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| Mol Biol Evol, 2000 Aug, 17(8), 1210 - 9 Distribution and abundance of microsatellites in the yeast genome can Be explained by a balance between slippage events and point mutations; Kruglyak S et al.; We fit a Markov chain model of microsatellite evolution introduced by Kruglyak et al . to data on all di-, tri-, and tetranucleotide repeats in the yeast genome . Our results suggest that many features of the distribution of abundance and length of microsatellites can be explained by this simple model, which incorporates a competition between slippage events and base pair substitutions, with no need to invoke selection or constraints on the lengths . Our results provide some new information on slippage rates for individual repeat motifs, which suggest that AT-rich trinucleotide repeats have higher slippage rates . As our model predicts, we found that many repeats were adjacent to shorter repeats of the same motif . However, we also found a significant tendency of microsatellites of different motifs to cluster. J Biol Chem, 2000 Oct 13, 275(41), 31641 - 7 Characterization of determinants for the specificity of Arabidopsis thioredoxins h in yeast complementation; Brehelin C et al.; The disruption of the two thioredoxin genes in Saccharomyces cerevisiae leads to a complex phenotype, including the inability to use methionine sulfoxide as sulfur source, modified cell cycle parameters, reduced H(2)O(2) tolerance, and inability to use sulfate as sulfur source . Expression of one of the multiple Arabidopsis thaliana thioredoxins h in this mutant complements only some aspects of the phenotype, depending on the expressed thioredoxin: AtTRX2 or AtTRX3 induce methionine sulfoxide assimilation and restore a normal cell cycle . In addition AtTRX2 also confers growth on sulfate but no H(2)O(2) tolerance . In contrast, AtTRX3 does not confer growth on sulfate but induces H(2)O(2) tolerance . We have constructed hybrid proteins between these two thioredoxins and show that all information necessary for sulfate assimilation is present in the C-terminal part of AtTRX2, whereas some information needed for H(2)O(2) tolerance is located in the N-terminal part of AtTRX3 . In addition, mutation of the atypical redox active site WCPPC to the classical site WCGPC restores some growth on sulfate . All these data suggest that the multiple Arabidopsis thioredoxins h originate from a totipotent ancestor with all the determinants necessary for interaction with the different thioredoxin target proteins . After duplications each member evolved by losing or masking some of the determinants. Semin Cell Dev Biol, 2000 Jun, 11(3), 173 - 9 Transport of proteins to the yeast vacuole: autophagy, cytoplasm-to-vacuole targeting, and role of the vacuole in degradation; Teter SA et al.; The vacuole/lysosome performs a central role in degradation . Proteins and organelles are transported to the vacuole by selective and non-selective pathways . Transport to the vacuole by autophagy is the primary mode for degradation of cytoplasmic constituents under starvation conditions . Autophagy overlaps mechanistically and genetically with a biosynthetic pathway termed Cvt (Cytoplasm-to-vacuole targeting) that operates under vegetative conditions to transport the resident vacuolar hydrolase aminopeptidase I (API) . API import has been dissected to reveal the action of a novel mechanism that transports cargo within double-membrane vesicles . Recent work has uncovered molecular components involved in autophagy and the Cvt pathway. Curr Genet, 2000 Jun, 37(6), 356 - 63 Phosphorylation is required for high-affinity binding of DBP, a yeast mitochondrial site-specific RNA binding protein; Li H et al.; All yeast mitochondrial mRNAs terminate at their 3' ends with a conserved dodecamer sequence, a site for high-affinity binding by DBP (dodecamer binding protein) . Using purified DBP, we show that binding requires an intact dodecamer site and is enhanced by the presence in an oligonucleotide of the immediate 4-5 upstream nucleotides . Binding affinity varied from 0.25 to 0.85 nM towards a set of RNA oligonucleotides containing messenger specific upstream sequences in addition to the dodecamer site . Furthermore, we show that phosphatase treatment of DBP abolishes its specific binding, indicating the involvement of reversible phosphorylation in the regulation of its binding activities . This finding will further our understanding of the mechanism of DBP in the regulation of RNA metabolism in yeast mitochondria. Mol Gen Genet, 2000 Jun, 263(5), 787 - 95 Telomere-based neo-Darwinian selection of yeast clonal subpopulations; Venditti S et al.; In Saccharomyces cerevisiae, imbalance of the genes coding for the heterochromatin components Sir3p and histone H4 (namely, overdosage of SIR3 and lack of one of the two genes coding for H4) causes modifications in telomere length and telomere sequence organization, favoring the insertion of Y' elements into a stably shortened (C1-3A)n repeat tract . We report here that the newly inserted Y' elements are unstable and are lost with high frequency, generating clonal subpopulations with short telomeres, as revealed by the analysis of a specific telomere (LIII) and of the overall population of telomeres . Moreover, the growth rates of the subpopulations with and without Y' elements on LIII are different, the Y'-less individuals reproducing 20% more slowly than individuals bearing Y' elements . When grown together with Y'-bearing individuals, the subpopulations with the normal LIII telomere (which are viable and genetically stable if grown alone) are rapidly competed out . Hence, genetic imbalance for the structural components of heterochromatin results in a complex and rapidly changing mixture of subpopulations in such cultures . Thus, in situations where subpopulations are allowed to compete, heterochromatin-based differential growth rates result in neo-Darwinian clonal selection. Mol Gen Genet, 2000 Jun, 263(5), 752 - 60 Characterisation of fission yeast alp11 mutants defines three functional domains within tubulin-folding cofactor B; Radcliffe PA et al.; The proper folding of tubulins prior to their incorporation into microtubules requires a group of conserved proteins called cofactors A to E . In fission yeast, homologues of these cofactors (at least B, D and E) are necessary for the biogenesis of microtubules and for cell viability . Here we show that the temperature-sensitive alp11-924 mutant, which is defective in the cofactor B homologue, contains an opal nonsense mutation, which results in the production of a truncated Alp11B protein (Alp11(1-118) . We isolated a tRNA(Trp) gene as a multicopy suppressor of this mutation, which rescues alp11-924 by read-through of the nonsense codon . The truncated Alp1-118 protein lacks the C-terminal half of Alp11B, consisting of a central coiled-coil region and the distal CLIP-170 domain found in a number of proteins involved in microtubule functions . Both of these domains are required for the maintenance of microtubule architecture in vivo . Detailed functional analyses lead us to propose that Alp11B comprises three functional domains: the N-terminal half executes the essential function, the central coiled-coil region is necessary for satisfactory maintenance of cellular alpha-tubulin levels, and the C-terminal CLIP-170 domain is required for efficient binding to alpha-tubulin. Biochem Biophys Res Commun, 2000 Jul 21, 274(1), 105 - 11 Hsl7p, the yeast homologue of human JBP1, is a protein methyltransferase; Lee JH et al.; The yeast protein Hsl7p is a homologue of Janus kinase binding protein 1, JBP1, a newly characterized protein methyltransferase . In this report, Hsl7p also is shown to be a methyltransferase . It can be crosslinked to {(3)H}S-adenosylmethionine and exhibits in vitro protein methylation activity . Calf histones H2A and H4 and bovine myelin basic protein were methylated by Hsl7p, whereas histones H1, H2B, and H3 and bovine cytochrome c were not . We demonstrated that JBP1 can complement Saccharomyces cerevisiae with a disrupted HSL7 gene as judged by a reduction of the elongated bud phenotype, and a point mutation in the JBP1 S-adenosylmethionine consensus binding sequence eliminated all complementation by JBP1 . Therefore, we conclude the yeast protein Hsl7p is a sequence and functional homologue of JBP1 . These data provide evidence for an intricate link between protein methylation and macroscopic changes in yeast morphology . Nucleic Acids Res, 2000 Aug 1, 28(15), 2873 - 81 The endonuclease activity of the yeast Dna2 enzyme is essential in vivo; Lee KH et al.; Dna2 is a multifunctional enzyme in yeast that possesses endonuclease activity well suited to remove RNA-DNA primers of Okazaki fragments, raising the question of whether endonuclease activity is essential for in vivo Dna2 function . Systematic site-directed mutations of amino acid residues in Saccharomyces cerevisiae DNA2 conserved in the central region of many eukaryotic DNA2 homologs allowed us to identify mutant dna2 alleles that were divided into three groups based on the viability of the mutant cells: (i) viable; (ii) inviable only when expression was repressed; (iii) inviable . Biochemical analyses of recombinant mutant Dna2 proteins isolated from the latter two groups revealed that they possessed normal ATPase/helicase activity, but were impaired in their endonuclease activity . Cells expressing mutant Dna2 enzymes partially impaired in endonuclease activity were viable, but were unable to grow when expression of their mutant Dna2 enzymes was further reduced . Their growth was restored when the mutant Dna2 proteins decreased in nuclease activity were induced to overexpress . In contrast, mutant Dna2 proteins lacking endonuclease activity did not allow cells to grow under any conditions tested . These in vivo and in vitro results demonstrate that the endonuclease activity of Dna2 is essential for Okazaki fragment processing. Nucleic Acids Res, 2000 Jul 15, 28(14), 2804 - 14 Recognition of protein coding genes in the yeast genome at better than 95% accuracy based on the Z curve; Zhang CT et al.; The Z curve is a three-dimensional space curve constituting the unique representation of a given DNA sequence in the sense that each can be uniquely reconstructed from the other . Based on the Z curve, a new protein coding gene-finding algorithm specific for the yeast genome at better than 95% accuracy has been proposed . Six cross-validation tests were performed to confirm the above accuracy . Using the new algorithm, the number of protein coding genes in the yeast genome is re-estimated . The estimate is based on the assumption that the unknown genes have similar statistical properties to the known genes . It is found that the number of protein coding genes in the 16 yeast chromosomes is </=5645, significantly smaller than the 5800-6000 which is widely accepted, and much larger than the 4800 estimated by another group recently . The mitochondrial genes were not included into the above estimate . A codingness index called the YZ score (YZ OE {0,1}) is proposed to recognize protein coding genes in the yeast genome . Among the ORFs annotated in the MIPS (Munich Information Centre for Protein Sequences) database, those recognized as non-coding by the present algorithm are listed in this paper in detail . The criterion for a coding or non-coding ORF is simply decided by YZ > 0.5 or YZ < 0.5, respectively . The YZ scores for all the ORFs annotated in the MIPS database have been calculated and are available on request by sending e-mail to the corresponding author. Nucleic Acids Res, 2000 Jul 15, 28(14), 2779 - 83 Requirement for the SRS2 DNA helicase gene in non-homologous end joining in yeast; Hegde V et al.; Mitotic cells experience double-strand breaks (DSBs) from both exogenous and endogenous sources . Since unrepaired DSBs can result in genome rearrangements or cell death, cells mobilize multiple pathways to repair the DNA damage . In the yeast Saccharomyces cerevisiae, mitotic cells preferentially use a homologous recombination repair pathway . However, when no significant homology to the DSB ends is available, cells utilize a repair process called non-homologous end joining (NHEJ), which can join ends with no homology through resection to uncover microhomologies of a few nucleotides . Although components of the homologous recombination repair system are also involved in NHEJ, the rejoining does not involve all of the homologous recombination repair genes . The SRS2 DNA helicase has been shown to be required for DSB repair when the homologous single-stranded regions are short . Here it is shown that SRS2 is also required for NHEJ, regardless of the cell mating type . Efficient NHEJ of sticky ends requires the Ku70 and Ku80 proteins and the silencing genes SIR2, SIR3 and SIR4 . However, NHEJ of blunt ends, while very inefficient, is not further reduced by mutations in YKU70, SIR2, SIR3, SIR4 or SRS2, suggesting that this rejoining process occurs by a different mechanism. Nucleic Acids Res, 2000 Jul 15, 28(14), 2690 - 4 Telomerase-dependent repeat divergence at the 3' ends of yeast telomeres; Forstemann K et al.; Yeast telomeres consist of approximately 300 nt of degenerate repeats with the consensus sequence G(2-3)(TG)(1-6) . We developed a method for the amplification of a genetically marked telomere by PCR, allowing precise length and sequence determination of the G-rich strand including the 3' terminus . We examined wild-type cells, telomerase RNA deficient cells and a strain deleted for YKU70, which encodes for a protein involved in telomere maintenance and DNA double strand break repair . The 3' end of the G-rich strand was found to be at a variable position within the telomeric repeat . No preference for either thymine or guanine as the 3' base was detected . Comparison of telomere sequences from clonal populations revealed that telomeres consist of a centromere-proximal region of stable sequence and a distal region with differing degenerate repeats . In wild-type as well as yku70-Delta cells, variation in the degenerate telomeric repeats was detected starting 40-100 nt from the 3' end . Sequence divergence was abolished after deletion of the telomerase RNA gene . Thus, this region defines the domain where telomere shortening and telomerase-mediated extension occurs . Since this domain is much larger than the number of nucleo-tides lost per generation in the absence of telomerase, we propose that telomerase does not extend a given telomere in every cell cycle. Eur J Biochem, 2000 Aug, 267(15), 4809 - 16 Diazaborine resistance in yeast involves the efflux pumps Ycf1p and Flr1p and is enhanced by a gain-of-function allele of gene YAP1; Jungwirth H et al.; We have found that YAP1-mediated diazaborine resistance in the yeast Saccharomyces cerevisiae requires two efflux pumps, i.e . the major-facilitator-superfamily transporter Flr1p, which is located in the cytoplasmic membrane and the ATP-binding-cassette transporter Ycf1p which is present in the vacuolar membrane . Both these transporters are known to be under the control of the transcriptional transactivator Yap1p which explains our earlier finding that overexpression of YAP1 mediates diazaborine resistance . Overexpression of YAP1 in a Deltaflr1Deltaycf1 double disruptant strain does not mediate any diazaborine resistance, showing that these pumps are the only ones involved in detoxification of this drug . We also found a new mechanism of diazaborine resistance which is caused by an allelic form of YAP1, designated YAP1-11 . This allele of YAP1 carries a mutation that leads to a C620F exchange in the C-terminal cysteine-rich-domain region and is the first mutant of YAP1 that was isolated by a conventional genetic screen for drug resistance . The protein encoded by the gain-of-function allele may transactivate by a different mechanism from the wild-type protein when overexpressed because it does not enhance YCF1 mRNA and still mediates diazaborine resistance in a Deltaflr1Deltaycf1 background. Gene, 2000 Jul 11, 252(1-2), 127 - 35 Exploring redundancy in the yeast genome: an improved strategy for use of the cre-loxP system; Delneri D et al.; Gene families having more than three members are a common phenomenon in the Saccharomyces cerevisiae genome . As yeast research enters the post-genome era, the development of existing deletion strategies is crucial for tackling this apparent redundancy, hence a method for performing rapid multiple gene disruptions in this organism has been developed . We constructed three replacement cassettes in which different selectable markers were placed between two loxP loci . Multiple deletions (of members of a gene family) were generated, in one strain, using sequential integration of different replacement markers (kanMX, LYS2, KlURA3 and SpHIS5) . Their excision from the genome was performed simultaneously, as the final step, using a new cre recombinase vector, which carries the cycloheximide-resistance gene from Candida maltosa as a selectable marker . Our multiple gene deletion system significantly accelerates and facilitates the functional analysis process and is particularly useful for studying gene families in either laboratory or industrial yeast strains. J Biol Chem, 2000 Oct 6, 275(40), 31488 - 95 A method for determining the in vivo topology of yeast polytopic membrane proteins demonstrates that Gap1p fully integrates into the membrane independently of Shr3p; Gilstring CF et al.; The general amino acid permease (Gap1p) of Saccharomyces cerevisiae is an integral membrane protein that contains 12 hydrophobic regions predicted to be membrane-spanning segments . A topological reporter construct, encoding an internal 53-amino acid peptide of invertase (Suc2p) containing three Asp-X-Ser/Thr glycosylation sites, was inserted in-frame into the hydrophilic NH(2)- and COOH-terminal domains and each of the 11 hydrophilic loops that separate the 12 hydrophobic segments of Gap1p . The resulting 13 gene sandwich fusion proteins were expressed in a gap1Delta null mutant strain; 9 of these retain amino acid transport activity and are folded and correctly targeted to the plasma membrane . The glycosylation state of each of the fusion proteins was monitored; the results indicate that all 12 hydrophobic segments of Gap1p span the membrane, and the NH(2) and COOH termini are cytoplasmically oriented . These results were independently tested by isolating sealed right-side-out microsomes from sec12-1 strains expressing six different Gap1p constructs containing functional factor Xa protease cleavage sites . The pattern of factor Xa protease cleavage was found to be consistent with the presence of 12 membrane-spanning domains . Gap1p exhibited the same membrane topology in strains lacking Shr3p; therefore, Gap1p fully integrates into the ER membrane independently of this permease-specific packaging chaperone. J Biol Chem, 2000 Nov 3, 275(44), 34054 - 9 Targeting of aminopeptidase I to the yeast vacuole is mediated by Ssa1p, a cytosolic member of the 70-kDa stress protein family; Silles E et al.; The two cytosolic members of the highly conserved 70-kDa stress protein family, Ssa1p and Ssa2p, were specifically retained by the prepro-NH(2) extension of the vacuolar aminopeptidase I precursor (pAPI) conjugated to agarose (Sulfolink) . A temperature-sensitive mutant strain a1(ts)a234 (ssa1(ts) ssa2 ssa3 ssa4), when incubated at the restrictive temperature, was able to assemble the API precursor into dodecamers, but failed to pack pAPI into vesicles and to convert it into mature API (mAPI), a process that occurs in the vacuole . Altogether these results indicate that Ssa1p mediates the targeting of pAPI to the vacuole. Pac Symp Biocomput . 2000;:479-90. Cluster, function and promoter: analysis of yeast expression array; Zhu J et al.; Gene clusters could be derived based on expression profiles, function categorization and promoter regions . To obtain thorough understanding of gene expression and regulation, the three aspects should be combined in an organic way . In this study, we explored the possible ways to analyze the large-scale gene expression data . Three approaches were used to analyze yeast temporal expression data: 1) start from clustering on the expression profiles followed by function categorization and promoter analysis, 2) start from function categorization followed by clustering on expression profiles and promoter analysis, and 3) start from clustering on the promoter region followed by clustering on expression profiles . For clustering analysis on the time-series data, we developed a largest-first algorithm, which provide a mechanism for quality control on clusters . For promoter analysis, we developed a core-extension algorithm. Yeast, 2000 Jun 30, 17(2), 88 - 94 Yeast two-hybrid systems and protein interaction mapping projects for yeast and worm; Walhout AJ et al.; The availability of complete genome sequences necessitates the development of standardized functional assays to analyse the tens of thousands of predicted gene products in high-throughput experimental settings . Such approaches are collectively referred to as 'functional genomics' . One approach to investigate the properties of a proteome of interest is by systematic analysis of protein-protein interactions . So far, the yeast two-hybrid system is the most commonly used method for large-scale, high-throughput identification of potential protein-protein interactions . Here, we discuss several technical features of variants of the two-hybrid systems in light of data recently obtained from different protein interaction mapping projects for the budding yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans . Proc Natl Acad Sci U S A, 2000 Aug 1, 97(16), 9088 - 92 Transcription-coupled repair in yeast is independent from ubiquitylation of RNA pol II: implications for Cockayne's syndrome; Lommel L et al.; Cockayne's syndrome cells lack transcription-coupled nucleotide excision repair (TCR) and ubiquitylation of RNA polymerase II large subunit (RNA pol II LS), suggesting that ubiquitylation of RNA pol II LS may be necessary for TCR in eukaryotes . Rsp5 is the sole yeast ubiquitin-protein ligase that ubiquitylates RNA pol II LS in cells exposed to DNA-damaging agents . In yeast lacking functional Rsp5, there is no ubiquitylation of RNA pol II LS . We show here that removal, repression, or over-expression of Rsp5 has no effect on TCR, demonstrating that ubiquitylation of the RNA pol II LS is not required for TCR . We infer that the lack of ubiquitylation of RNA pol II LS in Cockayne's syndrome cells does not cause their defect in TCR. Arch Biochem Biophys, 2000 Aug 1, 380(1), 165 - 73 Interaction of alcohol dehydrogenase with tert-butylhydroperoxide: stimulation of the horse liver and inhibition of the yeast enzymes; Tkachenko AG et al.; Preincubation of horse liver alcohol dehydrogenase (HLADH) with the oxidative agent, tert-butyl hydroperoxide (tBOOH) results in a twofold stimulation of the ethanol dehydrogenase activity of this enzyme . This stimulation was dependent on tBOOH concentration up to 100 mM; above this concentration tBOOH did not further stimulate ethanol oxidation by HLADH . Active-site-directed reagents and classical ADH binary complexes were used to probe the possible mechanism of this activating effect . The rate and extent of stimulation by tBOOH is strongly reduced by binary complexes with NAD(+) or NADH, whose pyrophosphate groups bind to Arg-47 and Arg-369 . In contrast stimulation by tBOOH was not prevented by AMP or the sulfhydryl reagents dithiothreitol and glutathione, suggesting, respectively, a lack of role for Lys-228 and sulfhydryl group oxidation in the stimulation by tBOOH . In contrast to the liver enzyme, treatment of yeast ADH (YADH) with tBOOH irreversibly inhibited its ethanol dehydrogenase activity . Inhibition of YADH by tBOOH approximated first-order rate kinetics with respect to enzyme at fixed concentrations of tBOOH between 0.5 to 300 mM . Four -SH groups per molecule of YADH were modified by tBOOH, whereas only two -SH groups were modified in HLADH . The stimulation of HLADH by tBOOH is suggested to be due to destabilization of the catalytic Zn-coordination sphere and amino acids associated with coenzyme binding in the active site, while inactivation of YADH appears to be associated with -SH group oxidation by the peroxide . EMBO J, 2000 Jul 17, 19(14), 3831 - 40 Telomere-led bouquet formation facilitates homologous chromosome pairing and restricts ectopic interaction in fission yeast meiosis; Niwa O et al.; A polarized chromosomal arrangement with clustered telomeres in a meiotic prophase nucleus is often called bouquet and is thought to be important for the pairing of homologous chromosomes . Fluorescence in situ hybridization in fission yeast indicated that chromosomal loci are positioned in an ordered manner as anticipated from the bouquet arrangement . Blocking the formation of the telomere cluster with the kms1 mutation created a disorganized chromosomal arrangement, not only for the regions proximal to the telomere but also for interstitial regions . The kms1 mutation also affected the positioning of a linear minichromosome . Consistent with this cytological observation, the frequency of ectopic homologous recombination between a linear minichromosome and a normal chromosome increased in the kms1 background . Intragenic recombination between allelic loci is reduced in the kms1 mutant, but those between non-allelic loci are unaffected or slightly increased . Thus, telomere-led chromosome organization facilitates homologous pairing and also restricts irregular chromosome pairing during meiosis. EMBO J, 2000 Jul 17, 19(14), 3822 - 30 Inverted Alu repeats unstable in yeast are excluded from the human genome; Lobachev KS et al.; The nearly one million ALU: repeats in human chromosomes are a potential threat to genome integrity . ALU:s form dense clusters where they frequently appear as inverted repeats, a sequence motif known to cause DNA rearrangements in model organisms . Using a yeast recombination system, we found that inverted ALU: pairs can be strong initiators of genetic instability . The highly recombinagenic potential of inverted ALU: pairs was dependent on the distance between the repeats and the level of sequence divergence . Even inverted ALU:s that were 86% homologous could efficiently stimulate recombination when separated by <20 bp . This stimulation was independent of mismatch repair . Mutations in the DNA metabolic genes RAD27 (FEN1), POL3 (polymerase delta) and MMS19 destabilized widely separated and diverged inverted ALU:s . Having defined factors affecting inverted ALU: repeat stability in yeast, we analyzed the distribution of ALU: pairs in the human genome . Closely spaced, highly homologous inverted ALU:s are rare, suggesting that they are unstable in humans . ALU: pairs were identified that are potential sites of genetic change. EMBO J, 2000 Jul 17, 19(14), 3778 - 87 Distinct roles of two Yth1p domains in 3'-end cleavage and polyadenylation of yeast pre-mRNAs; Barabino SM et al.; Yth1p is the yeast homologue of the 30 kDa subunit of mammalian cleavage and polyadenylation specificity factor (CPSF) . The protein is part of the cleavage and polyadenylation factor CPF, which includes cleavage factor II (CF II) and polyadenylation factor I (PF I), and is required for both steps in pre-mRNA 3'-end processing . Yth1p is an RNA-binding protein that was previously shown to be essential for polyadenylation . Here, we demonstrate that Yth1p is also required for the cleavage reaction and that two protein domains have distinct roles in 3'-end processing . The C-terminal part is required in polyadenylation to tether Fip1p and poly(A) polymerase to the rest of CPF . A single point mutation in the highly conserved second zinc finger impairs both cleavage and polyadenylation, and affects the ability of Yth1p to interact with the pre-mRNA and other CPF subunits . Finally, we find that Yth1p binds to CYC1 pre-mRNA in the vicinity of the cleavage site . Our results indicate that Yth1p is important for the integrity of CPF and participates in the recognition of the cleavage site. EMBO J, 2000 Jul 17, 19(14), 3750 - 61 The forkhead protein Fkh2 is a component of the yeast cell cycle transcription factor SFF; Pic A et al.; In the yeast Saccharomyces cerevisiae, the MADS-box protein Mcm1, which is highly related to mammalian SRF (serum response factor), forms a ternary complex with SFF (Swi five factor) to regulate the cell cycle expression of genes such as SWI5, CLB2 and ACE2 . Here we show that the forkhead protein Fkh2 is a component of SFF and is essential for ternary complex formation on the SWI5 and ACE2 promoters . Fkh2 is essential for the correct cell cycle periodicity of SWI5 and CLB2 gene expression and is phosphorylated with a timing that is consistent with a role in this expression . Furthermore, investigation of the relationship between Fkh2 and a related forkhead protein Fkh1 demonstrates that these proteins act in overlapping pathways to regulate cell morphology and cell separation . This is the first example of a eukaryotic transcription factor complex containing both a MADS-box and a forkhead protein, and it has important implications for the regulation of mammalian gene expression. EMBO J, 2000 Jul 17, 19(14), 3657 - 67 Role of the spindle pole body of yeast in mediating assembly of the prospore membrane during meiosis; Knop M et al.; Spindle pole bodies (SPBs) are the centrosome equivalents in yeast, required for microtubule organization . In yeast, the SPB further serves as the attachment sites of the prospore membrane during meiosis . Here we report the identification of two new meiosis-specific components of the SPB, Mpc54p and Mpc70p, and the first protein specific for the prospore membrane, Don1p . Mpc54p and Mpc70p are not present in mitotic SPBs, and during meiosis II they are components of a meiosis-specific structural alteration of the outer plaque of the SPB . Both proteins are dispensable for the meiotic divisions but are essentially required for the formation of the prospore membrane . In the mpc54 and mpc70 mutants, the Don1p-containing precursors of the prospore membrane can still be found in the cytoplasm and associated with the SPB . Unexpectedly, however, the assembly of the precursors to a continuous membrane system is affected . Thus, the meiotic SPB is directly involved in the formation of a specialized membrane system, the membrane of the prospore. Curr Biol, 2000 Jun 29, 10(13), 809 - 12 The Est3 protein is a subunit of yeast telomerase; Hughes TR et al.; EST1, EST2, EST3 and TLC1 function in a single pathway for telomere replication in the yeast Saccharomyces cerevisiae {1} {2}, as would be expected if these genes all encode components of the same complex . Est2p, the reverse transcriptase protein subunit, and TLC1, the templating RNA, are subunits of the catalytic core of yeast telomerase {3} {4} {5} . In contrast, mutations in EST1, EST3 or CDC13 eliminate telomere replication in vivo {1} {6} {7} {8} but are dispensable for in vitro telomerase catalytic activity {2} {9} . Est1p and Cdc13p, as components of telomerase and telomeric chromatin, respectively, cooperate to recruit telomerase to the end of the chromosome {7} {10} . However, Est3p has not yet been biochemically characterized and thus its specific role in telomere replication is unclear . We show here that Est3p is a stable component of the telomerase holoenzyme and furthermore, association of Est3p with the enzyme requires an intact catalytic core . As predicted for a telomerase subunit, fusion of Est3p to the high affinity Cdc13p telomeric DNA binding domain greatly increases access of telomerase to the telomere . Est1p is also tightly associated with telomerase; however, Est1p is capable of forming a stable TLC1-containing complex even in the absence of Est2p or Est3p . Yeast telomerase therefore contains a minimum of three Est proteins for which there is both in vivo and in vitro evidence for their role in telomere replication as subunits of the telomerase complex. Science, 2000 Jul 14, 289(5477), 300 - 3 Requirement of the spindle checkpoint for proper chromosome segregation in budding yeast meiosis; Shonn MA et al.; The spindle checkpoint was characterized in meiosis of budding yeast . In the absence of the checkpoint, the frequency of meiosis I missegregation increased with increasing chromosome length, reaching 19% for the longest chromosome . Meiosis I nondisjunction in spindle checkpoint mutants could be prevented by delaying the onset of anaphase . In a recombination-defective mutant (spo11Delta), the checkpoint delays the biochemical events of anaphase I, suggesting that chromosomes that are attached to microtubules but are not under tension can activate the spindle checkpoint . Spindle checkpoint mutants reduce the accuracy of chromosome segregation in meiosis I much more than that in meiosis II, suggesting that checkpoint defects may contribute to Down syndrome. J Bacteriol, 2000 Aug, 182(15), 4188 - 97 HARO7 encodes chorismate mutase of the methylotrophic yeast Hansenula polymorpha and is derepressed upon methanol utilization; Krappmann S et al.; The HARO7 gene of the methylotrophic, thermotolerant yeast Hansenula polymorpha was cloned by functional complementation . HARO7 encodes a monofunctional 280-amino-acid protein with chorismate mutase (EC 5.4 . 99.5) activity that catalyzes the conversion of chorismate to prephenate, a key step in the biosynthesis of aromatic amino acids . The HARO7 gene product shows strong similarities to primary sequences of known eukaryotic chorismate mutase enzymes . After homologous overexpression and purification of the 32-kDa protein, its kinetic parameters (k(cat) = 319.1 s(-1), n(H) = 1.56, {S}(0.5) = 16.7 mM) as well as its allosteric regulatory properties were determined . Tryptophan acts as heterotropic positive effector; tyrosine is a negative-acting, heterotropic feedback inhibitor of enzyme activity . The influence of temperature on catalytic turnover and the thermal stability of the enzyme were determined and compared to features of the chorismate mutase enzyme of Saccharomyces cerevisiae . Using the Cre-loxP recombination system, we constructed mutant strains carrying a disrupted HARO7 gene that showed tyrosine auxotrophy and severe growth defects . The amount of the 0.9-kb HARO7 mRNA is independent of amino acid starvation conditions but increases twofold in the presence of methanol as the sole carbon source, implying a catabolite repression system acting on HARO7 expression. Nature, 2000 Jul 6, 406(6791), 90 - 4 Two yeast forkhead genes regulate the cell cycle and pseudohyphal growth; Zhu G et al.; There are about 800 genes in Saccharomyces cerevisiae whose transcription is cell-cycle regulated . Some of these form clusters of co-regulated genes . The 'CLB2' cluster contains 33 genes whose transcription peaks early in mitosis, including CLB1, CLB2, SWI5, ACE2, CDC5, CDC20 and other genes important for mitosis . Here we find that the genes in this cluster lose their cell cycle regulation in a mutant that lacks two forkhead transcription factors, Fkh1 and Fkh2 . Fkh2 protein is associated with the promoters of CLB2, SWI5 and other genes of the cluster . These results indicate that Fkh proteins are transcription factors for the CLB2 cluster . The fkh1 fkh2 mutant also displays aberrant regulation of the 'SIC1' cluster, whose member genes are expressed in the M-G1 interval and are involved in mitotic exit . This aberrant regulation may be due to aberrant expression of the transcription factors Swi5 and Ace2, which are members of the CLB2 cluster and controllers of the SIC1 cluster . Thus, a cascade of transcription factors operates late in the cell cycle . Finally, the fkh1 fkh2 mutant displays a constitutive pseudohyphal morphology, indicating that Fkh1 and Fkh2 may help control the switch to this mode of growth. J Chromatogr B Biomed Sci Appl, 2000 May 26, 742(1), 13 - 24 Identification of yeast species by fatty acid profiling as measured by gas-liquid chromatography; El Menyawi I et al.; An improved rapid method for the identification of yeasts and yeast-like fungi from clinical sources which is based on fatty acid profiles obtained by gas-liquid chromatography (GLC) is described . The fatty acid profile database is based upon internal standardisation and using the relative retention times and the retention index of the analysed fatty acids . Differentiation between yeast species was achieved by the quantitative and qualitative comparison of measured fatty acid profiles with those in the database . A total of 1024 clinical isolates were analysed by GLC to test the validity of the database . 