRNAs that cannot be fully translated are degraded by the nonsense-mediated mRNA decay pathway (NMD) . RNA surveillance ensures that potentially deleterious truncated proteins are seldom made . NMD pathways that promote surveillance have been found in a wide range of eukaryotes . In Saccharomyces cerevisiae, the proteins encoded by the UPF1, UPF2, and UPF3 genes catalyze steps in NMD and are required for RNA surveillance . In this report, we show that the Upf proteins are also required to control the total accumulation of a large number of mRNAs in addition to their role in RNA surveillance . High-density oligonucleotide arrays were used to monitor global changes in the yeast transcriptome caused by loss of UPF gene function . Null mutations in the UPF genes caused altered accumulation of hundreds of mRNAs . The majority were increased in abundance, but some were decreased . The same mRNAs were affected regardless of which of the three UPF gene was inactivated . The proteins encoded by UPF-dependent mRNAs were broadly distributed by function but were underrepresented in two MIPS (Munich Information Center for Protein Sequences) categories: protein synthesis and protein destination . In a UPF(+) strain, the average level of expression of UPF-dependent mRNAs was threefold lower than the average level of expression of all mRNAs in the transcriptome, suggesting that highly abundant mRNAs were underrepresented . We suggest a model for how the abundance of hundreds of mRNAs might be controlled by the Upf proteins.

Mol Cell Biol, 1999 Oct, 19(10), 6575 - 84
Rpn9 is required for efficient assembly of the yeast 26S proteasome; Takeuchi J et al.; We have isolated the RPN9 gene by two-hybrid screening with, as bait, RPN10 (formerly SUN1), which encodes a multiubiquitin chain receptor residing in the regulatory particle of the 26S proteasome . Rpn9 is a nonessential subunit of the regulatory particle of the 26S proteasome, but the deletion of this gene results in temperature-sensitive growth . At the restrictive temperature, the Deltarpn9 strain accumulated multiubiquitinated proteins, indicating that the RPN9 function is needed for the 26S proteasome activity at a higher temperature . We analyzed the proteasome fractions separated by glycerol density gradient centrifugation by native polyacrylamide gel electrophoresis and found that a smaller amount of the 26S proteasome was produced in the Deltarpn9 cells and that the 26S proteasome was shifted to lighter fractions than expected . The incomplete proteasome complexes were found to accumulate in the Deltarpn9 cells . Furthermore, Rpn10 was not detected in the fractions containing proteasomes of the Deltarpn9 cells . These results indicate that Rpn9 is needed for incorporating Rpn10 into the 26S proteasome and that Rpn9 participates in the assembly and/or stability of the 26S proteasome.

Mol Cell Biol, 1999 Oct, 19(10), 6543 - 53
Genetic and physical interactions involving the yeast nuclear cap-binding complex; Fortes P et al.; Yeast strains lacking the yeast nuclear cap-binding complex (yCBC) are viable, although impaired in growth . We have taken advantage of this observation to carry out a genetic screen for components that show synthetic lethality (SL) with a cbp20-Delta cbp80-Delta double mutation . One set of SL interactions was due to mutations that were complemented by components of U1 small nuclear RNP (snRNP) and the yeast splicing commitment complex . These interactions confirm the role of yCBC in commitment complex formation . Physical interaction of yCBC with the commitment complex components Mud10p and Mud2p, which may directly mediate yCBC function, was demonstrated . Unexpectedly, we identified multiple SL mutations that were complemented by Cbf5p and Nop58p . These are components of the two major classes of yeast small nucleolar RNPs, which function in the maturation of rRNA precursors . Mutants lacking yCBC were found to be defective in rRNA processing . Analysis of the yCBC deletion phenotype suggests that this is likely to be due to a defect in the splicing of a subset of ribosomal protein mRNA precursors.

Biotechniques, 1999 Sep, 27(3), 520 - 5
Application of in situ PCR to yeast cells for screening YAC libraries; Aytay S et al.; We have used in situ PCR technology in yeast cells with the ultimate goal of cloning and screening genomic yeast artificial chromosome (YAC) libraries . The target sequences in YAC clones were amplified "in situ" in yeast cells by the same set of microsatellite primers used in solution-based PCR screening . The method is fast and sensitive and obviates the steps required for individual isolation of DNAs from hundreds to thousands of YAC clones and thus has an advantage over conventional solution-based PCR screening . This approach can conceivably be applied to the products of automated robotic workstations.

