Yeast, 2003 Apr 30, 20(6), 463 - 70 The Gts1 protein stabilizes the autonomous oscillator in yeast; Adams CA et al.; Continuous cultures of Saccharomyces cerevisiae show a robust autonomous temperature compensated oscillation in many metabolic functions . Respiratory activity, a convenient output to measure, oscillates with a period of 40 min . Deletion of GTS1, whose protein product has homology to the circadian per protein, has been implicated in temporal events within yeast, causes a reduction in periodicity to 18 min (wild-type period 40-60 min) . The dilution rate was steadily increased from 0.04/h to 0.085/h and the oscillation stabilized after four to six dilutions . However, Gts1p's involvement in the maintenance and generation of metabolic synchrony, and in the central oscillating loop, appear to be minimal, as the mutant oscillation was robust and autonomous . Deletion of GTS1 did cause decreased temperature compensation of the period of the oscillation from Q(10) = 1.07 for the wild-type to Q(10) = 1.6 for the mutant . Also the degree of nutrient compensation observed for the wild-type was not observed in the GTS1-null mutant strain . It is postulated that Gts1p is involved in the mechanism that communicates external conditions, such as temperature, to the central oscillating loop . J Cell Biol, 2003 Apr 28, 161(2), 359 - 69 Stu2p, the budding yeast member of the conserved Dis1/XMAP215 family of microtubule-associated proteins is a plus end-binding microtubule destabilizer; van Breugel M et al.; The Dis1/XMAP215 family of microtubule-associated proteins conserved from yeast to mammals is essential for cell division . XMAP215, the Xenopus member of this family, has been shown to stabilize microtubules in vitro, but other members of this family have not been biochemically characterized . Here we investigate the properties of the Saccharomyces cerevisiae homologue Stu2p in vitro . Surprisingly, Stu2p is a microtubule destabilizer that binds preferentially to microtubule plus ends . Quantitative analysis of microtubule dynamics suggests that Stu2p induces microtubule catastrophes by sterically interfering with tubulin addition to microtubule ends . These results reveal both a new biochemical activity for a Dis1/XMAP215 family member and a novel mechanism for microtubule destabilization. J Cell Biol, 2003 Apr 28, 161(2), 295 - 307 Sim4: a novel fission yeast kinetochore protein required for centromeric silencing and chromosome segregation; Pidoux AL et al.; Fission yeast centromeres are composed of two domains: the central core and the outer repeats . Although both regions are required for full centromere function, the central core has a distinct chromatin structure and is likely to underlie the kinetochore itself, as it is associated with centromere-specific proteins . Genes placed within either region are transcriptionally silenced, reflecting the formation of a functional kinetochore complex and flanking centromeric heterochromatin . Here, transcriptional silencing was exploited to identify components involved in central core silencing and kinetochore assembly or structure . The resulting sim (silencing in the middle of the centromere) mutants display severe chromosome segregation defects . sim2+ encodes a known kinetochore protein, the centromere-specific histone H3 variant Cnp1CENP-A . sim4+ encodes a novel essential coiled-coil protein, which is specifically associated with the central core region and is required for the unusual chromatin structure of this region . Sim4 coimmunoprecipitates with the central core component Mis6 and, like Mis6, affects Cnp1CENP-A association with the central domain . Functional Mis6 is required for Sim4 localization at the kinetochore . Our analyses illustrate the fundamental link between silencing, chromatin structure, and kinetochore function, and establish defective silencing as a powerful approach for identifying proteins required to build a functional kinetochore. J Biol Chem, 2003 Jul 11, 278(28), 26238 - 48 Epub 2003 Apr 28. Biochemical analysis of the yeast condensin Smc2/4 complex: an ATPase that promotes knotting of circular DNA; Stray JE et al.; To better understand the contributions that the structural maintenance of chromosome proteins (SMCs) make to condensin activity, we have tested a number of biochemical, biophysical, and DNA-associated attributes of the Smc2p-Smc4p pair from budding yeast . Smc2p and Smc4p form a stable heterodimer, the "Smc2/4 complex," which upon analysis by sedimentation equilibrium appears to reversibly self-associate to form heterotetramers . Individually, neither Smc2p nor Smc4p hydrolyzes ATP; however, ATPase activity is recovered by equal molar mixing of both purified proteins . Hydrolysis activity is unaffected by the presence of DNA . Smc2/4 binds both linearized and circular plasmids, and the binding appears to be independent of adenylate nucleotide . High mole ratios of Smc2/4 to plasmid promote a geometric change in circular DNA that can be trapped as knots by type II topoisomerases but not as supercoils by a type I topoisomerase . Binding titration analyses reveal that two Smc2/4-DNA-bound states exist, one disrupted by and one resistant to salt challenge . Competition-displacement experiments show that Smc2/4-DNA-bound species formed at even high protein to DNA mole ratios remain reversible . Surprisingly, only linear and supercoiled DNA, not nicked-circular DNA, can completely displace Smc2/4 prebound to a labeled, nicked-circular DNA . To explain this geometry-dependent competition, we present two models of DNA binding by SMCs in which two DNA duplexes are captured within the inter-coil space of an Smc2/4 heterodimer . Based on these models, we propose a DNA displacement mechanism to explain how differences in geometry could affect the competitive potential of DNA. Mol Cell, 2003 Apr, 11(4), 927 - 38 Fission yeast COP9/signalosome suppresses cullin activity through recruitment of the deubiquitylating enzyme Ubp12p; Zhou C et al.; The COP9/signalosome (CSN) is known to remove the stimulatory NEDD8 modification from cullins . The activity of the fission yeast cullins Pcu1p and Pcu3p is dramatically stimulated when retrieved from csn mutants but inhibited by purified CSN . This inhibition is independent of cullin deneddylation but mediated by the CSN-associated deubiquitylating enzyme Ubp12p, which forms a complex with Pcu3p in a CSN-dependent manner . In ubp12 mutants, as in csn mutants, Pcu3p activity is stimulated . CSN is required for efficient targeting of Ubp12p to the nucleus, where both cullins reside . Finally, the CSN/Ubp12p pathway maintains the stability of the Pcu1p-associated substrate-specific adaptor protein Pop1p . We propose that CSN/Ubp12p-mediated deubiquitylation creates an environment for the safe de novo assembly of cullin complexes by counteracting the autocatalytic destruction of adaptor proteins. World J Gastroenterol, 2003 May, 9(5), 1008 - 13 A small yeast RNA inhibits HCV IRES mediated translation and inhibits replication of poliovirus in vivo; Liang XS et al.; AIM: To investigate the anti-virus infection activity of internal ribosome entry site (IRES) specific inhibitor RNA (IRNA) . METHODS: IRNA eukaryotic vector pcRz-IRNA or mIRNA eukaryotic vector pcRz-mIRNA was transfected into human hepatocarcinoma cells (HHCC), then selected with neomycin G418 for 4 to 8 weeks, and then infected with polio virus vaccines line . The cytopethogenesis effect was investigated and the cell extract was collected . At last the polio virus titer of different cells was determined by plaque assay . RESULTS: Constructive expression of IRNA was not detrimental to cell growth . HCV IRES-mediated cap-independent translation was markedly inhibited in cells constructively expressing IRNA compared to control hepatoma cells . However, cap-dependent translation was not significantly affected in these cell line . Additionally, HHCC cells constitutively expressing IRNA became refractory to infection of polio virus . CONCLUSION: IRES specific IRNA can inhibit HCV IRES mediated translation and poliovirus replication. Oncogene, 2003 Apr 24, 22(16), 2452 - 6 Separation of long-range human TERT gene haplotypes by transformation-associated recombination cloning in yeast; Kim JH et al.; The hTERT gene encoding a catalytic subunit of human telomerase contains four blocks of variable number of tandem repeats (VNTRs)--two in intron 2 and two in intron 6 . The segregation of hTERT VNTRs was analysed in families, revealing that all of them were transmitted through meiosis following a Mendelian inheritance . The work reports a further characterization of the minisatellites in hTERT . We employed transformation-associated recombination (TAR) cloning to isolate parental hTERT alleles and determined the specific combination of minisatellites at each of the polymorphic sites . A long-range haplotyping of hTERT determined by