96.2% of all tested samples were identified correctly to the species level by the improved GLC method. Mol Cell Biol, 2000 Aug, 20(15), 5777 - 87 Architecture of the replication fork stalled at the 3' end of yeast ribosomal genes; Gruber M et al.; Every unit of the rRNA gene cluster of Saccharomyces cerevisiae contains a unique site, termed the replication fork barrier (RFB), where progressing replication forks are stalled in a polar manner . In this work, we determined the positions of the nascent strands at the RFB at nucleotide resolution . Within an HpaI-HindIII fragment essential for the RFB, a major and two closely spaced minor arrest sites were found . In the majority of molecules, the stalled lagging strand was completely processed and the discontinuously synthesized nascent lagging strand was extended three bases farther than the continuously synthesized leading strand . A model explaining these findings is presented . Our analysis included for the first time the use of T4 endonuclease VII, an enzyme recognizing branched DNA molecules . This enzyme cleaved predominantly in the newly synthesized homologous arms, thereby specifically releasing the leading arm. Mol Cell Biol, 2000 Aug, 20(15), 5447 - 53 Shared roles of yeast glycogen synthase kinase 3 family members in nitrogen-responsive phosphorylation of meiotic regulator Ume6p; Xiao Y et al.; Nitrogen limitation activates meiosis and meiotic gene expression in yeast, but nitrogen-responsive signal transduction mechanisms that govern meiotic gene expression are poorly understood . We show here that Ume6p, a subunit of the Ume6p-Ime1p meiotic transcriptional activator, undergoes increased phosphorylation in vivo in response to nitrogen limitation . Phosphorylation depends on an N-terminal glycogen synthase kinase 3 (GSK3) target site in which substitutions cause reduced Ume6p-Ime1p interaction and meiotic gene expression, thus arguing that phosphorylation promotes functional Ume6p-Ime1p interaction . Phosphorylation of this site depends on two GSK3 homologs, Rim11p and Mck1p . Prior studies indicate that Rim11p phosphorylates both Ume6p and Ime1p in vitro and is required for Ume6p-Ime1p interaction, but no evidence has linked Mck1p function to Ume6p activity . Here we find that Mck1p-Ume6p interaction is detectable by two-hybrid assays and that meiosis in a partially defective rim11-K68R mutant is completely dependent on Mck1p . These findings argue that nitrogen limitation governs Rim11p/Mck1p-dependent phosphorylation of Ume6p, which in turn is required for Ume6p-Ime1p interaction and meiotic gene activation. Mol Cell Biol, 2000 Aug, 20(15), 5404 - 14 Stimulation of mitotic recombination events by high levels of RNA polymerase II transcription in yeast; Saxe D et al.; The impact of high levels of RNA polymerase II transcription on mitotic recombination was examined using lys2 recombination substrates positioned on nonhomologous chromosomes . Substrates were used that could produce Lys(+) recombinants by either a simple (noncrossover) gene conversion event or a crossover-associated recombination event, by only a simple gene conversion event, or by only a crossover event . Transcription of the lys2 substrates was regulated by the highly inducible GAL1-10 promoter or the low-level LYS2 promoter, with GAL1-10 promoter activity being controlled by the presence or absence of the Gal80p negative regulatory protein . Transcription was found to stimulate recombination in all assays used, but the level of stimulation varied depending on whether only one or both substrates were highly transcribed . In addition, there was an asymmetry in the types of recombination events observed when one substrate versus the other was highly transcribed . Finally, the lys2 substrates were positioned as direct repeats on the same chromosome and were found to exhibit a different recombinational response to high levels of transcription from that exhibited by the repeats on nonhomologous chromosomes . The relevance of these results to the mechanisms of transcription-associated recombination are discussed. Mol Cell Biol Res Commun, 2000 Apr, 3(4), 212 - 7 Identification of proteassemblin, a mammalian homologue of the yeast protein, Ump1p, that is required for normal proteasome assembly; Griffin TA et al.; We have identified a mammalian homologue of yeast Ump1p by searching for similar proteins in human and mouse expressed sequence tag (EST) databases . Ump1p is an accessory protein that is required for normal proteasome assembly in yeast (1) . A mammalian homologue, which we refer to as "proteassemblin," is a constituent of proteasome assembly intermediates (preproteasomes), but not fully assembled 20S proteasomes, as is Ump1p in yeast . We also provide evidence that proteassemblin is a constituent of pre-immunoproteasomes that contain the precursor of the interferon-gamma-inducible subunit LMP2 . By analogy with Ump1p, we hypothesize that proteassemblin is required for normal mammalian proteasome assembly . Cell Signal, 2000 Jun, 12(6), 381 - 90 Mkp1 of Pneumocystis carinii associates with the yeast transcription factor Rlm1 via a mechanism independent of the activation state; Fox D et al.; The mitogen-activated protein (MAP) kinase Mkp1 of the fungal pathogen Pneumocystis carinii is a functional MAP kinase that complements the loss of Slt2p, the MAP kinase component of the cell integrity pathway of Saccharomyces cerevisiae, and is activated within P . carinii in response to oxidative stress . Mkp1 displays an unusual feature in that it contains a phosphorylation motif repeat (TEYMTEY) within the activation loop not present in any other fungal MAPK identified to date . Mutagenesis of the T186,Y188 phosphorylation motif within the activation domain of Mkp1 results in the loss of detectable kinase activity but still retains partial complementation function . In addition to the ability of Mkp1 to restore partial activity to the cell integrity pathway in the absence of phosphorylatable residues within the activation loop, the association of Mkp1 with a substrate of Slt2p, the transcription factor Rlm1p, can also occur in the absence of MAP kinase activation . The results of this study suggest that the presence of phosphorylatable residues within the activation loop of Mkp1 is not absolutely required for functional (complementation) activity or for the association of Mkp1 with the transcription factor Rlm1p . In contrast, the catalytic lysine of the ATP-binding domain of Mkp1 is necessary for both complementation function and interaction with Rlm1p. Mol Biol Evol, 2000 Jul, 17(7), 1061 - 74 Evolution of cyclin-dependent kinases (CDKs) and CDK-activating kinases (CAKs): differential conservation of CAKs in yeast and metazoa; Liu J et al.; Cyclin-dependent kinases (CDKs) function as central regulators of both the cell cycle and transcription . CDK activation depends on phosphorylation by a CDK-activating kinase (CAK) . Different CAKs have been identified in budding yeast, fission yeast, and metazoans . All known CAKs belong to the extended CDK family . The sole budding yeast CAK, CAK1, and one of the two CAKs in fission yeast, csk1, have diverged considerably from other CDKs . Cell cycle regulatory components have been largely conserved in eukaryotes; however, orthologs of neither CAK1 nor csk1 have been identified in other species to date . To determine the evolutionary relationships of yeast and metazoan CAKs, we performed a phylogenetic analysis of the extended CDK family in budding yeast, fission yeast, humans, the fruit fly Drosophila melanogaster, and the nematode Caenorhabditis elegans . We observed that there were 10 clades for CDK-related genes, of which seven appeared ancestral, containing both yeast and metazoan genes . The four clades that contain CDKs that regulate transcription by phosphorylating the carboxyl-terminal domain (CTD) of RNA Polymerase II generally have only a single orthologous gene in each species of yeast and metazoans . In contrast, the ancestral cell cycle CDK (analogous to budding yeast CDC28) gave rise to a number of genes in metazoans, as did the ancestor of budding yeast PHO85 . One ancestral clade is unique in that there are fission yeast and metazoan members, but there is no budding yeast ortholog, suggesting that it was lost subsequent to evolutionary divergence . Interestingly, CAK1 and csk1 branch together with high bootstrap support values . We used both the relative apparent synapomorphy analysis (RASA) method in combination with the S-F method of sampling reduced character sets and gamma-corrected distance methods to confirm that the CAK1/csk1 association was not an artifact of long-branch attraction . This result suggests that CAK1 and csk1 are orthologs and that a central aspect of CAK regulation has been conserved in budding and fission yeast . Although there are metazoan CDK-family members for which we could not define ancestral lineage, our analysis failed to identify metazoan CAK1/csk1 orthologs, suggesting that if the CAK1/csk1 gene existed in the metazoan ancestor, it has not been conserved. Mol Biol Cell, 2000 Jul, 11(7), 2429 - 43 Luv1p/Rki1p/Tcs3p/Vps54p, a yeast protein that localizes to the late Golgi and early endosome, is required for normal vacuolar morphology; Conboy MJ et al.; We have characterized LUV1/RKI1/TCS3/VPS54, a novel yeast gene required to maintain normal vacuolar morphology . The luv1 mutant was identified in a genetic screen for mutants requiring the phosphatase calcineurin for vegetative growth . luv1 mutants lack a morphologically intact vacuole and instead accumulate small vesicles that are acidified and contain the vacuolar proteins alkaline phosphatase and carboxypeptidase Y and the vacuolar membrane H(+)-ATPase . Endocytosis appears qualitatively normal in luv1 mutants, but some portion (28%) of carboxypeptidase Y is secreted . luv1 mutants are sensitive to several ions (Zn(2+), Mn(2+), and Cd(2+)) and to pH extremes . These mutants are also sensitive to hygromycin B, caffeine, and FK506, a specific inhibitor of calcineurin . Some vacuolar protein-sorting mutants display similar drug and ion sensitivities, including sensitivity to FK506 . Luv1p sediments at 100,000 x g and can be solubilized by salt or carbonate, indicating that it is a peripheral membrane protein . A Green Fluorescent Protein-Luv1 fusion protein colocalizes with the dye FM 4-64 at the endosome, and hemagglutinin-tagged Luv1p colocalizes with the trans-Golgi network/endosomal protease Kex2p . Computer analysis predicts a short coiled-coil domain in Luv1p . We propose that this protein maintains traffic through or the integrity of the early endosome and that this function is required for proper vacuolar morphology. J Agric Food Chem, 2000 Jun, 48(6), 2062 - 70 Chemical speciation influences comparative activity of selenium-enriched garlic and yeast in mammary cancer prevention; Ip C et al.; A recent human intervention trial showed that daily supplementation with selenized yeast (Se-yeast) led to a decrease in the overall cancer morbidity and mortality by nearly 50%; past research has also demonstrated that selenized garlic (Se-garlic) is very effective in mammary cancer chemoprevention in the rat model . The goal of this study was to compare certain biological activities of Se-garlic and Se-yeast and to elucidate the differences based on the chemical forms of selenium found in these two natural products . Characterization of organic selenium compounds in yeast (1922 microg/g Se) and garlic (296 microg/g Se) was carried out by high-performance liquid chromatography with inductively coupled plasma mass spectrometry or with electrospray mass spectrometry . Analytical speciation studies showed that the bulk of the selenium in Se-garlic and Se-yeast is in the form of gamma-glutamyl-Se-methylselenocysteine (73%) and selenomethionine (85%), respectively . The above methodology has the sensitivity and capability to account for >90% of total selenium . In the rat feeding studies, supplementation of Se-garlic in the diet at different levels consistently caused a lower total tissue selenium accumulation when compared to Se-yeast . On the other hand, Se-garlic was significantly more effective in suppressing the development of premalignant lesions and the formation of adenocarcinomas in the mammary gland of carcinogen-treated rats . Given the present finding on the identity of selenomethionine and gamma-glutamyl-Se-methylselenocysteine as the major form of selenium in Se-yeast and Se-garlic, respectively, the metabolism of these two compounds is discussed in an attempt to elucidate how their disposition in tissues might account for the differences in cancer chemopreventive activity. Biosci Rep, 2000 Feb, 20(1), 41 - 9 Increased stability and catalytic efficiency of yeast hexokinase upon interaction with zwitterionic micelles . Kinetics and conformational studies; Guerra R et al.; The effect of ligands (glucose, ATP and Mg2+) and zwitterionic micelles of lysophosphatidylcholine (LPC) or N-hexadecyl-N,N-dimethyl-3-ammonium propanesulfonate (HPS) in the yeast hexokinase (HK) stability was studied at 35 degrees C . The thermal inactivation kinetics followed one-exponential decay . The effect of ligands on protecting the enzyme against inactivation followed the order: glucose > glucose/Mg2+ >ATP/Mg2+ approximately or approximately equal to Mg2+l approximately or approximately equal to buffer only . Both LPC and HPS micelles increased the enzyme stability only when the incubation medium contained glucose or glucose/Mg2+, suggesting that the protein conformation is a key prerequisite for the enzyme-micelle interaction to take place . This enzyme-micelle interaction resulted in an increased catalytic efficiency (with a decrease in Km for ATP and increase in Vmax as well as in changes on the tertiary (intrinsic fluorescence) structure of the yeast hexokinase. Yi Chuan Xue Bao, 2000, 27(3), 219 - 26 {Phylogenetic relationships and evolution of yeast-like symbionts of the small brown planthoppers based on partial 18S rDNA sequences}; Yan J et al.; The yeast-like symbionts are isolated and purified from the small brown planthoppers collected from five areas in China . The 18S rDNA sequences of them are determined . Combined with the sequences of other fungi, we have constructed a comprehensive phylogenetic tree . The results suggested that the taxonomic positions of the YLS are in the class Pyrenomycetes in the subphylum Ascomycotina and the YLS have the closest relationship with H . chrysospermus . The YLS of different areas in China and Japan may belong to different geographical populations of one species . We could also draw a conclusion that there are independent acquisitions of various symbionts in differentiated hosts. J Biol Chem, 2000 Sep 22, 275(38), 29193 - 9 Functional independence of the two cysteine-rich activation domains in the yeast Mac1 transcription factor; Keller G et al.; Mac1 is a transcriptional activator whose activity is inhibited by copper ions . Mutagenesis studies were carried out to map residues important in the copper inhibition of Mac1 activity . Seven new missense mutations were identified that resulted in copper-independent Mac1 transcriptional activation . All seven mutations were clustered in one of two C-terminal cysteine-rich motifs, designated the C1 motif . All but one of the constitutive Mac1 mutations occurred in one of the conserved six residues in the (264)CXC{(X)(4)}CXC{(X)(2)}C{(X)(2)}{H(279)}C1 motif . The lone exception was a L260S substitution . Two additional MAC1 mutations exhibiting constitutive activity were in-frame deletions encompassing portions C1 . Engineered mutations in the second cysteine-rich motif did not yield a constitutively active Mac1 . These results are consistent with the C1 motif being the copper-regulatory switch . Both cysteine-rich motifs exhibited transactivation activity, although the C1 activator was weak relative to the C2 activator . Limited copper metalloregulation of Mac1 was observed with only the C1 activator fused to the N-terminal DNA binding domain . Thus, the two Cys-rich motifs appear to function independently . The C1 motif appears to be a functional copper-regulatory domain. FEBS Lett, 2000 Jun 23, 475(3), 187 - 91 Heterocomplex formation between metastasis-related protein S100A4 (Mts1) and S100A1 as revealed by the yeast two-hybrid system; Tarabykina S et al.; S100A4 (Mts1) is a Ca(2+)-binding protein of the S100 family . This protein plays an important role in promoting tumor metastasis . In order to identify S100A4 interacting proteins, we have applied the yeast two-hybrid system as an in vivo approach . By screening a mouse mammary adenocarcinoma library, we have demonstrated that S100A4 forms a heterocomplex with S100A1, another member of the S100 family . The non-covalent heterodimerization was confirmed by fluorescence spectroscopy and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry . Mutational analysis revealed that replacement of Cys(76) and/or Cys(81) of S100A4 by Ser abolishes the S100A4/S100A1 heterodimerization, but does not affect the S100A4 homodimerization in vivo. Proc Natl Acad Sci U S A, 2000 Jul 5, 97(14), 7957 - 62 Genome-wide characterization of the Zap1p zinc-responsive regulon in yeast; Lyons TJ et al.; The Zap1p transcription factor senses cellular zinc status and increases expression of its target genes in response to zinc deficiency . Previously known Zap1p-regulated genes encode the Zrt1p, Zrt2p, and Zrt3p zinc transporter genes and Zap1p itself . To allow the characterization of additional genes in yeast important for zinc homeostasis, a systematic study of gene expression on the genome-wide scale was used to identify other Zap1p target genes . Using a combination of DNA microarrays and a computer-assisted analysis of shared motifs in the promoters of similarly regulated genes, we identified 46 genes that are potentially regulated by Zap1p . Zap1p-regulated expression of seven of these newly identified target genes was confirmed independently by using lacZ reporter fusions, suggesting that many of the remaining candidate genes are also Zap1p targets . Our studies demonstrate the efficacy of this combined approach to define the regulon of a specific eukaryotic transcription factor. Proc Natl Acad Sci U S A, 2000 Jul 5, 97(14), 7865 - 70 Modeling the fission yeast cell cycle: quantized cycle times in wee1- cdc25Delta mutant cells; Sveiczer A et al.; A detailed mathematical model for the fission yeast mitotic cycle is developed based on positive and negative feedback loops by which Cdc13/Cdc2 kinase activates and inactivates itself . Positive feedbacks are created by Cdc13/Cdc2-dependent phosphorylation of specific substrates: inactivating its negative regulators (Rum1, Ste9 and Wee1/Mik1) and activating its positive regulator (Cdc25) . A slow negative feedback loop is turned on during mitosis by activation of Slp1/anaphase-promoting complex (APC), which indirectly re-activates the negative regulators, leading to a drop in Cdc13/Cdc2 activity and exit from mitosis . The model explains how fission yeast cells can exit mitosis in the absence of Ste9 (Cdc13 degradation) and Rum1 (an inhibitor of Cdc13/Cdc2) . We also show that, if the positive feedback loops accelerating the G(2)/M transition (through Wee1 and Cdc25) are weak, then cells can reset back to G(2) from early stages of mitosis by premature activation of the negative feedback loop . This resetting can happen more than once, resulting in a quantized distribution of cycle times, as observed experimentally in wee1(-) cdc25Delta mutant cells . Our quantitative description of these quantized cycles demonstrates the utility of mathematical modeling, because these cycles cannot be understood by intuitive arguments alone. J Biol Chem, 2000 Oct 13, 275(41), 31972 - 8 Regulation of the yeast transcriptional factor PHO2 activity by phosphorylation; Liu C et al.; The induction of yeast Saccharomyces cerevisiae gene PHO5 expression is mediated by transcriptional factors PHO2 and PHO4 . PHO4 protein has been reported to be phosphorylated and inactivated by a cyclin-CDK (cyclin-dependent kinase) complex, PHO80-PHO85 . We report here that PHO2 can also be phosphorylated . A Ser-230 to Ala mutation in the consensus sequence (SPIK) recognized by cdc2/CDC28-related kinase in PHO2 protein led to complete loss of its ability to activate the transcription of PHO5 gene . Further investigation showed that the Pro-231 to Ser mutation inactivated PHO2 protein as well, whereas the Ser-230 to Asp mutation did not affect PHO2 activity . Since the PHO2 Asp-230 mutant mimics Ser-230-phosphorylated PHO2, we postulate that only phosphorylated PHO2 protein could activate the transcription of PHO5 gene . Two hybrid assays showed that yeast CDC28 could interact with PHO2 . CDC28 immunoprecipitate derived from the YPH499 strain grown under low phosphate conditions phosphorylated GST-PHO2 in vitro . A phosphate switch regulates the transcriptional activation activity of PHO2, and mutations of the (SPIK) site affect the transcriptional activation activity of PHO2 and the interaction between PHO2 and PHO4 . BIAcore(R) analysis indicated that the negative charge in residue 230 of PHO2 was sufficient to help PHO2 interact with PHO4 in vitro. Naturwissenschaften, 2000 May, 87(5), 236 - 40 Fungal spore germination into yeast or mycelium: possible implications of dimorphism in evolution and human pathogenesis; Ghormade V et al.; The ability of dimorphism in fungi is conventionally regarded as a reversible change between the two vegetative forms, yeast and mycelium, in response to environmental change . A zygomycetous isolate, Benjaminiella poitrasii, exhibited yeast-mycelium transition in response to the change in temperature (37-28 degrees C) and decrease in glucose concentration . For the first time the presence of dimorphic response during asexual and sexual spore germination is reported under the dimorphism-triggering conditions in B . poitrasii . The zygospores germinated into budding yeast when subjected to yeast-form supporting conditions . The mycelium-form favoring conditions gave rise to true mycelium . Similarly, the asexual spores displayed a dimorphic response during germination . Our observations suggest that dimorphism is an intrinsic ability present in the vegetative, asexual, and sexual forms of the fungus . As dimorphic fungi are intermediate to the unicellular yeast and the filamentous forms, understanding of the dimorphic character could be useful to trace the evolutionary relationships among taxonomically different fungi . Moreover, the implications of spore germination during the onset of pathogenesis and in drug development for human health care are discussed. J Biol Chem, 2000 Sep 22, 275(38), 29187 - 92 Yeast lacking superoxide dismutase(s) show elevated levels of "free iron" as measured by whole cell electron paramagnetic resonance; Srinivasan C et al.; A current hypothesis explaining the toxicity of superoxide anion in vivo is that it oxidizes exposed {4Fe-4S} clusters in certain vulnerable enzymes causing release of iron and enzyme inactivation . The resulting increased levels of "free iron" catalyze deleterious oxidative reactions in the cell . In this study, we used low temperature Fe(III) electron paramagnetic resonance (EPR) spectroscopy to monitor iron status in whole cells of the unicellular eukaryote, Saccharomyces cerevisiae . The experimental protocol involved treatment of the cells with desferrioxamine, a cell-permeant, Fe(III)-specific chelator, to promote oxidation of all of the "free iron" to the Fe(III) state wherein it is EPR-detectable . Using this method, a small amount of EPR-detectable iron was detected in the wild-type strain, whereas significantly elevated levels were found in strains lacking CuZn-superoxide dismutase (CuZn-SOD) (sod1 delta), Mn-SOD (sod2 delta), or both SODs, throughout their growth but particularly in stationary phase . The accumulation was suppressed by expression of wild-type human CuZn-SOD (in the sod1 delta mutant), by pmr1, a genetic suppressor of the sod delta mutant phenotype (in the sod1 delta sod2 delta double knockout strain), and by anaerobic growth . In wild-type cells, an increase in the EPR-detectable iron pool could be induced by treatment with paraquat, a redox-cycling drug that generates superoxide . Cells that were not pretreated with desferrioxamine had Fe(III) EPR signals that were equally as strong as those from treated cells, indicating that "free iron" accumulated in the ferric form in our strains in vivo . Our results indicate a relationship between superoxide stress and iron handling and support the above hypothesis for superoxide-related oxidative damage. Mol Cell, 2000 Mar, 5(3), 501 - 11 Exit from mitosis in budding yeast: biphasic inactivation of the Cdc28-Clb2 mitotic kinase and the role of Cdc20; Yeong FM et al.; Cdc20, an activator of the anaphase-promoting complex (APC), is also required for the exit from mitosis in Saccharomyces cerevisiae . Here we show that during mitosis, both the inactivation of Cdc28-Clb2 kinase and the degradation of mitotic cyclin Clb2 occur in two steps . The first phase of Clb2 proteolysis, which commences at the metaphase-to-anaphase transition when Clb2 abundance is high, is dependent on Cdc20 . The second wave of Clb2 destruction in telophase requires activation of the Cdc20 homolog, Hct1/Cdh1 . The first phase of Clb2 destruction, which lowers the Cdc28-Clb2 kinase activity, is a prerequisite for the second . Thus, Clb2 proteolysis is not solely mediated by Hct1 as generally believed; instead, it requires a sequential action of both Cdc20 and Hct1. Mol Cell, 2000 Mar, 5(3), 489 - 99 The yeast hnRNP-like protein Hrp1/Nab4 marks a transcript for nonsense-mediated mRNA decay; Gonzalez CI et al.; The nonsense-mediated mRNA decay (NMD) pathway monitors premature translation termination and degrades aberrant mRNAs . In yeast, it has been proposed that a surveillance complex searches 3' of a nonsense codon for a downstream sequence element (DSE) associated with RNA-binding proteins . An interaction between the complex and the DSE-binding protein(s) triggers NMD . Here we describe the identification and characterization of the Hrp1/Nab4 protein as a DSE-binding factor that activates NMD . Mutations in HRP1 stabilize nonsense-containing transcripts without affecting the decay of wild-type mRNAs . Hrp1p binds specifically to a DSE-containing RNA and interacts with Upf1p, a component of the surveillance complex . A mutation in HRP1 that stabilizes nonsense-containing mRNAs abolishes its affinity for the DSE and fails to interact with Upf1p . We present a model describing how Hrp1p marks a transcript for rapid decay. Mol Cell, 2000 May, 5(5), 865 - 76 Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast; Mossessova E et al.; Modification of cellular proteins by the ubiquitin-like protein SUMO is essential for nuclear processes and cell cycle progression in yeast . The Ulp1 protease catalyzes two essential functions in the SUMO pathway: (1) processing of full-length SUMO to its mature form and (2) deconjugation of SUMO from targeted proteins . Selective reduction of the proteolytic reaction produced a covalent thiohemiacetal transition state complex between a Ulp1 C-terminal fragment and its cellular substrate Smt3, the yeast SUMO homolog . The Ulp1-Smt3 crystal structure and functional testing of elements within the conserved interface elucidate determinants of SUMO recognition, processing, and deconjugation . Genetic analysis guided by the structure further reveals a regulatory element N-terminal to the proteolytic domain that is required for cell growth in yeast. Mol Cell, 2000 May, 5(5), 841 - 51 Compartmentalization of the cell cortex by septins is required for maintenance of cell polarity in yeast; Barral Y et al.; Formation and maintenance of specialized plasma membrane domains are crucial for many biological processes, such as cell polarization and signaling . During isotropic bud growth, the yeast cell periphery is divided into two domains: the bud surface, an active site of exocytosis and growth, and the relatively quiescent surface of the mother cell . We found that cells lacking septins at the bud neck failed to maintain the exocytosis and morphogenesis factors Spa2, Sec3, Sec5, and Myo2 in the bud during isotropic growth . Furthermore, we found that septins were required for proper regulation of actin patch stability; septin-defective cells permitted to enter isotropic growth lost actin and growth polarity . We propose that septins maintain cell polarity by specifying a boundary between cortical domains. J Hum Virol, 2000 May-Jun, 3(3), 113 - 24 Binding of the human papillomavirus type 16 E7 oncoprotein and the adeno-associated virus Rep78 major regulatory protein in vitro and in yeast and the potential for downstream effects; Hermonat PL et al.; OBJECTIVE: Both human papillomavirus (HPV) and adeno-associated virus (AAV) are common anogenital viruses and likely co-infect the epithelium in vivo . However, whereas HPVs are positively associated with cervical cancer, AAV appears to be negatively associated . In tissue culture, AAV-encoded Rep78--which is essential for AAV--inhibits gene expression and oncogenic transformation by HPV-16/18 and bovine papillomavirus type 1 . Here we observed whether the HPV-16 E7 oncoprotein might recognize and bind Rep78 . Further, upon finding Rep78-E7 binding, we investigated some of the potential downstream effects such an interaction might have . E7 is capable of recognizing a variety of proteins, including RB105, TATA box-binding protein (TBP), TBP-associated factor (TAF)(II)110, E2F, cyclins A and D, and c-jun . Some of these interactions are likely responsible for E7's cancer-promoting activity . STUDY DESIGN/METHODS: Rep78-E7 interaction was investigated in vitro by West(far)-Western and affinity chromatography analysis and in vivo in living yeast by the GAL4 two-hybrid cDNA assay . Mapping of the E7 binding domain within Rep78 was carried out using a series of amino- and carboxy-truncated Rep78 proteins in a West(far)-Western assay . Downstream effects of the interaction were analyzed by competitive affinity chromatography (protein-protein) and competitive electrophoretic mobility shift assay (protein-DNA) . RESULTS: E7 and Rep78 were found to interact both in vitro and in vivo (yeast) in all assays attempted . The E7 binding domain within Rep78 was found to reside within amino acids 121-370 . Regarding downstream effects of this interaction, Rep78 was found to mildly inhibit E7-TAF(II)110 and E7-RB105 interaction in vitro but to have little affect on E7-TBP interaction . Finally, it was found that E7 was able to affect Rep78's interaction with AAV's terminal repeat (TR) DNA in vitro, reducing the formation of the largest sized Rep78-TR complexes in a dosage-dependent manner . CONCLUSIONS: These data suggest that the Rep78-E7 interaction may have repercussions for both viruses . The Rep78-E7 interaction may be a second mechanism, in addition to Rep78 regulation of the p97 promoter, by which AAV inhibits HPV-16 oncogenic transformation . These data also suggest that HPV-16 may affect the AAV life cycle by altering Rep78-TR interaction. Genetics, 2000 Jul, 155(3), 1069 - 81 MPH1, a yeast gene encoding a DEAH protein, plays a role in protection of the genome from spontaneous and chemically induced damage; Scheller J et al.; We have characterized the MPH1 gene from Saccharomyces cerevisiae . mph1 mutants display a spontaneous mutator phenotype . Homologs were found in archaea and in the EST libraries of Drosophila, mouse, and man . Mph1 carries the signature motifs of the DEAH family of helicases . Selected motifs were shown to be necessary for MPH1 function by introducing missense mutations . Possible indirect effects on translation and splicing were excluded by demonstrating nuclear localization of the protein and splicing proficiency of the mutant . A mutation spectrum did not show any conspicuous deviations from wild type except for an underrepresentation of frameshift mutations . The mutator phenotype was dependent on REV3 and RAD6 . The mutant was sensitive to MMS, EMS, 4-NQO, and camptothecin, but not to UV light and X rays . Epistasis analyses were carried out with representative mutants from various repair pathways (msh6, mag1, apn1, rad14, rad52, rad6, mms2, and rev3) . No epistatic interactions were found, either for the spontaneous mutator phenotype or for MMS, EMS, and 4-NQO sensitivity . mph1 slightly increased the UV sensitivity of mms2, rad6, and rad14 mutants, but no effect on X-ray sensitivity was observed . These data suggest that MPH1 is not part of a hitherto known repair pathway . Possible functions are discussed. Genetics, 2000 Jul, 155(3), 1045 - 54 Functional interaction of CCR4-NOT proteins with TATAA-binding protein (TBP) and its associated factors in yeast; Badarinarayana V et al.; The CCR4-NOT transcriptional regulatory complex affects expression of a number of genes both positively and negatively . We report here that components of the CCR4-NOT complex functionally and physically interact with TBP and TBP-associated factors . First, mutations in CCR4-NOT components suppressed the his4-912delta insertion in a manner similar to that observed for the defective TBP allele spt15-122 . Second, using modified HIS3 promoter derivatives containing specific mutations within the TATA sequence, we found that the NOT proteins were general repressors that disrupt TBP function irrespective of the DNA sequence . Third, increasing the dosage of NOT1 specifically inhibited the ability of spt15-122 to suppress the his4-912delta insertion but did not affect the Spt- phenotype of spt3 or spt10 at this locus . Fourth, spt3, spt8, and spt15-21 alleles (all involved in affecting interaction of SPT3 with TBP) suppressed ccr4 and caf1 defects . Finally, we show that NOT2 and NOT5 can be immunoprecipitated by TBP . NOT5 was subsequently shown to associate with TBP and TAFs and this association was dependent on the integrity of TFIID . These genetic and physical interactions indicate that one role of the CCR4-NOT proteins is to inhibit functional TBP-DNA interactions, perhaps by interacting with and modulating the function of TFIID. Genetics, 2000 Jul, 155(3), 1005 - 18 Defects in protein glycosylation cause SHO1-dependent activation of a STE12 signaling pathway in yeast; Cullen PJ et al.; In haploid Saccharomyces cerevisiae, mating occurs by activation of the pheromone response pathway . A genetic selection for mutants that activate this pathway uncovered a class of mutants defective in cell wall integrity . Partial loss-of-function alleles of PGI1, PMI40, PSA1, DPM1, ALG1, MNN10, SPT14, and OCH1, genes required for mannose utilization and protein glycosylation, activated a pheromone-response-pathway-dependent reporter (FUS1) in cells lacking a basal signal (ste4) . Pathway activation was suppressed by the addition of mannose to hexose isomerase mutants pgi1-101 and pmi40-101, which bypassed the requirement for mannose biosynthesis in these mutants . Pathway activation was also suppressed in dpm1-101 mutants by plasmids that contained RER2 or PSA1, which produce the substrates for Dpm1 . Activation of FUS1 transcription in the mannose utilization/protein glycosylation mutants required some but not all proteins from three different signaling pathways: the pheromone response, invasive growth, and HOG pathways . We specifically suggest that a Sho1 --> Ste20/Ste50 --> Ste11 --> Ste7 --> Kss1 --> Ste12 pathway is responsible for activation of FUS1 transcription in these mutants . Because loss of pheromone response pathway components leads to a synthetic growth defect in mannose utilization/protein glycosylation mutants, we suggest that the Sho1 --> Ste12 pathway contributes to maintenance of cell wall integrity in vegetative cells. EMBO J, 2000 Jul 3, 19(13), 3475 - 84 Covalent modifier NEDD8 is essential for SCF ubiquitin-ligase in fission yeast; Osaka F et al.; A ubiquitin-like modifier, NEDD8, is covalently attached to cullin-family proteins, but its physiological role is poorly understood . Here we report that the NEDD8-modifying pathway is essential for cell viability and function of Pcu1 (cullin-1 orthologue) in fission yeast . Pcu1 assembled on SCF ubiquitin-ligase was completely modified by NEDD8 . Pcu1(K713R) defective for NEDD8 conjugation lost the ability to complement lethality due to pcu1 deletion . Forced expression of Pcu1(K713R) or depletion of NEDD8 in cells resulted in impaired cell proliferation and marked stabilization of the cyclin-dependent kinase inhibitor Rum1, which is a substrate of the SCF complex . Based on these findings, we propose that covalent modification of cullin-1 by the NEDD8 system plays an essential role in the function of SCF in fission yeast. EMBO J, 2000 Jul 3, 19(13), 3215 - 22 The yeast prion {URE3} can be greatly induced by a functional mutated URE2 allele; Fernandez-Bellot E et al.; The non-Mendelian element {URE3} of yeast is considered to be a prion form of the Ure2 protein . The {URE3} phenotype occurs at a frequency of 10(-5) in haploid yeast strains, is reversible, and its frequency is increased by overexpressing the URE2 gene . We created a new mutant of the Ure2 protein, called H2p, which results in a 1000-fold increase in the rate of {URE3} occurrence . To date, only the overexpression of various C-terminal truncated mutants of Ure2p gives rise to a comparable level . The h2 allele is, thus, the first characterized URE2 allele that induces prion formation when expressed at a low level . By shuffling mutated and wild-type domains of URE2, we also created the first mutant Ure2 protein that is functional and induces prion formation . We demonstrate that the domains of URE2 function synergistically in cis to induce {URE3} formation, which highlights the importance of intramolecular interactions in Ure2p folding . Additionally, we show using a green fluorescent protein (GFP) fusion protein that the h2 allele exhibits numerous filiform structures that are not generated by the wild-type protein. J Photochem Photobiol B, 2000 Mar, 55(1), 74 - 9 Mathematical description of synergistic interaction of UV light and hyperthermia for yeast cells; Petin VG et al.; A new mathematical model for the synergistic interaction of lesions produced by ultraviolet (UV) light and high temperature has been proposed . The model suggests that synergism is expected from the additional lethal lesions arising from the interaction of sublesions induced by both agents . These sublesions are considered noneffective after each agent is taken alone . The model predicts the dependence of the synergistic interaction on the ratio of lethal lesions produced by each agent applied, the greatest value of the synergistic effect as well as the conditions under which it can be achieved, and the dependence of synergistic effect on UV light fluence rate . These predictions of the model have been tested for the simultaneous combined action of UV light (wavelength 254 nm) and heat (45-57.5 degrees C) on two strains of wild-type diploid yeast cells of Saccharomyces cerevisiae . The theory appears to be appropriate and the conclusions valid. J Photochem Photobiol B, 2000 Mar, 55(1), 20 - 6 Time-resolved fluorescence of tryptophans in yeast hexokinase-PI: effect of subunit dimerization and ligand binding; Maity H et al.; Time-resolved and steady-state fluorescence measurements have been performed on monomeric and dimeric forms of yeast hexokinase-PI . Observation of similar emission spectra and fluorescence decay parameters for both the forms of the enzyme suggests that tryptophan residue(s) are not likely to be present at the subunit-subunit interface and the process of dimerization does not perturb the local environment of tryptophan(s) . The fluorescence decay of tryptophans in enzyme could be fitted to a bi-exponential function with two lifetime components, tau1 approximately 2.2 ns and tau2 approximately 3.9 ns . Binding of glucose, which is known to convert the 'open' conformation of the enzyme to a 'closed' active conformation, results in approximately 30% reduction in emission intensity and a selective decrease in tau1 from approximately 2.2 to approximately 1.1 ns . These effects can be reversed by the addition of trehalose 6-phosphate (an inhibitor of yeast hexokinase), suggesting that the trehalose 6-phosphate inhibits the enzyme by binding to its 'open' inactive conformation rather than competing with glucose to bind to the 'closed' active conformation . Binding of nucleotide ligands (ATP, ADP and adenyl-(beta,gamma-methylene)-diphosphate (AMPPCP)) to the monomeric or dimeric form of enzyme quenched the steady-state fluorescence by approximately 4-8%, but had no measurable effect on the distribution of lifetimes or on their magnitudes . Addition of nucleotides to the enzyme-glucose complex also did not produce any further change in fluorescence decay parameters . These results indicate that it is highly unlikely that the formation of a ternary enzyme-glucose-nucleotide complex from the binary enzyme-glucose complex is accompanied by a large conformational change in the enzyme, as has been surmised in some earlier studies. Yi Chuan Xue Bao, 1999, 26(6), 731 - 7 {Study on gene-dosage effect of high level expression of the yeast glucoamylase genes}; Li CL et al.; Diploid strains homozygous for both MAT allele and STA genes (a/a, STA1/STA1 or STA2/STA2 or STA3/STA3) and diploid strains homozygous for MAT allele but intercombinative for STA genes (a/a, STA1/STA2 or STA2/STA3 or STA1/STA3) were constructed by means of the protoplast fusion or the colchicine treatment . According to glucoamylase activity in YPS medium, we studied the gene-dosage effect and their interrelation of these three polymeric genes coding for glucoamylase . The results of the glucoamylase activity determination showed that the gene-dosage effect of glucoamylase is obvious in diploid and triploid strains homozygous for both MAT allele and STA gene, such as the glucoamylase activity of a diploid strain SFY56-6 and a triploid strain SFY56-104 homozygous for both MAT allele and STA genes were respectively 2.35 and 3.18-fold as compared with that of the their parental strain IATA-Y56 that is a haploid Saccharomyces diastaticus . Moreover, the glucoamylase activity of diploid strains homozygous for MAT allele but intercombinative for STA genes also showed the combinative gene-dosage effect to a certain extent. Curr Opin Cell Biol, 2000 Aug, 12(4), 457 - 66 Sorting in the endosomal system in yeast and animal cells; Lemmon SK et al.; The endosomal system is a major membrane-sorting apparatus . New evidence reveals that novel coat proteins assist specific sorting steps and docking factors ensure the vectorial nature of trafficking in the endosomal compartment . There is also good evidence for ubiquitin regulating passage of certain proteins into multivesicular late endosomes, which mature by accumulating invaginated membrane . Lipids play a central role in this involution process, as do the class E vacuolar protein-sorting proteins. Curr Opin Cell Biol, 2000 Aug, 12(4), 509 - 16 Divide and multiply: organelle partitioning in yeast; Catlett NL et al.; The mechanisms ensuring accurate partitioning of yeast vacuoles and mitochondria are distinct, yet they share common elements . Both organelles move along actin filaments, and both organelles require fusion and fission to maintain normal morphology . Recent studies have revealed that while vacuolar inheritance requires a processive myosin motor, mitochondrial inheritance requires controlled actin polymerization . Distinct sets of proteins required for the fusion and fission of each organelle have also been identified. Curr Biol, 2000 Jun 15, 10(12), 727 - 30 A myosin light chain mediates the localization of the budding yeast IQGAP-like protein during contractile ring formation; Shannon KB et al.; Cytokinesis in animal cells is accomplished through constriction of an actomyosin ring {1} {2} {3}, which must assemble at the correct time and place in order to ensure proper division of genetic material and organelles . Budding yeast is a useful model system for determining the biochemical pathway of contractile ring assembly . The budding yeast IQGAP-like protein, Cyk1/Iqg1p, has multiple roles in the assembly and contraction of the actomyosin ring {4} {5} {6} . Previously, the IQ motifs of Cyk1/Iqg1p were shown to be required for the localization of this protein at the bud neck {6} . We have investigated the binding partner of the IQ motifs, which are predicted to interact with calmodulin-like proteins . Mlc1p was originally identified as a light chain for a type V myosin, Myo2p; however, a cytokinesis defect associated with disruption of the MLC1 gene suggested that the essential function of Mlc1p may involve interactions with other proteins {7} . We show that Mlc1p binds the IQ motifs of Cyk1/Iqg1p and present evidence that this interaction recruits Cyk1/Iqg1p to the bud neck . Immunofluorescence staining shows that Mlc1p is localized to sites of polarized cell growth as well as the bud neck before and independently of Cyk1p . These results demonstrate that Mlc1p is important for the assembly of the actomyosin ring in budding yeast and that this function is mediated through interaction with Cyk1/Iqg1p. Virology, 2000 Jul 5, 272(2), 315 - 25 A mutagenic analysis of the E5 protein of human papillomavirus type 16 reveals that E5 binding to the vacuolar H+-ATPase is not sufficient for biological activity, using mammalian and yeast expression systems; Adam JL et al.; The E5 gene of human papillomavirus type 16 encodes a highly hydrophobic membrane protein previously shown to inhibit endosomal acidification, presumably by binding to the 16-kDa pore-forming subunit of the vacuolar H(+)-ATPase (v-ATPase) . The role of this interaction in the disruption of v-ATPase activity was explored through extensive mutagenesis of E5 to identify residues that mediate binding to the 16-kDa subunit . Coimmunoprecipitations revealed that the hydrophobic span between residues 41 and 54 is primarily responsible for this interaction and can be replaced with random hydrophobic amino acids . Studies using mutated 16-kDa proteins indicated that the fourth transmembrane domain of the pore subunit mediates binding to E5 . Analysis of the E5 mutants in a yeast expression system revealed that several mutants that retained the capacity to bind to the 16-kDa subunit in COS-1 cells failed to disrupt vacuolar acidification . These data argue that E5 binding to the pore subunit is not sufficient for the associated activity of disruption of v-ATPase function . Biochem Biophys Res Commun, 2000 Jul 5, 273(2), 509 - 15 Rapid isolation and characterization of the yeast proteasome regulatory complex; Saeki Y et al.; The 26S proteasome, which catalyzes degradation of ubiquitinated proteins, is composed of the 20S proteasome and the 19S complex . Recently, it has been reported that the 26S complex can be dissociated into the lid complex and the 20S-proteasome-base complex in a mutant yeast and that the lid complex is required for ubiquitin-dependent proteolysis . In the present study, we established methods for rapid isolation of the 19S complex, the lid complex, and the base complex from wild-type yeast . The isolated 19S complex was capable of binding to the 20S proteasome to reconstitute the 26S proteasome . In contrast with the previously reported result showing that Rpn10, a multiubiquitin chain binding subunit, is a component of the base complex, we present