Mol Cell, 1999 Aug, 4(2), 187 - 97
Yeast Sec14p deficient in phosphatidylinositol transfer activity is functional in vivo; Phillips SE et al.; Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi secretory function . It is widely accepted, though unproven, that phosphatidylinositol transfer between membranes represents the physiological activity of phosphatidylinositol transfer proteins (PITPs) . We report that Sec14pK66,239A is inactivated for phosphatidylinositol, but not phosphatidylcholine (PC), transfer activity . As expected, Sec14pK66,239A fails to meet established criteria for a PITP in vitro and fails to stimulate phosphoinositide production in vivo . However, its expression efficiently rescues the lethality and Golgi secretory defects associated with sec14-1ts and sec14 null mutations . This complementation requires neither phospholipase D activation nor the involvement of a novel class of minor yeast PITPs . These findings indicate that PI binding/transfer is remarkably dispensable for Sec14p function in vivo.

J Biol Chem, 1999 Sep 24, 274(39), 27823 - 8
Yeast carboxyl-terminal domain kinase I positively and negatively regulates RNA polymerase II carboxyl-terminal domain phosphorylation; Patturajan M et al.; Monoclonal antibodies that recognize specific carboxyl-terminal domain (CTD) phosphoepitopes were used to examine CTD phosphorylation in yeast cells lacking carboxyl-terminal domain kinase I (CTDK-I) . We show that deletion of the kinase subunit CTK1 results in an increase in phosphorylation of serine in position 5 (Ser(5)) of the CTD repeat (Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7)) during logarithmic growth . This result indicates that CTDK-I negatively regulates CTD Ser(5) phosphorylation . We also show that CTK1 deletion (ctk1Delta) eliminates the transient increase in CTD serine 2 (Ser(2)) phosphorylation observed during the diauxic shift . This result suggests that CTDK-I may play a direct role in phosphorylating CTD Ser(2) in response to nutrient depletion . Northern blot analysis was used to show that genes normally induced during the diauxic shift are not properly induced in a ctk1Delta strain . Glycogen synthase (GSY2) and cytosolic catalase (CTT1) mRNA levels increase about 10-fold in wild-type cells, but this increase is not observed in ctk1Delta cells suggesting that increased message levels may require Ser(2) phosphorylation . Heat shock also induces Ser(2) phosphorylation, but we show here that this change in CTD modification and an accompanying induction of heat shock gene expression is independent of CTDK-I . The observation that SSA3/SSA4 expression is increased in ctk1Delta cells grown at normal temperature suggests a possible role for CTDK-I in transcription repression . We discuss several possible positive and negative roles for CTDK-I in regulating CTD phosphorylation and gene expression.

Yeast, 1999 Sep 15, 15(12), 1269 - 74
A strong carbon source effect is mediated by pUC plasmid sequences in a series of classical yeast vectors designed for promoter characterization; Pauwels K et al.; While using YIp356 and YEp356R lacZ reporter plasmids, we found lacZ expression driven by the ARG2 promoter to be much higher in cells grown on a non-glucose carbon source than in glucose-grown cells (5-10-fold higher on galactose and up to 40-fold higher on ethanol) . Furthermore, expression increased 30-fold upon shifting from a high-glucose to a low-glucose medium . This carbon source regulation requires Snf1p and possibly Ssn6p . It appears, however, to be artefactually mediated by plasmid sequences located upstream from the multicloning site . This emerged from the following observations: (a) the derepressive effect disappears if any extra piece of DNA is inserted upstream from the ARG2 promoter; and (b) similar derepression on low glucose is observed with another yeast promoter (ARG11), provided that the flanking 5' region is short . We determined that the cis-elements responsible for this physiologically irrelevant glucose regulation are located between positions 636 and 879 of the pUC18 DNA sequence .