TAR cloning was verified by classical Mendelian analysis . Since such a strategy can be applied for any chromosomal locus, we conclude that recombinational gene capture could greatly facilitate haplotypes analysis. Mol Genet Genomics, 2003 Apr, 269(1), 68 - 77 Epub 2003 Mar 04. Gal11 is a general activator of basal transcription, whose activity is regulated by the general repressor Sin4 in yeast; Mizuno T et al.; Mutations in SIN4, which encodes a global transcriptional regulator in Saccharomyces cerevisiae, have been suggested to lead to an increase in basal transcription of various genes by causing an alteration in chromatin structure . We reported previously that this activation of basal transcription occurs via a mechanism that differs from activator-mediated transcriptional enhancement . This finding prompted us to seek general activators of basal transcription by screening for extragenic suppressors of a sin4 mutation using PHO5, which is activated by the transcriptional activator Pho4, as a reporter gene . One of the mutations found, the semi-dominant ABE1-1, is described here . The ABE1-1 mutation reduced the enhanced basal transcription of PHO5 caused by the sin4 mutation, but did not impair Pho4-mediated activation of PHO5 . The ABE1-1 mutation also suppressed the aggregation phenotype and the rough colony morphology of the sin4 mutant cells, while it exacerbated temperature sensitive growth and telomere shortening, suggesting that Abe1p is involved in the basal transcription not only of PHO5 but also of other diversely regulated genes . SWI1, which encodes a component of the Swi-Snf complex that has chromatin remodeling activity, was identified as a gene-dosage suppressor of the ABE1-1 mutation . ABE1-1 was found to be allelic to GAL11 . These observations suggest that Gal11 acts as a general activator for the basal transcription of various genes, possibly by relieving torsional stress in chromatin, and that its function is repressed by the Sin4 protein. Plant J, 2003 May, 34(3), 257 - 67 The translation initiation factor eIF1A is an important determinant in the tolerance to NaCl stress in yeast and plants; Rausell A et al.; Protein synthesis is very sensitive to NaCl . However, the molecular targets responsible for this sensitivity have not been described . A cDNA library of the halotolerant plant sugar beet was functionally screened in a sodium-sensitive yeast strain . We obtained a cDNA clone (BveIF1A) encoding the eukaryotic translation initiation factor eIF1A . BveIF1A was able to partially complement the yeast eIF1A-deficient strain . Overexpression of the sugar beet eIF1A specifically increased the sodium and lithium salt tolerance of yeast . This phenotype was not accompanied by changes in sodium or potassium homeostasis . Under salt stress conditions, yeast cells expressing BveIF1A presented a higher rate of amino acid incorporation into proteins than control cells . In an in vitro protein synthesis system from wheat germ, the BveIF1A recombinant protein improved translation in the presence of NaCl . Finally, transgenic Arabidopsis plants expressing BveIF1A exhibited increased tolerance to NaCl . These results suggest that the translation initiation factor eIF1A is an important determinant of sodium tolerance in yeast and plants. Nucleic Acids Res, 2003 May 1, 31(9), 2443 - 50 Topological structure analysis of the protein-protein interaction network in budding yeast; Bu D et al.; Interaction detection methods have led to the discovery of thousands of interactions between proteins, and discerning relevance within large-scale data sets is important to present-day biology . Here, a spectral method derived from graph theory was introduced to uncover hidden topological structures (i.e . quasi-cliques and quasi-bipartites) of complicated protein-protein interaction networks . Our analyses suggest that these hidden topological structures consist of biologically relevant functional groups . This result motivates a new method to predict the function of uncharacterized proteins based on the classification of known proteins within topological structures . Using this spectral analysis method, 48 quasi-cliques and six quasi-bipartites were isolated from a network involving 11,855 interactions among 2617 proteins in budding yeast, and 76 uncharacterized proteins were assigned functions. J Biol Chem, 2003 Jul 4, 278(27), 24476 - 80 Epub 2003 Apr 23. Directed evolution of a yeast arsenate reductase