evidence that the lid complex isolated from wild-type yeast contains Rpn10 . J Mol Biol, 2000 Jun 23, 299(5), 1313 - 24 The free yeast aspartyl-tRNA synthetase differs from the tRNA(Asp)-complexed enzyme by structural changes in the catalytic site, hinge region, and anticodon-binding domain; Sauter C et al.; Aminoacyl-tRNA synthetases catalyze the specific charging of amino acid residues on tRNAs . Accurate recognition of a tRNA by its synthetase is achieved through sequence and structural signalling . It has been shown that tRNAs undergo large conformational changes upon binding to enzymes, but little is known about the conformational rearrangements in tRNA-bound synthetases . To address this issue the crystal structure of the dimeric class II aspartyl-tRNA synthetase (AspRS) from yeast was solved in its free form and compared to that of the protein associated to the cognate tRNA(Asp) . The use of an enzyme truncated in N terminus improved the crystal quality and allowed us to solve and refine the structure of free AspRS at 2.3 A resolution . For the first time, snapshots are available for the different macromolecular states belonging to the same tRNA aminoacylation system, comprising the free forms for tRNA and enzyme, and their complex . Overall, the synthetase is less affected by the association than the tRNA, although significant local changes occur . They concern a rotation of the anticodon binding domain and a movement in the hinge region which connects the anticodon binding and active-site domains in the AspRS subunit . The most dramatic differences are observed in two evolutionary conserved loops . Both are in the neighborhood of the catalytic site and are of importance for ligand binding . The combination of this structural analysis with mutagenesis and enzymology data points to a tRNA binding process that starts by a recognition event between the tRNA anticodon loop and the synthetase anticodon binding module . EMBO J, 1983, 2(8), 1249 - 54 Control-mechanisms acting at the transcriptional and post-transcriptional levels are involved in the synthesis of the arginine pathway carbamoylphosphate synthase of yeast; Messenguy F et al.; In Saccharomyces cerevisiae, the synthesis of the arginine pathway enzyme carbamoylphosphate synthase (CPSase A) is subject to two control mechanisms . One mechanism, the general control of amino acid biosynthesis, influences the expression of both CPA1 and CPA2 genes, the structural genes for the two subunits of the enzyme . The second mechanism, the specific control of arginine biosynthesis, only affects the expression of CPA1 . To study these mechanisms in more detail, we have cloned the CPA1 and CPA2 genes and used their DNA to measure the CPA1 and CPA2 mRNA content of cells grown under various conditions . A close coordination was observed in the variation of the levels of CPA1 and CPA2 mRNAs and polypeptide products under conditions where the general control of amino acid biosynthesis operates . In contrast, little correlation was found between the levels of CPA1 mRNA and the corresponding protein for conditions affecting repression by arginine: the total amplitude of variation was 6-fold higher for the CPA1 protein than for the CPA1 messenger transcript . Such findings are consistent with the conclusion that the general control operates at the transcriptional level and that the specific arginine control acts primarily at a post-transcriptional level. J Cell Biol, 2000 Jun 26, 149(7), 1361 - 76 Cdc28 activates exit from mitosis in budding yeast; Rudner AD et al.; The activity of the cyclin-dependent kinase 1 (Cdk1), Cdc28, inhibits the transition from anaphase to G1 in budding yeast . CDC28-T18V, Y19F (CDC28-VF), a mutant that lacks inhibitory phosphorylation sites, delays the exit from mitosis and is hypersensitive to perturbations that arrest cells in mitosis . Surprisingly, this behavior is not due to a lack of inhibitory phosphorylation or increased kinase activity, but reflects reduced activity of the anaphase-promoting complex (APC), a defect shared with other mutants that lower Cdc28/Clb activity in mitosis . CDC28-VF has reduced Cdc20- dependent APC activity in mitosis, but normal Hct1- dependent APC activity in the G1 phase of the cell cycle . The defect in Cdc20-dependent APC activity in CDC28-VF correlates with reduced association of Cdc20 with the APC . The defects of CDC28-VF suggest that Cdc28 activity is required to induce the metaphase to anaphase transition and initiate the transition from anaphase to G1 in budding yeast. Yeast, 2000 Jul, 16(10), 967 - 70 Identifying tagged transposon insertion sites in yeast by direct genomic sequencing; Horecka J et al.; Tagged transposons are powerful tools for large-scale studies of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae . The current techniques used to identify transposon insertion sites in the yeast genome require a DNA amplification step that can be time-consuming and problematic . We show that the DNA amplification step can be bypassed . Insertion sites can be identified rapidly and reliably by direct genomic sequencing using a transposon-specific primer, BigDye-labelled terminators, and an automated sequencer . Direct genomic sequencing can also save time on the genetic analysis phase of transposon-based projects . Nucleic Acids Res, 2000 Jul 1, 28(13), 2519 - 26 Identification of high affinity Tbf1p-binding sites within the budding yeast genome; Koering CE et al.; The yeast TBF1 gene is essential for mitotic growth and encodes a protein that binds the human telomere repeats in vitro, although its cellular function is unknown . The sequence of the DNA-binding domain of Tbf1p is more closely related to that of the human telomeric proteins TRF1 and TRF2 than to any yeast protein sequence, yet the functional homologue of TRF1 and TRF2 is thought to be Rap1p . In this study we show that the Tbf1p DNA-binding domain can target the Gal4 transactivation domain to a (TTAGGG)(n) sequence inserted in the yeast genome, supporting the model that Tbf1p binds this sub-telomeric repeat motif in vivo . Immunofluorescence of Tbf1p shows a spotty pattern throughout the interphase nucleus and along synapsed chromosomes in meiosis, suggesting that Tbf1p binds internal chromosomal sites in addition to sub-telomeric regions . PCR-assisted binding site selection was used to define a consensus for high affinity Tbf1p-binding sites . Compilation of 50 selected oligonucleotides identified the consensus TAGGGTTGG . Five potential Tbf1p-binding sites resulting from a search of the total yeast genome were tested directly in gel shift assays and shown to bind Tbf1p efficiently in vitro, thus confirming this as a valid consensus for Tbf1p recognition. Genetika, 2000 May, 36(5), 622 - 9 {TAR-cloning of the short arm of human chromosome 7 in yeast and search for terminal sequences}; Glazkova DV et al.; A partial clone library of the short arm of human chromosome 7 was created in yeast artificial chromosomes (YAC) using TAR-cloning . The DNA of monochromosome somatic hybrid cells (mouse/human) RuRag 14-4-7-44 containing short arm human chromosome 7 was used for cloning . The clone library was screened for YACs with the human DNA; the mitotic stability of these YACs, the sizes of cloned fragments, and an independent clonal distribution in the chromosome were determined . Human YACs were tested for the presence of chromosome 7p telomeric sequences. J Biol Chem, 2000 Sep 22, 275(38), 29238 - 43 A single amino acid change in subunit 6 of the yeast mitochondrial ATPase suppresses a null mutation in ATP10; Paul MF et al.; In an earlier study, the ATP10 gene of Saccharomyces cerevisiae was shown to code for an inner membrane protein required for assembly of the F(0) sector of the mitochondrial ATPase complex (Ackerman, S., and Tzagoloff, A . (1990) J . Biol . Chem . 265, 9952-9959) . To gain additional insights into the function of Atp10p, we have analyzed a revertant of an atp10 null mutant that displays partial recovery of oligomycin-sensitive ATPase and of respiratory competence . The suppressor mutation in the revertant has been mapped to the OLI2 locus in mitochondrial DNA and shown to be a single base change in the C-terminal coding region of the gene . The mutation results in the substitution of a valine for an alanine at residue 249 of subunit 6 of the ATPase . The ability of the subunit 6 mutation to compensate for the absence of Atp10p implies a functional interaction between the two proteins . Such an interaction is consistent with evidence indicating that the C-terminal region with the site of the mutation and the extramembrane domain of Atp10p are both on the matrix side of the inner membrane . Subunit 6 has been purified from the parental wild type strain, from the atp10 null mutant, and from the revertant . The N-terminal sequences of the three proteins indicated that they all start at Ser(11), the normal processing site of the subunit 6 precursor . Mass spectral analysis of the wild type and mutants subunit 6 failed to reveal any substantive difference of the wild type and mutant proteins when the mass of the latter was corrected for Ala --> Val mutation . These data argue against a role of Atp10p in post-translational modification of subunit 6 . Although post-translational modification of another ATPase subunit interacting with subunit 6 cannot be excluded, a more likely function for Atp10p is that it acts as a subunit 6 chaperone during F(0) assembly. Mol Cell Biol, 2000 Jul, 20(14), 5350 - 9 Akr1p and the type I casein kinases act prior to the ubiquitination step of yeast endocytosis: Akr1p is required for kinase localization to the plasma membrane; Feng Y et al.; Ubiquitination of the plasma membrane-localized yeast a-factor receptor (Ste3p) triggers a rapid, ligand-independent endocytosis leading to its vacuolar degradation . This report identifies two mutants that block uptake by blocking ubiquitination, these being mutant either for the ankyrin repeat protein Akr1p or for the redundant type I casein kinases Yck1p and Yck2p . While no obvious defect was seen for wild-type Ste3p phosphorylation in akr1 or yck mutant backgrounds, examination of the Delta320-413 Ste3p deletion mutant phosphorylation did reveal a clear defect in both mutants . The Delta320-413 deletion removes 18 Ser-Thr residues (possible YCK-independent phosphorylation sites) yet retains the 15 Ser-Thr residues of the Ste3p PEST-like ubiquitination-endocytosis signal . Two other phenotypes link akr1 and yck mutants: both are defective in phosphorylation of wild-type alpha-factor receptor, and while both are defective for Ste3p constitutive internalization, both remain partially competent for the Ste3p ligand-dependent uptake mode . Yck1p-Yck2p may be the function responsible in phosphorylation of the PEST-like ubiquitination-endocytosis signal . Akr1p appears to function in localizing Yck1p-Yck2p to the plasma membrane, a localization that depends on prenylation of C-terminal dicysteinyl motifs . In akr1Delta cells, Yck2p is mislocalized, showing a diffuse cytoplasmic localization identical to that seen for a Yck2p mutant that lacks the C-terminal Cys-Cys, indicating a likely Akr1p requirement for the lipid modification of Yck2p, for prenylation, or possibly for palmitoylation. Mol Cell Biol, 2000 Jul, 20(14), 5269 - 75 TATA binding protein can stimulate core-directed transcription by yeast RNA polymerase I; Aprikian P et al.; The TATA binding protein (TBP) interacts with two transcription factor complexes, upstream activating factor (UAF) and core factor (CF), to direct transcription by RNA polymerase I (polI) in the yeast Saccharomyces cerevisiae . Previous work indicates that one function of TBP is to serve as a bridge, enabling UAF to recruit and stabilize the binding of CF (23, 24) . In this work we show that, in addition to aiding recruitment, TBP also directly aids CF function . Overexpression of TBP in strains with UAF components deleted will stimulate CF-directed transcription nearly to wild-type levels in vivo . In vitro, increasing the concentration of TBP stimulates CF-directed transcription in the absence of either UAF or its DNA binding site . This dual function of TBP, serving as a critical member of a core promoter complex as well as a contact point for upstream activators, appears similar to the dual roles that TBP also plays in transcription by RNA polII. Science, 2000 Jun 23, 288(5474), 2215 - 9 Requirement of Mis6 centromere connector for localizing a CENP-A-like protein in fission yeast; Takahashi K et al.; Mammalian kinetochores contain the centromere-specific histone H3 variant CENP-A, whose incorporation into limited chromosomal regions may be important for centromere function and chromosome segregation during mitosis . However, regulation of CENP-A localization and its role have not been clear . Here we report that the fission yeast homolog SpCENP-A is essential for establishing centromere chromatin associated with equal chromosome segregation . SpCENP-A binding to the nonrepetitious inner centromeres depended on Mis6, an essential centromere connector protein acting during G1-S phase of the cell cycle . Mis6 is likely required for recruiting SpCENP-A to form proper connection of sister centromeres. RNA, 2000 Jun, 6(6), 826 - 43 Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2; Bousquet-Antonelli C et al.; Chemical modifications and processing of the 18S, 5.8S, and 25S ribosomal RNAs from the 35S pre-ribosomal RNA depend on an important set of small nucleolar ribonucleoprotein particles (snoRNPs) . Genetic depletion of yeast Gar1p, an essential common component of H/ACA snoRNPs, leads to inhibition of uridine isomerizations to pseudo-uridines on the 35S pre-rRNA and of the early pre-rRNA cleavages at sites A1 and A2, resulting in a loss of mature 18S rRNA synthesis . To identify Gar1p functional partners, we screened for mutations that are synthetically lethal with a gar1 mutant allele encoding a Gar1p mutant protein lacking its two glycine/arginine-rich (GAR) domains . We identified a previously uncharacterized Saccharomyces cerevisiae open reading frame, YDR083W (now designated RRP8), that encodes a highly conserved protein containing motifs found in methyltransferases . Rrp8p localizes to the nucleolus . A yeast strain lacking this protein is viable at 30 degrees C but displays strong growth impairment at lower temperatures . In this strain, cleavage of the pre-rRNA at site A2 is strongly affected whereas cleavages at sites A0 and A1 are only slightly inhibited or delayed. Enzyme Microb Technol, 2000 Jun 1, 26(9-10), 793 - 800 The methylotrophic yeast Hansenula polymorpha: a versatile cell factory; van Dijk R et al.; The development of heterologous overexpression systems for soluble proteins has greatly advanced the study of the structure/function relationships of these proteins and their biotechnological and pharmaceutical applications . In this paper we present an overview on several aspects of the use of the methylotrophic yeast Hansenula polymorpha as a host for heterologous gene expression . H . polymorpha has been successfully exploited as a cell factory for the large-scale production of such components . Stable, engineered strains can be obtained by site-directed integration of expression cassettes into the genome, for which various constitutive and inducible promoters are available to control the expression of the foreign genes . New developments have now opened the way to additional applications of H . polymorpha, which are unprecedented for other organisms . Most importantly, it may be the organism of choice for reliable, large-scale production of heterologous membrane proteins, using inducible intracellular membranes and targeting sequences to specifically insert these proteins stably into these membranes . Furthermore, the use of H . polymorpha offers the possibility to accumulate the produced components into specific compartments, namely peroxisomes . These organelles are massively induced during growth of the organism on methanol and may occupy up to 80% of the cell volume . Accumulation inside peroxisomes prevents undesired modifications (e.g . proteolytic processing or glycosylation) and is also in particular advantageous when proteins are produced which are toxic or harmful for the host. J Biol Chem, 2000 Sep 1, 275(35), 26898 - 905 Interaction between yeast sgs1 helicase and DNA topoisomerase III; Bennett RJ et al.; The Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ family of DNA helicases that includes the human Bloom's syndrome and Werner's syndrome proteins . In this work, we report studies on the interaction between Sgs1 and DNA topoisomerase III in vitro and in vivo . Affinity chromatography experiments with various fragments of Sgs1, a 1447-amino acid polypeptide, suggested that its N-terminal one-fifth was sufficient for interaction with DNA topoisomerase III . Gel electrophoretic mobility shift assays also indicated that a fragment Sgs1(1-283), containing residues 1-283, inhibited the binding of DNA topoisomerase III to single-stranded DNA . A shorter protein fragment containing residues 1-107 also showed partial inhibition in these assays . Studies of a sgs1 top1 double mutant lacking both Sgs1 and DNA topoisomerase I showed that the slow growth phenotype of this double mutant is suppressed by expressing full-length Sgs1, but not Sgs1 without the N-terminal 107 amino acid residues . In sgs1 top3 cells devoid of DNA topoisomerase III, however, expression of full-length Sgs1 or Sgs1 lacking the N-terminal 107 amino acid residues has the same effect of reducing the growth rate of the double mutant . These in vitro and in vivo data indicate that Sgs1 and DNA topoisomerase III physically interact and that this interaction is physiologically significant. Yeast, 2000 Jun 30, 16(9), 857 - 60 Rapid and reliable protein extraction from yeast; Kushnirov VV; The methods currently used for protein extraction from yeast are either laborious or insufficiently reliable . Here I report a method for protein extraction for electrophoretic analysis that is both easy and reliable . In this method, yeast cells are subjected to mild alkali treatment and then boiled in a standard electrophoresis loading buffer . The method was tested for different strains of Saccharomyces cerevisiae and for yeast Hansenula polymorpha DL-1 . It yields virtually complete extraction independently of the strain, growth conditions and protein molecular weight and allows working with small amounts of yeast cells grown on agar plates . Yeast, 2000 Jun 30, 16(9), 811 - 27 Mutational analysis of the karmellae-inducing signal in Hmg1p, a yeast HMG-CoA reductase isozyme; Profant DA et al.; In response to elevated levels of HMG-CoA reductase, an integral endoplasmic reticulum (ER) membrane protein, cells assemble novel ER arrays . These membranes provide useful models for exploration of ER structure and function, as well as general features of membrane biogenesis and turnover . Yeast express two functional HMG-CoA reductase isozymes, Hmg1p and Hmg2p, each of which induces morphologically different ER arrays . Hmg1p induces stacks of paired nuclear-associated membranes called karmellae . In contrast, Hmg2p induces peripheral ER membrane arrays and short nuclear-associated membrane stacks . In spite of their ability to induce different cellular responses, both Hmg1p and Hmg2p have similar structures, including a polytopic membrane domain containing eight predicted transmembrane helices . By examining a series of recombinant HMG-CoA reductase proteins, our laboratory previously demonstrated that the last ER-lumenal loop (Loop G) of the Hmg1p membrane domain contains a signal needed for proper karmellae assembly . Our goal was to examine the primary sequence requirements within Loop G that were critical for proper function of this signal . To this end, we randomly mutagenized the Loop G sequence, expressed the mutagenized Hmg1p in yeast, and screened for inability to generate karmellae at wild-type levels . Out of approximately 4000 strains with Loop G mutations, we isolated 57 that were unable to induce wild-type levels of karmellae assembly . Twenty-nine of these mutants contained one or more point mutations in the Loop G sequence, including nine single point mutants, four of which had severe defects in karmellae assembly . Comparison of these mutations to single point mutations that did not affect karmellae assembly did not reveal obvious patterns of sequence requirements . For example, both conservative and non-conservative changes were present in both groups and changes that altered the total charge of the Loop G region were observed in both groups . Our hypothesis is that Loop G serves as a karmellae-inducing signal by mediating protein-protein or protein-lipid interactions and that amino acids revealed by this analysis may be important for maintaining the proper secondary structure needed for these interactions . J Med Virol, 2000 Jul, 61(3), 298 - 302 Analysis of relative binding affinity of E7-pRB of human papillomavirus 16 clinical variants using the yeast two-hybrid system; Choo KB et al.; A number of genotypes of the human papillomaviruses (HPV) are associated with malignancies of the uterine cervix . Sequencing work has revealed the existence of intratype HPV variants with minor differences in the nucleotide sequence . More recent data suggest the possibility that some of the variants may have different modes of clinical manifestation . In this study, sequences of the E6 and E7 oncogenes of 17 HPV16 isolates derived from PAP smear samples of Taiwanese patients were analyzed . A number of E6 and E7 novel variants were found . Particularly, a prevalent (64.7%) E6 polymorphic site A442C with an E113D amino acid substitution seems specific to Taiwanese patients . In E7, two novel but silent polymorphic sites G663A (41.2%) and T846C (88.2%) were also prevalent in the samples analyzed . The yeast two-hybrid system was adopted for rapid assessment of relative E7-pRb binding affinity in the variants . The relative binding affinities of the E7 proteins of different HPV types to pRB were in close agreement with previous biochemical data . A T663G/C24W polymorphic change in E7 correlated with a decrease in E7-pRb relative binding affinity the significance of which remains to be clarified . This semi-quantitative biochemical and genetic approach may be useful as a first step in the development of clinical protocols for the screening and identification of important HPV variants for clinical interpretation and for further functional analysis by transfection or other bioassays . Biochem J, 2000 Jul 1, 349(Pt 1), 211 - 5 Investigation of the slow inhibition of almond beta-glucosidase and yeast isomaltase by 1-azasugar inhibitors: evidence for the 'direct binding' model; Lohse A et al.; (-)-1-Azafagomine {(3R,4R,5R)-4,5-dihydroxy-3-hydroxymethylhexahydropyridazine; inhibitor 1} is a potent glycosidase inhibitor designed to mimic the transition state of a substrate undergoing glycoside cleavage . The inhibition of glycosidases by inhbitor 1 and analogues has been found to be a relatively slow process . This 'slow inhibition' process was investigated in the inhibition of almond beta-glucosidase and yeast isomaltase by inhibitor 1 and analogues . Progress-curve experiments established that the time-dependent inhibition of both enzymes by inhibitor 1 was a consequence of relatively slow dissociation and association of the inhibitor from and to the enzyme, and not a result of slow interchanges between protein conformations . A number of hydrazine-containing analogues of inhibitor 1 also inhibited beta-glucosidase and isomaltase slowly, while the amine isofagomine {(3R,4R,5R)-3,4-dihydroxy-5-hydroxymethylpiperidine; inhibitor 5} only inhibited beta-glucosidase slowly . Inhibitor 1 and related inhibitors were found to leave almond beta-glucosidase with almost identical rate constants, so that the difference in K(i) values depended almost entirely on changes in the binding rate constant, k(on) . The same trend was observed for the inhibition of yeast isomaltase by inhibitor 1 and a related inhibitor . The values of the rate constants were obtained at 25 degrees C and at pH 6.8. Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 7214 - 9 Homologs of the yeast Sec complex subunits Sec62p and Sec63p are abundant proteins in dog pancreas microsomes; Tyedmers J et al.; Cotranslational protein transport into dog pancreas microsomes involves the Sec61p complex plus a luminal heat shock protein 70 . Posttranslational protein transport into the yeast endoplasmic reticulum (ER) involves the so-called Sec complex in the membrane, comprising a similar Sec61p subcomplex, the putative signal peptide receptor subcomplex, and the heat shock protein 40-type subunit, Sec63p, plus a luminal heat shock protein 70 . Recently, human homologs of yeast proteins Sec62p and Sec63p were discovered . Here we determined the concentrations of these two membrane proteins in dog pancreas microsomes and observed that the canine homologs of yeast proteins Sec62p and Sec63p are abundant proteins, present in almost equimolar concentrations as compared with Sec61alphap monomers . Furthermore, we detected fractions of these two proteins in association with each other as well as with the Sec61p complex . The J domain of the human Sec63p was shown to interact with immunoglobulin heavy chain binding protein . Thus, the membrane of the mammalian ER contains components, known from the posttranslationally operating protein translocase in yeast . We suggest that these components are required for efficient cotranslational protein transport into the mammalian ER as well as for other transport processes. J Mol Biol, 2000 Jun 2, 299(2), 405 - 19 Evidence from mutational specificity studies that yeast DNA polymerases delta and epsilon replicate different DNA strands at an intracellular replication fork; Karthikeyan R et al.; Although polymerases delta and epsilon are required for DNA replication in eukaryotic cells, whether each polymerase functions on a separate template strand remains an open question . To begin examining the relative intracellular roles of the two polymerases, we used a plasmid-borne yeast tRNA gene and yeast strains that are mutators due to the elimination of proofreading by DNA polymerases delta or epsilon . Inversion of the tRNA gene to change the sequence of the leading and lagging strand templates altered the specificities of both mutator polymerases, but in opposite directions . That is, the specificity of the polymerase delta mutator with the tRNA gene in one orientation bore similarities to the specificity of the polymerase epsilon mutator with the tRNA gene in the other orientation, and vice versa . We also obtained results consistent with gene orientation having a minor influence on mismatch correction of replication errors occurring in a wild-type strain . However, the data suggest that neither this effect nor differential replication fidelity was responsible for the mutational specificity changes observed in the proofreading-deficient mutants upon gene inversion . Collectively, the data argue that polymerases delta and epsilon each encounter a different template sequence upon inversion of the tRNA gene, and so replicate opposite strands at the plasmid DNA replication fork . J Biol Chem, 2000 Sep 22, 275(38), 29623 - 7 Transcription elongation factor S-II confers yeast resistance to 6-azauracil by enhancing expression of the SSM1 gene; Shimoaraiso M et al.; Loss of function of S-II makes yeast sensitive to 6-azauracil . Here, we identified a multi-copy suppressor gene of this phenotype, termed SSM1 (suppressor of 6-azauracil sensitivity of the S-II null mutant 1), that encodes a novel protein consisting of 280 amino acid residues . Although both the SSM1 null mutant and the S-II/SSM1 double null mutant were viable under normal growth conditions, they resembled the S-II null mutant in being sensitive to 6-azauracil . Expression of the SSM1 gene was found to be repressed in the S-II null mutant but was restored by overexpression of chimeric S-II molecules that were able to stimulate transcription elongation by RNA polymerase II in vitro . Furthermore, we identified two transcription arrest sites within the transcription unit of the SSM1 gene in vitro that could be relieved by S-II . These results indicate that S-II confers yeast resistance to 6-azauracil by stimulating transcription elongation of the SSM1 gene. Mutat Res, 2000 Jun 30, 460(1), 41 - 52 Identification of factors interacting with hMSH2 in the fetal liver utilizing the yeast two-hybrid system . In vivo interaction through the C-terminal domains of hEXO1 and hMSH2 and comparative expression analysis; Rasmussen LJ et al.; Mutations in DNA mismatch repair (MMR) genes have been shown to segregate with Hereditary Nonpolyposis Colorectal Cancer (HNPCC) . However, because many HNPCC families fail to display mutations in known MMR genes, we argued that changes in other components of the MMR pathway may be responsible . The increasing number of proteins reported to interact in the MMR pathway suggests that larger complexes are formed, the composition of which may differ among cell types and tissues . In an attempt to identify tissue-specific MMR-associated factors, we employed the yeast two-hybrid system, using the human hMSH2 as bait and a human fetal liver library as prey . We demonstrate that hMSH2 interacts with a human 5'-3' exonuclease 1 (hEXO1/HEX1) and that this interaction is mediated through their C-terminal domains . The hMSH6 protein does not interact with hEXO1 in the two-hybrid system . Dot-blot analysis of multiple tissue RNA revealed that hMSH2 and hEXO1 are coexpressed at high levels in fetal liver as well as in adult testis and thymus . Northern blot analysis also revealed that hEXO1/HEX1 is highly expressed in several liver cancer cell lines as well as in colon and pancreas adenocarcinomas, but not in the corresponding non-neoplastic tissue. EMBO J, 2000 Jun 15, 19(12), 3016 - 27 Structure of the C-terminal domain of Tup1, a corepressor of transcription in yeast; Sprague ER et al.; The Tup1-Ssn6 corepressor complex regulates the expression of several sets of genes, including genes that specify mating type in the yeast Saccharomyces cerevisiae . Repression of mating-type genes occurs when Tup1-Ssn6 is brought to the DNA by the Matalpha2 DNA-binding protein and assembled upstream of a- and haploid-specific genes . We have determined the 2.3 A X-ray crystal structure of the C-terminal domain of Tup1 (accesion No . 1ERJ), a 43 kDa fragment that contains seven copies of the WD40 sequence motif and binds to the Matalpha2 protein . Moreover, this portion of the protein can partially substitute for full-length Tup1 in bringing about transcriptional repression . The structure reveals a seven-bladed beta propeller with an N-terminal subdomain that is anchored to the side of the propeller and extends the beta sheet of one of the blades . Point mutations in Tup1 that specifically affect the Tup1-Matalpha2 interaction cluster on one surface of the propeller . We identified regions of Tup1 that are conserved among the fungal Tup1 homologs and may be important in protein-protein interactions with additional components of the Tup1-mediated repression pathways. Biochim Biophys Acta, 2000 Jun 26, 1486(1), 55 - 71 Phosphatidylinositol/phosphatidylcholine transfer proteins in yeast; Li X et al.; Phosphatidylinositol transfer proteins (PITPs) are now becoming widely recognized as intriguing proteins that participate in the coordination and coupling of phospholipid metabolism with vesicle trafficking, and in the regulation of important signaling cascades . Yet, only in one case is there a large body of evidence that speaks to the precise identities of PITP-dependent cellular reactions, and to the mechanisms by which PITPs execute function in eukaryotic cells . At present, yeast provide the most powerful system for analysis of the physiology of PITP function in vivo, and the mechanism by which this function is carried out . Here, we review the recent progress and remaining questions in the area of PITP function in yeast. Chromosoma, 2000, 109(1-2), 110 - 6 Methods for immunoelectron microscopic and fine structural analysis of synaptonemal complexes and nodules in yeast; Schmekel K; Several gene products involved in meiotic chromosome pairing and recombination in yeast have been identified in recent years . Two nuclear structures play key roles in the meiotic processes: the synaptonemal complex (SC), which is essential for the pairing of the chromosomes, and the recombination nodules (RNs), which mark the sites of recombination . Good morphological representation of the yeast SC and RNs is needed in order to show structural changes caused by specific mutations in protein-coding genes and for fine localization of proteins using immunoelectron microscopy (immuno-EM) . This paper presents a newly developed preparation method for EM and immuno-EM that allows analysis of fine structural details and localization of proteins in the SC and RNs in yeast . Structural components of the SC are clearly seen and appear strikingly similar to those in the SC in other organisms . Antibodies against the SC protein Zip1, a transverse filament protein, label the central region of the SC strongly and specifically as expected . The improved method will be an important tool in high-resolution determination of the location of proteins in the meiotic yeast nucleus. Chromosoma, 2000, 109(1-2), 62 - 71 A homologue of the yeast HOP1 gene is inactivated in the Arabidopsis meiotic mutant asy1; Caryl AP et al.; Synapsis of homologous chromosomes is a key event in meiosis as it is essential for normal chromosome segregation and is implicated in the regulation of crossover frequency . We have previously reported the identification and cytological characterisation of a T-DNA-tagged asynaptic mutant of Arabidopsis thaliana . We have demonstrated that this mutant, asy1, is defective in meiosis in both males and females . Cloning and nucleotide sequencing of the ASY1 gene has revealed that it encodes a polypeptide of 596 amino acids that exhibits similarity to the HOP1 gene of Saccharomyces cerevisiae, which is known to encode a protein essential for synaptonemal complex assembly and normal synapsis . Expression studies indicate that, in common with a number of other Arabidopsis meiotic genes, ASY1 exhibits low-level expression in a range of plant tissues . Southern analysis coupled with database searching has resulted in the identification of an ASY1 homologue, ASY2 . Although asy1 exhibits a strong asynaptic phenotype, a residual low level of synapsis indicates that ASY1 and ASY2 may exhibit a low degree of functional redundancy. Chromosoma, 2000, 109(1-2), 27 - 34 Destruction of the securin Pds1p occurs at the onset of anaphase during both meiotic divisions in yeast; Salah SM et al.; Sister chromatid cohesion is established during DNA replication and depends on a multiprotein complex called cohesin . At the onset of anaphase the cohesive structures that hold sisters together must be destroyed to allow segregation of sisters . In the budding yeast Saccharomyces cerevisiae loss of sister chromatid cohesion depends on a separating protein (separin) called Esp1 . At the metaphase to anaphase transition, separin is activated by proteolysis of its inhibitory subunit (securin) called Pds1 . This process is mediated by the anaphase promoting complex and an accessory protein Cdc20 . In meiosis a single round of DNA replication is followed by two successive rounds of segregation . Thus loss of cohesion is spun out over two divisions . By studying the mechanisms that initiate anaphase in meiotic division we show that the yeast securin Pds1p is present in meiotic nuclei and is destroyed at the onset of each meiotic division . We also show that securin destruction depends on Cdc20p which accumulates within nuclei around the time of Pds1p's disappearance. Gene, 2000 May 30, 250(1-2), 1 - 14 The continued evolution of two-hybrid screening approaches in yeast: how to outwit different preys with different baits; Fashena SJ et al.; The original two-hybrid system, an experimental approach designed to detect protein interactions, exploited the modular nature of many transcription factors . It has provided the intellectual and technical seed for the evolution of an array of innovative approaches, the application of which broadens the scope of experimentally feasible questions to include the interaction of proteins with diverse binding partners . The available array of modified and alternative approaches facilitates the analysis of complex cellular machinery and signaling networks that rely on multiple protein interactions . Such advances have facilitated the functional analysis of proteins on the genome level, a feat considered untenable a decade ago. Curr Genet, 2000 May, 37(5), 292 - 7 Influence of homology size and polymorphism on plasmid integration in the yeast CYC1 DNA region; Koren P et al.; We studied the influence of homology size and polymorphism on the integration of circular plasmids into the yeast CYC1 region . The plasmids used also contained the URA3 gene, and the proportion of Ura+ transformants resulting from plasmid integration into the CYC1 region was determined by Southern-blot analysis . A size-dependent decrease in integration into the CYC1 region was observed from 858 bp to 363 bp of homology . However, with a homology size of 321, 259 or 107 bp, about 2% of the transformants still contained plasmid molecules integrated in the CYC1 region . A single point mutation in the 858-bp fragment decreased the proportion of integrations to the CYC1 gene, but the presence of additional mutations did not have a cumulative effect . For plasmids isolated in a single-stranded (ss) form, the presence of two or six point mutations did not influence integration . These results were compared with those obtained in other assays designed to study substrate requirements for homologous recombination. Curr Genet, 2000 May, 37(5), 277 - 84 A screen of yeast respiratory mutants for sensitivity against the mycotoxin citrinin identifies the vacuolar ATPase as an essential factor for the toxicity mechanism; Ammar H et al.; In countries with a hot climate the mycotoxin citrinin represents a serious problem in fungal food-poisoning . In humans the renal system is affected the most and the mitochondrial respiratory chain was identified as a possible sensitive target for this toxin . In addition, citrinin has an antifungal activity that also inhibits the growth of the yeast Saccharomyces cerevisiae . So far the precise mode of action and the subcellular targets for citrinin have not been identified . Therefore, we decided to use the model organism yeast for a genetic approach to identify genes that play a role in the sensitivity against this mycotoxin . A large collection of conditional respiratory deficient yeast mutants was screened for sensitivity against citrinin . One special pet-ts mutant was identified that exhibited a higher sensitivity against citrinin . The genetic system of yeast allowed the isolation of the respective wild-type gene . This yeast gene encodes the Vph2p subunit that is essential for the correct assembly of the vacuolar ATPase . Isolation of the mutated gene and gene-disruption experiments of VPH2 and the partially overlapping small YKL118W gene verified this finding . The wild-type VPH2 gene restores all defects of the mutants . In contrast to this, YKL118W gave no complementation and the null mutant showed no phenotype . Thereby the yeast vacuolar ATPase was found to be important for the toxic effect of citrinin in yeast cells . The consequences of this finding for the molecular mechanism of citrinin action and its relation to the mitochondrial respiratory chain are discussed. Oncogene, 2000 Jun 1, 19(24), 2855 - 66 Yeast homolog of human SAG/ROC2/Rbx2/Hrt2 is essential for cell growth, but not for germination: chip profiling implicates its role in cell cycle regulation; Swaroop M et al.; In an attempt to understand the signaling pathway mediating redox-induced apoptosis, we cloned SAG, an evolutionarily conserved zinc RING finger gene that, when overexpressed, protects cells from apoptosis induced by redox agents . Here we report functional characterization of SAG by the use of yeast genetics approach . Targeted disruption of ySAG, yeast homolog of human SAG, and subsequent tetrad analysis revealed that ySAG is required for yeast viability . Complementation experiment showed that the lethal phenotype induced by the ySAG deletion is fully rescued by wildtype SAG, but not by several hSAG mutants . Complementation experiment has also confirmed that ySAG is essential for normal vegetative growth, rather than being required for sporulation . Furthermore, cell death induced by SAG deletion was accompanied by cell enlargement and abnormal cell cycle profiling with an increased DNA content . Importantly, SAG was found to be the second family member of Rbx (RING box protein) or ROC (Regulator of cullins) or Hrt that is a component of SCF E3 ubiquitin ligase . Indeed, like ROC1/Rbx1/Hrt1, SAG binds to Cul1 and SAG-Cul1 complex has ubiquitin ligase activity to promote poly-ubiquitination of E2/Cdc34 . This ligase activity is required for complementation of death phenotype induced by ySAG disruption . Finally, chip profiling of the entire yeast genome revealed induction of several G1/S as well as G2/M checkpoint control genes upon SAG withdrawal . Thus, SAG appears to control cell cycle progression in yeast by promoting ubiquitination and degradation of cell cycle regulatory proteins . Oncogene (2000) 19, 2855 - 2866 J Bacteriol, 2000 Jul, 182(13), 3619 - 25 Tol1, a fission yeast phosphomonoesterase, is an in vivo target of lithium, and its deletion leads to sulfite auxotrophy; Miyamoto R et al.; Lithium is the drug of choice for the treatment of bipolar affective disorder . The identification of an in vivo target of lithium in fission yeast as a model organism may help in the understanding of lithium therapy . For this purpose, we have isolated genes whose overexpression improved