Yeast, 1999 Sep 15, 15(12), 1171 - 81
A mutant for the yeast scERV1 gene displays a new defect in mitochondrial morphology and distribution; Becher D et al.; The yeast scERV1 gene is the best characterized representative of a new gene family found in different lower and higher eukaryotes . The gene product is essential for the yeast cell and has a complex influence on different aspects of mitochondrial biogenesis . The homologous mammalian ALR(Augmenter of Liver Regeneration) genes from man, mouse and rat are important at different developmental stages of the organism as, for example, in spermatogenesis and liver regeneration . In this study the influence of scERV1 on the morphology of mitochondria and its submitochondrial localization are investigated . A temperature-sensitive mutant of the gene was stained with a mitochondria-specific dye and fluorescence was inspected at the permissive and restrictive temperature . A new phenotype for morphological defects of mitochondria was identified . Already at the permissive temperature mitochondrial vesicles accumulate at defined positions in the cell . After shift to the restrictive temperature, morphological changes, and finally complete loss of mitochondrial structures, are observed . Ultrastructural studies confirm these findings and demonstrate the loss of the mitochondrial inner membrane and at the final stage a drastic reduction or complete absence of mitochondria from the cell . GFP fusion experiments with the scERV1 gene and subcellular localization by fractionation experiments identify the gene product inside mitoplasts and the cytosol . Re-investigation of the mutant phenotype demonstrates that after longer incubation of the mutant at the restrictive temperature an irreversible defect of the cells, even on glucose complete medium, is found that is in accordance with a complete loss or irreversible damage of mitochondria .

Genes Dev, 1999 Sep 1, 13(17), 2271 - 83
Fission yeast condensin complex: essential roles of non-SMC subunits for condensation and Cdc2 phosphorylation of Cut3/SMC4; Sutani T et al.; The condensin complex in frog extracts, containing two SMC (structural maintenance of chromosomes) and three non-SMC subunits, promotes mitotic chromosome condensation, and its supercoiling activity increases during mitosis by Cdc2 phosphorylation . Here, we report that fission yeast has the same five-member condensin complex, each of which is essential for mitotic condensation . The condensin complex was purified and the subunits were identified by microsequencing . Cnd1, Cnd2, and Cnd3, three non-SMC subunits showing a high degree of sequence conservation to frog subunits, are essential for viability, and their gene disruption leads to a phenotype indistinguishable from that observed in cut3-477 and cut14-208, known mutations in SMC4 and SMC2-like subunits . Condensin subunits tagged with GFP were observed to alter dramatically their localization during the cell cycle, enriched in the nucleus during mitosis, but cytoplasmic during other stages . This stage-specific alteration in localization requires mitosis-specific phosphorylation of the T19 Cdc2 site in Cut3 . The T19 site is phosphorylated in vitro by Cdc2 kinase and shows the maximal phosphorylation in metaphase in vivo . Its alanine substitution mutant fails to suppress the temperature-sensitive phenotype of cut3-477, and shows deficiency in condensation, probably because Cut3 T19A remains cytoplasmic . Therefore, direct Cdc2 phosphorylation of fission yeast condensin may facilitate its nuclear accumulation during mitosis.

Mol Gen Genet, 1999 Jul, 261(6), 977 - 84
TRI12, a trichothecene efflux pump from Fusarium sporotrichioides: gene isolation and expression in yeast; Alexander NJ et al.; Many of the genes involved in trichothecene toxin biosynthesis in Fusarium sporotrichioides are present within a gene cluster . Here we report the complete sequence for TRI12, a gene encoding a trichothecene efflux pump that is located within the trichothecene gene cluster of F . sporotrichioides . TRI12 encodes a putative polypeptide of 598 residues with sequence similarities to members of the major facilitator superfamily (MFS) and is predicted to contain 14 transmembrane-spanning segments . Disruption of TRI12 results in both reduced growth on complex media and reduced levels of trichothecene production . Growth of tri12 mutants on trichothecene-containing media is inhibited, suggesting that TRI12 may play a role in F . sporotrichioides self-protection against trichothecenes . Functional analysis of TRI12 was performed by expressing it in yeast strains that were co-transformed with a gene (TRI3) encoding a trichothecene 15-O-acetyltransferase . In the presence of the TRI3 substrate, 15-decalonectrin, cultures of yeast strains carrying TRI12 and TRI3 accumulated much higher levels of the acetylated product, calonectrin, than was observed for strains carrying TRI3 alone . PDR5, a transporter of the ABC superfamily, which is known to mediate trichothecene resistance in yeast, increased calonectrin accumulation in TRI12/TRI3 yeast strains but not in TRI3 strains . These results confirm the involvement of TRI12 in the trichothecene efflux associated with toxin biosynthesis, and demonstrate the usefulness of yeast as a host system for studies of MFS-type transporters.

