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.<