cell growth under high LiCl concentrations . Overexpression of tol1(+), one of the isolated genes, increased the tolerance of wild-type yeast cells for LiCl but not for NaCl . tol1(+) encodes a member of the lithium-sensitive phosphomonoesterase protein family, and it exerts dual enzymatic activities, 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase . tol1(+) gene-disrupted cells required high concentrations of sulfite in the medium for growth . Consistently, sulfite repressed the sulfate assimilation pathway in fission yeast . However, tol1(+) gene-disrupted cells could not fully recover from their growth defect and abnormal morphology even when the medium was supplemented with sulfite, suggesting the possible implication of inositol polyphosphate 1-phosphatase activity for cell growth and morphology . Given the remarkable functional conservation of the lithium-sensitive dual-specificity phosphomonoesterase between fission yeast and higher-eukaryotic cells during evolution, it may represent a likely in vivo target of lithium action across many species. Arch Tierernahr, 2000, 53(2), 157 - 77 Chromium yeast affects growth performance and plasma traits but not carcass characteristics of growing-finishing pigs depending on the glycemic index; Lemme A et al.; Forty Swiss Large White pigs (barrows with 31.7 kg initial to 103.7 kg final BW) were equally and randomly assigned to one of four treatments (H0, H200, L0, L200) involving a combination of chromium supplementation (0 or 200 micrograms/kg) and glycemic index (high GI (H) or low GI (L)) . Growth performance, energy and protein digestibility, carcass composition, and some plasma traits were investigated . The data indicated, that the substitution of dietary carbohydrates with fat and crude fibre (low GI) resulted in lower growth performance due to impaired energy digestibility . Moreover, the strong stimulation of insulin secretion due to the high and rapid availability of carbohydrates of the diets H0 and H200 caused increased carcass fat deposition . Chromium supplementation also affected plasma insulin and glucagon concentrations . Depending on glycemic index, chromium affected the growth performance . Daily gain was reduced in pigs of the L200 treatment compared to the L0 group . This finding indicated that the energy availability expressed as GI is one of several nutritional factors, which determine the efficacy of dietary chromium . We could not corroborate evidences that dietary chromium modifies the chemical composition of the whole carcass, but depending on GI, chemical composition of the longissimus muscle was affected. Adv Exp Med Biol, 2000, 475, 185 - 95 Rox1 mediated repression . Oxygen dependent repression in yeast; Kastaniotis AJ et al.; For a large number of oxygen-regulated genes in the facultative aerobe, Saccharomyces cerevisiae, the presence of oxygen is sensed through the ability to use oxygen for heme biosynthesis . Heme induces the transcription of oxygen-induced genes and represses the transcription of hypoxic genes . Repression is mediated by the Rox1 protein in conjunction with the Ssn6/Tup1 general repression complex . The differential repression of hypoxic genes results from a combination of the tightness of Rox1 binding to the regulatory region of specific hypoxic genes and the presence or absence of binding sites for Mot3 which enhances Rox1 repression. Adv Exp Med Biol, 2000, 475, 177 - 84 Mitochondrial-nuclear crosstalk is involved in oxygen-regulated gene expression in yeast; Poyton RO et al.; The expression of several oxygen-regulated nuclear genes in the yeast Saccharomyces cerevisiae is affected by the mitochondrion . Recent evidence suggests two levels of mitochondrial involvement . On the one hand, mitochondrial respiratory function is essential for the anoxic induction of some hypoxic genes . On the other hand, the mitochondrial genome itself functions independently of its respiratory function, in the optimal expression of some aerobic genes . These findings suggest that the mitochondrion release at least two types of 'signals' that function in the expression of oxygen-regulated genes. Plant J, 2000 May, 22(3), 187 - 98 Arabidopsis thaliana poly (A) binding protein 2 (PAB2) functions in yeast translational and mRNA decay processes; Palanivelu R et al.; The single yeast gene (PAB1) encoding poly (A) binding protein (PABP) has several roles in post-transcriptional processes, including translation initiation and mRNA decay . PABP is encoded by a large gene family in plants . Within Arabidopsis thaliana, the several characterized PABP genes exhibit an extreme degree of sequence divergence and are differentially expressed . Arabidopsis PAB2 is expressed in distinct tissues or during defined developmental windows in most plant organs . In this study we demonstrate that PAB2 restores viability to a yeast pab1 mutant strain . Yeast strains containing wild-type, null (PAB2s) and temperature sensitive (PAB2ts) alleles of PAB2 were used to explore the molecular functions of the plant protein . PAB2 can participate in poly (A) tail shortening, thus demonstrating that it interacts with the yeast poly(A) nuclease complex . PAB2 is required for translation, helping to maintain intact polysome structures . Consistent with its role in translation initiation, poly (A) was found to enhance PAB2 binding to Arabidopsis eIF-iso4G in vitro . In addition, PAB2 can partially restore the linkage between deadenylation, decapping and mRNA decay in yeast . Taken together, our results suggest that Arabidopsis PAB2 participates in many of the same complex post-transcriptional processes identified for yeast PAB1, and is functionally distinct from other characterized Arabidopsis PABPs. J Appl Microbiol, 2000 Jun, 88(6), 961 - 7 Characterization of an extracellular enzyme system produced by Micromonospora chalcea with lytic activity on yeast cells; Gacto M et al.; Growth of Micromonospora chalcea on a defined medium containing laminarin as the sole carbon source induced the production of an extracellular enzyme system capable of lysing cells of various yeast species . Production of the lytic enzyme system was repressed by glucose . Incubation of sensitive cells with the active component enzymes of the lytic system produced protoplasts in high yield . Analysis of the enzyme composition indicated that beta(1-->3) glucanase and protease were the most prominent hydrolytic activities present in the culture fluids . The system also displayed weak chitinase and beta(1-->6) glucanase activities whilst devoid of mannanase activity . Our observations suggest that the glucan supporting the cell wall framework of susceptible yeast cells is not directly accessible to the purified endo-beta(1-->3) glucanase and that external proteinaceous components prevent breakdown of this polymer in whole cells . We propose that protease acts in synergy with beta(1-->3) glucanase and that the primary action of the former on surface components allows subsequent solubilization of inner glucan leading to lysis. Mol Biol Cell, 2000 Jun, 11(6), 1989 - 2005 Identification of a novel family of nonclassic yeast phosphatidylinositol transfer proteins whose function modulates phospholipase D activity and Sec14p-independent cell growth; Li X et al.; Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi function and cell viability . We now report a characterization of five yeast SFH (Sec Fourteen Homologue) proteins that share 24-65% primary sequence identity with Sec14p . We show that Sfh1p, which shares 64% primary sequence identity with Sec14p, is nonfunctional as a Sec14p in vivo or in vitro . Yet, SFH proteins sharing low primary sequence similarity with Sec14p (i.e., Sfh2p, Sfh3p, Sfh4p, and Sfh5p) represent novel phosphatidylinositol transfer proteins (PITPs) that exhibit phosphatidylinositol- but not phosphatidylcholine-transfer activity in vitro . Moreover, increased expression of Sfh2p, Sfh4p, or Sfh5p rescues sec14-associated growth and secretory defects in a phospholipase D (PLD)-sensitive manner . Several independent lines of evidence further demonstrate that SFH PITPs are collectively required for efficient activation of PLD in vegetative cells . These include a collective requirement for SFH proteins in Sec14p-independent cell growth and in optimal activation of PLD in Sec14p-deficient cells . Consistent with these findings, Sfh2p colocalizes with PLD in endosomal compartments . The data indicate that SFH gene products cooperate with "bypass-Sec14p" mutations and PLD in a complex interaction through which yeast can adapt to loss of the essential function of Sec14p . These findings expand the physiological repertoire of PITP function in yeast and provide the first in vivo demonstration of a role for specific PITPs in stimulating activation of PLD. Mol Cell Biol, 2000 Jul, 20(13), 4782 - 90 Conservation and function of a potential substrate-binding domain in the yeast Clb5 B-type cyclin; Cross FR et al.; Cyclin A contains a region implicated in binding to the p27 inhibitor and to substrates . There is strong evolutionary conservation of surface residues contributing to this region in many cyclins, including yeast B-type cyclins, despite the absence of a yeast p27 homolog . The yeast S-phase B-type cyclin Clb5p interacted with mammalian p27 in a two-hybrid assay . This interaction was disrupted by mutations designed to disrupt hydrophobic interactions (hpm mutation) or hydrogen bonding (Q241A mutation) based on the cyclin A-p27 crystal structure . In contrast, mutation of the Clb5p p27-binding domain only slightly reduced binding and inhibition by the Sic1p Clb-Cdc28p kinase inhibitor . Mutations disrupting the p27-binding domain strongly reduced Clb5p biological activity in diverse assays without reducing Clb5p-associated kinase activity . An analogous hpm mutation in the mitotic cyclin Clb2p reduced mitotic function, but in some assays this mutation increased the ability of Clb2p to perform functions normally restricted to Clb5p . These results support the idea of a modular, structurally conserved cyclin domain involved in substrate targeting. Cell, 2000 Apr 28, 101(3), 307 - 17 A chromodomain protein, Swi6, performs imprinting functions in fission yeast during mitosis and meiosis; Nakayama J et al.; Inheritance of stable states of gene expression is essential for cellular differentiation . In fission yeast, an epigenetic imprint marking the mating-type (mat2/3) region contributes to inheritance of the silenced state, but the nature of the imprint is not known . We show that a chromodomain-containing Swi6 protein is a dosage-critical component involved in imprinting the mat locus . Transient overexpression of Swi6 alters the epigenetic imprint at the mat2/3 region and heritably converts the expressed state to the silenced state . The establishment and maintenance of the imprint are tightly coupled to the recruitment and the persistence of Swi6 at the mat2/3 region during mitosis as well as meiosis . Remarkably, Swi6 remains bound to the mat2/3 interval throughout the cell cycle and itself seems to be a component of the imprint . Our analyses suggest that the unit of inheritance at the mat2/3 locus comprises the DNA plus the associated Swi6 protein complex. Genes Genet Syst, 2000 Feb, 75(1), 33 - 9 An improved integration replacement/disruption method for mutagenesis of yeast essential genes; Tohe A et al.; We improved the integration replacement/disruption method (Shortle, D., Novic, P., and Botstein, D . Proc . Natl . Acad . Sci . USA 81: 4889-4893, 1984) for isolating mutants in any of essential genes of the yeast Saccharomyces cerevisiae by integrating mutagenized DNA into the wild type gene of interest . We adopted this method to isolate temperature-sensitive mutants of the MPC1 gene encoding the YLL031C ORF . To facilitate integration of the mutagenic plasmid at a site near the 5' end of the ORF, a BamHI site was created at 300 bp downstream of the 5' end of the truncated ORF to be mutagenized . The MPC1 gene was disrupted in the wild type haploid strain by integrating a 5'-truncated derivative of the gene with mutations induced by in vitro mutagenesis . Transformants thus obtained were subjected for diagnosis of conditional lethality by replica-plating onto an appropriate selection medium to detect mutants . A primary mutant isolated by this method reverted in a high frequency due to a tandem repeat created by mutagenic integration . We deviced a method to obtain a stable temperature-sensitive strain by disrupting the tandem duplication . Two stable temperature-sensitive mutants thus obtained were found to be remedial either with 1 M sorbitol or with 0.1 M Mg2+ and to be sensitive to local anestheticum, tetracaine, at 25 degrees C. Mol Microbiol, 2000 May, 36(4), 830 - 45 A large-scale study of Yap1p-dependent genes in normal aerobic and H2O2-stress conditions: the role of Yap1p in cell proliferation control in yeast; Dumond H et al.; Yeast genes regulated by the transcriptional activator Yap1p were screened by two independent methods: (i) use of a LacZ-fused gene library and (ii) high-density membrane hybridization . Changes in transcriptome profile were examined in the presence and in the absence of Yap1p, as well as under normal and H2O2-mediated stress conditions . Both approaches gave coherent results, leading to the identification of many genes that appear to be directly or indirectly regulated by Yap1p . Promoter sequence analysis of target genes revealed that this regulatory effect is not always dependent upon the presence of a Yap1p binding site . The results show that the regulatory role of Yap1p is not restricted to the activation of stress response but that this factor can act as a positive or a negative regulator, both under normal and oxidative stress conditions . Among the targets, a few genes participating in growth control cascades were detected . In particular, the RPI1 gene, a repressor of the ras-cAMP pathway, was found to be downregulated by Yap1p during the early phase of growth, but upregulated in the stationary phase or after oxidative stress. Nucleosides Nucleotides Nucleic Acids, 2000 Mar, 19(3), 515 - 31 Synthesis and properties of uniquely modified oligoribonucleotides: yeast tRNA(Phe) fragments with 6-methyluridine and 5,6-dimethyluridine at site-specific positions; Sochacka E et al.; The phosphoramidites of 6-methyluridine and 5,6-dimethyluridine were synthesized and the modified uridines site-selectively incorporated into heptadecamers corresponding in sequence to the yeast tRNA(Phe) anticodon and TpsiC domains . The oligoribonucleotides were characterized by NMR, MALDI-TOF MS and UV-monitored thermal denaturations . The 6-methylated uridines retained the syn conformation at the polymer level and in each sequence location destabilized the RNAs compared to that of the unmodified RNA . The decrease in RNA duplex stability is predictable . However, loss of stability when the modified uridine is in a loop is sequence context dependent, and can not, at this time, be predicted from the location in the loop. Int J Syst Evol Microbiol, 2000 May, 50 Pt 3, 1373 - 80 Phylogenetic analysis of the ballistoconidium-forming yeast genus Sporobolomyces based on 18S rDNA sequences; Hamamoto M et al.; The 18S rDNA nucleotide sequences of 25 Sporobolomyces species and five Sporidiobolus species were determined . Those of Sporobolomyces dimmenae JCM 8762T, Sporobolomyces ruber JCM 6884T, Sporobolomyces sasicola JCM 5979T and Sporobolomyces taupoensis JCM 8770T showed the presence of intron-like regions with lengths of 1586, 324, 322 and 293 nucleotides, respectively, which were presumed to be group I introns . A total of 63 18S rDNA nucleotide sequences was analysed, including 33 published reference sequences . Sporobolomyces species and the other basidiomycetes species were distributed throughout the phylogenetic tree . The resulting phylogeny indicated that Sporobolomyces is polyphyletic . Sporobolomyces species were mainly divided into four groups within the Urediniomycetes . The groups are designated as the Sporidiales, Agaricostilbum/Bensingtonia, Erythrobasidium and subbrunneus clusters . The last group, comprising four species, Sporobolomyces coprosmicola, Sporobolomyces dimmenae, Sporobolomyces linderae and Sporobolomyces subbrunneus, forms a new and distinct cluster in the phylogenetic tree in this study. Schriftenr Ver Wasser Boden Lufthyg, 2000, 105, 371 - 4 Evaluation of the oestrogen-receptor activating potency of environmental samples by means of yeast oestrogen assays; Rehmann K et al.; A bioassay based on genetically modified yeast strains was applied to screen environmental samples for their oestrogen-like activity . The yeast system was selected from the vast number of "oestrogen bioassays" available because it permits rapid and easy handling at low costs . The test protocol developed allowed the examination of dimethyl sulfoxide-dissolved samples within one day, with an EC50 between 1.5 nM and 3.5 nM 17 beta-oestradiol and a detection limit between 0.3 nM and 0.5 nM 17 beta-oestradiol for yeast strain I . Advantages and disadvantages as well as future prospects of this kind of oestrogen bioassay will be considered by discussing selected results. Biochemistry, 2000 Jun 13, 39(23), 6898 - 909 A limited spectrum of mutations causes constitutive activation of the yeast alpha-factor receptor; Sommers CM et al.; Activation of G protein coupled receptors (GPCRs) by binding of ligand is the initial event in diverse cellular signaling pathways . To examine the frequency and diversity of mutations that cause constitutive activation of one particular GPCR, the yeast alpha-factor receptor, we screened libraries of random mutations for constitutive alleles . In initial screens for mutant receptor alleles that exhibit signaling in the absence of added ligand, 14 different point mutations were isolated . All of these 14 mutants could be further activated by alpha-factor . Ten of the mutants also acquired the ability to signal in response to binding of desTrp(1) inverted question markAla(3)alpha-factor, a peptide that acts as an antagonist toward normal alpha-factor receptors . Of these 10 mutants, at least eight alleles residing in the third, fifth, sixth, and seventh transmembrane segments exhibit bona fide constitutive signaling . The remaining alleles are hypersensitive to alpha-factor rather than constitutive . They can be activated by low concentrations of endogenous alpha-factor present in MATa cells . The strongest constitutively active receptor alleles were recovered multiple times from the mutational libraries, and extensive mutagenesis of certain regions of the alpha-factor receptor did not lead to recovery of any additional constitutive alleles . Thus, only a limited number of mutations is capable of causing constitutive activation of this receptor . Constitutive and hypersensitive signaling by the mutant receptors is partially suppressed by coexpression of normal receptors, consistent with preferential association of the G protein with unactivated receptors. Microbiol Mol Biol Rev, 2000 Jun, 64(2), 281 - 315 Maintenance and integrity of the mitochondrial genome: a plethora of nuclear genes in the budding yeast; Contamine V et al.; Instability of the mitochondrial genome (mtDNA) is a general problem from yeasts to humans . However, its genetic control is not well documented except in the yeast Saccharomyces cerevisiae . From the discovery, 50 years ago, of the petite mutants by Ephrussi and his coworkers, it has been shown that more than 100 nuclear genes directly or indirectly influence the fate of the rho(+) mtDNA . It is not surprising that mutations in genes involved in mtDNA metabolism (replication, repair, and recombination) can cause a complete loss of mtDNA (rho(0) petites) and/or lead to truncated forms (rho(-)) of this genome . However, most loss-of-function mutations which increase yeast mtDNA instability act indirectly: they lie in genes controlling functions as diverse as mitochondrial translation, ATP synthase, iron homeostasis, fatty acid metabolism, mitochondrial morphology, and so on . In a few cases it has been shown that gene overexpression increases the levels of petite mutants . Mutations in other genes are lethal in the absence of a functional mtDNA and thus convert this petite-positive yeast into a petite-negative form: petite cells cannot be recovered in these genetic contexts . Most of the data are explained if one assumes that the maintenance of the rho(+) genome depends on a centromere-like structure dispensable for the maintenance of rho(-) mtDNA and/or the function of mitochondrially encoded ATP synthase subunits, especially ATP6 . In fact, the real challenge for the next 50 years will be to assemble the pieces of this puzzle by using yeast and to use complementary models, especially in strict aerobes. Rev Bras Biol, 2000 Feb, 60(1), 113 - 21 Growth of Nile tilapia Oreochromis niloticus fed with different levels of alcohol yeast; Medri V et al.; Two hundred and forty 45-day-old fingerlings of Nile tilapia (Oreochromis niloticus), sexually reverted with an initial average weight of 1.25 +/- 0.14 g, distributed in a totally casualized delineation, during 330 days . The effect of the substitution of 10%, 20% and 30% of the ration by distillery yeast (Saccharomiyces cerevisae) in balanced experimental rations on the development in the breeding of tilapias did not show a harmful effect up to the maximum tested level of 30%, showing that the choice of the yeast in the ration for these fishes depends on the availability and occasional cost . The yeast provides fish with good resistance to unfavorable environmental conditions, which could be verified since there were no statistical differences among the treatments with different levels of yeast and the witness . The long growth rate in terms of length and weight can be attributed to the little available space for each specimen, not obeying the limit of populational density, as well as reduction of food ingestion, which coincide with the winter season, above of the absence of natural feeding. Results Probl Cell Differ, 2000, 29, 21 - 44 Coordination of metabolic activity and stress resistance in yeast longevity; Jazwinski SM; The genetic analysis of longevity in yeast has revealed the importance of metabolic control and resistance to stress in aging . It has also shown that these two physiological processes are interwoven . Molecular mechanisms underlying the longevity effects of metabolic control and stress resistance, as well as genetic stability, are emerging . The yeast RAS genes play a substantial role in coordinating at least the first two of these processes . Numerous correlates can be found between the physiological processes involved in yeast aging and aging in Caenorhabditis elegans and in Drosophila, and the dietary restriction paradigm in mammals. Trends Biochem Sci, 2000 Jun, 25(6), 277 - 83 Transcriptional regulation through Mediator-like coactivators in yeast and metazoan cells; Malik S et al.; A novel multiprotein complex has recently been identified as a coactivator for transcriptional control of protein-encoding genes by RNA polymerase II in higher eukaryotic cells . This complex is evolutionarily related to the Mediator complex from yeast and, on the basis of its structural and functional characteristics, promises to be a key target of diverse regulatory circuits. Mol Biochem Parasitol, 2000 May, 108(2), 225 - 36 SmMAK16, the Schistosoma mansoni homologue of MAK16 from yeast, targets protein transport to the nucleolus; Milhon JL et al.; The SmMAK16 gene from Schistosoma mansoni was cloned by chance when an adult worm cDNA library was probed with antiserum to affinity-purified S . mansoni GSH S-transferases . SmMAK16 encodes a hydrophilic protein of 259 amino acids with a molecular mass of 31 kDa . The protein shares 43% sequence identity and 66% similarity to the nuclear protein MAK16 of Saccharomyces cerevisiae that has been implicated both in cell cycle progression and biogenesis of 60S ribosomal subunits . Both proteins display a similar degree of sequence similar to the hypothetical protein CeMAK16 from Caenorhabditis elegans . These proteins share a number of apparent protein motifs, including two nuclear localization signals (NLS), multiple sites for phosphorylation by protein kinase CK2 and four conserved cysteine residues that resemble a zinc binding domain . SmMAK16 mRNA is more highly expressed in adult female worm than males . Recombinant SmMAK16 was phosphorylated by human protein kinase CK2 . When chimeric constructs containing SmMAK16 fused the green fluorescent protein (GFP) were transiently transfected into COS-7s cells, the reporter was localized not in nuclei, but exclusively in nucleoli . The yeast and nematode homologues were likewise able to direct nucleolar accumulation of the fluorescent reporter . The high degree of sequence conservation together with the ability to direct nucleolar protein transport supports the hypothesis that MAK16 proteins play a key role in the biogenesis of 60S subunits. Biochim Biophys Acta, 2000 May 31, 1458(2-3), 443 - 56 Organisation of the yeast ATP synthase F(0):a study based on cysteine mutants, thiol modification and cross-linking reagents; Velours J et al.; A topological study of the yeast ATP synthase membranous domain was undertaken by means of chemical modifications and cross-linking experiments on the wild-type complex and on mutated enzymes obtained by site-directed mutagenesis of genes encoding ATP synthase subunits . The modification by non-permeant maleimide reagents of the Cys-54 of mutated subunit 4 (subunit b), of the Cys-23 in the N-terminus of subunit 6 (subunit a) and of the Cys-91 in the C-terminus of mutated subunit f demonstrated their location in the mitochondrial intermembrane space . Near-neighbour relationships between subunits of the complex were demonstrated by means of homobifunctional and heterobifunctional reagents . Our data suggest interactions between the first transmembranous alpha-helix of subunit 6, the two hydrophobic segments of subunit 4 and the unique membrane-spanning segments of subunits i and f . The amino acid residue 174 of subunit 4 is close to both oscp and the beta-subunit, and the residue 209 is close to oscp . The dimerisation of subunit 4 in the membrane revealed that this component is located in the periphery of the enzyme and interacts with other ATP synthase complexes. Biochim Biophys Acta, 2000 May 31, 1458(2-3), 428 - 42 Insights into ATP synthase assembly and function through the molecular genetic manipulation of subunits of the yeast mitochondrial enzyme complex; Devenish RJ et al.; Development of an increasingly detailed understanding of the eucaryotic mitochondrial ATP synthase requires a detailed knowledge of the stoichiometry, structure and function of F(0) sector subunits in the contexts of the proton channel and the stator stalk . Still to be resolved are the precise locations and roles of other supernumerary subunits present in mitochondrial ATP synthase complexes, but not found in the bacterial or chloroplast enzymes . The highly developed system of molecular genetic manipulation available in the yeast Saccharomyces cerevisiae, a unicellular eucaryote, permits testing for gene function based on the effects of gene disruption or deletion . In addition, the genes encoding ATP synthase subunits can be manipulated to introduce specific amino acids at desired positions within a subunit, or to add epitope or affinity tags at the C-terminus, enabling questions of stoichiometry, structure and function to be addressed . Newly emerging technologies, such as fusions of subunits with GFP are being applied to probe the dynamic interactions within mitochondrial ATP synthase, between ATP synthase complexes, and between ATP synthase and other mitochondrial enzyme complexes. Curr Biol, 2000 May 18, 10(10), 615 - 8 Cdc14 activates cdc15 to promote mitotic exit in budding yeast; Jaspersen SL et al.; Inactivation of mitotic cyclin-dependent kinases (Cdks) is required for cells to exit mitosis {1} {2} . In the budding yeast Saccharomyces cerevisiae, Cdk inactivation is triggered by the phosphatase Cdc14, which is activated by a complex network of regulatory proteins that includes the protein kinase Cdc15 {3} {4} {5} {6} . Here we show that the ability of Cdc15 to promote mitotic exit is inhibited by phosphorylation . Cdc15 is phosphorylated in vivo at multiple Cdk-consensus sites during most of the cell cycle, but is transiently dephosphorylated in late mitosis . Although phosphorylation appears to have no effect on Cdc15 kinase activity, a non-phosphorylatable mutant of Cdc15 is a more potent stimulator of mitotic exit than wild-type Cdc15, indicating that phosphorylation inhibits Cdc15 function in vivo . Interestingly, inhibitory phosphorylation of Cdc15 is removed by the phosphatase Cdc14 in vitro, and overproduction of Cdc14 leads to Cdc15 dephosphorylation in vivo . Thus, Cdc15 serves both as an activator and substrate of Cdc14 . Although this scheme raises the possibility that positive feedback promotes Cdc14 activation, we present evidence that such feedback is not essential for Cdc14 activation in vivo . Instead, Cdc15 dephosphorylation may promote some additional function of Cdc15 that is independent of its effects on Cdc14 activation. Org Lett, 1999 Dec 16, 1(12), 1879 - 80 Chemo- and stereoselective reduction of an alpha-cyanoketone by bakers' yeast at low temperature; Florey P et al.; {formula: see text} The bakers' yeast mediated reduction of 3-oxo-3-phenylpropanenitrile (1) proceeds at 4 degrees C to give exclusively (S)-3-hydroxy-3-phenylpropanenitrile (3) in 59% yield . This is in contrast to the corresponding reaction at room temperature which yields a mixture of reduction and alkylation products . This work demonstrates the use of low temperature to improve yeast selectivity. Org Lett, 1999 Dec 2, 1(11), 1839 - 42 A highly enantioselective conjugate reduction of 3-arylinden-1-ones using bakers' yeast for the preparation of (S)-3-arylindan-1-ones; Clark WM et al.; {formula: see text} The bakers' yeast reduction of 3-(1,3-benzodioxol-5-yl)-6-propoxy-1H-inden-1-one 4 has been shown to give (S)-3-(1,3-benzodioxol-5-yl)-2,3-dihydro-6-propoxy-1H-indan-1-one 6 in 65% yield with high enantioselectivity (> 99.0% ee), a key intermediate for the synthesis of the endothelin receptor antagonist SB 217242 . In addition, the substituted 3-arylinden-1-ones 10a-e gave equally high enantioselectivity for the 3-arylindan-1-one products 13a-e . Mechanistic studies of the reaction indicate the operative pathway to be an asymmetric conjugate reduction, wherein the hydride transfer from NAD(P)H occurs from the Re-face of the indenone substrate. Genetics, 2000 Jun, 155(2), 611 - 22 Loss of Rhb1, a Rheb-related GTPase in fission yeast, causes growth arrest with a terminal phenotype similar to that caused by nitrogen starvation; Mach KE et al.; The Rheb GTPase is most similar in primary sequence to the Ras, Rap, R-Ras, and Ral GTPases, which regulate cell growth and differentiation in many cell types . A likely fission yeast homologue of mammalian Rheb, which we designated Rhb1, was identified by genome sequencing . Our investigation of rhb1 showed that rhb1(-) cells arrested cell growth and division with a terminal phenotype similar to that of nitrogen-starved cells . In particular, cells depleted of Rhb1 arrested as small, round cells with 1N DNA content, arrested more quickly in low-nitrogen medium, and induced expression of fnx1 and mei2 mRNA, two mRNAs that were normally induced by nitrogen starvation . Since mammalian Rheb binds and may regulate Raf-1, a Ras effector, we tested for functional overlap between Ras1 and Rhb1 in fission yeast . This analysis showed that Ras1 overexpression did not suppress rhb1(-) mutant phenotypes, Rhb1 overexpression did not suppress ras1(-) mutant phenotypes, and ras1(-) rhb1(-) double mutants had phenotypes equal to the sum of the corresponding single-mutant phenotypes . Hence, there is no evidence for overlapping functions between Ras1 and Rhb1 . On the basis of this study, we hypothesize that Rhb1 negatively regulates entry into stationary phase when extracellular nitrogen levels are adequate for growth . If this hypothesis is correct, then Rhb1 and Ras1 regulate alternative responses to limiting nutrients. Genetics, 2000 Jun, 155(2), 601 - 9 The {KIL-d} element specifically regulates viral gene expression in yeast; Talloczy Z et al.; The cytoplasmically inherited {KIL-d} element epigenetically regulates killer virus gene expression in Saccharomyces cerevisiae . {KIL-d} results in variegated defects in expression of the M double-stranded RNA viral segment in haploid cells that are "healed" in diploids . We report that the {KIL-d} element is spontaneously lost with a frequency of 10(-4)-10(-5) and reappears with variegated phenotypic expression with a frequency of > or =10(-3) . This high rate of loss and higher rate of reappearance is unlike any known nucleic acid replicon but resembles the behavior of yeast prions . However, {KIL-d} is distinct from the known yeast prions in its relative guanidinium hydrochloride incurability and independence of Hsp104 protein for its maintenance . Despite its transmissibility by successive cytoplasmic transfers, multiple cytoplasmic nucleic acids have been proven not to carry the {KIL-d} trait . {KIL-d} epigenetically regulates the expression of the M double-stranded RNA satellite virus genome, but fails to alter the expression of M cDNA . This specificity remained even after a cycle of mating and meiosis . Due to its unique genetic properties and viral RNA specificity, {KIL-d} represents a new type of genetic element that interacts with a viral RNA genome. EMBO J, 2000 Jun 1, 19(11), 2641 - 51 Locus specificity determinants in the multifunctional yeast silencing protein Sir2; Cuperus G et al.; Yeast SIR2, the founding member of a conserved gene family, acts to modulate chromatin structure in three different contexts: silent (HM) mating-type loci, telomeres and rDNA . At HM loci and telomeres, Sir2p forms a complex with Sir3p and Sir4p . However, Sir2p's role in rDNA silencing is Sir3/4 independent, requiring instead an essential nucleolar protein, Net1p . We describe two novel classes of SIR2 mutations specific to either HM/telomere or rDNA silencing . Despite their opposite effects, both classes of mutations cluster in the same two regions of Sir2p, each of which borders on a conserved core domain . A surprising number of these mutations are dominant . Several rDNA silencing mutants display a Sir2p nucleolar localization defect that correlates with reduced Net1p binding . Although the molecular defect in HM/telomere-specific mutants is unclear, they mimic an age-related phenotype where Sir3p and Sir4p relocalize to the nucleolus . Artificial targeting can circumvent the silencing defect in a subset of mutants from both classes . These results define distinct functional domains of Sir2p and provide evidence for additional Sir2p-interacting factors with locus-specific silencing functions. EMBO J, 2000 Jun 1, 19(11), 2515 - 24 Membrane hyperpolarization and salt sensitivity induced by deletion of PMP3, a highly conserved small protein of yeast plasma membrane; Navarre C et al.; Yeast plasma membranes contain a small 55 amino acid hydrophobic polypeptide, Pmp3p, which has high sequence similarity to a novel family of plant polypeptides that are overexpressed under high salt concentration or low temperature treatment . The PMP3 gene is not essential under normal growth conditions . However, its deletion increases the plasma membrane potential and confers sensitivity to cytotoxic cations, such as Na(+) and hygromycin B . Interestingly, the disruption of PMP3 exacerbates the NaCl sensitivity phenotype of a mutant strain lacking the Pmr2p/Enap Na(+)-ATPases and the Nha1p Na(+)/H(+) antiporter, and suppresses the potassium dependency of a strain lacking the K(+) transporters, Trk1p and Trk2p . All these phenotypes could be reversed by the addition of high Ca(2+) concentration to the medium . These genetic interactions indicate that the major effect of the PMP3 deletion is a hyperpolarization of the plasma membrane potential that probably promotes a non-specific influx of monovalent cations . Expression of plant RCI2A in yeast could substitute for the loss of Pmp3p, indicating a common role for Pmp3p and the plant homologue. J Mol Biol, 2000 Jun 9, 299(3), 601 - 13 TFIIIC-independent in vitro transcription of yeast tRNA genes; Dieci G et al.; The most peculiar transcriptional property of eukaryotic tRNA genes, as well as of other genes served by RNA polymerase III, is their complete dependence on the intragenic interaction platform provided by transcription factor IIIC (TFIIIC) for the productive assembly of the TBP-containing initiation factor TFIIIB . The sole exception, in yeast, is the U6 RNA gene, which is able to exploit a TATAAATA element, 30 bp upstream of the transcription start site, for the TFIIIC-independent assembly of TFIIIB . To find out whether this extragenic core promoter organization and autonomous TFIIIB assembly capacity are unique features of the U6 gene or also apply to other genes transcribed by RNA polymerase III, we scanned the 5'-flanking regions (up to position -100) of the entire tRNA gene set of Saccharomyces cerevisiae searching for U6-like TATA motifs . Four tRNA genes harboring such a sequence motif around position -30 were identified and found to be transcribed in vitro by a minimal system only composed of TFIIIB and RNA polymerase III . In this system, start site selection is not at all affected by the absence of TFIIIC, which, when added, significantly stimulates transcription by determining an increase in the number, rather than in the efficiency of utilization, of productive initiation complexes . A specific TBP-TATA element interaction is absolutely required for TFIIIC-independent transcription, but the nearby sequence context also contributes to the efficiency of autonomous TFIIIB assembly . The existence of a TFIIIB assembly pathway leading to the faithful transcription of natural eukaryotic tRNA genes in the absence of TFIIIC provides novel insights into the functional flexibility of the eukaryotic tRNA gene transcription machinery and on its evolution from an ancestral RNA polymerase III system relying on upstream, TATA- centered control elements . J Biol Chem, 2000 Aug 25, 275(34), 26428 - 35 Persistent yeast single-stranded RNA viruses exist in vivo as genomic RNA.RNA polymerase complexes in 1:1 stoichiometry; Solorzano A et al.; Yeast narnavirus 20 S and 23 S RNAs encode RNA-dependent RNA polymerases p91 and p104, respectively, but do not encode coat proteins . Both RNAs form ribonucleoprotein complexes with their cognate polymerases . Here we show that these complexes are not localized in mitochondria, unlike the closely related mitoviruses, which reside in these organelles . Cytoplasmic localization of these polymerases was demonstrated by immunofluorescence and by fluorescence emitted from green fluorescent protein-fused polymerases . These fusion proteins were able to form ribonucleoprotein complexes as did the wild-type polymerases . Fluorescent observations and cell fractionation experiments suggested that the polymerases were stabilized by complex formation with their viral RNA genomes . Immunoprecipitation experiments with anti-green fluorescent protein antibodies demonstrated that a single polymerase molecule binds to a single viral RNA genome in the complex . Moreover, the majority (if not all) of 20 S and 23 S RNA molecules were found to form complexes with their cognate RNA polymerases . Since these viral RNAs were not encapsidated, ribonucleoprotein complex formation with their cognate RNA polymerases appears to be their strategy to survive in the host as persistent viruses. J Biochem (Tokyo), 2000 Jun, 127(6), 1109 - 13 Characterization of recombinant human DNA topoisomerase IIIalpha activity expressed in yeast; Hotoda N et al.; Recombinant human DNA topoisomerase IIIalpha was expressed in mutant yeast cells devoid of both topoisomerases I and III, and the gene product was partially purified . The activity of the protein in supercoil removal was found to be limited and also biphasic: in the first phase it processively changed the linking-number of hypernegatively supercoiled DNA, but only to the superhelicity of a regular negative supercoil; in the second phase the enzyme relaxed the DNA further, but only slightly and slowly . The optimal solution conditions for the enzyme activity were found to be physiological . The assay results with a truncation mutant showed that the C-terminal 334 amino acids are unnecessary for the activity, suggesting that this region, and perhaps the entire protein, is involved in a function other than supercoil removal. J Biochem (Tokyo), 2000 Jun, 127(6), 1041 - 6 Inhibitory effects of 1,4-naphthoquinone derivatives on rat cytochrome P4501A1-dependent monooxygenase activity in recombinant yeast microsomes; Inouye K et al.; We reported previously that various naphthoquinone derivatives inhibited cytochrome P450-dependent monooxygenase of liver and placenta microsomes {Muto, N . et al . (1987) Biochem . Biophys . Res . Commun . 