Antibiot Khimioter, 1999, 44(4), 21 - 4
{Comparative cytotoxic characteristics of the interferon-inducing yeast RNA-tilorone complex}; Zholobak NM et al.; Cytotoxicity of the yeast RNA-tilorona molecular complex (MC) with interferonogenic properties and its influence on the DNA replicative synthesis were studied in experiments with human lymphocytes and 3 cell lines . It was shown that the MC doses of 25, 100 and 250 micrograms/ml were absolutely nontoxic for all the cell lines . The main parameters of the MC toxicity based on the cell viability were calculated . The parameters were found to correlate in the order of their magnitude with those relating to interferonogens of the polynucleotide nature . Within the dose ranges of 10 to 100 micrograms/ml the MC had a stimulating effect on replicative processes in the cells . It was concluded that the use of the MC as an inductor in large-scale manufacture of human and animal interferons of type 1 was promising.

J Biotechnol, 1999 May 28, 71(1-3), 133 - 41
Modeling and optimization of alpha-amylase production in a recombinant yeast fed-batch culture taking account of the cell cycle population distribution; Uchiyama K et al.; A simple mathematical model describing the cell cycle dependency of rice alpha-amylase production by a recombinant yeast was constructed to investigate the efficiency of cell cycle population control . First, the effects of the glucose concentration and cultivation temperature on the specific growth rate, the specific production rate of rice alpha-amylase, and the distribution of the cell cycle population were studied under balanced growth conditions . On the basis of the results, parameter values for the mathematical model were then estimated . The proposed model was shown to be applicable for unbalanced as well as balanced growth phases . The optimal control strategy in respect of temperature and glucose concentration for maximum rice alpha-amylase production, taking into account the cell cycle population, was determined and the result was compared with that obtained by a simple mathematical model in which cell cycle distribution was not considered . Finally, the effect of the initial population of each cell cycle phase on the final amount of the product under optimal operational conditions was investigated . The simulation and experimental data coincided well with each other, and the model was used to optimize the control strategy for maximum alpha-amylase production.

J Biotechnol, 1999 May 28, 71(1-3), 17 - 24
Modelling the effect of osmolality on the bursting strength of yeast cells; Zhang Z et al.; When yeast cells are resuspended in buffer prior to homogenisation, the diluent osmotic pressure can have a significant effect on cell mechanical strength . In this paper a model is proposed which describes the relationship between the cell bursting force and the osmotic pressure of the diluent, using chemical potential and force balance analyses . Yeast cells were exposed for 1 h to diluents with osmolalities varying from almost 0 to 700 mmol kg-1 before their bursting strengths were measured by micromanipulation . The experimental data were compared with the predictions made from the model and in general they were in good agreement . It is expected that this model might be used to understand cell disruption behaviour in downstream processing equipment such as homogenisers.

Plant Physiol, 1999 Sep, 121(1), 45 - 52
Roles of sugar alcohols in osmotic stress adaptation . Replacement of glycerol by mannitol and sorbitol in yeast; Shen B et al.; For many organisms there is a correlation between increases of metabolites and osmotic stress tolerance, but the mechanisms that cause this protection are not clear . To understand the role of polyols, genes for bacterial mannitol-1-P dehydrogenase and apple sorbitol-6-P dehydrogenase were introduced into a Saccharomyces cerevisiae mutant deficient in glycerol synthesis . Sorbitol and mannitol provided some protection, but less than that generated by a similar concentration of glycerol generated by glycerol-3-P dehydrogenase (GPD1) . Reduced protection by polyols suggested that glycerol had specific functions for which mannitol and sorbitol could not substitute, and that the absolute amount of the accumulating osmoticum might not be crucial . The retention of glycerol and mannitol/sorbitol, respectively, was a major difference . During salt stress, cells retained more of the six-carbon polyols than glycerol . We suggest that the loss of >98% of the glycerol synthesized could provide a safety valve that dissipates reducing power, while a similar high intracellular concentration of retained polyols would be less protective . To understand the role of glycerol in salt tolerance, salt-tolerant suppressor mutants were isolated from the glycerol-deficient strain . One mutant, sr13, partially suppressed the salt-sensitive phenotype of the glycerol-deficient line, probably due to a doubling of {K(+)} accumulating during stress . We compare these results to the "osmotic adjustment" concept typically applied to accumulating metabolites in plants . The accumulation of polyols may have dual functions: facilitating osmotic adjustment and supporting redox control.