into a protein-tyrosine phosphatase; Mukhopadhyay R et al.; Arsenic, which is ubiquitous in the environment and comes from both geochemical and anthropogenic sources, has become a worldwide public health problem . Every organism studied has intrinsic or acquired mechanisms for arsenic detoxification . In Saccharomyces cerevisiae arsenate is detoxified by Acr2p, an arsenate reductase . Acr2p is not a phosphatase but is a homologue of CDC25 phosphatases . It has the HCX5R phosphatase motif but not the glycine-rich phosphate binding motif (GXGXXG) that is found in protein-tyrosine phosphatases . Here we show that creation of a phosphate binding motif through the introduction of glycines at positions 79, 81, and 84 in Acr2p resulted in a gain of phosphotyrosine phosphatase activity and a loss of arsenate reductase activity . Arsenate likely achieved geochemical abundance only after the atmosphere became oxidizing, creating pressure for the evolution of an arsenate reductase from a protein-tyrosine phosphatase . The ease by which an arsenate reductase can be converted into a protein-tyrosine phosphatase supports this hypothesis. Analyst, 2003 Mar, 128(3), 220 - 4 Detection of selenocompounds in a tryptic digest of yeast selenoprotein by MALDI time-of-flight MS prior to their structural analysis by electrospray ionization triple quadrupole MS; Encinar JR et al.; MALDI-TOFMS was proposed as a key technique to a novel generic approach for the speciation analysis of selenium in yeast supplements . Owing to a lower detection limit and superior matrix tolerance to electrospray MS it allowed a successful detection of selenocompounds in samples for which electrospray MS had failed . The analytical approach developed was applied to the identification of a previously unreported selenopentapeptide (m/z 596) in the tryptic digest of a water-soluble selenoprotein fraction isolated by size-exclusion chromatography . The information on the mass of the protonated molecular ion obtained from MALDI allowed the optimization of the conditions for collision induced dissociation MS using a triple quadrupole spectrometer that enabled the determination of the amino acid sequence SeMet-Asn-Ala-Gly-Arg of the selenopeptide. Boll Chim Farm, 2003 Mar-Apr, 142(2), 66 - 8 The kinetic study of the selenium yeast stability; Szulc B et al.; The kinetics of degradation of selenium yeast was investigated at different temperatures . The effect of temperature has been determined and from this data, applying of Arrhenius-law, the stability of selenium yeast at 25 degrees C has been predicted and the t10 was determined . The changes in selenium content of the selenium yeast were determined by the fluorimetric method . It was proved, that the decomposition observed the first-order kinetic equations . The shelf life of selenium yeast predicted from the Arrhenius plot exceeded 1126 days . In the other hand the stability of selenium preparation in normal storage conditions (T = 25 degrees C) was determined to compare with predicted values . These results were found in agreement with experimental ones obtained at room temperature . No discernible physical changes of selenium preparations were observed after the storage in normal conditions (25 degrees C). Science, 2003 Apr 18, 300(5618), 492 - 5 Regulation of elongating RNA polymerase II by forkhead transcription factors in yeast; Morillon A et al.; The elongation phase of transcription by RNA polymerase II (RNAPII) is highly regulated and tightly linked to pre-messenger RNA (pre-mRNA) processing . Recent studies have implicated an early elongation checkpoint that facilitates the link to pre-mRNA processing . Here we show that the yeast forkhead transcription factors, Fkh1p and Fkh2p, associate with the coding regions of active genes and influence, in opposing ways, transcriptional elongation and termination . These events are coordinated with serine-5 and -2 phosphorylation of the heptad repeat of the carboxy-terminal domain (CTD) of RNAPII . Our results suggest that, in addition to their documented promoter function, Fkh1p and Fkh2p coordinate early transcription elongation and pre-mRNA processing . This may reflect a general feature of gene regulation in eukaryotes. FEMS Yeast Res, 2001 Apr, 1(1), 15 - 22 Trichosporon porosum comb . nov., an anamorphic basidiomycetous yeast inhabiting soil, related to the loubieri/laibachii group of species that assimilate hemicelluloses and phenolic