146, 487-494} . To understand the complex inhibitory behaviors that were observed, it is desirable to study the relationship between structure and inhibitory activity of naphthoquinones in a simplified system containing a single P450 species . In the present study, the inhibitory effects of six derivatives of 1,4-naphthoquinone (hereafter referred to as NQ) on rat cytochrome P4501A1-dependent 7-ethoxycoumarin O-deethylation were examined using yeast microsomes containing overexpressed rat P4501A1 . Of these, 2-methyl-5-hydroxy-NQ, 2-methyl-NQ, 2-hydroxy-NQ, and NQ showed competitive inhibition, whereas 5,8-dihydroxy-NQ and 5-hydroxy-NQ showed noncompetitive inhibition . Judging from the inhibitor constant (K(i)), the binding affinity of the four competitive inhibitors for the substrate-binding pocket of P4501A1 is in the order: 2-CH(3)-5-OH-NQ > 2-CH(3)-NQ > NQ >> 2-OH-NQ . On binding with P4501A1, 2-CH(3)-5-OH-NQ, 2-CH(3)-NQ, and NQ induced distinct Type II, Type I, and reverse Type I spectra, respectively . These results indicate that methyl and hydroxyl groups introduced into NQ have unique effects on their binding mode and binding affinity. J Cell Biol, 2000 May 29, 149(5), 1027 - 38 Yeast nucleoporins involved in passive nuclear envelope permeability; Shulga N et al.; The vertebrate nuclear pore complex (NPC) harbors an approximately 10-nm diameter diffusion channel that is large enough to admit 50-kD polypeptides . We have analyzed the permeability properties of the Saccharomyces cerevisiae nuclear envelope (NE) using import (NLS) and export (NES) signal-containing green fluorescent protein (GFP) reporters . Compared with wild-type, passive export rates of a classical karyopherin/importin (Kap) Kap60p/Kap95p-targeted NLS-GFP reporter (cNLS-GFP) were significantly faster in nup188-Delta and nup170-Delta cells . Similar results were obtained using two other NLS-GFP reporters, containing either the Kap104p-targeted Nab2p NLS (rgNLS) or the Kap121p-targeted Pho4p NLS (pNLS) . Elevated levels of Hsp70 stimulated cNLS-GFP import, but had no effect on the import of rgNLS-GFP . Thus, the role of Hsp70 in NLS-directed import may be NLS- or targeting pathway-specific . Equilibrium sieving limits for the diffusion channel were assessed in vivo using NES-GFP reporters of 36-126 kD and were found to be greater than wild-type in nup188-Delta and nup170-Delta cells . We propose that Nup170p and Nup188p are involved in establishing the functional resting diameter of the NPC's central transport channel. Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6487 - 92 Phospholipid:diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants; Dahlqvist A et al.; Triacylglycerol (TAG) is known to be synthesized in a reaction that uses acyl-CoA as acyl donor and diacylglycerol (DAG) as acceptor, and which is catalyzed by the enzyme acyl-CoA:diacylglycerol acyltransferase . We have found that some plants and yeast also have an acyl-CoA-independent mechanism for TAG synthesis, which uses phospholipids as acyl donors and DAG as acceptor . This reaction is catalyzed by an enzyme that we call phospholipid:diacylglycerol acyltransferase, or PDAT . PDAT was characterized in microsomal preparations from three different oil seeds: sunflower, castor bean, and Crepis palaestina . We found that the specificity of the enzyme for the acyl group in the phospholipid varies between these species . Thus, C . palaestina PDAT preferentially incorporates vernoloyl groups into TAG, whereas PDAT from castor bean incorporates both ricinoleoyl and vernoloyl groups . We further found that PDAT activity also is present in yeast microsomes . The substrate specificity of this PDAT depends on the head group of the acyl donor, the acyl group transferred, and the acyl chains of the acceptor DAG . The gene encoding the enzyme was identified . The encoded PDAT protein is related to lecithin:cholesterol acyltransferase, which catalyzes the acyl-CoA-independent synthesis of cholesterol esters . However, budding yeast PDAT and its relatives in fission yeast and Arabidopsis form a distinct branch within this protein superfamily, indicating that a separate PDAT enzyme arose at an early point in evolution. Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6421 - 6 Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway; Beaudoin F et al.; A Caenorhabditis elegans ORF encoding the presumptive condensing enzyme activity of a fatty acid elongase has been characterized functionally by heterologous expression in yeast . This ORF (F56H11 . 4) shows low similarity to Saccharomyces cerevisiae genes involved in fatty acid elongation . The substrate specificity of the C . elegans enzyme indicated a preference for Delta(6)-desaturated C18 polyunsaturated fatty acids . Coexpression of this activity with fatty acid desaturases required for the synthesis of C20 polyunsaturated fatty acids resulted in the accumulation of arachidonic acid from linoleic acid and eicosapentaenoic acid from alpha-linolenic acid . These results demonstrate the reconstitution of the n-3 and n-6 polyunsaturated fatty acid biosynthetic pathways . The C . elegans ORF is likely to interact with endogenous components of a yeast elongation system, with the heterologous nematode condensing enzyme F56H11.4 causing a redirection of enzymatic activity toward polyunsaturated C18 fatty acid substrates. Biochim Biophys Acta, 2000 Jul 3, 1475(2), 151 - 6 Vanadate and copper induce overlapping oxidative stress responses in the vanadate-tolerant yeast Hansenula polymorpha; Mannazzu I et al.; The mechanisms by which vanadate exerts a toxic effect on living organisms are not completely understood . This is principally due to the variety of intracellular targets of the metal and to the changes in the chemical form and oxidation states that vanadate can undergo, both in the external environment and intracellularly . In order to further elucidate the reasons for vanadate toxicity, and assuming that common detoxification mechanisms can be evoked by a general heavy metal response, we have compared some aspects of the cellular responses to vanadate and copper in the yeast Hansenula polymorpha . By means of 2D electrophoresis we show the existence of common determinants in the responses to vanadate- and copper-induced stresses . Moreover, we demonstrate that both metals induce significant increases in antioxidant enzyme levels, and that there are significant overlaps in the heavy metal and oxidative stress responses . Interestingly, vanadate induces an increase in catalase activity that is much higher than that seen with copper and, unlike copper, does not cause lipid peroxidation of cellular membranes . This suggests that H . polymorpha cells activate a further specific detoxification pathway against vanadate-induced oxidative insults. J Biol Chem, 2000 Aug 11, 275(32), 24686 - 92 The inositol hexakisphosphate kinase family . Catalytic flexibility and function in yeast vacuole biogenesis; Saiardi A et al.; Saiardi et al . (Saiardi, A., Erdjument-Bromage, H., Snowman, A., Tempst, P., and Snyder, S . H . (1999) Curr . Biol . 9, 1323-1326) previously described the cloning of a kinase from yeast and two kinases from mammals (types 1 and 2), which phosphorylate inositol hexakisphosphate (InsP(6)) to diphosphoinositol pentakisphosphate, a "high energy" candidate regulator of cellular trafficking . We have now studied the significance of InsP(6) kinase activity in Saccharomyces cerevisiae by disrupting the kinase gene . These ip6kDelta cells grew more slowly, their levels of diphosphoinositol polyphosphates were 60-80% lower than wild-type cells, and the cells contained abnormally small and fragmented vacuoles . Novel activities of the mammalian and yeast InsP(6) kinases were identified; inositol pentakisphosphate (InsP(5)) was phosphorylated to diphosphoinositol tetrakisphosphate (PP-InsP(4)), which was further metabolized to a novel compound, tentatively identified as bis-diphosphoinositol trisphosphate . The latter is a new substrate for human diphosphoinositol polyphosphate phosphohydrolase . Kinetic parameters for the mammalian type 1 kinase indicate that InsP(5) (K(m) = 1.2 micrometer) and InsP(6) (K(m) = 6.7 micrometer) compete for phosphorylation in vivo . This is the first time a PP-InsP(4) synthase has been identified . The mammalian type 2 kinase and the yeast kinase are more specialized for the phosphorylation of InsP(6) . Synthesis of the diphosphorylated inositol phosphates is thus revealed to be more complex and interdependent than previously envisaged. Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 389 - 94 Candida tartarivorans sp . nov., an anamorphic ascomycetous yeast with the capacity to degrade L(+)- and meso-tartaric acid; Fonseca A et al.; An undescribed anamorphic yeast species of ascomycetous affinity, for which the name Candida tartarivorans is proposed, was isolated from dried wine lees in Portugal using a selective medium with L(+)-tartaric acid as the sole source of carbon and energy . The single isolate (IGC 4854T) showed the following characteristics: sympodial holoblastic conidiogenesis, absence of asci with ascospores, a negative colour reaction with Diazonium Blue B, production of elaborate pseudomycelium and ability to grow with inositol as sole source of carbon . Analysis of the physiological data pointed to a close relationship with other inositol-assimilating taxa, namely the genera Arxula, Stephanoascus, Sympodiomyces, Zygoascus and selected Candida species . Comparative analysis of the D1/D2 variable domain of the 26S rRNA gene of all available sequences for ascomycetous yeasts showed that strain IGC 4854T did not match with any other species in the database . The closest relative was Candida auringiensis Santa Maria, but the two species differed in 24 nucleotide positions . A description of the new species is given. Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 381 - 7 Trichosporon veenhuisii sp . nov., an alkane-assimilating anamorphic basidiomycetous yeast; Middelhoven WJ et al.; A morphological and physiological description of an alkane-assimilating anamorphic basidiomycetous yeast species, named Trichosporon veenhuisii, 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 . The type strain is CBS 7136T. Org Lett, 1999 Oct 21, 1(8), 1153 - 5 Improving the stereoselectivity of bakers' yeast reductions by genetic engineering; Rodriguez S et al.; {formula: see text} The stereoselectivities of bakers' yeast catalyzed reductions of beta-keto esters can be manipulated by genetic design . Strains in which two major beta-keto ester reductases are either knocked out or overexpressed have been constructed . The former approach results in whole cell biocatalysts with reversed stereoselectivity from unmodified bakers' yeast while the latter shows useful improvements in stereoselectivity . These results indicate that the "designer yeast" approach can provide useful biocatalysts for these transformations. Mol Cell Biol, 2000 Jun, 20(12), 4411 - 9 Sli2 (Ypk1), a homologue of mammalian protein kinase SGK, is a downstream kinase in the sphingolipid-mediated signaling pathway of yeast; Sun Y et al.; ISP-1 is a new type of immunosuppressant, the structure of which is homologous to that of sphingosine . In a previous study, ISP-1 was found to inhibit mammalian serine palmitoyltransferase, the primary enzyme involved in sphingolipid biosynthesis, and to reduce the intracellular pool of sphingolipids . ISP-1 induces the apoptosis of cytotoxic T cells, which is triggered by decreases in the intracellular levels of sphingolipids . In this study, the inhibition of yeast (Saccharomyces cerevisiae) proliferation by ISP-1 was observed . This ISP-1-induced growth inhibition was also triggered by decreases in the intracellular levels of sphingolipids . In addition, DNA duplication without cytokinesis was detected in ISP-1-treated yeast cells on flow cytometry analysis . We have cloned multicopy suppressor genes of yeast which overcome the lethal sphingolipid depletion induced by ISP-1 . One of these genes, SLI2, is synonymous with YPK1, which encodes a serine/threonine kinase . Kinase-dead mutants of YPK1 did not show any resistance to ISP-1, leading us to predict that the kinase activity of the Ypk1 protein should be essential for this resistance to ISP-1 . Ypk1 protein overexpression had no effect on sphingolipid biosynthesis by the yeast . Furthermore, both the phosphorylation and intracellular localization of the Ypk1 protein were regulated by the intracellular sphingolipid levels . These data suggest that the Ypk1 protein is a downstream kinase in the sphingolipid-mediated signaling pathway of yeast . The Ypk1 protein was reported to be a functional homologue of the mammalian protein kinase SGK, which is a downstream kinase of 3-phosphoinositide-dependent kinase 1 (PDK1) . PDK1 phosphotidylinositol (PI) is regulated by PI-3,4,5-triphosphate and PI-3,4-bisphosphate through the pleckstrin homology (PH) domain . Overexpression of mammalian SGK also overcomes the sphingolipid depletion in yeast . Taking both the inability to produce PI-3,4, 5-triphosphate and PI-3,4-bisphosphate and the lack of a PH domain in the yeast homologue of PDK1, the Pkh1 protein, into account, these findings further suggest that yeast may use sphingolipids instead of inositol phospholipids as lipid mediators. Mol Cell Biol, 2000 Jun, 20(12), 4295 - 308 Yeast Ran-binding protein 1 (Yrb1) shuttles between the nucleus and cytoplasm and is exported from the nucleus via a CRM1 (XPO1)-dependent pathway; Kunzler M et al.; The RanGTP-binding protein RanBP1, which is located in the cytoplasm, has been implicated in release of nuclear export complexes from the cytoplasmic side of the nuclear pore complex . Here we show that Yrb1 (the yeast homolog of RanBP1) shuttles between the nucleus and the cytoplasm . Nuclear import of Yrb1 is a facilitated process that requires a short basic sequence within the Ran-binding domain (RBD) . By contrast, nuclear export of Yrb1 requires an intact RBD, which forms a ternary complex with the Xpo1 (Crm1) NES receptor in the presence of RanGTP . Nuclear export of Yrb1, however, is insensitive towards leptomycin B, suggesting a novel type of substrate recognition between Yrb1 and Xpo1 . Taken together, these data suggest that ongoing nuclear import and export is an important feature of Yrb1 function in vivo. J Biol Chem, 2000 Aug 11, 275(32), 24928 - 34 The yeast histone acetyltransferase A2 complex, but not free Gcn5p, binds stably to nucleosomal arrays; Sendra R et al.; We have investigated the structural basis for the differential catalytic function of the yeast Gcn5p-containing histone acetyltransferase (HAT) A2 complex and free recombinant yeast Gcn5p (rGcn5p) . HAT A2 is shown to be a unique complex that contains Gcn5p, Ada2p, and Ada3p, but not proteins specific to other related HAT A complexes, e.g . ADA, SAGA . Nevertheless, HAT A2 produces the same unique polyacetylation pattern of nucleosomal substrates reported previously for ADA and SAGA, demonstrating that proteins specific to the ADA and SAGA complexes do not influence the enzymatic activity of Gcn5p within the HAT A2 complex . To investigate the role of substrate interactions in the differential behavior of free and complexed Gcn5p, sucrose density gradient centrifugation was used to characterize the binding of HAT A2 and free rGcn5p to intact and trypsinized nucleosomal arrays, H3/H4 tetramer arrays, and nucleosome core particles . We find that HAT A2 forms stable complexes with all nucleosomal substrates tested . In distinct contrast, rGcn5p does not interact stably with nucleosomal arrays, despite being able to specifically monoacetylate the H3 N terminus of nucleosomal substrates . Our data suggest that the ability of the HAT A2 complex to bind stably to nucleosomal arrays is functionally related to both local and global acetylation by the complexed and free forms of Gcn5p. Acta Biochim Pol, 1999, 46(4), 862 - 72 Involvement of the essential yeast DNA polymerases in induced gene conversion; Halas A et al.; In the yeast Saccharomyces cerevisiae three different DNA polymerases alpha, delta and epsilon are involved in DNA replication . DNA polymerase alpha is responsible for initiation of DNA synthesis and polymerases delta and epsilon are required for elongation of DNA strand during replication . DNA polymerases delta and epsilon are also involved in DNA repair . In this work we studied the role of these three DNA polymerases in the process of recombinational synthesis . Using thermo-sensitive heteroallelic mutants in genes encoding DNA polymerases we studied their role in the process of induced gene conversion . Mutant strains were treated with mutagens, incubated under permissive or restrictive conditions and the numbers of convertants obtained were compared . A very high difference in the number of convertants between restrictive and permissive conditions was observed for polymerases alpha and delta, which suggests that these two polymerases play an important role in DNA synthesis during mitotic gene conversion . Marginal dependence of gene conversion on the activity of polymerase epsilon indicates that this DNA polymerase may be involved in this process but rather as an auxiliary enzyme. Eur J Biochem, 2000 Jun, 267(11), 3167 - 80 Biogenesis of the yeast frataxin homolog Yfh1p . Tim44-dependent transfer to mtHsp70 facilitates folding of newly imported proteins in mitochondria; Geissler A et al.; Tim44 is an essential component of the mitochondrial inner membrane protein import machinery . In this study we asked if Tim44 is of relevance in intramitochondrial protein folding . We investigated the role of Tim44 in the biogenesis of the authentic mitochondrial protein Yfh1p, the yeast homolog of mammalian frataxin, which was recently implicated in Friedreich ataxia . After inactivation of Tim44, binding of mitochondrial heat shock protein (mtHsp)70 to translocating Yfh1p and subsequent folding to the native state was nearly completely blocked . Residual amounts of imported Yfh1p showed an increased tendency to aggregate . To further characterize the functions of Tim44 in the matrix, we imported dihydrofolate reductase (DHFR) as a model protein . Depletion of Tim44 allowed import of DHFR, although folding of the newly imported DHFR was delayed . Moreover, the depletion of Tim44 caused a strongly reduced binding of mtHsp70 and Mge1 to the translocating polypeptide . Subsequent dissociation of mtHsp70 from imported DHFR was delayed, indicating that mtHsp70-substrate complexes formed independently of Tim44 differ from the complexes that form under the control of Tim44 . We conclude that Tim44 not only plays a role in protein translocation but also in the pathways of mitochondrial protein folding. J Virol, 2000 Jun, 74(12), 5486 - 94 A yeast-based genetic system for functional analysis of viral mRNA capping enzymes; Ho CK et al.; Virus-encoded mRNA capping enzymes are attractive targets for antiviral therapy, but functional studies have been limited by the lack of genetically tractable in vivo systems that focus exclusively on the RNA-processing activities of the viral proteins . Here we have developed such a system by engineering a viral capping enzyme-vaccinia virus D1(1-545)p, an RNA triphosphatase and RNA guanylyltransferase-to function in the budding yeast Saccharomyces cerevisiae in lieu of the endogenous fungal triphosphatase (Cet1p) and guanylyltransferase (Ceg1p) . This was accomplished by fusion of D1(1-545)p to the C-terminal guanylyltransferase domain of mammalian capping enzyme, Mce1(211-597)p, which serves as a vehicle to target the viral capping enzyme to the RNA polymerase II elongation complex . An inactivating mutation (K294A) of the mammalian guanylyltransferase active site in the fusion protein had no impact on genetic complementation of cet1Deltaceg1Delta cells, thus proving that (i) the viral guanylyltransferase was active in vivo and (ii) the mammalian domain can serve purely as a chaperone to direct other proteins to the transcription complex . Alanine scanning had identified five amino acids of vaccinia virus capping enzyme-Glu37, Glu39, Arg77, Glu192, and Glu194-that are essential for gamma phosphate cleavage in vitro . Here we show that the introduction of mutation E37A, R77A, or E192A into the fusion protein abrogates RNA triphosphatase function in vivo . The essential residues are located within three motifs that define a family of viral and fungal metal-dependent phosphohydrolases with a distinctive capacity to hydrolyze nucleoside triphosphates to nucleoside diphosphates in the presence of manganese or cobalt . The acidic residues Glu37, Glu39, and Glu192 likely comprise the metal-binding site of vaccinia virus triphosphatase, insofar as their replacement by glutamine abolishes the RNA triphosphatase and ATPase activities. J Biol Chem, 2000 Aug 11, 275(32), 24458 - 65 YLL031c belongs to a novel family of membrane proteins involved in the transfer of ethanolaminephosphate onto the core structure of glycosylphosphatidylinositol anchors in yeast; Flury I et al.; MCD4 and GPI7 are important for the addition of glycosylphosphatidylinositol (GPI) anchors to proteins in the yeast Saccharomyces cerevisiae . Mutations in these genes lead to a reduction of GPI anchoring and cell wall fragility . Gpi7 mutants accumulate a GPI lipid intermediate of the structure Manalpha1-2{NH(2)-(CH(2))(2)-PO(4)-->}Manalpha1-2Manalpha 1-6{NH(2)-(C H(2))(2)-PO(4)-->}Manalpha1-4GlcNalpha1-6{acyl-->}inositol-P O(4)-lipi d, which, in comparison with the complete GPI precursor lipid CP2, lacks an HF-sensitive side chain on the alpha1-6-linked mannose . In contrast, mcd4-174 accumulates only minor amounts of abnormal GPI intermediates . Here we investigate whether YLL031c, an open reading frame predicting a further homologue of GPI7 and MCD4, plays any role in GPI anchoring . YLL031c is an essential gene . Its depletion results in a reduction of GPI anchor addition to GPI proteins as well as to cell wall fragility . YLL031c-depleted cells accumulate GPI intermediates with the structures Manalpha1-2Manalpha1-2Manalpha1-6{NH(2)-(CH(2))(2)-PO( 4)-->}Manalpha1 -4GlcNalpha1-6{acyl-->}inositol-PO(4)-lipid and Manalpha1-2Manalpha1-2Manalpha1-6Manalpha1-4G lcNalpha1-6{acyl-->}inos itol-PO(4)-lipid . Subcellular localization studies of a tagged version of YLL031c suggest that this protein is mainly in the ER, in contrast to Gpi7p, which is found at the cell surface . The data are compatible with the idea that YLL031c transfers the ethanolaminephosphate to the inner alpha1-2-linked mannose, i.e . the group that links the GPI lipid anchor to proteins, whereas Mcd4p and Gpi7p transfer ethanolaminephosphate onto the alpha1-4- and alpha1-6-linked mannoses of the GPI anchor, respectively. Org Lett, 1999 Sep 9, 1(5), 815 - 7 Yeast protein farnesyltransferase . Binding of S-alkyl peptides and related analogues Rozema DB, Phillips ST, Poulter CD. {formula: see text} Protein farnesyltransferase (PFTase) catalyzes alkylation of cysteine residues by farnesyl diphosphate (FPP) . The dissociation constants for the PFTase.peptide analogue complexes for the series of analogues fl-RTRC(X)VIA (X = H, methyl, dodecyl, farnesyl) were measured by fluorescence anisotropy . The results indicate that an ionizable sulfhydryl moiety is important for substrate binding and the farnesyl group in the product facilitates binding. Mol Gen Genet, 2000 Apr, 263(3), 520 - 6 The yeast peptidyl proline isomerases FPR3 and FPR4, in high copy numbers, suppress defects resulting from the absence of the E3 ubiquitin ligase TOM1; Davey M et al.; Tom1p is a 3268-amino acid protein with extensive homology to the hect-domain class of E3 ubiquitin ligases . Disruption of the TOMI gene results in temperature sensitivity for growth . Genes encoding the peptidyl proline isomerases Fpr3p and Fpr4p, when present on multicopy plasmids, will suppress this temperature-sensitive growth phenotype . FPR3 can also suppress the mating defect seen in tom1 strains . Suppression is specific for disruption of TOM1, since FPR3 does not restore wild-type growth to strains lacking the E2 ubiquitin-conjugating enzyme Rad6p or the transcriptional regulator Ngglp . Interestingly, the peptidyl proline isomerase domains of Fpr3p and Fpr4p are not required for suppression; rather the essential sequences include about 170 highly conserved residues at the proteins' N-termini . Previously we found that Tomlp plays a role in gene regulation . Since overexpression of FPR4 does not suppress the reduced expression of the ARG1 promoter found in tom1 deletion strains, Tom1p probably has one or more functions beyond its involvement in gene expression. Mol Gen Genet, 2000 Apr, 263(3), 483 - 91 Som1, a third component of the yeast mitochondrial inner membrane peptidase complex that contains Imp1 and Imp2; Jan PS et al.; The mitochondrial inner membrane peptidase Imp is required for proteolytic processing of the mitochondrially encoded protein Cox2, the nucleus-encoded Cyt b2, Mcr1, and Cyt c1, and possibly other proteins, during their transport across the mitochondrial membranes . The peptidase contains two catalytic subunits, Imp1 and Imp2 . The small protein Soml was previously shown to affect the function of Imp1, but the precise role of Soml remained unknown . Using mutants deleted for IMP1, IMP2 and SOM1, we show here that the Som1 protein is absent in the imp1delta mutant, whereas the level of the Imp1 subunit of the peptidase is only slightly reduced in the soml null mutant . The Soml protein is not essential for proteolytic processing of Cyt b2, while the two other known Imp1 substrates, Cox2 and Mcr1, are not processed in the absence of Som1 . Proteolytic processing of Cyt c1 by the Imp2 subunit, and of Ccp by an as yet unidentified peptidase, is not impaired in the som1 deletion mutant . By crosslinking and co-immunoprecipitation assays we demonstrate that the Imp1 and Som1 proteins physically interact . We conclude from our results that stabilisation of Som1 and correct Imp1 function is mediated by a direct interaction between the Imp1 and Som1 proteins, suggesting that Som1 represents a third subunit of the Imp peptidase complex. J Biol Inorg Chem, 2000 Apr, 5(2), 189 - 203 The metal binding properties of the zinc site of yeast copper-zinc superoxide dismutase: implications for amyotrophic lateral sclerosis; Lyons TJ et al.; We have investigated factors that influence the properties of the zinc binding site in yeast copper-zinc superoxide dismutase (CuZnSOD) . The properties of yeast CuZnSOD are essentially invariant from pH 5 to pH 9 . However, below this pH range there is a change in the nature of the zinc binding site which can be interpreted as either (1) a change in metal binding affinity from strong to weak, (2) the expulsion of the metal bound at this site, or (3) a transition from a normal distorted tetrahedral ligand orientation to a more symmetric arrangement of ligands . This change is strongly reminiscent of a similar pH-induced transition seen for the bovine protein and, based on the data presented herein, is proposed to be a property that is conserved among CuZnSODs . The transition demonstrated for the yeast protein is not only sensitive to the pH of the buffering solution but also to the occupancy and redox status of the adjacent copper binding site . Furthermore, we have investigated the effect of single site mutations on the pH- and redox-sensitivity of Co2+ binding at the zinc site . Each of the mutants H46R, H48Q, H63A, H63E, H80C, G85R, and D83H is capable of binding Co2+ to a zinc site with a distorted tetrahedral geometry similar to that of wild-type . However, they do so only if Cu+ is bound at the copper site or if the pH in raised to near physiological levels, indicating that the change at the zinc binding site seen in the wild-type is conserved in the mutants, albeit with an altered pKa . The mutants H71C and D83A did not bind Co2+ in a wild-type-like fashion under any of the conditions tested . This study reveals that the zinc binding site is exquisitely sensitive to changes in the protein environment . Since three of the mutant yeast proteins investigated here contain mutations analogous to those that cause ALS (amyotrophic lateral sclerosis) in humans, this finding implicates improper metal binding as a mechanism by which CuZnSOD mutants exert their toxic gain of function. Science, 2000 May 19, 288(5469), 1242 - 4 Distinct classes of yeast promoters revealed by differential TAF recruitment; Li XY et al.; The transcription factor TFIID contains the TATA box binding protein (TBP) and multiple TBP-associated factors (TAFs) . Here, the association of TFIID components with promoters that either are dependent on multiple TAFs (TAFdep) or have no apparent TAF requirement (TAFind) is analyzed in yeast . At TAFdep promoters, TAFs are present at levels comparable to that of TBP, whereas at TAFind promoters, TAFs are present at levels that approximate background . After inactivation of several general transcription factors, including TBP, TAFs are still recruited by activators to TAFdep promoters . The results reveal two classes of promoters: at TAFind promoters, TBP is recruited in the apparent absence of TAFs, whereas at TAFdep promoters, TAFs are co-recruited with TBP in a manner consistent with direct activator-TAF interactions. Genes Dev, 2000 May 15, 14(10), 1261 - 8 (CA/GT)(n) microsatellites affect homologous recombination during yeast meiosis; Gendrel CG et al.; One of the most common microsatellites in eukaryotes consists of tandem arrays of the dinucleotide GT . Although the study of the instability of such repetitive DNA has been extremely fruitful over the last decade, no biological function has been demonstrated for these sequences . We investigated the genetic behavior of a region of the yeast Saccharomyces cerevisiae genome containing a 39-CA/GT dinucleotide repeat sequence . When the microsatellite sequence was present at the ARG4 locus on homologous chromosomes, diploid cells undergoing meiosis generated an excess of tetrads containing a conversion of the region restricted to the region of the microsatellite close to the recombination-initiation double-strand break . Moreover, whereas the repetitive sequence had no effect on the frequency of single crossover, its presence strongly stimulated the formation of multiple crossovers . The combined data strongly suggest that numerous recombination events are restricted to the initiation side of the microsatellite as though progression of the strand exchange initiated at the ARG4 promoter locus was impaired by the repetitive sequence . This observation corroborates in vitro experiments that demonstrated that RecA-promoted strand exchange is inhibited by CA/GT dinucleotide tracts . Surprisingly, meiotic instability of the microsatellite was very high (>0.1 alterations per tetrad) in all the spores with parental and recombinant chromosomes. Genes Dev, 2000 May 15, 14(10), 1229 - 35 Two protein tyrosine phosphatases, Ptp2 and Ptp3, modulate the subcellular localization of the Hog1 MAP kinase in yeast; Mattison CP et al.; The MAP kinase Hog1 transiently accumulates in the nucleus upon activation . Although Hog1 nuclear export correlates with its dephosphorylation, we find that dephosphorylation is not necessary for export . Unexpectedly, a strain lacking the nuclear protein tyrosine phosphatase, Ptp2, showed decreased Hog1 nuclear retention, while a strain lacking the cytoplasmic Ptp3 showed prolonged Hog1 nuclear accumulation, consistent with Ptp2 being a nuclear tether for Hog1 and Ptp3 being a cytoplasmic anchor . In support of this result PTP2 overexpression sequestered Hog1 in the nucleus while PTP3 overexpression restricted Hog1 to the cytoplasm . Thus, Ptp2 and Ptp3 regulate Hog1 localization by binding Hog1. Genes Dev, 2000 May 15, 14(10), 1196 - 208 The something about silencing protein, Sas3, is the catalytic subunit of NuA3, a yTAF(II)30-containing HAT complex that interacts with the Spt16 subunit of the yeast CP (Cdc68/Pob3)-FACT complex; John S et al.; We have purified and characterized a Gcn5-independent nucleosomal histone H3 HAT complex, NuA3 (Nucleosomal Acetyltransferase of histone H3) . Peptide sequencing of proteins from the purified NuA3 complex identified Sas3 as the catalytic HAT subunit of the complex . Sas3 is the yeast homolog of the human MOZ oncogene . Sas3 is required for both the HAT activity and the integrity of the NuA3 complex . In addition, NuA3 contains the TBP- associated factor, yTAF(II)30, which is also a component of the TFIID, TFIIF, and SWI/SNF complexes . Sas3 mediates interaction of the NuA3 complex with Spt16 both in vivo and in vitro . Spt16 functions as a component of the yeast CP (Cdc68/Pob3) and mammalian FACT (facilitates chromatin transcription) complexes, which are involved in transcription elongation and DNA replication . This interaction suggests that the NuA3 complex might function in concert with FACT-CP to stimulate transcription or replication elongation through nucleosomes by providing a coupled acetyltransferase activity. Arch Microbiol, 2000 Apr, 173(4), 253 - 61 Morphology-related effects on gene expression and protein accumulation of the yeast Arxula adeninivorans LS3; Wartmann T et al.; The dimorphism of the yeast Arxula adeninivorans LS3 is regulated by cultivation temperatures . Up to 42 degrees C the yeast grows as budding cells, which turn to mycelia at higher temperatures . To test whether the dimorphism is exclusively induced by high temperatures or also by other conditions, mutants were selected with an altered behaviour with respect to dimorphism . After mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, five of 25,000 colonies formed a very rough surface consisting of mycelia at 30 degrees C, in contrast to the wild-type . These mutants allow temperature-mediated and morphology-related effects on gene expression and protein accumulation to be distinguished . Budding cells and mycelia showed different expression of genes encoding secretory proteins at the same temperature . Mycelia secreted two-fold more protein than budding cells, including the enzymes glucoamylase and invertase . This indicated that morphology, rather than temperature, is the decisive factor in the analysed processes. Biochem Biophys Res Commun, 2000 May 19, 271(3), 796 - 800 A possible interaction of thioredoxin with VDUP1 in HeLa cells detected in a yeast two-hybrid system; Yamanaka H et al.; Human thioredoxin (hTrx), a small ubiquitous protein with strong reducing potential, has multiple biological functions, including signal transduction and regulation of the activity of transcription factors . hTrx expression is enhanced in HPV-transformed cancer cells; however, the role of hTrx in the malignant cells is not fully understood . We employed a yeast two-hybrid system to search for proteins that bind to hTrx in HeLa cells, a type of HPV-transformed human cervical cancer cell . In a screen of 1.62 x 10(6) yeast cotransformed with a HeLa cDNA library and an hTrx vector, 13 clones were identified as candidates for hTrx-binding proteins . Among them, 3 clones were found to code in frame for the carboxyl-terminal portion of VDUP1 protein, lacking at most the first 155 residues from the start codon . A reconstructed clone carrying the full-length VDUP1 coding sequence also showed the ability to bind to an hTrx fusion protein . Loss of interaction between VDUP1 and hTrx was observed either when two cysteines (Cys 32 and 35) in hTrx were substituted by serines or when the deletion in VDUP1 was extended from amino acid position 155 to 225 or beyond . The 71-mer peptide fragment (position 155-225) of VDUP-1 alone did not bind to hTrx . J Org Chem, 2000 Jan 14, 65(1), 129 - 35 Access to Wieland-Miescher ketone in an enantiomerically pure form by a kinetic resolution with yeast-mediated reduction; Fuhshuku K et al.; Both enantiomers of Wieland-Miescher ketone {3,4,8, 8a-tetrahydro-8a-methyl-1,6(2H,7H)-naphthalenedione}, in a highly enantiomerically enriched form, became readily available by a newly developed kinetic resolution with yeast-mediated reduction . From a screening of yeast strains, Torulaspora delbrueckii IFO 10921 was selected . The collected cells of this strain, obtained by an incubation in a glucose medium, smoothly reduced only the isolated carbonyl group of the (S)-enantiomer, while the (R)-enantiomer remained intact . Starting from both enantiomers ( approximately 70% ee) prepared by an established proline-mediated asymmetric Robinson annulation, the reduction with T . delbrueckii gave the (R)-enantiomer (98% ee) and the corresponding alcohol (4aS,5S)-4,4a, 5,6,7,8-hexahydro-5-hydroxy-4a-methyl-2(3H)-naphthalenone (94% ee, 94% de) in preparative scale in nearly quantitative yields . An approach for the asymmetric synthesis of the Wieland-Miescher ketone was also successful . 2-Methyl-2-(3-oxobutyl)-1,3-cyclohexanedione, the prochiral precursor, was reduced with this strain to give a cyclic acetal form of (2S, 3S)-3-hydroxy-2-methyl-2-(3-oxobutyl)cyclohexanone, in a stereomerically pure form. Mutat Res, 2000 Apr 28, 459(3), 173 - 86 The pattern of sensitivity of yeast dna2 mutants to DNA damaging agents suggests a role in DSB and postreplication repair pathways; Budd ME et al.; The Saccharomyces cerevisiae DNA2 gene encodes a DNA-stimulated ATPase and DNA helicase/nuclease essential for DNA replication . In characterizing dna2 mutants, we have found that Dna2p also participates in DNA repair or in damage avoidance mechanisms . dna2 mutants are sensitive to X rays, although they are less sensitive than rad52 mutants . The X-ray sensitivity of dna2 mutants is suppressed by overexpression of a 5' to 3' exonuclease, the yeast FEN-1 structure-specific nuclease, encoded by the RAD27 gene, which also suppresses the growth defect of dna2-ts mutants . SGS1 encodes a helicase with similar properties to Dna2 protein . Although sgs1Delta mutants are resistant to X rays, dna2-2 sgs1Delta double mutants are more sensitive to X rays than the dna2-2 mutant . Temperature sensitive dna2 mutants are only slightly sensitive to UV light, show normal levels of spontaneous and UV induced mutagenesis, and have only a 2.5-fold elevated level of dinucleotide tract instability compared to wildtype . However, dna2Delta strains kept alive by overproduction of RAD27 are highly sensitive to UV light . These phenotypes, in addition to the epistasis analysis reported, allow us to propose that Dna2 is involved in postreplication and DSB repair pathways. J Gen Virol, 2000 Jun, 81(Pt 6), 1587 - 91 The GCD10 subunit of yeast eIF-3 binds the methyltransferase-like domain of the 126 and 183 kDa replicase proteins of tobacco mosaic virus in the yeast two-hybrid system; Taylor DN et al.; The tobacco mosaic virus (TMV) replicase complex contains virus- and host-encoded proteins . In tomato, one of these host proteins was reported previously to be related serologically to the GCD10 subunit of yeast eIF-3 . The yeast two-hybrid system has now been used to show that yeast GCD10 interacts selectively with the methyltransferase domain shared by the 126 and 183 kDa TMV replicase proteins . These findings are consistent with a role for a GCD10-like protein in the TMV replicase complex and suggest that, in TMV-infected cells, the machinery of virus replication and protein synthesis may be closely connected. Proc Natl Acad Sci U S A, 2000 May 23, 97(11), 5984 - 8 The yeast A kinases differentially regulate iron uptake and respiratory function; Robertson LS et al.; Yeast has three A kinase catalytic subunits, which have greater than 75% identity and are encoded by the TPK genes (TPK1, TPK2, and TPK3) {Toda, T., Cameron, S., Sass, P., Zoller, M . & Wigler, M . (1987) Cell 50, 277-287} . Although they are redundant for viability, the three A kinases are not redundant for pseudohyphal growth {Robertson, L . S . & Fink, G . R . (1998) Proc . Natl . Acad . Sci . USA 95, 13783-13787; Pan, X . & Heitman, J . (1999) Mol . Cell . Biol . 