Cell, 1999 Aug 20, 98(4), 501 - 12
Nuclear shuttling of yeast scaffold Ste5 is required for its recruitment to the plasma membrane and activation of the mating MAPK cascade; Mahanty SK et al.; Localization of Ste5 to GP at the plasma membrane is essential for transmission of the pheromone signal to associated MAP kinase cascade enzymes . Here, we show that this crucial localization requires prior shuttling of Ste5 through the nucleus . Ste5 shuttles through the nucleus constitutively during vegetative growth . Pheromone enhances nuclear export of Ste5, and this pool translocates vectorially to the cell periphery . Remarkably, Ste5 that cannot transit the nucleus is unable to localize at the periphery and activate the pathway, while Ste5 with enhanced transit through the nucleus has enhanced ability to localize to the periphery and activate the pathway . This novel regulatory scheme may ensure that cytoplasmic Ste5 does not activate downstream kinases in the absence of pheromone and could be applicable to other membrane-recruited signaling proteins.

Nucleic Acids Res, 1999 Oct 1, 27(19), 3921 - 30
The inefficient replication origin from yeast ribosomal DNA is naturally impaired in the ARS consensus sequence and in DNA unwinding; Miller CA et al.; Ribosomal DNA (rDNA) replication origins of Saccharomyces cerevisiae are known to function inefficiently, both in the context of the tandem rDNA repeats in the chromosome and as single copy autonomously replicating sequences (ARSs) in plasmids . Here we examined components of the rDNA ARS that might contribute to inefficient extrachromosomal replication . Like the efficient H4 ARS, the rDNA ARS requires a match to the 11 bp ARS consensus sequence (ACS) and a broad non-conserved region that may contain multiple elements, including a DNA unwinding element (DUE) . Using a single-strand-specific nuclease hypersensitivity assay and by determining the superhelical density required for stable DNA unwinding, we found that the DNA of the rDNA ARS is not as easily unwound as the H4 ARS . Unwinding of the rDNA ARS required additional energy, similar to the unwinding of mutations in the H4 ARS that stabilize the double helix in the DUE region and impair replication . In vivo extrachromosomal replication of the rDNA ARS was cold sensitive, like H4 ARS mutants that require additional energy to unwind the DUE region but unlike the easily unwound, wild-type H4 ARS . Impairment of replication function at reduced temperature suggests that the elevated energy requirement for DNA unwinding inherent in the wild-type rDNA ARS contributes to inefficient replication function . We also examined the essential ACS match in the rDNA ARS, which is known to be imperfect at one position . A point mutation in the essential ACS that corrects the imperfect match increased the efficiency of extrachromosomal replication . Our results reveal that the essential ACS element and DNA unwinding in the rDNA ARS are naturally impaired, suggesting that inefficient function of the rDNA replication origin has a biological purpose.

J Biol Chem, 1999 Sep 17, 274(38), 26962 - 7
The yeast transcription factor Mac1 binds to DNA in a modular fashion; Jamison McDaniels CP et al.; Mac1 is a metalloregulatory protein that regulates expression of the high affinity copper transport system in the yeast Saccharomyces cerevisiae . Under conditions of high copper concentration, Mac1 represses transcription of genes coding for copper transport proteins . Mac1 binds to DNA sequences called copper response elements (CuREs), which have the consensus sequence 5'-TTTGC(T/G)C(A/G)-3' . Mac1 contains two zinc binding sites, a copper binding site, and the sequence motif RGRP, which has been found in other proteins to mediate binding to the minor groove of A/T-rich sequences in DNA . We have used hydroxyl radical footprinting, missing nucleoside, and methylation interference experiments to investigate the structure of the complex of the DNA binding domain of Mac1 (called here Mac1(t)) with the two CuRE sites found in the yeast CTR1 promoter . We conclude from these experiments that Mac1(t) binds in a modular fashion to DNA, with its RGRP AT-hook motif interacting with the TTT sequence at the 5' end of the CTR1 CuRE site, and with another DNA-binding module(s) binding in the adjacent major groove in the GCTCA sequence.