19, 4874-4887}; Tpk2, but not Tpk1 or Tpk3, is required for pseudohyphal growth . Genome-wide transcriptional profiling has revealed unique signatures for each of the three A kinases leading to the identification of additional functional diversity among these proteins . Tpk2 negatively regulates genes involved in iron uptake and positively regulates genes involved in trehalose degradation and water homeostasis . Tpk1 is required for the derepression of branched chain amino acid biosynthesis genes that seem to have a second role in the maintenance of iron levels and DNA stability within mitochondria . The fact that TPK2 mutants grow better than wild types on nonfermentable carbon sources and on media deficient in iron supports the unique role of Tpk2 in respiratory growth and carbon source use. J Biol Chem, 2000 Jul 21, 275(29), 22048 - 55 Kinetic mechanism of the histone acetyltransferase GCN5 from yeast; Tanner KG et al.; The transcriptional coactivator GCN5 from yeast (yGCN5) is a histone acetyltransferase that is essential for activation of target genes . GCN5 is a member of a large family of histone acetyltransferases that are conserved between yeast and humans . To understand the molecular mechanisms of histone/protein acetylation, a detailed kinetic analysis was performed . Bi-substrate kinetic analysis using acetyl-coenzyme A (AcCoA) and an H3 histone synthetic peptide indicated that both substrates must bind to form a ternary complex before catalysis . Product inhibition studies revealed that the product CoA was a competitive inhibitor versus AcCoA . Desulfo-CoA, a dead-end inhibitor, also demonstrated simple competitive inhibition versus AcCoA . Acetylated (Lys14Ac) H3 peptide displayed noncompetitive inhibition against both H3 peptide and AcCoA . These results support a sequential ternary complex (ordered Bi-Bi) kinetic mechanism, where AcCoA binds first, followed by H3 histone . Acetylated (Lys14Ac) H3 product is released first, and CoA is the last product to leave . Also, two methods were developed to measure the binding affinities of AcCoA/CoA for GCN5 . Employing the fluorescent CoA analog etheno-CoA (epsilonCoA, 1-N(6)-etheno-CoA), a K(d) for epsilonCoA of 5.1 +/- 1.1 microm was determined by fluorescence anisotropy . This value was similar to the K(d) value of 8.5 +/- 2.6 microm for AcCoA obtained using equilibrium dialysis and to the K(i) (inhibition constant) of 6.7 microm for CoA obtained from steady-state kinetic assays . Together, these data suggest that the acetyl moiety of AcCoA contributes little to the binding energy. J Biol Chem, 2000 Aug 4, 275(31), 23608 - 14 Casein kinase I-dependent phosphorylation within a PEST sequence and ubiquitination at nearby lysines signal endocytosis of yeast uracil permease; Marchal C et al.; Uracil uptake by Saccharomyces cerevisiae is mediated by the FUR4-encoded uracil permease . The modification of uracil permease by phosphorylation at the plasma membrane is a key mechanism for regulating endocytosis of this protein . This modification in turn facilitates its ubiquitination and internalization . Following endocytosis, the permease is targeted to the lysosome/vacuole for proteolysis . We have previously shown that uracil permease is phosphorylated at several serine residues within a well characterized N-terminal PEST sequence . In this report, we provide evidence that lysine residues 38 and 41, adjacent to the PEST sequence, are the target sites for ubiquitination of the permease . Conservative substitutions at both Lys(38) and Lys(41) give variant permeases that are phosphorylated but fail to internalize . The PEST sequence contains potential phosphorylation sites conforming to the consensus sequences for casein kinase 1 . Casein kinase 1 (CK1) protein kinases, encoded by the redundant YCKI and YCK2 genes, are located at the plasma membrane . Either alone supports growth, but loss of function of both is lethal . Here, we show that in CK1-deficient cells, the permease is poorly phosphorylated and poorly ubiquitinated . Moreover, CK1 overproduction rescued the defective endocytosis of a mutant permease in which the serine phosphoacceptors were replaced by threonine (a less effective phosphoacceptor), which suggests that Yck activity may play a direct role in phosphorylating the permease . Permease internalization was not greatly affected in CK1-deficient cells, despite the low level of ubiquitination of the protein . This may be due to CK1 having a second counteracting role in endocytosis as shown by the higher turnover of variant permeases with unphosphorylatable versions of the PEST sequence. EMBO J, 2000 May 15, 19(10), 2381 - 90 Recombination-induced CAG trinucleotide repeat expansions in yeast involve the MRE11-RAD50-XRS2 complex; Richard GF et al.; Recombination induced by double-strand breaks (DSBs) in yeast leads to a higher proportion of expansions to contractions than does replication-associated tract length changes . Expansions are apparently dependent on the property of the repeat array to form hairpins, since DSB repair of a CAA(87) repeat induces only contractions of the repeat sequence . DSB-repair efficiency is reduced by 40% when DNA synthesis must traverse a CAG(98) array, as compared with a CAA(87) array . These data indicate that repair- associated DNA synthesis is inhibited by secondary structures formed by CAG(98) and that these structures promote repeat expansions during DSB repair . Overexpression of Mre11p or Rad50p suppresses the inhibition of DSB repair by CAG(98) and significantly increases the average size of expansions found at the recipient locus . Both effects are dependent on the integrity of the Mre11p-Rad50p-Xrs2p complex . The Mre11 complex thus appears to be directly involved in removing CAG or CTG hairpins that arise frequently during DNA synthesis accompanying gene conversion of these trinucleotide repeats. EMBO J, 2000 May 15, 19(10), 2323 - 31 The chromo domain protein chd1p from budding yeast is an ATP-dependent chromatin-modifying factor; Tran HG et al.; CHD proteins are members of the chromo domain family, a class of proteins involved in transcription, DNA degradation and chromatin structure . In higher eukaryotes, there are two distinct subfamilies of CHD proteins: CHD1 and CHD3/4 . Analyses carried out in vitro indicate that the CHD3/4 proteins may regulate transcription via alteration of chromatin structure . However, little is known about the role of CHD proteins in vivo, particularly the CHD1 subfamily . To understand better the cellular function of CHD proteins, we initiated a study on the Chd1p protein from budding yeast . Using genomic DNA arrays, we identified genes whose expression is affected by the absence of Chd1p . A synthetic-lethal screen uncovered genetic interactions between SWI/SNF genes and CHD1 . Biochemical experiments using Chd1p purified from yeast showed that it reconfigures the structure of nucleosome core particles in a manner distinct from the SWI-SNF complex . Taken together, these results suggest that Chd1p functions as a nucleosome remodeling factor, and that Chd1p may share overlapping roles with the SWI-SNF complex to regulate transcription. Biochem Pharmacol, 2000 Jan 15, 59(2), 177 - 85 Agonistic and synergistic activity of tamoxifen in a yeast model system; Graumann K et al.; The background of agonist/antagonist behaviour of the non-steroidal antiestrogen tamoxifen is still not fully understood . Depending on cell type, its activities range from full agonistic to antagonistic in different tissues . We investigated the transactivational properties of tamoxifen in a basic yeast model system which reconstitutes ligand-dependent human estrogen receptor-alpha (hER alpha) gene activation . Tamoxifen exerted low agonist activity in this system compared to 17 beta-estradiol (E2) . Efficiencies and potencies of several isomers were calculated by fitting experimental data with a logistic dose-response function . Cis-, trans- and cis-transtamoxifen and trans-4-hydroxytamoxifen (4-OHT) showed comparable efficiencies and potencies in yeast . When subeffective doses of trans-, cis-/trans-, or trans-4-OH tamoxifen were combined with increasing 17 beta-estradiol concentrations, even a synergistic increase in efficiencies could be observed . Interestingly, the cis-isomer did not show this synergistic effect . Mutation of the N-terminus of the estrogen receptor changed the transactivational behaviour of tamoxifen and abolished the synergistic action with 17 beta-estradiol . Except for 4-OHT, the potencies of the investigated isomers, defined as ligand concentrations with half-maximal response, highly correlated with the binding affinities to hER alpha . Therefore, cis-, trans-, and cis-/trans-tamoxifen could be regarded as full agonists in yeast, while 4-OHT was regarded as a partial antagonist in yeast . Furthermore, these results indicate that the functional difference between trans-tamoxifen and trans-4-OHT is not due to their different affinities for the receptor protein. J Biol Chem, 2000 May 19, 275(20), 15246 - 53 Substitutions of Asn-726 in the active site of yeast DNA topoisomerase I define novel mechanisms of stabilizing the covalent enzyme-DNA intermediate; Fertala J et al.; Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology and is the cellular target of camptothecin . Recent reports of enzyme structure highlight the importance of conserved amino acids N-terminal to the active site tyrosine and the involvement of Asn-726 in mediating Top1p sensitivity to camptothecin . To investigate the contribution of this residue to enzyme catalysis, we evaluated the effect of substituting His, Asp, or Ser for Asn-726 on yeast Top1p . Top1N726S and Top1N726D mutant proteins were resistant to camptothecin, although the Ser mutant was distinguished by a lack of detectable changes in activity . Thus, a basic residue immediately N-terminal to the active site tyrosine is required for camptothecin cytotoxicity . However, replacing Asn-726 with Asp or His interfered with distinct aspects of the catalytic cycle, resulting in cell lethality . In contrast to camptothecin, which inhibits enzyme-catalyzed religation of DNA, the His substituent enhanced the rate of DNA scission, whereas the Asp mutation diminished the enzyme binding of DNA . Yet, these effects on enzyme catalysis were not mutually exclusive as the His mutant was hypersensitive to camptothecin . These results suggest distinct mechanisms of poisoning DNA topoisomerase I may be explored in the development of antitumor agents capable of targeting different aspects of the Top1p catalytic cycle. J Biol Chem, 2000 May 19, 275(20), 15182 - 92 Analysis of a gene encoding Rpn10 of the fission yeast proteasome reveals that the polyubiquitin-binding site of this subunit is essential when Rpn12/Mts3 activity is compromised; Wilkinson CR et al.; Substrates are targeted for proteolysis by the ubiquitin pathway by the addition of a polyubiquitin chain before being degraded by the 26 S proteasome . Previously, a subunit of the proteasome, S5a, was identified that was able to bind to polyubiquitin in vitro and thus proposed to act as a substrate recognition component . Deletion of the corresponding Saccharomyces cerevisiae gene, MCB1/RPN10, rendered cells viable indicating that other proteasomal polyubiquitin receptors must exist . In this study, we describe pus1(+), the fission yeast homologue of RPN10 . This gene is also not required for cell viability; however, the Deltapus1 mutant is synthetically lethal with mutations in other proteasomal component-encoding genes, namely mts3, pad1, and mts4 (RPN12, RPN11, and RPN1) . Overexpression of pus1(+) is able to rescue mts3-1 at 32 degrees C but overexpression of a cDNA encoding a version of Pus1 that does not bind to polyubiquitin cannot and leads to greatly reduced viability when used to rescue the mts3-1Deltapus1 double mutant . The Mts3 protein was unable to bind to polyubiquitin in vitro, but the Pus1 and Mts3 proteins were found to bind to one another in vitro, which taken together with the genetic data suggests that they are also closely associated in vivo. Yeast, 2000 May, 16(7), 667 - 74 Current awareness on yeast; Alberti A et al.; In order to keep subscribers up-to-date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly-published material on yeasts . Each bibliography is divided into 10 sections . 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology . Within each section, articles are listed in alphabetical order with respect to author . If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted . (4 weeks journals - search completed 16th Feb 2000) Eur J Biochem, 2000 May, 267(10), 3090 - 8 Functional expression of mung bean Ca2+/H+ antiporter in yeast and its intracellular localization in the hypocotyl and tobacco cells; Ueoka-Nakanishi H et al.; The Ca2+-transport activity and intracellular localization of the translation product of cDNA for mung bean Ca2+/H+ antiporter (VCAX1) were examined . When the cDNA was expressed in Saccharomyces cerevisiae that lacked its own genes for vacuolar Ca2+-ATPase and the antiporter, VCAX1 complemented the active Ca2+ transporters, and the microsomal membranes from the transformant showed high activity of the Ca2+/H+ antiporter . Treatment of the vacuolar membranes with a cross-linking reagent resulted in a clear band of the dimer detected with antibody specific for VCAX1p . The antibody was also used for immunolocalization of the antiporter in fractions obtained by sucrose-density-gradient centrifugation of the microsomal fraction from mung bean . The immunostained band was detected in the vacuolar membrane fraction and the slightly heavy fractions that exhibited activity of the Golgi marker enzyme . A fusion protein of VCAX1p and green fluorescent protein was expressed in tobacco cells . The green fluorescence was clearly observed on the vacuolar membrane and, in some cases, in the small vesicles . The subcellular fractionation of transformed tobacco cells confirmed the vacuolar membrane localization of the fusion protein . These results confirm that VCAX1p functions in the vacuolar membrane as a Ca2+/H+ antiporter and also suggest that VCAX1p may exist in the Golgi apparatus. Eur J Biochem, 2000 May, 267(10), 2973 - 80 Binding of rat brain hexokinase to recombinant yeast mitochondria: identification of necessary physico-chemical determinants; Azoulay-Zohar H et al.; The association of rat brain hexokinase with heterologous recombinant yeast mitochondria harboring human porin (Yh) is comparable to that with rat liver mitochondria in terms of cation requirements, cooperativity in binding, and the effect of amphipathic compounds . Mg2+, which is required for hexokinase binding to all mitochondria, can be replaced by other cations . The efficiency of hexokinases, however, depends on the valence of hydrophilic cations, or the partition of hydrophobic cations in the membrane, implying that these act by reducing a prohibitive negative surface charge density on the outer membrane rather than fulfilling a specific structural requirement . Macromolecular crowding (using dextran) has dual effects . Dextran added in excess increases hexokinase binding to yeast mitochondria, according to the porin molecule they harbor . This effect, significant with wild-type yeast mitochondria, is only marginal with Yh as well as rat mitochondria . On the other hand, an increase in the number of hexokinase binding sites on mitochondria is also observed . This increase, moderate in wild-type organelles, is more pronounced with Yh . Finally, dextran, which has no effect on the modulation of hexokinase binding by cations, abolishes the inhibitory effect of amphipathic compounds . Thus, while hexokinase binding to mitochondria is predetermined by the porin molecule, the organization of the latter in the membrane plays a critical role as well, indicative that porin must associate with other mitochondrial components to form competent binding sites on the outer membrane. J Biol Chem, 2000 Jul 21, 275(29), 22427 - 34 Apurinic endonuclease activity of yeast Apn2 protein; Unk I et al.; Abasic (apurinic/apyrimidinic; AP) sites are generated in vivo through spontaneous base loss and by enzymatic removal of bases damaged by alkylating agents and reactive oxygen species . In Saccharomyces cerevisiae, the APN1 and APN2 genes function in alternate pathways of AP site removal . Apn2-like proteins have been identified in other eukaryotes including humans, and these proteins form a distinct subfamily within the exonuclease III (ExoIII)/Ape1/Apn2 family of proteins . Apn2 and other members of this subfamily contain a carboxyl-terminal extension not present in the ExoIII/Ape1-like proteins . Here, we purify the Apn2 protein from yeast and show that it is a class II AP endonuclease . Deletion of the carboxyl terminus does not affect the AP endonuclease activity of the protein, but this protein is defective in the removal of AP sites in vivo . The carboxyl terminus may enable Apn2 to complex with other proteins, and such a multiprotein assembly may be necessary for the efficient recognition and cleavage of AP sites in vivo. J Cell Sci, 2000 Jun, 113 ( Pt 11), 1923 - 8 Yeast colonies synchronise their growth and development; Palkova Z et al.; The ability to emit and receive signals over long distances is one of the characteristic attributes of multicellular organisms . Such communication can be mediated in different manners (by chemical compounds, light waves, acoustic waves etc.) and usually is reflected in the behaviour of the communicating organisms . Recently, we reported that individual yeast colonies, organised multicellular structures, can also communicate at long distance by means of volatile ammonia, which is produced by colonies in pulses separated by acidification of the medium . Here, we demonstrate that the colony that first reached the stage of intense ammonia production induces ammonia production response in surrounding colonies regardless of their age, causing the synchronisation of their NH(3) pulses and, consequently, the mutual affection of their growth . Also an artificial source of ammonia (but neither NH(4)(+) nor NaOH gradients) can immediately induce the ammonia production even in the colony starting its acidic stage of the development . The repeated transition of Candida mogii colonies from the acidic phase to the phase of intensive ammonia production is accompanied by dramatic changes in colony morphology and also in cell morphology and growth . Relatively smooth colonies in the acidic phase are formed by growing pseudohyphae . After ammonia induction, pseudohyphae decompose into non-dividing yeast-like cells, which rearrange themselves into ruffled spaghetti-like structures . The synchronisation of colony growth, that also exists between yeast colonies of different genera, could be important in establishing their optimal distribution in a natural habitat. J Cell Sci, 2000 Jun, 113 ( Pt 11), 1903 - 12 Centromere clustering is a major determinant of yeast interphase nuclear organization; Jin QW et al.; During interphase in the budding yeast, Saccharomyces cerevisiae, centromeres are clustered near one pole of the nucleus as a rosette with the spindle pole body at its hub . Opposite to the centromeric pole is the nucleolus . Chromosome arms extend outwards from the centromeric pole and are preferentially directed towards the opposite pole . Centromere clustering is reduced by the ndc10 mutation, which affects a kinetochore protein, and by the microtubule poison nocodazole . This suggests that clustering is actively maintained or enforced by the association of centromeres with microtubules throughout interphase . Unlike the Rabl-orientation known from many higher eukaryotes, centromere clustering in yeast is not only a relic of anaphase chromosome polarization, because it can be reconstituted without the passage of cells through anaphase . Within the rosette, homologous centromeres are not arranged in a particular order that would suggest somatic pairing or genome separation. J Struct Biol, 2000 Apr, 129(2-3), 295 - 305 Comparative spatial localization of protein-A-tagged and authentic yeast nuclear pore complex proteins by immunogold electron microscopy; Fahrenkrog B et al.; The nuclear pore complex (NPC) mediates protein and RNP import in and RNA and RNP export out of the nucleus of eukaryotic cells . Due to its genetic tractability, yeast offers a versatile system for investigating the chemical composition and molecular architecture of the NPC . In this context, protein A tagging is a commonly used tool for characterizing and localizing yeast NPC proteins (nucleoporins) . By preembedding anti-protein A immunogold electron microscopy (immunogold EM), we have localized two yeast nucleoporins, Nsp1p and Nic96p, in mutant yeast strains recombinantly expressing these nucleoporins tagged with four (Nsp1p) or two (Nic96p) IgG binding domains of protein A (i.e., ProtA-Nsp1p and ProtA-Nic96p) . We have compared the location of the recombinant fusion proteins ProtA-Nsp1p and ProtA-Nic96p (i.e., as specified by their protein A tag) to the location of authentic Nsp1p and Nic96p (i.e., as defined by the epitopes recognized by corresponding nucleoporin antibodies) and found all of them to reside at the same three NPC sites . Hence, recombinant expression and protein A tagging of the nucleoporins Nsp1p and Nic96p have not caused any significant mislocation of the fusion proteins and thus enabled mapping of these two yeast nucleoporins at the ultrastructural level in a faithful manner . J Struct Biol, 2000 Apr, 129(2-3), 159 - 74 The dynamics of yeast telomeres and silencing proteins through the cell cycle; Laroche T et al.; Genes integrated near the telomeres of budding yeast have a variegated pattern of gene repression that is mediated by the silent information regulatory proteins Sir2p, Sir3p, and Sir4p . Immunolocalization and fluorescence in situ hybridization (FISH) reveal 6-10 perinuclear foci in which silencing proteins and subtelomeric sequences colocalize, suggesting that these are sites of Sir-mediated repression . Telomeres lacking subtelomeric repeat elements and the silent mating locus, HML, also localize to the periphery of the nucleus . Conditions that disrupt telomere proximal repression disrupt the focal staining pattern of Sir proteins, but not necessarily the localization of telomeric DNA . To monitor the telomere-associated pools of heterochromatin-binding proteins (Sir and Rap1 proteins) during mitotic cell division, we have performed immunofluorescence and telomeric FISH on populations of yeast cells synchronously traversing the cell cycle . We observe a partial release of Rap1p from telomeres in late G2/M, although telomeres appear to stay clustered during G2-phase and throughout mitosis . A partial release of Sir3p and Sir4p during mitosis also occurs . This is not observed upon HU arrest, although other types of DNA damage cause a dramatic relocalization of Sir and Rap1 proteins . The observed cell cycle dynamics were confirmed by direct epifluorescence of a GFP-Rap1p fusion . Using live GFP fluorescence we show that the diffuse mitotic distribution of GFP-Rap1p is restored to the interphase pattern of foci in early G1-phase . Mol Cell Biol, 2000 Jun, 20(11), 3880 - 6 Multiple signals regulate GAL transcription in yeast; Rohde JR et al.; Gal4p activates transcription of the Saccharomyces GAL genes in response to galactose and is phosphorylated during interaction with the RNA polymerase II (Pol II) holoenzyme . One phosphorylation at S699 is necessary for full GAL induction and is mediated by Srb10p/CDK8 of the RNA Pol II holoenzyme mediator subcomplex . Gal4p S699 phosphorylation is necessary for sensitive response to inducer, and its requirement for GAL induction can be abrogated by high concentrations of galactose in strains expressing wild-type GAL2 and GAL3 . Gal4p S699 phosphorylation occurs independently of Gal3p and is responsible for the long-term adaptation response observed in gal3 yeast . SRB10 and GAL3 are shown to represent parallel mechanisms for GAL gene induction . These results demonstrate that Gal4p activity is controlled by two independent signals: one that acts through Gal3p-galactose and a second that is mediated by the holoenzyme-associated cyclin-dependent kinase Srb10p . Since Srb10p is regulated independently of galactose, our results suggest a function for CDK8 in coordinating responses to specific inducers with the environment through the phosphorylation of gene-specific activators. Mol Cell Biol, 2000 Jun, 20(11), 3795 - 806 Hierarchy of S-phase-promoting factors: yeast Dbf4-Cdc7 kinase requires prior S-phase cyclin-dependent kinase activation; Nougarede R et al.; In all eukaryotes, the initiation of DNA synthesis requires the formation of prereplicative complexes (pre-RCs) on replication origins, followed by their activation by two S-T protein kinases, an S-phase cyclin-dependent kinase (S-CDK) and a homologue of yeast Dbf4-Cdc7 kinase (Dbf4p-dependent kinase {DDK}) . Here, we show that yeast DDK activity is cell cycle regulated, though less tightly than that of the S-CDK Clb5-Cdk1, and peaks during S phase in correlation with Dbf4p levels . Dbf4p is short-lived throughout the cell cycle, but its instability is accentuated during G(1) by the anaphase-promoting complex . Downregulating DDK activity is physiologically important, as joint Cdc7p and Dbf4p overexpression is lethal . Because pre-RC formation is a highly ordered process, we asked whether S-CDK and DDK need also to function in a specific order for the firing of origins . We found that both kinases are activated independently, but we show that DDK can perform its function for DNA replication only after S-CDKs have been activated . Cdc45p, a protein needed for initiation, binds tightly to chromatin only after S-CDK activation (L . Zou and B . Stillman, Science 280:593-596, 1998) . We show that Cdc45p is phosphorylated by DDK in vitro, suggesting that it might be one of DDK's critical substrates after S-CDK activation . Linking the origin-bound DDK to the tightly regulated S-CDK in a dependent sequence of events may ensure that DNA replication initiates only at the right time and place. J Biomol NMR, 2000 Mar, 16(3), 183 - 96 The influence of DNA binding on the backbone dynamics of the yeast cell-cycle protein Mbp1; McIntosh PB et al.; Mbp1 is a transcription factor involved in the regulation of the cell cycle in yeast . The N-terminus of this protein contains a DNA binding domain that includes a winged helix-turn-helix motif . The C-terminal 24 residues of this domain (the 'tail') are disordered in the crystal state, but are important for DNA binding . We have measured 15N NMR relaxation rates at 11.75 and 14.1 T to determine the dynamics of the free protein and in its complex with a specific DNA duplex . The dynamics data were quantitatively analysed using both spectral density mapping and the Lipari-Szabo formalism including the effects of chemical exchange and rotational anisotropy . A detailed analysis has been made of the effect of anisotropy, exchange and experimental precision on the recovered motional parameters . The backbone NH relaxation is affected by motions on a variety of time scales from millisecond to tens of picoseconds . The relaxation data show a structured core of 100 residues corresponding to that observed in the crystal state . Within the core of the protein, two regions on either side of the putative recognition helix (helix B) show slow (ca . 0.2 ms) conformational exchange dynamics that are quenched upon DNA binding . The C-terminal 24 residues are generally more dynamic than in the core . However, in the free protein, a stretch of approximately 8 residues in the middle of the tail show relaxation behaviour similar to that in the core, indicating a structured region . NOEs between Ala 114 in this structured part of the tail and residues in the N-terminal beta strand of the core of the protein demonstrate that the tail folds back onto the core of the protein . In the complex with DNA, the structured part of the tail extends by ca . 3 residues . These data provide a framework for understanding the biochemical data on the mechanism and specificity of DNA binding. Curr Genet, 2000 Apr, 37(4), 221 - 33 Characterization of novel rad6/ubc2 ubiquitin-conjugating enzyme mutants in yeast; Freiberg G et al.; Null mutations in the RAD6/UBC2 gene encoding an E2 ubiquitin-conjugating enzyme cause deficiencies in DNA repair, N-end-rule protein degradation, sporulation and telomeric silencing, and alter the preferred integration positions for Ty1 retrotransposons . Here we selected for mutants of RAD6 that cause a release of telomeric silencing . Some alleles retained nearly wild-type ability for sporulation, DNA repair and the degradation of proteins . Alteration in Ty1 integration-site bias accompanied some of these alleles . The possibility that some mutations specifically affect binding of an unknown protein that works with Rad6 in its silencing role, but is not required for DNA repair or N-end-rule activity, is discussed in terms of the Rad6 crystal structure. J Biomol Screen, 2000 Apr, 5(2), 71 - 6 Miniaturization of a functional transcription assay in yeast (human progesterone receptor) in the 384- and 1536-well plate format; Berg M et al.; Miniaturization of high throughput screening assays to high-density microplate formats (384 or 1536 wells) is currently the focus of research activity in modern drug discovery facilities . In this article, we describe the adaptation of a fluorescence-based functional transcription assay in yeast for assessing modulators of human progesterone receptor to the 384- and 1536-well microplate format, comparing the experimental results to those obtained in the well-established 96-well format . The experiences gained from the optimization of the liquid-handling procedures and the miniaturization of an enzyme assay (beta-galactosidase) were implemented . Thus optimized pipetting protocols were developed to perform a reporter gene assay in yeast in microplate formats of higher density . In the functional transcription assay in yeast, the reporter gene expression showed the expected dependence on the ligand's dose and affinity in principle in all three microplate formats . For the first time, this assay system has been established in the 1536-well microplate format using CyBi-Well 96/384/1536 as the liquid-handling unit . The comparison of the signal:background ratios showed a lower sensitivity of the assay in the microplate formats of higher density . This study is an example of a successful miniaturization of a yeast cell-based assay to high-density plate formats on the basis of a careful adaptation procedure and optimized liquid-handling conditions. FEBS Lett, 2000 May 4, 473(1), 6 - 9 Apoptosis in yeast--a monocellular organism exhibits altruistic behaviour; Frohlich KU et al.; Apoptosis is a highly regulated form of programmed cell death crucial for life and health in metazoan animals . Apoptosis is defined by a set of cytological alterations . The recent discovery of these markers in yeast indicates the presence of the basic mechanisms of apoptosis already in unicellular eukaryotes . Oxygen radicals regulate both mammalian and yeast apoptosis . We suggest that apoptosis originated in unicellular organisms as an altruistic response to severe oxidative damage . Later, cells developed mechanisms to purposely produce reactive oxygen species as a regulator of apoptosis . Yeast may become an important model to investigate the conserved steps of apoptosis. J Nutr, 2000 May, 130(5), 1274 - 9 High chromium yeast supplementation improves glucose tolerance in pigs by decreasing hepatic extraction of insulin; Guan X et al.; Twenty Landrace x Yorkshire cross pigs (body wt, 47.9+/-2.9 kg) were used to evaluate effects of dietary high chromium (Cr) yeast supplementation on plasma kinetics of glucose, insulin and C-peptide . Pigs were provided free access to either a control diet (C) containing 204 microg Cr/kg or a diet supplemented with an additional 200 microg Cr/kg as high Cr yeast (CR) for between 23 and 30 d . After overnight food deprivation, dextrose (500 g/L) was infused through a jugular vein catheter at a dose of 0.5 g glucose/kg body weight with an infusion rate of 10 g glucose/min within 6 min . High Cr yeast supplementation did not affect body weight gain or food intake . There were no differences in fasting plasma concentrations of either glucose or C-peptide, although basal plasma concentration of insulin tended to be higher in pigs fed CR (P<0.10) . Plasma glucose concentrations were lower (P<0.01) at postinfusion times 5, 10, 15 and 20 min in pigs fed CR . Plasma insulin concentrations in pigs fed CR were higher (P<0.05) at 2 and 0 min before the completion of dextrose infusion . However, the increase in plasma insulin concentrations was not accompanied by a comparable elevation in plasma C-peptide concentrations . The 30-min (postinfusion) area of plasma glucose concentrations tended to be lower (P<0.10) in pigs fed CR, but there were no differences in 30-min areas of either plasma insulin or plasma C-peptide concentrations between treatments . Plasma clearance rates of glucose, insulin and C-peptide were higher and their half-lives shorter (P<0.05) in pigs fed CR . In conclusion, dietary high Cr yeast supplementation improved glucose tolerance, possibly through a decrease in hepatic extraction of insulin. J Biol Chem, 2000 Jul 21, 275(29), 22075 - 81 Subunit D (Vma8p) of the yeast vacuolar H+-ATPase plays a role in coupling of proton transport and ATP hydrolysis; Xu T et al.; To investigate the function of subunit D in the vacuolar H(+)-ATPase (V-ATPase) complex, random and site-directed mutagenesis was performed on the VMA8 gene encoding subunit D in yeast . Mutants were selected for the inability to grow at pH 7.5 but the ability to grow at pH 5.5 . Mutations leading to reduced levels of subunit D in whole cell lysates were excluded from the analysis . Seven mutants were isolated that resulted in pH-dependent growth but that contained nearly wild-type levels of subunit D and nearly normal assembly of the V-ATPase as assayed by subunit A levels associated with isolated vacuoles . Each of these mutants contained 2-3 amino acid substitutions and resulted in loss of 60-100% of proton transport and 58-93% of concanamycin-sensitive ATPase activity . To identify the mutations responsible for the observed effects on activity, 14 single amino acid substitutions and 3 double amino acid substitutions were constructed by site-directed mutagenesis and analyzed as described above . Six of the single mutations and all three of the double mutations led to significant (>30%) loss of activity, with the mutations having the greatest effects on activity clustering in the regions Val(71)-Gly(80) and Lys(209)-Met(221) . In addition, both M221V and the double mutant V71D/E220V led to significant uncoupling of proton transport and ATPase activity, whereas the double mutant G80D/K209E actually showed increased coupling efficiency . Both a mutant showing reduced coupling and a mutant with only 6% of wild-type proton transport activity showed normal dissociation of the V-ATPase complex in vivo in response to glucose deprivation . These results suggest that subunit D plays an important role in coupling of proton transport and ATP hydrolysis and that only low rates of turnover of the enzyme are required to support in vivo dissociation. J Biol Chem, 2000 Aug 4, 275(31), 23933 - 8 Manganese selectivity of pmr1, the yeast secretory pathway ion pump, is defined by residue gln783 in transmembrane segment 6 . Residue Asp778 is essential for cation transport; Mandal D et al.; We have solubilized and purified the histidine-tagged yeast secretory pathway/Golgi ion pump Pmr1 to near homogeneity in one step, using nickel affinity chromatography . The purified pump demonstrates both Ca(2+)- and Mn(2+)-dependent ATP hydrolysis and phosphoenzyme intermediate formation in forward (ATP) and reverse (P(i)) directions . This preparation has allowed us to examine, in detail, the properties of mutations D778A and Q783A in transmembrane segment M6 of Pmr1 . In phenotypic screens of Ca(2+) chelator and Mn(2+) toxicity reported separately (Wei, Y., Chen, J., Rosas, G., Tompkins, D.A., Holt, P.A., and Rao, R . (2000) J . Biol . Chem . 275, XXXX-XXXX), D778A was a loss-of-function mutant apparently defective for transport of both Ca(2+) and Mn(2+), whereas mutant Q783A displayed a differential sensitivity consistent with the selective loss of Mn(2+) transport . We show that mutant D778A is devoid of cation-dependent ATP hydrolytic activity and phosphoenzyme formation from ATP . However, reverse phosphorylation from P(i) is preserved but is insensitive to inhibition by Ca(2+) or Mn(2+) ions, which is evidence for a specific inability to bind cations in this mutant . We also show that Ca(2+) can activate ATP hydrolysis in the purified Q783A mutant, with a half-maximal concentration of 0.06 micrometer, essentially identical to that of wild type (0.07 micrometer) . Mn(2+) activation of ATP hydrolysis was half-maximal at 0.02 micrometer in wild type, establishing a normal selectivity profile of Mn(2+) > Ca(2+) . Strikingly, Mn(2+)-ATPase in the Q783A mutant was nearly abolished, even at concentrations of up to 10 micrometer . These results were confirmed in assays of phosphoenzyme intermediates . Molecular modeling of the packing between helices M4 and M6 suggests that residue Gln(783) in M6 may form a critical hydrophobic interaction with Val(335) in M4, such that the Ala substitution modifies the packing or tilt of the helices and thus the ion pore . The data emphasize the critical role of transmembrane segment M6 in defining the cation binding pocket of P-type ATPases. J Biol Chem, 2000 Aug 4, 275(31), 23927 - 32 Phenotypic screening of mutations in Pmr1, the yeast secretory pathway Ca2+/Mn2+-ATPase, reveals residues critical for ion selectivity and transport; Wei Y et al.; Thirty-five mutations were generated in the yeast secretory pathway/Golgi ion pump, Pmr1, targeting oxygen-containing side chains within the predicted transmembrane segments M4, M5, M6, M7, and M8, likely to be involved in coordination of Ca(2+) and Mn(2+) ions . Mutants were expressed in low copy number in a yeast strain devoid of endogenous Ca(2+) pumps and screened for loss of Ca(2+) and Mn(2+) transport on the basis of hypersensitivity to 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and Mn(2+) toxicity, respectively . Three classes of mutants were found: mutants indistinguishable from wild type (Class 1), mutants indistinguishable from the pmr1 null strain (Class 2), and mutants with differential sensitivity to BAPTA and Mn(2+) toxicity (Class 3) . We show that Class 1 mutants retain normal/near normal properties, including (45)Ca transport, Golgi localization, and polypeptide conformation . In contrast, Class 2 mutants lacked any detectable (45)Ca transport; of these, a subset also showed defects in trafficking and protein folding, indicative of structural problems . Two residues identified as Class 2 mutants in this screen, Asn(774) and Asp(778) in M6, also play critical roles in related ion pumps and are therefore likely to be common architectural components of the cation-binding site . Class 3 mutants appear to have altered selectivity for Ca(2+) and Mn(2+) ions, as exemplified by mutant Q783A in M6 . These results demonstrate the utility of phenotypic screening in the identification of residues critical for ion transport and selectivity in cation pumps. J Biol Chem, 2000 Jul 28, 275(30), 22678 - 85 Transcriptional regulation of the yeast PHO8 promoter in comparison to the coregulated PHO5 promoter; Munsterkotter M et al.; Expression of the PHO8 and PHO5 genes that encode a nonspecific alkaline and acid phosphatase, respectively, is regulated in response to the P(i) concentration in the medium by the same transcription factors . Upon induction by phosphate starvation, both promoters undergo characteristic chromatin remodeling, yet the extent of remodeling at the PHO8 promoter is significantly lower than at PHO5 . Despite the coordinate regulation of the two promoters, the PHO8 promoter is almost 10 times weaker than PHO5 . Here we show that of two Pho4 binding sites that had been previously mapped at the PHO8 promoter in vitro, only the high affinity one, UASp2, is functional in vivo . Activation of the PHO8 promoter is partially Pho2-dependent . However, unlike at PHO5, Pho4 can bind strongly to its binding site in the absence of Pho2 and remodel chromatin in a Pho2-independent manner . Replacement of the inactive UASp1 element by the UASp1 element from the PHO5 promoter results in more extensive chromatin remodeling and a concomitant 2-fold increase in promoter activity . In contrast, replacement of the high affinity UASp2 site with the corresponding site from PHO5 precludes chromatin remodeling completely and as a consequence promoter activation, despite efficient binding of Pho4 to this site . Deletion of the promoter region normally covered by nucleosomes -3 and -2 results in a 2-fold increase in promoter activity, further supporting a repressive role of these nucleosomes . These data show that there can be strong binding of Pho4 to a UAS element without any chromatin remodeling and promoter activation . The close correlation between promoter activity and the extent of chromatin disruption strongly suggests that the low level of PHO8 induction in comparison with PHO5 is partly due to the inability of Pho4 to achieve complete chromatin remodeling at this promoter. J Biol Chem, 2000 Jul 7, 275(27), 20243 - 6 GI domain-mediated association of the eukaryotic initiation factor 2alpha kinase GCN2 with its activator GCN1 is required for general amino acid control in budding yeast; Kubota H et al.; In response to the starvation of a single amino acid, the budding yeast Saccharomyces cerevisiae activates numerous genes involved in various amino acid biosynthetic pathways, all of which are under the control of transcription factor GCN4 . This general amino acid control response is based on de-repressed translation of GCN4 mRNA, which is induced by the activation of the eIF2alpha kinase, GCN2 . Although it is known that in vivo activation of GCN2 requires GCN1, the mode of GCN1 action remains to be elucidated at the molecular level . Here, we show that GCN2 interacts with GCN1 via the GI domain, a novel protein-binding module that occurs at the N terminus; mutations to conserved residues of this domain abolish its binding to GCN1 . Furthermore, the yeast cells with GCN2 defective in interaction with GCN1 fail to display general control response . A similar phenotype is observed in cells overexpressing the GI domain of GCN2 or its target region on GCN1 . Thus, GI domain-mediated association of GCN2 to GCN1 is required for general amino acid control . This finding provides the first insight into the molecular mechanism for the activation of GCN2 by GCN1. Curr Opin Cell Biol, 2000 Jun, 12(3), 334 - 9 Transcriptional regulation of meiosis in yeast; Vershon AK et al.; The genes required for meiosis and sporulation in yeast are expressed at specific points in a highly regulated temporal pathway . Recent experiments using DNA microarrays to examine gene expression during meiosis and the identification of many regulatory factors have provided important advances in our understanding of how genes are regulated at the different stages of meiosis. Biochem Biophys Res Commun, 2000 May 10, 271(2), 464 - 8 Purification and polymerization properties of two lethal yeast actin mutants; Frieden C et al.; The budding yeast Saccharomyces cerevisiae contains a single actin gene and the gene product, actin, is essential for growth . Two mutants of yeast actin that do not support yeast growth were prepared from yeast by coexpressing the mutant and a 6-histidine-tagged wild-type actin followed by separation of the wild-type and mutant actin using Ni-NTA chromatography as described elsewhere {Buzan, J., Du, J., Karpova, T., and Frieden, C . (1999) Proc . Natl . Acad . Sci . USA 96, 2823-2827} . The mutations, in muscle actin numbering, were at positions 334 (Glu334Lys) and 168 (Gly168Arg) and were chosen based on phenotypic changes observed in the behavior of actin mutants of Caenorhabditis elegans . Glu334 is located on the surface of actin between subdomains 1 and 3 while Gly168 is located in a region near actin-actin contacts in the actin filament . The Glu334Lys mutant polymerized slightly faster than wild-type yeast actin, suggesting that loss of interactions with some actin binding protein, rather than loss of actin-actin contacts, was responsible for its inability to support yeast growth . The Gly168Arg mutant polymerized at a rate similar to wild-type but the extent was considerably less, kinetic characteristics suggesting a high critical concentration (ca . 4 microM) without a large change in the ability to form nuclei for the nucleation-elongation process . Biochem Biophys Res Commun, 2000 May 10, 271(2), 392 - 400 Overexpression of human UDP-glucose pyrophosphorylase rescues galactose-1-phosphate uridyltransferase-deficient yeast; Lai K et al.; To better understand the pathophysiology of galactose-1-phosphate uridyltransferase (GALT) deficiency in humans, we studied the mechanisms by which a GALT-deficient yeast survived on galactose medium . Under normal conditions, GALT-deficient yeast cannot grow in medium that contains 0.2% galactose as the sole carbohydrate, a phenotype of Gal(-) . We isolated revertants from a GALT-deficient yeast by direct selection for growth in galactose, a phenotype of Gal(+) . Comparison of gene expression profiles among wild-type and revertant strains on galactose medium revealed that the revertant down-regulated genes encoding enzymes including galactokinase, galactose permease, and UDP-galactose-4-epimerase (the GAL regulon) . By contrast, the revertant strain up-regulated the gene for UDP-glucose pyrophosphorylase, UGP1 . There was reduced accumulation of galactose-1-phosphate in the galactose-grown revertant cells when compared to the GALT-deficient parent cells . In vitro biochemical analysis showed that UDP-glucose pyrophosphorylase had bifunctional properties and could catalyze the conversion of galactose-1-phosphate to UDP-galactose in the presence of UTP . To test if augmented expression of this gene could produce a Gal(+) phenotype in the GALT-deficient parent cells, we overexpressed the yeast UGP1 and the human homolog, hUGP2 in the mutant strain . The Gal(-) yeast transformed with either UGP1 or hUGP2 regained their ability to grow on galactose . We conclude that revertant can grow on galactose medium by reducing the accumulation of toxic precursors through down-regulation of the GAL regulon and up-regulation of the UGP1 gene . We speculate that increased expression of hUGP2 in humans could alleviate poor outcomes in humans with classic galactosemia . Science, 2000 May 5, 288(5467), 863 - 7 Template boundary in a yeast telomerase specified by RNA structure; Tzfati Y et al.; The telomerase ribonucleoprotein has a phylogenetically divergent RNA subunit, which contains a short template for telomeric DNA synthesis . To understand how telomerase RNA participates in mechanistic aspects of telomere synthesis, we studied a conserved secondary structure adjacent to the template . Disruption of this structure caused DNA synthesis to proceed beyond the normal template boundary, resulting in altered telomere sequences, telomere shortening, and cellular growth defects . Compensatory mutations restored normal telomerase function . Thus, the RNA structure, rather than its sequence, specifies the template boundary . This study reveals a specific function for an RNA structure in the enzymatic action of telomerase. Plant Mol Biol, 2000 Jan, 42(2), 335 - 44 The yeast polyadenylate-binding protein (PAB1) gene acts as a disease lesion mimic gene when expressed in plants; Li Q et al.; We have expressed the gene (PAB1) encoding the yeast polyadenylate-binding protein (Pab1p) in tobacco . Plants that accumulate the Pab1p display a range of abnormalities, ranging from a characteristic chlorosis in leaves to a necrosis and large inhibition of growth . The severity of these abnormalities reflects the levels of yeast Pab1p expression in the transgenic plants . In contrast, no obvious differences could be seen in callus cultures between the transgene and vector control . Plants that display PAB-associated abnormalities were resistant to a range of plant pathogens, and had elevated levels of expression of a pathogenesis-related gene . These two properties--impairment of growth and induction of defense responses--indicate that the yeast PAB1 gene can act as a disease lesion mimic gene in plants. Syst Appl Microbiol, 1999 Dec, 22(4), 619 - 25 Kurtzmanomyces insolitus sp . nov., a new anamorphic heterobasidiomycetous yeast species; Sampaio JP et al.; A new anamorphic heterobasidiomycetous yeast species, Kurtzmanomyces insolitus, is described using a polyphasic taxonomic approach . The new species has the salient characteristics of the genus Kurtzmanomyces and, additionally, the ability to produce ballistoconidia . Data derived from comparative micromorphological studies, physiological characterisation, ultrastructure and nucleic acid analyses led to assigning the new species to Kurtzmanomyces rather than to the currently accepted genera of ballistoconidia-forming fungi . An emendation of the genus Kurtzmanomyces is proposed to allow the inclusion of the new species. Mol Biol Cell, 2000 May, 11(5), 1887 - 903 Structure-function relationships in yeast tubulins; Richards KL et al.; A comprehensive set of clustered charged-to-alanine mutations was generated that systematically alter TUB1, the major alpha-tubulin gene of Saccharomyces cerevisiae . A variety of phenotypes were observed, including supersensitivity and resistance to the microtubule-destabilizing drug benomyl, lethality, and cold- and temperature-sensitive lethality . Many of the most benomyl-sensitive tub1 alleles were synthetically lethal in combination with tub3Delta, supporting the idea that benomyl supersensitivity is a rough measure of microtubule instability and/or insufficiency in the amount of alpha-tubulin . The systematic tub1 mutations were placed, along with the comparable set of tub2 mutations previously described, onto a model of the yeast alpha-beta-tubulin dimer based on the three-dimensional structure of bovine tubulin . The modeling revealed a potential site for binding of benomyl in the core of beta-tubulin . Residues whose mutation causes cold sensitivity were concentrated at the lateral and longitudinal interfaces between adjacent subunits . Residues that affect binding of the microtubule-binding protein Bim1p form a large patch across the exterior-facing surface of alpha-tubulin in the model . Finally, the positions of the mutations suggest that proximity to the alpha-beta interface may account for the finding of synthetic lethality of five viable tub1 alleles with the benomyl-resistant but otherwise entirely viable tub2-201 allele. Mol Biol Cell, 2000 May, 11(5), 1753 - 64 The yeast heat shock transcription factor changes conformation in response to superoxide and temperature; Lee S et al.; In vitro DNA-binding assays demonstrate that the heat shock transcription factor (HSF) from the yeast Saccharomyces cerevisiae can adopt an altered conformation when stressed . This conformation, reflected in a change in electrophoretic mobility, requires that two HSF trimers be bound to DNA . Single trimers do not show this change, which appears to represent an alteration in the cooperative interactions between trimers . HSF isolated from stressed cells displays a higher propensity to adopt this altered conformation . Purified HSF can be stimulated in vitro to undergo the conformational change by elevating the temperature or by exposing HSF to superoxide anion . Mutational analysis maps a region critical for this conformational change to the flexible loop between the minimal DNA-binding domain and the flexible linker that joins the DNA-binding domain to the trimerization domain . The significance of these findings is discussed in the context of the induction of the heat shock response by ischemic stroke, hypoxia, and recovery from anoxia, all known to stimulate the production of superoxide. Mol Biol Cell, 2000 May, 11(5), 1739 - 51 Complex regulation of the yeast heat shock transcription factor; Bonner JJ et al.; The yeast heat shock transcription factor (HSF) is regulated by posttranslational modification . Heat and superoxide can induce the conformational change associated with the heat shock response . Interaction between HSF and the chaperone hsp70 is also thought to play a role in HSF regulation . Here, we show that the Ssb1/2p member of the hsp70 family can form a stable, ATP-sensitive complex with HSF-a surprising finding because Ssb1/2p is not induced by heat shock . Phosphorylation and the assembly of HSF into larger, ATP-sensitive complexes both occur when HSF activity decreases, whether during adaptation to a raised temperature or during growth at low glucose concentrations . These larger HSF complexes also form during recovery from heat shock . However, if HSF is assembled into ATP-sensitive complexes (during growth at a low glucose concentration), heat shock does not stimulate the dissociation of the complexes . Nor does induction of the conformational change induce their dissociation . Modulation of the in vivo concentrations of the SSA and SSB proteins by deletion or overexpression affects HSF activity in a manner that is consistent with these findings and suggests the model that the SSA and SSB proteins perform distinct roles in the regulation of HSF activity. Mol Biol Cell, 2000 May, 11(5), 1611 - 30 Glycosylphosphatidylinositol biosynthesis defects in Gpi11p- and Gpi13p-deficient yeast suggest a branched pathway and implicate gpi13p in phosphoethanolamine transfer to the third mannose; Taron CH et al.; Glycosylphosphatidylinositols (GPIs) are critical for membrane anchoring and intracellular transport of certain secretory proteins . GPIs have a conserved trimannosyl core bearing a phosphoethanolamine (EthN-P) moiety on the third mannose (Man-3) through which the glycolipid is linked to protein, but diverse GPI precursors with EthN-Ps on Man-1 and Man-2 have also been described . We report on two essential yeast genes whose products are required late in GPI assembly . GPI11 (YDR302w) encodes a homologue of human Pig-Fp, a protein implicated in the addition of EthN-P to Man-3 . PIG-F complements the gpi11 deletion, but the rescued haploids are temperature sensitive . Abolition of Gpi11p or Pig-Fp function in GPI11 disruptants blocks GPI anchoring and formation of complete GPI precursors and leads to accumulation of two GPIs whose glycan head groups contain four mannoses but differ in the positioning and number of side chains, probably EthN-Ps . The less polar GPI bears EthN-P on Man-2, whereas the more polar lipid has EthN-P on Man-3 . The latter finding indicates that Gpi11p is not required for adding EthN-P to Man-3 . Gpi13p (YLL031cp), a member of a family of phosphoryltransferases, is a candidate for the enzyme responsible for adding EthN-P to Man-3 . Depletion of Gpi13p in a Gpi11p-defective strain prevents formation of the GPI bearing EthN-P on Man-3, and Gpi13p-deficient strains accumulate a Man(4)-GPI isoform that bears EthN-P on Man-1 . We further show that the lipid accumulation phenotype of Gpi11p-deficient cells resembles that of cells lacking Gpi7p, a sequence homologue of Gpi13p known to add EthN-P to Man-2 of a late-stage GPI precursor . This result suggests that in yeast a Gpi11p-deficiency can affect EthN-P addition to Man-2 by Gpi7p, in contrast to the Pig-Fp defect in mammalian cells, which prevents EthN-P addition to Man-3 . Because Gpi11p and Pig-Fp affect EthN-P transfer to Man-2 and Man-3, respectively, these proteins may act in partnership with the GPI-EthN-P transferases, although their involvement in a given EthN-P transfer reaction varies between species . Possible roles for Gpi11p in the supply of the EthN-P donor are discussed . Because Gpi11p- and Gpi13p-deficient cells accumulate isoforms of Man(4)-GPIs with EthN-P on Man-2 and on Man-1, respectively, and because the GPIs that accumulate in Gpi11p-defective strains are likely to have been generated independently of one another, we propose that the yeast GPI assembly pathway is branched. J Cell Biol, 2000 May 1, 149(3), 575 - 90 Association of yeast RNA polymerase I with a nucleolar substructure active in rRNA synthesis and processing; Fath S et al.; A novel ribonucleoprotein complex enriched in nucleolar proteins was purified from yeast extracts and constituents were identified by mass spectrometry . When isolated from rapidly growing cells, the assembly contained ribonucleic acid (RNA) polymerase (pol) I, and some of its transcription factors like TATA-binding protein (TBP), Rrn3p, Rrn5p, Rrn7p, and Reb1p along with rRNA processing factors, like Nop1p, Cbf5p, Nhp2p, and Rrp5p . The small nucleolar RNAs (snoRNAs) U3, U14, and MRP were also found to be associated with the complex, which supports accurate transcription, termination, and pseudouridylation of rRNA . Formation of the complex did not depend on pol I, and the complex could efficiently recruit exogenous pol I into active ribosomal DNA (rDNA) transcription units . Visualization of the complex by electron microscopy and immunogold labeling revealed a characteristic cluster-forming network of nonuniform size containing nucleolar proteins like Nop1p and Fpr3p and attached pol I . Our results support the idea that a functional nucleolar subdomain formed independently of the state of rDNA transcription may serve as a scaffold for coordinated rRNA synthesis and processing. J Biol Chem, 2000 Jul 7, 275(27), 20545 - 50 Stalk segment 4 of the yeast plasma membrane H+-ATPase . Mutational evidence for a role in the E1-E2 conformational change; Ambesi A et al.; In the P(2)-type ATPases, there is growing evidence that four alpha-helical stalk segments connect the cytoplasmic part of the molecule, responsible for ATP binding and hydrolysis, to the membrane-embedded part that mediates cation transport . The present study has focused on stalk segment 4, which displays a significant degree of sequence conservation among P(2)-ATPases . When site-directed mutants in this region of the yeast plasma membrane H(+)-ATPase were constructed and expressed in secretory vesicles, more than half of the amino acid substitutions led to a severalfold decrease in the rate of ATP hydrolysis, although they had little or no effect on the coupling between hydrolysis and transport . Strikingly, mutant ATPases bearing single substitutions of 13 consecutive residues from Ile-359 through Gly-371 were highly resistant to inorganic orthovanadate, with IC(50) values at least 10-fold above those seen in the wild-type enzyme . Most of the same mutants also displayed a significant reduction in the K(m) for MgATP and an increase in the pH optimum for ATP hydrolysis . Taken together, these changes in kinetic behavior point to a shift in equilibrium from the E(2) conformation of the ATPase toward the E(1) conformation . The residues from Ile-359 through Gly-371 would occupy three full turns of an alpha-helix, suggesting that this portion of stalk segment 4 may provide a conformationally active link between catalytic sites in the cytoplasm and cation-binding sites in the membrane. Cell Struct Funct, 2000 Feb, 25(1), 11 - 20 Overexpression of PRA2, a Rab/Ypt-family small GTPase from Pea Pisum sativum, aggravates the growth defect of yeast ypt mutants; Matsuda N et al.; A large number of Rab/Ypt-family small GTPases have been identified from higher plants . While some of them can complement yeast ypt mutants, the expression of Arabidopsis Ara4 protein aggravated the growth defect of a subset of ypt mutants, probably because of the titration of common regulator(s) of yeast Ypt proteins {Ueda, T . et al . (1996) Plant Cell, 8: 2079-20911 . PRA2 from pea Pisum sativum encodes an interesting Rab GTPase whose expression is regulated by light {Yoshida, K . et al . (1993) Proc . Natl . Acad . Sci . USA, 90: 6636-6640} . We examined whether PRA2 complements any of the yeast ypt mutants and found again that PRA2 does not complement but rather confers the growth defect to some of the ypt mutants . No growth defect was observed when PRA2 was expressed in the wild-type yeast cells . Unlike the case of Ara4, neither Arabidopsis nor yeast GDI remedied the growth defect by Pra2, indicating that the mechanism of the exacerbation is different . Mutational analysis of PRA2 suggests that the growth inhibition can be ascribed to unidentified factor(s) which prefers the GTP-bound form of Pra2 . This yeast system will be useful for identifying such putative regulatory factor(s) from yeast and plants and analyzing their interactions with Pra2. Genetics, 2000 May, 155(1), 475 - 9 The Mre11p/Rad50p/Xrs2p complex and the Tel1p function in a single pathway for telomere maintenance in yeast; Ritchie KB et al.; The Mre11p/Rad50p/Xrs2p complex is involved in the repair of double-strand DNA breaks, nonhomologous end joining, and telomere length regulation . TEL1 is primarily involved in telomere length regulation . By an epistasis analysis, we conclude that Tel1p and the Mre11p/Rad50p/Xrs2p complex function in a single pathway of telomere length regulation. Genetics, 2000 May, 155(1), 31 - 42 Inhibition of mRNA turnover in yeast by an xrn1 mutation enhances the requirement for eIF4E binding to eIF4G and for proper capping of transcripts by Ceg1p; Brown JT et al.; Null mutants of XRN1, encoding the major cytoplasmic exoribonuclease in yeast, are viable but accumulate decapped, deadenylated transcripts . A screen for mutations synthetic lethal with xrn1Delta identified a mutation in CDC33, encoding eIF4E . This mutation (glutamate to glycine at position 72) affected a highly conserved residue involved in interaction with eIF4G . Synthetic lethality between xrn1 and cdc33 was not relieved by high-copy expression of eIF4G or by disruption of the yeast eIF4E binding protein Caf20p . High-copy expression of a mutant eIF4G defective for eIF4E binding resulted in a dominant negative phenotype in an xrn1 mutant, indicating the importance of this interaction in an xrn1 mutant . Another allele of CDC33, cdc33-1, along with mutations in CEG1, encoding the nuclear guanylyltransferase, were also synthetic lethal with xrn1Delta, whereas mutations in PRT1, encoding a subunit of eIF3, were not . Mutations in CDC33, CEG1, PRT1, PAB1, and TIF4631, encoding eIF4G1, have been shown to lead to destabilization of mRNAs . Although such destabilization in cdc33, ceg1, and pab1 mutants can be partially suppressed by an xrn1 mutation, we observed synthetic lethality between xrn1 and either cdc33 or ceg1 and no suppression of the inviability of a pab1 null mutation by xrn1Delta . Thus, the inhibition of mRNA turnover by blocking Xrn1p function does not suppress the lethality of defects upstream in the turnover pathway but it does enhance the requirement for (7)mG caps and for proper formation of the eIF4E/eIF4G cap recognition complex. EMBO J, 2000 May 2, 19(9), 1942 - 52 Dependence and independence of {PSI(+)} and {PIN(+)}: a two-prion system in yeast? Derkatch IL, Bradley ME, Masse SV, Zadorsky SP, Polozkov GV, Inge-Vechtomov SG, Liebman SW. The {PSI(+)} prion can be induced by overproduction of the complete Sup35 protein, but only in strains carrying the non-Mendelian {PIN(+)} determinant . Here we demonstrate that just as {psi (-)} strains can exist as {PIN(+)} and {pin(-)} variants, {PSI(+)} can also exist in the presence or absence of {PIN(+)} . {PSI(+)} and {PIN(+)} tend to be cured together, but can be lost separately . {PSI(+)}-related phenotypes are not affected by {PIN(+)} . Thus, {PIN(+)} is required for the de novo formation of {PSI(+)}, not for {PSI(+)} propagation . Although {PSI(+)} induction is shown to require {PIN(+)} even when the only overexpressed region of Sup35p is the prion domain, two altered prion domain fragments circumventing the {PIN(+)} requirement are characterized . Finally, in strains cured of {PIN(+)}, prolonged incubation facilitates the reappearance of {PIN(+)} . Newly appearing {PIN(+)} elements are often unstable but become stable in some mitotic progeny . Such reversibility of curing, together with our previous demonstration that the inheritance of {PIN(+)} is non-Mendelian, supports the hypothesis that {PIN(+)} is a prion . Models for {PIN(+)} action, which explain these findings, are discussed. J Biol Chem, 2000 May 5, 275(18), 13835 - 41 Multiple growth factor induction of a murine early response gene that complements a lethal defect in yeast ribosome biogenesis; Nelson SA et al.; Identification of the transcriptionally activated targets of receptor tyrosine kinases is critical to understanding biologic programs directing both normal and neoplastic growth . To elucidate these molecular processes, we identified genes induced by a potent mesenchymal mitogen, platelet-derived growth factor (PDGF) . Using differential display reverse transcription-polymerase chain reaction technology, we isolated a novel growth factor-induced cDNA, San5 . San5 transcript induction occurred within 60 min in NIH 3T3 fibroblasts and proceeded in the presence of cycloheximide . Maximal induction of the San5 transcript occurred between 8 and 16 h, concurrent with passage of fibroblasts through G(1) . San5 message was potently induced by PDGF AA and BB and acidic and basic fibroblast growth factors, all strong activators of fibroblast proliferation, but not by epidermal growth factor and interleukin-4 . In a murine hematopoietic progenitor cell line, San5 transcript induction strictly correlated with {(3)H}thymidine uptake . Isolation and sequencing of the murine San5 cDNA revealed amino acid sequence homology to yeast Nop5p, a nucleolar protein required for pre-rRNA processing and ribosome assembly . Strikingly, SAN5 was able to rescue a nop5 null mutant, implicating SAN5 in the process of ribosome biogenesis . Consistent with this result, SAN5 was localized to the nucleolus in both yeast and mouse . Thus, San5 may provide a link between growth factor receptor activation and the cellular translational machinery. J Biol Chem, 2000 May 5, 275(18), 13780 - 8 Zinc stoichiometry of yeast RNA polymerase II and characterization of mutations in the zinc-binding domain of the largest subunit; Donaldson IM et al.; Atomic absorption spectroscopy demonstrated that highly purified RNA polymerase II from the yeast Saccharomyces cerevisiae binds seven zinc ions . This number agrees with the number of potential zinc-binding sites among the 12 different subunits of the enzyme and with our observation that the ninth largest subunit alone is able to bind two zinc ions . The zinc-binding motif in the largest subunit of the enzyme was investigated using mutagenic analysis . Altering any one of the six conserved residues in the zinc-binding motif conferred either a lethal or conditional phenotype, and zinc blot analysis indicated that mutant forms of the domain had a 2-fold reduction in zinc affinity . Mutations in the zinc-binding domain reduced RNA polymerase II activity in cell-free extracts, even though protein blot analysis indicated that the mutant subunit was present in excess of wild-type levels . Purification of one mutant RNA polymerase revealed a subunit profile that was wild-type like with the exception of two subunits not required for core enzyme activity (Rpb4p and Rpb7p), which were missing . Core activity of the mutant enzyme was reduced 20-fold . We conclude that mutations in the zinc-binding domain can reduce core activity without altering the association of any of the subunits required for this activity. J Biol Chem, 2000 May 5, 275(18), 13597 - 604 Mammalian homologues of yeast sec31p . An ubiquitously expressed form is localized to endoplasmic reticulum (ER) exit sites and is essential for ER-Golgi transport; Tang BL et al.; The yeast coat protein II (COPII) is responsible for vesicle budding from the endoplasmic reticulum (ER) . Mammalian functional homologues for all yeast COPII components, except for Sec31p, have been reported . We have cloned a mammalian cDNA whose product (Sec31A) is about 26% identical to Saccharomyces cerevisiae Sec31p . Data base searches also revealed another partial sequence encoding a polypeptide (Sec31B) that is 40% identical to Sec31A . Northern analysis revealed that Sec31A transcripts are ubiquitously and abundantly expressed, while Sec31B transcripts are particularly enriched in the testis and thymus, but present in very low levels in other tissues . Sec31A is localized to vesicular structures that scatter throughout the cell but are concentrated at the perinuclear region . The structures marked by Sec31A contain Sec13, a component of COPII that is well characterized to mark the ER exit sites . Immunoelectron microscopy revealed that Sec31A colocalizes with Sec13 in structures with extensive vesicular-tubular profiles . Antibodies raised against a C-terminal portion of Sec31A co-precipitate Sec13 and inhibit ER-Golgi transport of temperature-arrested vesicular stomatitis G protein in a semi-intact cell assay . Cytosol immunodepleted of Sec31A failed to support vesicular stomatitis G protein transport, which can be rescued by a high molecular weight fraction of the cytosol containing both Sec31A and Sec13 . We conclude that Sec31A represents a functional mammalian homologue of yeast Sec31p. J Biol Chem, 2000 May 5, 275(18), 13291 - 6 Expression of Arabidopsis thaliana mitochondrial alanyl-tRNA synthetase is not sufficient to trigger mitochondrial import of tRNAAla in yeast; Mireau H et al.; It has often been suggested that precursors to mitochondrial aminoacyl-tRNA synthetases are likely carriers for mitochondrial import of tRNAs in those organisms where this process occurs . In plants, it has been shown that mutation of U(70) to C(70) in Arabidopsis thaliana tRNA(Ala)(UGC) blocks aminoacylation and also prevents import of the tRNA into mitochondria . This suggests that interaction of tRNA(Ala) with alanyl-tRNA synthetase (AlaRS) is necessary for import to occur . To test whether this interaction is sufficient to drive import, we co-expressed A . thaliana tRNA(Ala)(UGC) and the precursor to the A . thaliana mitochondrial AlaRS in Saccharomyces cerevisiae . The A . thaliana enzyme and its cognate tRNA were correctly expressed in yeast in vivo . However, although the plant AlaRS was efficiently imported into mitochondria in the transformed strains, we found no evidence for import of the A . thaliana tRNA(Ala) nor of the endogenous cytosolic tRNA(Ala) isoacceptors . We conclude that at least one other factor besides the mitochondrial AlaRS precursor must be involved in mitochondrial import of tRNA(Ala) in plants. J Mol Biol, 2000 May 12, 298(4), 567 - 75 The human homologue of the yeast splicing factor prp6p contains multiple TPR elements and is stably associated with the U5 snRNP via protein-protein interactions; Makarov EM et al.; An essential step of pre-mRNA spliceosome assembly is the interaction between the snRNPs U4/U6 and U5, to form the {U4/U6.U5} tri-snRNP . While the tri-snRNP protein Prp6p appears to play an important role for tri-snRNP formation in yeast, little is known about the interactions that connect the two snRNP particles in human tri-snRNPs . Here, we describe the molecular characterisation of a 102kD protein form HeLa tri-snRNPs . The 102kD protein exhibits a significant degree of overall homology with the yeast Prp6p, including the conservation of multiple tetratrico peptide repeats (TPR), making this the likely functional homologue of Prp6p . However, while the yeast Prp6p is considered to be a U4/U6-specific protein, the human 102kD protein was found to be tightly associated with purified 20 S U5 snRNPs . This association appears to be primarily due to protein-protein interactions . Interestingly, antibodies directed against the C-terminal TPR elements of the 102kD protein specifically and exclusively immunoprecipitate free U5 snRNPs, but not {U4/U6.U5} tri-snRNPs, from HeLa nuclear extract, suggesting that the C-terminal region of the 102kD protein is covered by U4/U6 or tri-snRNP-specific proteins . Since proteins containing TPR elements are typically involved in multiple protein-protein interactions, we suggest that the 102kD protein interacts within the tri-snRNP with both the U5 and U4/U6 snRNPs, thus bridging the two particles . Consistent with this idea, we show that in vitro translated U5-102kD protein binds to purified 13S U4/U6 snRNPs, which contain, in addition to the Sm proteins, all known U4/U6-specific proteins . J Biol Chem, 2000 Jun 30, 275(26), 20168 - 78 Regulation of peroxisome size and number by fatty acid beta -oxidation in the yeast yarrowia lipolytica; Smith JJ et al.; The Yarrowia lipolytica MFE2 gene encodes peroxisomal beta-oxidation multifunctional enzyme type 2 (MFE2) . MFE2 is peroxisomal in a wild-type strain but is cytosolic in a strain lacking the peroxisomal targeting signal-1 (PTS1) receptor . MFE2 has a PTS1, Ala-Lys-Leu, that is essential for targeting to peroxisomes . MFE2 lacking a PTS1 can apparently oligomerize with full-length MFE2 to enable targetting to peroxisomes . Peroxisomes of an oleic acid-induced MFE2 deletion strain, mfe2-KO, are larger and more abundant than those of the wild-type strain . Under growth conditions not requiring peroxisomes, peroxisomes of mfe2-KO are larger but less abundant than those of the wild-type strain, suggesting a role for MFE2 in the regulation of peroxisome size and number . A nonfunctional version of MFE2 did not restore normal peroxisome morphology to mfe2-KO cells, indicating that their phenotype is not due to the absence of MFE2 . mfe2-KO cells contain higher amounts of beta-oxidation enzymes than do wild-type cells . We also show that increasing the level of the beta-oxidation enzyme thiolase results in enlarged peroxisomes . Our results implicate peroxisomal beta-oxidation in the control of peroxisome size and number in yeast. Biochim Biophys Acta, 2000 Jan 31, 1490(1-2), 106 - 8 cDNA cloning of the mammalian sterol C5-desaturase and the expression in yeast mutant; Nishi S et al.; Sterol C5-desaturase (SC5D) cDNA is an enzyme that catalyzes the dehydrogenation to introduce C5-6 double bond into lathosterol in cholesterol biosynthesis . We have isolated and sequenced the cDNA clones encoding human and mouse SC5D . The functional complementation of a defective yeast mutant proves that the human and mouse cDNA clones encode SC5D . Mammalian SC5D was presumed as an integral membrane protein containing histidine residues conserved also in yeasts and plant. Bioseparation, 1999, 8(6), 293 - 306 Purification of alpha-glucosidae and invertase from bakers' yeast on modified polymeric supports; Lothe RR et al.; In the present work Amberlite XAD-16 and Indion NPA-1, Polystyrene Divinylbenzene macroreticular spherical resins, have been evaluated quantitatively as supports for the adsorption and isolation of the yeast proteins and the enzymes, invertase and alpha-glucosidase . Modification of these supports has been carried out by surface grafting using acrylate polymers to reduce the hydrophobicity and nonspecific adsorption of proteins . Good grafting efficiency, in excess of 90%, has been obtained using ultrasonic irradiation for the surface activation of polystyrene resins . XAD-16 has higher adsorption capacities for the total yeast proteins as well as for both the enzymes, alpha-glucosidase and invertase, than NPA-1 in its respective native and grafted form . Adsorption capacities of XAD-16 and NPA-1 in their respective native and grafted forms for alpha-glucosidase are higher than the capacities for invertase . Nonspecific adsorption of total proteins has been reduced considerably after the grafting of acrylate polymers on hydrophobic supports . At the same time selectivity for the adsorption of both the enzymes has been enhanced on grafted supports . The overall solid-liquid adsorption mass transfer coefficient values (Kla) estimated for adsorption of invertase on XAD are lower than those for alpha-glucosidase . Native and grafted resins could be regenerated and reused for adsorption of alpha-glucosidase for two regeneration cycles studied . Storage stability of invertase and alpha-glucosidase is the same on native and grafted form of XAD-16 and is more than the enzymes in the free form. Rev Argent Microbiol, 2000 Jan-Mar, 32(1), 1 - 6 {Identification and typing of yeast isolates from hospital patients in Mexico City}; Manzano-Gayosso P et al.; Nosocomial yeast infections have become a major cause of morbidity and mortality in immunocompromised patients . This study presents the frequency of yeasts isolated from different biological products from a Mexico City hospital's patients during one year . C . albicans was isolated in 72.3% of patients, followed by C . glabrata (13.4%) and C . parapsilosis (8%) . Twenty different morphotypes were identified and the serotype B was the most frequent (79%). Eur J Biochem, 2000 May, 267(9), 2666 - 71 A novel function of yeast fatty acid synthase . Subunit alpha is capable of self-pantetheinylation; Fichtlscherer F et al.; The prosthetic group of yeast fatty acid synthase (FAS), 4'-phosphopantetheine, is covalently linked to Ser180 of subunit alpha . It originates from coenzyme A and is transferred to the enzyme by a specific phosphopantetheine:protein transferase (PPTase) . The present study demonstrates that the FAS-activating PPTase of yeast represents a distinct catalytic domain of the FAS complex and resides within the C-terminal portion of subunit alpha . The autoactivation capacity of yeast FAS became evident from in vitro pantetheinylation studies using purified apo-FAS preparations . These were readily converted to pantetheinylated holo-FAS simply upon addition of free coenzyme A . Pantetheinylation-competent apo-FAS was prepared in vitro by constructing hybrid oligomers containing alpha-subunits from two different pantetheine-less FAS-mutants . The respective mutants were selected according to their ability to complement each other, in vivo . In vitro formation of hybrid apo-FAS complexes was achieved by dimethylmaleic anhydride (DMMA) -induced reversible dissociation of mixtures of the two constituent mutant enzymes . This treatment was both necessary and sufficient to produce pantetheinylation-competent apo-FAS . Specific FAS activities were comparable independent of whether the apo-enzymes were pantetheinylated in vivo or in vitro . Apart from the induction of overall FAS activity, incorporation of phosphopantetheine into apo-FAS was also demonstrated by the use of 3H-labelled coenzyme A, leading to the formation of radioactively labelled FAS . It is concluded that pantetheinylation of yeast FAS is performed by an intrinsic catalytic activity of the apo-enzyme proper . The endogenous PPTase acts in trans between different subunits alpha in the alpha6beta6 oligomer . The self-pantetheinylation of yeast FAS represents the first example of an apo-enzyme being capable of post-translational autoactivitation. Microbiology, 2000 Apr, 146 ( Pt 4), 877 - 84 Cellular lipid composition influences stress activation of the yeast general stress response element (STRE); Chatterjee MT et al.; The heat inducibility of the yeast heat-shock response (HSR) pathway has been shown to be critically dependent on the level of unsaturated fatty acids present in the cell . Here the inducibility by heat or salt of the independently regulated general stress response (GSR) pathway is shown to be affected in the same way . An increase in the percentage of unsaturated fatty acids in heat- or salt-acclimated cells correlated with a decrease in the induction of a general stress-response-promoter-element (STRE)-driven reporter gene by either stress . Despite inducing reporter gene expression, sorbic acid treatment did not confer salt cross-tolerance on the cells . This failure correlated with a failure to increase the percentage of unsaturated fatty acids in the cells, suggesting that GSR pathway induction, in the absence of lipid changes, is insufficient for the induction of cross-tolerance . Cells grown with fatty acid supplements under anaerobic conditions provided further evidence for a potential role for lipids in the acquisition of stress resistance . These cells contained different fatty acid profiles depending on the fatty acid supplement supplied, exhibited differential sensitivity to both heat and salt stress, but had not undergone STRE induction . These results suggest that heat- and salt-stress induction of the GSR are sensitive to the level of unsaturated fatty acids present in the cell and that stress cross-tolerance may be a lipid-mediated phenomenon . Given that an increased level of unsaturated fatty acids also down-regulates heat induction of the HSR pathway, these observations lead to the provocative hypothesis that lipid modifications, rather than HSR or GSR pathway induction, are a major contributor to the induced heat and salt tolerance of yeast cells. Genes Dev, 2000 Apr 15, 14(8), 951 - 62 Bromodomain factor 1 corresponds to a missing piece of yeast TFIID; Matangkasombut O et al.; The basal transcription factor TFIID consists of the TATA-binding protein (TBP) and TBP-associated factors (TAFs) . Yeast Taf67 is homologous to mammalian TAF(II)55 . Using a yeast two-hybrid screen to identify proteins that interact with Taf67, we isolated Bromodomain factor 1 (Bdf1) and its homolog (Bdf2) . The Bdf proteins are genetically redundant, as cells are inviable without at least one of the two BDF genes . Both proteins contain two bromodomains, a motif found in several proteins involved in transcription and chromatin modification . The BDF genes interact genetically with TAF67 . Furthermore, Bdf1 associates with TFIID and is recruited to a TATA-containing promoter . Deletion of Bdf1 or the Taf67 Bdf-interacting domain leads to defects in gene expression . Interestingly, the higher eukaryotic TAF(II)250 has an acetyltransferase activity, two bromodomains, and an associated kinase activity . Its yeast homolog, Taf145, has acetyltransferase activity but lacks the bromodomains and kinase . Bdf1, like TAF(II)250, has a kinase activity that maps carboxy-terminal to the bromodomains . The structural and functional similarities suggest that Bdf1 corresponds to the carboxy-terminal region of higher eukaryotic TAF(II)250 and that the interaction between TFIID and Bdf1 is important for proper gene expression. Trends Biochem Sci, 2000 May, 25(5), 229 - 35 Phosphoinositide signaling and the regulation of membrane trafficking in yeast; Odorizzi G et al.; Phosphoinositides are key regulators of diverse cellular processes in eukaryotic cells . Genetic studies in yeast have advanced our understanding of how phosphoinositide-signaling pathways regulate membrane trafficking . Enzymes required for the synthesis (kinases) and turnover (phosphatases) of distinct phosphoinositides have been identified and several downstream effector molecules linked to phosphoinositide signaling have recently been characterized. FEBS Lett, 2000 Apr 21, 472(1), 159 - 65 Stimulation of the yeast high osmolarity glycerol (HOG) pathway: evidence for a signal generated by a change in turgor rather than by water stress; Tamas MJ et al.; The Saccharomyces cerevisiae HOG pathway controls responses to osmotic shock such as production of the osmolyte glycerol . Here we show that the HOG pathway can be stimulated by addition of glycerol . This stimulation was strongly diminished in cells expressing an unregulated Fps1p glycerol channel, presumably because glycerol rapidly equilibrated across the plasma membrane . Ethanol, which passes the plasma membrane readily and causes water stress by disturbing the hydration of biomolecules, did not activate the HOG pathway . These observations suggest that stimulation of the HOG pathway is mediated by a turgor change and not by water stress per se. J Biol Chem, 2000 Jul 14, 275(28), 21761 - 7 The H subunit (Vma13p) of the yeast V-ATPase inhibits the ATPase activity of cytosolic V1 complexes; Parra KJ et al.; V-ATPases are composed of a peripheral complex containing the ATP-binding sites, the V(1) sector, attached to a membrane complex containing the proton pore, the V(o) sector . In vivo, free, inactive V(1) and V(o) sectors exist in dynamic equilibrium with fully assembled, active V(1) V(o) complexes, and this equilibrium can be perturbed by changes in carbon source . Free V(1) complexes were isolated from the cytosol of wild-type yeast cells and mutant strains lacking V(o) subunit c (Vma3p) or V(1) subunit H (Vma13p) . V(1) complexes from wild-type or vma3Delta mutant cells were very similar, and contained all previously identified yeast V(1) subunits except subunit C (Vma5p) . These V(1) complexes hydrolyzed CaATP but not MgATP, and CaATP hydrolysis rapidly decelerated with time . V(1) complexes from vma13Delta cells contained all V(1) subunits except C and H, and had markedly different catalytic properties . The initial rate of CaATP hydrolysis was maintained for much longer . The complexes also hydrolyzed MgATP, but showed a rapid deceleration in hydrolysis . These results indicate that the H subunit plays an important role in silencing unproductive ATP hydrolysis by cytosolic V(1) complexes, but suggest that other mechanisms, such as product inhibition, may also play a role in silencing in vivo. J Bacteriol, 2000 May, 182(10), 2746 - 52 The promoter of the yeast INO4 regulatory gene: a model of the simplest yeast promoter; Robinson KA et al.; In Saccharomyces cerevisiae, the phospholipid biosynthetic genes are transcriptionally regulated in response to inositol and choline . This regulation requires the transcriptional activator proteins Ino4p and Ino2p, which form a heterodimer that binds to the UAS(INO) element . We have previously shown that the promoters of the INO4 and INO2 genes are among the weakest promoters characterized in yeast . Because little is known about the promoters of weakly expressed yeast genes, we report here the analysis of the constitutive INO4 promoter . Promoter deletion constructs scanning 1,000 bp upstream of the INO4 gene identified a small region (-58 to -46) that is absolutely required for expression . S1 nuclease mapping shows that this region contains the transcription start sites for the INO4 gene . An additional element (-114 to -86) modestly enhances INO4 promoter activity (fivefold) . Thus, the region required for INO4 transcription is limited to 68 bp . These studies also found that INO4 gene expression is not autoregulated by Ino2p and Ino4p, despite the presence of a putative UAS(INO) element in the INO4 promoter . We further report that the INO4 steady-state transcript levels and Ino4p levels are regulated twofold in response to inositol and choline, suggesting a posttranscriptional mechanism of regulation. Mol Cell Biol, 2000 May, 20(10), 3538 - 49 Deletion of the PAT1 gene affects translation initiation and suppresses a PAB1 gene deletion in yeast; Wyers F et al.; The yeast poly(A) binding protein Pab1p mediates the interactions between the 5' cap