J Biol Chem, 1999 Sep 17, 274(38), 26654 - 60
The yeast Hsp110 family member, Sse1, is an Hsp90 cochaperone; Liu XD et al.; In eukaryotes, production of the diverse repertoire of molecular chaperones during normal growth and in response to stress is governed by the heat shock transcription factor HSF . The HSC82 and HSP82 genes, encoding isoforms of the yeast Hsp90 molecular chaperone, were recently identified as targets of the HSF carboxyl-terminal activation domain (CTA), whose expression is required for cell cycle progression during prolonged heat stress conditions . In the present study, we have identified additional target genes of the HSF CTA, which include nearly all of the heat shock-inducible members of the Hsp90 chaperone complex, demonstrating coordinate regulation of these components by HSF . Heat shock induction of SSE1, encoding a member of the Hsp110 family of heat shock proteins, was also dependent on the HSF CTA . Disruption of SSE1 along with STI1, encoding an established subunit of the Hsp90 chaperone complex, resulted in a severe synthetic growth phenotype . Sse1 associated with partially purified Hsp90 complexes and deletion of the SSE1 gene rendered cells susceptible to the Hsp90 inhibitors macbecin and geldanamycin, suggesting functional interaction between Sse1 and Hsp90 . Sse1 is required for function of the glucocorticoid receptor, a model substrate of the Hsp90 chaperone machinery, and Hsp90-based repression of HSF under nonstress conditions . Taken together, these data establish Sse1 as an integral new component of the Hsp90 chaperone complex in yeast.

Hokkaido Igaku Zasshi, 1999 Jul, 74(4), 301 - 14
{Establishment of mouse p53 yeast functional assay and evaluation of its detectability of p53 gene mutation}; Tsunematsu I; Mice have widely been used as an experimental model for carcinogenesis induced by chemicals or irradiation . Recently, transgenic mice expressing oncogene proteins and knockout mice lacking for tumor suppressor genes are available, and used for the analysis of mechanisms underlying carcinogenesis . In such experimental carcinogenesis, a rapid and sensitive method for screening p53 mutations is desired . In human carcinogenesis, p53 yeast functional assay has been proved to be a very useful method for screening p53 mutations . However, the p53 yeast functional assay has been unsuccessful in mice because of the high background mutations in them . In the present study, the author developed a mouse p53 yeast functional assay by reducing the background mutations . Initially, 25.8 +/- 2.8% of background mutant red colonies were given by total RNA from normal mouse liver . The background level was lowered to 13.6 +/- 3.3% by improvement of RT-PCR conditions . Then the p53 cDNA-containing plasmids were rescued from the red colonies and the cDNA sequences were determined . The analysis revealed that many background mutations were caused by insertions of extra adenine (A) and thymine (T) at A and T homopolymeric runs, respectively . Majority of the insertion mutations occurred at 5' terminus of murine p53 cDNA . Based on these findings, we constructed a new vector and designed an optimal PCR primer set to exclude 5' terminal sequence in the yeast assay . Finally, the background mutation rate was reduced to 8.0 +/- 1.4%, which was comparable with the rate of 5.2 +/- 2.7% in human p53 yeast functional assay . Using the murine p53 yeast functional assay, we screened several cell lines for p53 mutations and determined those mutations by DNA sequencing . Furthermore, we investigated p53 mutations in UV-irradiated skins of XPC-gene-knockout mice . The yeast functional assay followed by DNA sequencing analysis revealed predominant mutations in dipyrimidines in the p53 coding sequence . These results indicate that mouse p53 yeast functional assay will be very useful for the analysis of p53 mutations in experimental carcinogenesis.

Hereditas, 1999, 130(2), 145 - 75
Environmental effects and the genetics of oviposition site preference for natural yeast substrates in Drosophila buzzatii
Barker JS, Starmer WT.
Habitat selection expressed as oviposition site preferences (OSP), is one component of the complex of behaviours of females seeking a place to oviposit . Drosophila buzzatii females lay their eggs in cactus necroses (rots), where the alternative oviposition sites are patches of adjacent or even partially mixed growing yeast species . The OSP exhibited by individual females is not absolute, but subject to environmental effects and the physiology of the fly, and may vary depending on the particular combination of yeast species present in a rot . Nevertheless, we have shown that OSP of D . buzzatii females is heritable, with evidence from variation among isofemale lines, direct estimation of heritability, generation means analysis and short term selection . Further, this genetic variation appears to be ubiquitous, polygenic and largely non-additive for all yeast species combinations . The consequences of such genotype-specific habitat selection for the maintenance of genetic variation are considered by an evaluation of our results in comparison with assumptions of models of habitat selection . As all assumed mechanisms of these models are apparently met, OSP for yeast species would seem to be a powerful force for the maintenance of genetic variation, and not only at loci affecting the choice of oviposition sites.

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