structure and the 3' poly(A) tail of mRNA, whose structures synergistically activate translation in vivo and in vitro . We found that deletion of the PAT1 (YCR077c) gene suppresses a PAB1 gene deletion and that Pat1p is required for the normal initiation of translation . A fraction of Pat1p cosediments with free 40S ribosomal subunits on sucrose gradients . The PAT1 gene is not essential for viability, although disruption of the gene severely impairs translation initiation in vivo, resulting in the accumulation of 80S ribosomes and in a large decrease in the amounts of heavier polysomes . Pat1p contributes to the efficiency of translation in a yeast cell-free system . However, the synergy between the cap structure and the poly(A) tail is maintained in vitro in the absence of Pat1p . Analysis of translation initiation intermediates on gradients indicates that Pat1p acts at a step before or during the recruitment of the 40S ribosomal subunit by the mRNA, a step which may be independent of that involving Pab1p . We conclude that Pat1p is a new factor involved in protein synthesis and that Pat1p might be required for promoting the formation or the stabilization of the preinitiation translation complexes. Mol Cell Biol, 2000 May, 20(10), 3522 - 8 Evidence for the involvement of nucleotide excision repair in the removal of abasic sites in yeast; Torres-Ramos CA et al.; In eukaryotes, DNA damage induced by ultraviolet light and other agents which distort the helix is removed by nucleotide excision repair (NER) in a fragment approximately 25 to 30 nucleotides long . In humans, a deficiency in NER causes xeroderma pigmentosum (XP), characterized by extreme sensitivity to sunlight and a high incidence of skin cancers . Abasic (AP) sites are formed in DNA as a result of spontaneous base loss and from the action of DNA glycosylases involved in base excision repair . In Saccharomyces cerevisiae, AP sites are removed via the action of two class II AP endonucleases, Apn1 and Apn2 . Here, we provide evidence for the involvement of NER in the removal of AP sites and show that NER competes with Apn1 and Apn2 in this repair process . Inactivation of NER in the apn1Delta or apn1Delta apn2Delta strain enhances sensitivity to the monofunctional alkylating agent methyl methanesulfonate and leads to further impairment in the cellular ability to remove AP sites . A deficiency in the repair of AP sites may contribute to the internal cancers and progressive neurodegeneration that occur in XP patients. Mol Cell Biol, 2000 May, 20(10), 3459 - 69 Fission yeast Eso1p is required for establishing sister chromatid cohesion during S phase; Tanaka K et al.; Sister chromatid cohesion is essential for cell viability . We have isolated a novel temperature-sensitive lethal mutant named eso1-H17 that displays spindle assembly checkpoint-dependent mitotic delay and abnormal chromosome segregation . At the permissive temperature, the eso1-H17 mutant shows mild sensitivity to UV irradiation and DNA-damaging chemicals . At the nonpermissive temperature, the mutant is arrested in M phase with a viability loss due to a failure to establish sister chromatid cohesion during S phase . The lethal M-phase arrest phenotype, however, is suppressed by inactivation of a spindle checkpoint . The eso1(+) gene is not essential for the onset and progression of DNA replication but has remarkable genetic interactions with those genes regulating the G(1)-S transition and DNA replication . The N-terminal two-thirds of Eso1p is highly homologous to DNA polymerase eta of budding yeast and humans, and the C-terminal one-third is homologous to budding yeast Eco1p (also called Ctf7p), which is required for the establishment of sister chromatid cohesion . Deletion analysis and determination of the mutation site reveal that the function of the Eco1p/Ctf7p-homologous domain is necessary and sufficient for sister chromatid cohesion . On the other hand, deletion of the DNA polymerase eta domain in Eso1p increases sensitivity to UV irradiation . These results indicate that Eso1p plays a dual role during DNA replication . The C-terminal region acts to establish sister chromatid cohesion, and the N-terminal region presumably catalyzes translesion DNA synthesis when template DNA contains lesions that block regular DNA replication. Mol Gen Genet, 2000 Mar, 263(2), 253 - 61 The protein kinases Rck1 and Rck2 inhibit meiosis in budding yeast; Ramne A et al.; The genes RCK1 and RCK2 of budding yeast were initially identified as suppressors of checkpoint mutations in fission yeast . Here, we show that homozygous diploid rck1/rck1 mutants in standard sporulation medium enter meiosis in about half the time required by wild-type cells . A similar, but weaker, effect is seen in rck2/rck2 mutants, whereas double homozygous rck1/rck1 rck2/rck2 mutants display a phenotype similar to that of the rck1/rck1 single mutants . In diploids with mutations in either of the meiotic checkpoint genes MEC1 and RAD24, overexpression of RCK1 or RCK2 reduces meiotic proficiency, most prominently seen with RCK2 . The rate of meiotic recombination was unaltered in rck1 and rck2 mutants . There is a transient shift in the relative abundance of the two RCK2 transcripts in meiotic cells . We propose that one function of Rck1 and Rck2 is to inhibit meiosis. Radiats Biol Radioecol, 2000 Jan-Feb, 40(1), 99 - 104 {Simultaneous action of UV light and hyperthermia on survival and recombination of yeast: effect of intensity of agents on their synergistic interaction}; Rassokhina AV et al.; Synergistic effects of simultaneous application of ultraviolet (UV) light and hyperthermia on survival and recombination of diploid yeast cells were studied . For both test-systems the dependence of the synergistic interaction on UV light fluence rate and exposure temperature was revealed: the temperature range synergistically increasing the action of UV light is shifted towards low temperature values with decreasing of UV light fluence rate . For cell survival, the dependence of the synergistic enhancement ratio on the exposure temperature passes through a maximum . A possible qualitative interpretation of these results is discussed. J Biol Chem, 2000 Jun 23, 275(25), 19352 - 60 Psr1p/Psr2p, two plasma membrane phosphatases with an essential DXDX(T/V) motif required for sodium stress response in yeast; Siniossoglou S et al.; Regulation of intracellular ion concentration is an essential function of all cells . In this study, we report the identification of two previously uncharacterized genes, PSR1 and PSR2, that perform an essential function under conditions of sodium ion stress in the yeast Saccharomyces cerevisiae . Psr1p and Psr2p are highly homologous and were identified through their homology with the endoplasmic reticulum membrane protein Nem1p . Localization and biochemical fractionation studies show that Psr1p is associated with the plasma membrane via a short amino-terminal sequence also present in Psr2p . Growth of the psr1psr2 mutant is severely inhibited under conditions of sodium but not potassium ion or sorbitol stress . This growth defect is due to the inability of the psr1psr2 mutant to properly induce transcription of ENA1/PMR2, the major sodium extrusion pump of yeast cells . We provide genetic evidence that this regulation is independent of the phosphatase calcineurin, previously implicated in the sodium stress response in yeast . We show that Psr1p contains a DXDX(T/V) phosphatase motif essential for its function in vivo and that a Psr1p-PtA fusion purified from yeast extracts exhibits phosphatase activity . Based on these data, we suggest that Psr1p/Psr2p, members of an emerging class of eukaryotic phosphatases, are novel regulators of salt stress response in yeast. Mikrobiologiia, 2000 Mar-Apr, 69(2), 280 - 5 {Discrimination of the soil yeast species Williopsis saturnus and Williopsis suaveolens by the polymerase chain reaction using nonspecific primers.}; Naumov GI et al.; Thirty-five yeast strains of the genus Williopsis, analyzed by the polymerase chain reaction with the universal primer N21, were found to belong to two sibling species, W . saturnus and W . suaveolens . Such affiliation of the strains studied agrees well with the results of genetic and physiological investigations. Mikrobiologiia, 2000 Mar-Apr, 69(2), 276 - 9 {A new yeast species Candida anutae sp.nov . from the fruiting bodies of agaricus}; Bab'eva IP et al.; Among the yeasts isolated from the fruiting bodies of different species of agarics picked in forests near Moscow and Turku (Finland) in 1995-1998, populations of an earlier unknown species, morphologically similar to Metschnikowia lunata but differing from it by physiological characteristics and the absence of asci with spores, were constantly found . Description of the new species is given within the genus Candida Berkhout. IUBMB Life, 2000 Feb, 49(2), 153 - 9 Epitope-tagged constructs of the yeast plasma-membrane H(+)-ATPase; dos Santos CF et al.; In this study, two different epitope tags (HA, c-myc) were introduced near the N terminus of the yeast PMA1 H(+)-ATPase . The resulting proteins were indistinguishable from the wild-type ATPase in their ability to travel through the secretory pathway, as judged by quantitative immunoblotting of isolated secretory vesicles . Furthermore, there were no significant abnormalities in ATPase activity (including K(m) for MgATP, Vmax, pH optimum, and IC50 for inhibition by vanadate) or in ATP-dependent proton pumping . Finally, the epitope-tagged ATPases could support normal growth and displayed the expected activation by glucose. J Agric Food Chem, 2000 Apr, 48(4), 1081 - 5 Characterization of the nitrogen compounds released during yeast autolysis in a model wine system; Martinez-Rodriguez AJ et al.; The nitrogen composition of wines aged with yeast for a long period of time, as in the case of sparkling wines, depends on the composition of the base wine and on the compounds released by the yeast . In this paper, the release of the different classes of nitrogen compounds during autolysis of one of the strains of yeast used in the manufacture of sparkling wines has been studied . The yeast, Saccharomyces bayanus, was suspended in a model wine buffer, pH 3.0 and 10% ethanol, and incubated at 30 degrees C . Samples of the autolysate were taken after 4, 24, 48, 72, 168, and 360 h of autolysis . An electrophoretic and chromatographic study was conducted of the proteins, peptides with molecular weights higher and lower than 700 Da, and amino acids released during the autolysis . Using SDS-PAGE, it was observed that it was predominantly polypeptides with molecular weights lower than 10 000 that were released . Through HPLC of the fraction lower than 10 000 Da, it was observed that it is polypeptides with molecular weights of between 10 000 and 700 Da that are released first and that these later break up to give rise to peptides with molecular weights lower than 700 Da, which in turn break down into amino acids . This indicates that the nature of the nitrogen compounds present in wines aged with yeast depends on the aging time, being less polymerized as the aging time increases. Mol Cells, 2000 Feb 29, 10(1), 113 - 8 Yeast asparagine (Asn) tRNA without Q base promotes eukaryotic frameshifting more efficiently than mammalian Asn tRNAs with or without Q base; Carlson BA et al.; In this study, we compare the efficiency of Asn tRNA from mammalian sources with and without the highly modified queuosine (Q) base in the wobble position of its anticodon and Asn tRNA from yeast, which naturally lacks Q base, to promote frameshifting . Interestingly, no differences in the ability of the two mammalian Asn tRNAs to promote frameshifting were observed, while yeast tRNA(ASn)(-Q) promoted frameshifting more efficiently than its mammalian counterparts in both rabbit reticulocyte lysates and wheat germ extracts . The shiftability of yeast Asn tRNA is therefore not due, or at least not completely, to the lack of Q base and most likely the shiftiness resides in structural differences elsewhere in the molecule . However, we cannot absolutely rule out a role of Q base in frameshifting as wheat germ extracts and a lysate depleted of most of its tRNA and supplemented with calf liver tRNA contain both Asn tRNA with or without Q base. Gene, 2000 Apr 18, 247(1-2), 129 - 36 Inactivation of the SNF5 transcription factor gene abolishes the lethal phenotype induced by the expression of HIV-1 integrase in yeast; Parissi V et al.; The ubiquitous human transcription factor Ini1 has been shown to interact with HIV-1 integrase (IN) and to stimulate in vitro the reactions catalyzed by this enzyme . We have previously used a yeast model to study the effect of HIV-1 IN expression (Caumont, A.B., Jamieson, G.A., Pichuantes, S., Nguyen, A.T., Litvak, S., Dupont, C . -H., 1996 . Expression of functional HIV-1 integrase in the yeast Saccharomyces cerevisiae leads to the emergence of a lethal phenotype: potential use for inhibitor screening . Curr . Genet . 29, 503-510) . Here, we describe the effect of the inactivation of the gene encoding for SNF5, a yeast transcription factor homologous to Ini1, on the lethality induced by the expression of HIV-1 IN in yeast . We observed that the retroviral IN was unable to perform its lethal activity in cells where the SNF5 gene has been disrupted, suggesting that SNF5 may play a role in the lethal effect induced by IN in yeast . SNF5 inactivation affects neither yeast viability nor expression of HIV-1 IN . Given the homology between SNF5 and its human counterpart Ini1, our results suggest that this factor may be important for IN activity in infected cells . Moreover, given the important role proposed for this transcription factor in the integration step and the fact that it is dispensable for cell viability, the interaction between Ini1/ySNF5 and HIV-1 IN should become a potential target in the search for new antiretroviral agents. Gene, 2000 Apr 18, 247(1-2), 53 - 61 Zero background yeast reporter plasmids; Melcher K et al.; UAS-less reporter plasmids are widespread and powerful tools for the identification and analysis of binding sites for transcriptional activators . The common reporter plasmids for the yeast Saccharomyces cerevisiae are multicopy (2mu) vectors with the CYC1 core promoter upstream of the lacZ gene . Insertion of putative or known activator binding sites upstream of the core promoter puts lacZ (beta-galactosidase) expression under the control of the corresponding activator . Although these constructs have proved to work well for most purposes, they have certain limitations: (1) they give significant and carbon-source-dependent lacZ background expression; (2) unlike most other yeast promoters, the CYC1 upstream region has a partially open chromatin structure with an accessible TATA box; (3) they use only a single, moderately sensitive reporter; and (4) the use of multicopy vectors can result in activator titration . Here, we introduce novel reporter plasmids based on the yeast MEL1 (alpha-galactosidase) gene that can overcome all of these limitations . It is also shown that background expression is due to fortuitous activator binding sites within the plasmid backbones that are insufficiently shielded from the core promoters in the common CYC1 reporter plasmids. Nucleic Acids Res, 2000 May 15, 28(10), 2060 - 8 DNA repair in a yeast origin of replication: contributions of photolyase and nucleotide excision repair; Suter B et al.; DNA damage formation and repair are tightly linked to protein-DNA interactions in chromatin . We have used minichromosomes in yeast as chromatin substrates in vivo to investigate how nucleotide excision repair (NER) and repair by DNA-photolyase (photoreactivation) remove pyrimidine dimers from an origin of replication ( ARS1 ) . The ARS1 region is nuclease sensitive and flanked by nucleosomes on both sides . Photoreactivation was generally faster than NER at all sites . Site-specific heterogeneity of repair was observed for both pathways . This heterogeneity was different for NER and photoreactivation and it was altered in a minichromosome where ARS1 was transcribed . The results indicate distinct inter-actions of the repair systems with protein complexes bound in the ARS region (ORC, Abf1) and a predominant role of photolyase in CPD repair of an origin of replication. IUBMB Life, 2000 Jan, 49(1), 17 - 22 Specific domain of cGMP-dependent protein kinase Ibeta but not Ialpha functions as a transcriptional activator in yeast; Yuasa K et al.; Recently, cyclic GMP-dependent protein kinase (cGK) was shown to translocate to the nucleus and regulate gene transcription . To determine whether cGK I proteins function as transcriptional activators, we produced the constructs of cGK Ialpha or Ibeta fused with the DNA binding domain of the yeast transcriptional activator GAL4 . Here, we demonstrate that the amino-terminal region of cGK Ibeta (amino acids 1-107) exhibits transcriptional activation in yeast . However, full-length cGK Ialpha and Ibeta and the amino-terminal region of cGK Ialpha had no transcriptional activation function . Amino acid replacement in the leucine zipper motif of the amino-terminal region of cGK Ibeta substantially reduced transcriptional activation . These results suggest that the Ibeta-specific region in cGK I proteins may interact with other proteins by way of the leucine zipper motif and has a transcriptional activation function. Biotechniques, 2000 Apr, 28(4), 668 - 70, 672, 674 DNA extraction method for screening yeast clones by PCR; Akada R et al.; A simple procedure for isolating yeast DNA suitable for use as a template for PCR amplification is described . SDS treatment alone is sufficient for extraction of chromosomal DNA from yeast cells . Cells of a yeast colony are suspended in a small volume (about 20 microL) of a 0.25% SDS solution, mixed vigorously and centrifuged . The supernatant can be directly used as a template after dilution to give an SDS concentration of less than 0.01% in the final PCR mixture. Biochem J, 2000 May 1, 347 Pt 3, 687 - 91 Oxidative phosphorylation in cardiolipin-lacking yeast mitochondria; Koshkin V et al.; The role of cardiolipin in mitochondrial energy transformation was studied by comparing oxidative phosphorylation in the Saccharomyces cerevisiae cardiolipin synthase null mutant crd1Delta, and in isogenic wild type . Oxygen consumption experiments and membrane potential kinetics during the phosphorylation cycle in isolated mitochondria indicated that the absence of cardiolipin causes only a moderate deficiency of mitochondrial energy-transforming machinery at 25 degrees C . However, at 40 degrees C, respiration was completely uncoupled from phosphorylation for the mutant mitochondria, in contrast with that for the wild-type . Membranepotential kinetics demonstrated an increased susceptibility of the mutant mitochondria to gradual deterioration during in vitro incubation . These results suggest that cardiolipin, although normally associated with several of the major enzymes of oxidative phosphorylation and required in vitro for their maximal activity, is not absolutely necessary for mitochondrial energy transformation under optimal conditions . The role of cardiolipin is, rather, to improve efficiency of oxidative phosphorylation and its resistance to unfavourable conditions, such as increased temperature. Biochemistry, 2000 Apr 25, 39(16), 4869 - 80 Participation of the amino-terminal domain in the self-association of the full-length yeast TATA binding protein; Daugherty MA et al.; The association of monomeric TATA binding protein with promoter DNA is an essential first step in many current models of eukaryotic transcription initiation . This step is followed by others in which additional transcription factors, and finally RNA polymerase, assemble at the promoter . Here we characterize the quaternary interactions of the Saccharomyces cerevisiae TATA-binding protein (yTBP), in the absence of other proteins or DNA . The data reveal a robust pattern in which yTBP monomers equilibrate with tetramers and octamers over a broad span of temperatures (4 degrees C </= T </= 37 degrees C) and salt concentrations (60 mM </= {KCl} </= 1 M), that includes the physiological range . Association is highly cooperative, with octamer formation favored by approximately 9 kcal/mol over tetramer formation . Changes in association constant with {KCl} are consistent with an assembly-linked release of ions at low salt and an assembly-linked uptake of ions at high salt, for both monomer right arrow over left arrow tetramer and tetramer right arrow over left arrow octamer reaction steps . Fluorescence emission spectra and steady-state anisotropies reveal that the amino-terminal domain changes conformation and dynamics at both association steps and that the polarity of the environment near tryptophan 26 is sensitive to changes in {KCl} in the monomeric and tetrameric states but not the octameric state . These results are consistent with a {salt}-dependent change in the assembly mechanism near 300 mM KCl and suggest that the amino-terminal domain may modulate the self-association of the full-length protein . TBP self-association may regulate many of its cellular functions, including transit of the nuclear membrane and participation in transcription initiation. Biochemistry, 2000 Apr 25, 39(16), 4711 - 21 Mixed disulfide with glutathione as an intermediate in the reaction catalyzed by glutathione reductase from yeast and as a major form of the enzyme in the cell; Arscott LD et al.; Glutathione reductase catalyzes the reduction of glutathione disulfide by NADPH . The FAD of the reductase is reduced by NADPH, and reducing equivalents are passed to a redox-active disulfide to complete the first half-reaction . The nascent dithiol of two-electron reduced enzyme (EH(2)) interchanges with glutathione disulfide forming two molecules of glutathione in the second half-reaction . It has long been assumed that a mixed disulfide (MDS) between one of the nascent thiols and glutathione is an intermediate in this reaction . In addition to the nascent dithiol composed of Cys(45) and Cys(50), the enzyme contains an acid catalyst, His(456), having a pK(a) of 9.2 that protonates the first glutathione (residue numbers refer to the yeast enzyme sequence) . Reduction of yeast glutathione reductase by glutathione and reoxidation of EH(2) by glutathione disulfide indicate that the mixed disulfide accumulates, in particular, at low pH . The reaction of glutathione disulfide with EH(2) is stoichiometric in the absence of an excess of glutathione . The equilibrium position among E(ox), MDS, and EH(2) is determined by the glutathione concentration and is not markedly influenced by pH between 6.2 and 8.5 . The mixed disulfide is the principal product in the reaction of glutathione with oxidized enzyme (E(ox)) at pH 6 . 2 . Its spectrum can be distinguished from that of EH(2) by a slightly lower thiolate (Cys(50))-FAD charge-transfer absorbance at 540 nm . The high GSH/GSSG ratio in the cytoplasm dictates that the mixed disulfide will be the major enzyme species. Mutat Res, 2000 Apr, 462(2-3), 293 - 301 The yeast p53 functional assay: a new tool for molecular epidemiology . Hopes and facts; Fronza G et al.; The assumption of molecular epidemiology that carcinogens leave fingerprints has suggested that analysis of the frequency, type, and site of mutations in genes frequently altered in carcinogenesis may provide clues to the identification of the factors contributing to carcinogenesis . In this mini-review, we revise the development, and validation of the yeast-based p53 functional assay as a new tool for molecular epidemiology . We show that this assay has some very interesting virtues but also has some drawbacks . The yeast functional assay can be used to determine highly specific mutation fingerprints in the human p53 cDNA sequence . Discrimination is possible when comparing mutation spectra induced by sufficiently different mutagens . However, we also reported that the same carcinogen may induce distinguishable mutation spectra due to known influencing factors. Gene, 2000 Apr 4, 246(1-2), 93 - 102 Transcription and proper splicing of a mammalian gene in yeast; Kunze B et al.; The house mouse strain C57BL/6 harbours 64 copies of the multicopy gene Sp100-rs . Three of these are contained in the yeast artificial chromosome (YAC) clone yMm75 . Four Sp100-rs transcripts of 3.0, 2.6, 1.6 and 1.3kb were detected by Northern hybridization in the yMm75-harbouring line of Saccharomyces cerevisiae . Additional and less abundant transcripts were detected by RT-PCR . With one exception, the YAC-derived Sp100-rs transcripts were a subset of those found in the C57BL/6 mouse . This indicates transcription and proper splicing of murine pre-mRNAs in yeast . Analysis of the splice sites shows that the yeast splicing machinery accepts splice sites that deviate from the standard yeast consensus sequences . It may be feasible, therefore, at least in a fair proportion of cases, to exploit the mammalian mRNAs present in transgenic yeast for gene recognition of YAC-inserts. FEBS Lett, 2000 Apr 14, 471(2-3), 235 - 9 A cytochrome c-GFP fusion is not released from mitochondria into the cytoplasm upon expression of Bax in yeast cells; Roucou X et al.; To study Bax-induced release of cytochrome c in vivo, we have expressed a cytochrome c-GFP (green fluorescent protein) fusion in Saccharomyces cerevisiae cells null for the expression of the endogenous cytochrome . We show here that cytochrome c-GFP is efficiently localised to mitochondria and able to function as an electron carrier between complexes III and IV of the respiratory chain . Strikingly, while natural cytochrome c is released into the cytoplasm upon expression of Bax, the cytochrome c-GFP fusion is not . Nevertheless, cells co-expressing Bax and the cytochrome c-GFP fusion die, indicating that mitochondrial release of cytochrome c is not essential for cell death to occur in yeast . The failure to release cytochrome c-GFP is presumed to arise from increased bulk due to the GFP moiety . We propose that in intact yeast cells, Bax-induced release of cytochrome c into the cytoplasm occurs through a selective pore and not as a consequence of the non-specific breakage of the mitochondrial outer membrane. FEBS Lett, 2000 Apr 14, 471(2-3), 224 - 8 Functional conservation between yeast and plant endosomal Na(+)/H(+) antiporters; Quintero FJ et al.; Vacuolar compartmentation of Na(+) is an essential mechanism for salinity tolerance since it lowers cytosolic Na(+) levels while contributing to osmotic adjustment for cell turgor and expansion . The AtNHX1 protein of Arabidopsis thaliana substituted functionally for ScNHX1, the endosomal Na(+)/H(+) antiporter of yeast . Ion tolerance conferred by AtNHX1 and ScNHX1 correlated with ion uptake into an intracellular pool that was energetically dependent on the vacuolar (H(+))ATPase . AtNHX1 localized to vacuolar membrane fractions of yeast . Hence, both transporters share an evolutionarily conserved function in Na(+) compartmentation . AtNHX1 mRNA levels were upregulated by ABA and NaCl treatment in leaf but not in root tissue. J Biol Chem, 2000 Apr 21, 275(16), 11645 - 9 Yeast lacking Cu-Zn superoxide dismutase show altered iron homeostasis . Role of oxidative stress in iron metabolism; De Freitas JM et al.; Saccharomyces cerevisiae lacking copper-zinc superoxide dismutase (sod1) shows a series of defects, including reduced rates of aerobic growth in synthetic glucose medium and reduced ability to grow by respiration in glycerol-rich medium . In this work, we observed that addition of iron improved the respiratory growth of the sod1 mutant and in glucose medium total intracellular iron content was higher in the sod1 mutant than in wild type cells . Transcription of the high affinity iron transporter gene, FET3, was enhanced in the sod1 mutant, suggesting that iron transport systems were up-regulated . An sod1/fet3 double mutant showed increased sensitivity to oxygen and increased transcription of FET4, an alternative, low affinity, iron transporter . We propose that this increased iron demand in the sod1 mutant may be a reflection of the cells' efforts to reconstitute iron-sulfur cluster-containing enzymes that are continuously inactivated in conditions of excess superoxide. J Biol Chem, 2000 Jun 23, 275(25), 19210 - 7 The GAG-like protein of the yeast Ty1 retrotransposon contains a nucleic acid chaperone domain analogous to retroviral nucleocapsid proteins; Cristofari G et al.; The reverse transcription process for retroviruses and retrotransposons takes place in a nucleocore structure in the virus or virus-like particle . In retroviruses the major protein of the nucleocore is the nucleocapsid protein (NC protein), which derives from the C-terminal region of GAG . Retroviral NC proteins are formed of either one or two CCHC zinc finger(s) flanked by basic residues and have nucleic acid chaperone and match-maker properties essential for virus replication . Interestingly, the GAG protein of a number of retroelements including Spumaviruses does not possess the hallmarks of retroviral GAGs and in particular lacks a canonical NC protein . In an attempt to search for a nucleic acid chaperone activity in this class of retroelements we used the yeast Ty1 retrotransposon as a model system . Results shows that the C-terminal region of Ty1 GAG contains a nucleic acid chaperone domain capable of promoting the annealing of primer tRNA(i)(Met) to the multipartite primer binding site, Ty1 RNA dimerization and initiation of reverse transcription . Moreover Ty1 RNA dimerization, in a manner similar to Ty3 but unlike retroviral RNAs, appears to be mediated by tRNA(i)(Met) . These findings suggest that nucleic acid chaperone proteins probably are general co-factors for reverse transcriptases. Genes Dev, 2000 Apr 1, 14(7), 830 - 40 An aminoacylation-dependent nuclear tRNA export pathway in yeast; Grosshans H et al.; Yeast Los1p, the homolog of human exportin-t, mediates nuclear export of tRNA . Using fluorescence in situ hybridization, we could show that the export of some intronless tRNA species is not detectably affected by the disruption of LOS1 . To find other factors that facilitate tRNA export, we performed a suppressor screen of a synthetically lethal los1 mutant and identified the essential translation elongation factor eEF-1A . Mutations in eEF-1A impaired nuclear export of all tRNAs tested, which included both spliced and intronless species . An even stronger defect in nuclear exit of tRNA was observed under conditions that inhibited tRNA aminoacylation . In all cases, inhibition of tRNA export led to nucleolar accumulation of mature tRNAs . Our data show that tRNA aminoacylation and eEF-1A are required for efficient nuclear tRNA export in yeast and suggest coordination between the protein translation and the nuclear tRNA processing and transport machineries. Nature, 2000 Apr 6, 404(6778), 625 - 8 The Cdt1 protein is required to license DNA for replication in fission yeast; Nishitani H et al.; To maintain genome stability in eukaryotic cells, DNA is licensed for replication only after the cell has completed mitosis, ensuring that DNA synthesis (S phase) occurs once every cell cycle . This licensing control is thought to require the protein Cdc6 (Cdc18 in fission yeast) as a mediator for association of minichromosome maintenance (MCM) proteins with chromatin . The control is overridden in fission yeast by overexpressing Cdc18 (ref . 11) which leads to continued DNA synthesis in the absence of mitosis . Other factors acting in this control have been postulated and we have used a re-replication assay to identify Cdt1 (ref . 14) as one such factor . Cdt1 cooperates with Cdc18 to promote DNA replication, interacts with Cdc18, is located in the nucleus, and its concentration peaks as cells finish mitosis and proceed to S phase . Both Cdc18 and Cdt1 are required to load the MCM protein Cdc21 onto chromatin at the end of mitosis and this is necessary to initiate DNA replication . Genes related to Cdt1 have been found in Metazoa and plants (A . Whitaker, I . Roysman and T . Orr-Weaver, personal communication), suggesting that the cooperation of Cdc6/Cdc18 with Cdt1 to load MCM proteins onto chromatin may be a generally conserved feature of DNA licensing in eukaryotes. J Biol Chem, 2000 Jun 16, 275(24), 18093 - 8 The cytochrome bc1 and cytochrome c oxidase complexes associate to form a single supracomplex in yeast mitochondria; Cruciat CM et al.; The mitochondrial electron transport chain complexes are large multisubunit complexes embedded in the inner membrane . We report here that in the yeast Saccharomyces cerevisiae, the cytochrome bc(1) and cytochrome c oxidase complexes co-exist as a larger complex of approximately 1000 kDa in the mitochondrial membrane . Following solubilization with a mild detergent, the cytochrome bc(1)-cytochrome c oxidase complex remains stable . It was analyzed using the techniques of gel filtration and blue native-polyacrylamide gel electrophoresis . Direct physical association of subunits of the cytochrome bc(1) complex with those of the cytochrome c oxidase complex was verified by co-immunoprecipitation analysis . Our data indicate that the cytochrome bc(1) complex is exclusively in association with the cytochrome c oxidase complex in yeast mitochondria . We term this complex the cytochrome bc(1)-cytochrome c oxidase supracomplex. J Anim Sci, 2000 Mar, 78(3), 668 - 74 A new phytase expressed in yeast effectively improves the bioavailability of phytate phosphorus to weanling pigs; Stahl CH et al.; We have recently expressed a new phytase enzyme in a yeast system . Three experiments with a total of 140 weanling crossbred pigs were conducted to examine the efficacy of this enzyme in improving the bioavailability of phytate-P in corn-soybean meal diets to young pigs . Experiment 1 compared the efficacy of this new phytase with a commercially available phytase (Natuphos, BASF) for 4 wk at an inclusion level of 1,200 U/kg of diet . Experiment 2 compared the responses of pigs to four doses of the new phytase supplementation (300, 600, 900, and 1,200 U/kg of diet) for 4 wk . Experiment 3 compared the efficacy of this new phytase and Natuphos at a marginally optimal dose (700 U/kg of diet) for 5 wk . A group of pigs were fed the P-deficient basal diet as a negative control in Exp . 1, and a group of pigs were fed the basal diet plus .17 or .22% inorganic P as a positive control in all experiments . In Exp . 1, pigs fed the two sources of phytase had similar ADG (564 vs 567 g), gain/feed (.597 vs .589), plasma inorganic P concentrations (8.9 vs 8.4 mg/dL), and mobility scores (4.25 vs 4.46) that were higher (P < .05) than those of the negative control . In Exp . 2, plasma inorganic P concentration was a fairly linear response to the phytase dose (r > .83) at wk 1 and 2 . Overall ADG of pigs also tended to increase with the phytase dose (P = .15) . In Exp . 3, pigs fed the two sources of phytase had ADG (483 vs 506 g) similar to that of the positive control (508 g) . These two groups also had similar plasma inorganic P concentrations (7.7 vs 7.4 mg/dL) that were lower (P < .05) than those of the positive control group (9.7 mg/dL) . There was no significant effect of dietary treatments on ADFI in all three experiments . In conclusion, our new phytase was as effective as Natuphos, at the inclusion level of 700 or 1,200 U/kg of a P-deficient, corn-soybean meal diet, in improving phytate-P utilization by young pigs. J Bacteriol, 2000 May, 182(9), 2376 - 86 A rac homolog is required for induction of hyphal growth in the dimorphic yeast Yarrowia lipolytica; Hurtado CA et al.; Dimorphism in fungi is believed to constitute a mechanism of response to adverse conditions and represents an important attribute for the development of virulence by a number of pathogenic fungal species . We have isolated YlRAC1, a gene encoding a 192-amino-acid protein that is essential for hyphal growth in the dimorphic yeast Yarrowia lipolytica and which represents the first Rac homolog described for fungi . YlRAC1 is not an essential gene, and its deletion does not affect the ability to mate or impair actin polarization in Y . lipolytica . However, strains lacking functional YlRAC1 show alterations in cell morphology, suggesting that the function of YlRAC1 may be related to some aspect of the polarization of cell growth . Northern blot analysis showed that transcription of YlRAC1 increases steadily during the yeast-to-hypha transition, while Southern blot analysis of genomic DNA suggested the presence of several RAC family members in Y . lipolytica . Interestingly, strains lacking functional YlRAC1 are still able to grow as the pseudohyphal form and to invade agar, thus pointing to a function for YlRAC1 downstream of MHY1, a previously isolated gene encoding a C(2)H(2)-type zinc finger protein with the ability to bind putative stress response elements and whose activity is essential for both hyphal and pseudohyphal growth in Y . lipolytica. Cell, 2000 Mar 17, 100(6), 619 - 33 Establishing biorientation occurs with precocious separation of the sister kinetochores, but not the arms, in the early spindle of budding yeast; Goshima G et al.; Sister kinetochores are bioriented toward the spindle poles in higher eukaryotic prometaphase before chromosome segregation . We show that, in budding yeast, the sister kinetochores are separated in the very early spindle, while the sister arms remain associated . Biorientation of the separated kinetochores is achieved already after replication . Mtw1p, a homolog of fission yeast Mis12 required for biorientation, locates at the centromeres in an Ndc10p-dependent manner . Mtw1p and the sequences 1.8 and 3.8 kb from CEN3 and CEN15, respectively, behave like the precociously separated kinetochores, whereas the sequences 23 and 35 kb distant from CEN3 and CEN5 previously used as the centromere markers behave like a part of the arm . Mtw1p and Ndc10p are identically located except for additional spindle localization of Ndc10p . A model explaining small centromeres and early spindle formation in budding yeast is proposed. Nature, 2000 Mar 30, 404(6777), 515 - 8 Yeast Sm-like proteins function in mRNA decapping and decay; Tharun S et al.; One of the main mechanisms of messenger RNA degradation in eukaryotes occurs by deadenylation-dependent decapping which leads to 5'-to-3' decay . A family of Sm-like (Lsm) proteins has been identified, members of which contain the 'Sm' sequence motif, form a complex with U6 small nuclear RNA and are required for pre-mRNA splicing . Here we show that mutations in seven yeast Lsm proteins (Lsm1-Lsm7) also lead to inhibition of mRNA decapping . In addition, the Lsm1-Lsm7 proteins co-immunoprecipitate with the mRNA decapping enzyme (Dcp1), a decapping activator (Pat1/Mrt1) and with mRNA . This indicates that the Lsm proteins may promote decapping by interactions with the mRNA and the decapping machinery . In addition, the Lsm complex that functions in mRNA decay appears to be distinct from the U6-associated Lsm complex, indicating that Lsm proteins form specific complexes that affect different aspects of mRNA metabolism. Jpn J Cancer Res, 2000 Feb, 91(2), 181 - 9 p53 status in multiple human urothelial cancers: assessment for clonality by the yeast p53 functional assay in combination with p53 immunohistochemistry; Yamamoto S et al.; Multifocal synchronous or metachronous tumor development is a common observation in human urothelial cancer cases . However, the underlying mechanism has remained obscure . We have employed a new tool to investigate the p53 gene status, the yeast p53 functional assay, in combination with immunohistochemistry in a total of 50 tumor samples from 32 cases with urothelial cancers, including 8 with multiple synchronous tumor development and 2 demonstrating metachronous tumors . p53 mutations were found in 13 cases (9 with missense mutations, 3 with deletion, 1 with splicing mutation) by the yeast p53 functional assay . p53 protein overexpression was seen in all 9 cases with missense mutations, but in only one of the 4 cases with nonsense mutations . Two tumors without p53 mutation also showed positive p53 immunoreactivity . Overall, p53 abnormalities including mutations and/or protein overexpression were found in 15 (47%) cases . p53 abnormalities were significantly more frequent in non-papillary and in high grade tumors . Loss of the wild type allele in addition to a p53 mutation was suggested in 8 of the 15 (53%) cases . All 4 cases with mutations in multiple synchronous tumors had identical p53 mutations in the separate urothelial cancers, strongly suggestive of monoclonality . The one case with multiple metachronous tumors, in contrast, was characterized by variation in the p53 status, indicative of different clonal origins . In conclusion, combined assessment for p53 status as used here (yeast p53 functional assay plus immunohistochemistry) may provide insights into the molecular mechanisms of urothelial carcinogenesis. Biochim Biophys Acta, 2000 Apr 25, 1491(1-3), 161 - 76 Novel roles for elongin C in yeast; Jackson T et al.; Mammalian Elongin C is a 112-amino acid protein that binds to the von Hippel-Lindau (VHL) tumor suppressor and to Elongin A, the transcriptionally active subunit of the RNA polymerase II elongation factor, SIII . It is conserved in eukaryotic cells, as homologs have been identified in Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans . The mammalian protein is thought to function as part of a ubiquitin targeting E3 ligase, yet the function in yeast has not been determined . In this report we examine the role of Elongin C in yeast and establish that yeast Elongin C may function in a mode distinct from its role as an E3 ligase . The RNA is expressed ubiquitously, albeit at low levels . Two hybrid analyses demonstrate that Elongin C in yeast interacts with a specific set of proteins that are involved in the stress response . This suggests a novel role for Elongin C and provides insights into additional potential functions in mammalian cells.
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