compounds; Middelhoven WJ et al.; Several isolates representing the genus Trichosporon were collected over a 6-year period from soils in The Netherlands . Based on classical growth tests with carbon and nitrogen compounds these were identical . Three of these (CBS 8396, CBS 8397 and CBS 8522) were subjected to molecular analysis of the D1/D2 region of the large subunit of rDNA . This confirmed that the three strains were identical, yet distinct from other members of the genus . Conspecificity was demonstrated with the type strain (CBS 2040) of Apiotrichum porosum Stautz (1931), with the exception that A . porosum, which had been isolated from exudate of a yew tree, differed morphologically from the soil strains . Based on the identity of DNA base sequences, morphology was not considered to be an adequate parameter to separate otherwise identical strains into two genera . Therefore, the new combination Trichosporon porosum is presented . Based on molecular sequence analysis, T . porosum may be related to T . sporotrichoides, within a weakly related clade that includes species such as Trichosporon laibachii and Trichosporon loubieri . The strains of T . porosum degrade phenolic compounds and hemicelluloses, which are characteristics with potential ecological importance in soil habitats . Characters distinguishing the nine species of the laibachii/loubieri group of species were listed . These include traditionally used tests as well as assimilation patterns of some aliphatic and phenolic compounds . Based on these tests, species such as Trichosporon multisporum and T . laibachii could be separated. FEMS Yeast Res, 2001 Apr, 1(1), 9 - 13 Phosphoinositides in yeast: genetically tractable signalling; Wera S et al.; Research on signalling through phosphoinositides has made tremendous advances over the last few years . Studies with budding yeast (Saccharomyces cerevisiae) combine the advantage of a eukaryotic system with those of a rapidly growing, genetically modifiable and tractable organism of which the genome is fully sequenced . Hence, despite some differences in phosphoinositide signalling between mammals and yeast (e.g . the absence of PtdIns(3,4,5)P(3)), this model organism is at the forefront of phosphoinositide research . In this review we will focus on recent discoveries concerning the role of phosphoinositides in yeast. FEMS Yeast Res, 2002 Jan, 1(4), 265 - 70 Recognition of the basidiomycetous yeast Sporobolomyces ruberrimus sp . nov . as a distinct species based on molecular and morphological analyses; Fell JW et al.; Sporobolomyces ruberrimus Yamasaki and Fujii nom . inval . is established as a distinct species by ribosomal-DNA base composition in the D1, D2 and ITS regions and by morphology . A Latin description is given to validate the name with CBS 7500 as the type strain. FEMS Yeast Res, 2002 Mar, 2(1), 59 - 71 Regulation of the yeast glycine cleavage genes is responsive to the availability of multiple nutrients; Piper MD et al.; The mitochondrial glycine cleavage complex has an important role in metabolism since it provides the cell with the ability to use glycine as sole one-carbon and sole nitrogen source . In previous studies we have found that the genes encoding the unique components of this complex (GCV1, GCV2 and GCV3) are regulated by the one-carbon status of the cell . Here we have examined the transcriptional regulation of these genes with respect to nitrogen source . Two controls are required for the full range of GCV2 expression . One acts through a GATA sequence known to be involved in the control of transcription in response to the quality of the available nitrogen source and the other acts through an eight-base palindrome immediately 5' of the GATA element . This palindromic sequence previously had no known function . These data expand the repertoire of nitrogen catabolite repression to include the poor nitrogen source glycine and the genes encoding the enzyme complex responsible for its catabolism . Furthermore, deletion analyses and mutagenesis have shown that the palindromic sequence is important for full repression of GCV2 in complex glucose-containing medium and functions in a manner that is different from control mediated by the GATA element. FEMS Yeast Res, 2002 May, 2(2), 183 - 201 Glucose-sensing and -signalling mechanisms in yeast; Rolland F et al.; Glucose has dramatic effects on the regulation of carbon metabolism and on many other properties of yeast cells . Several sensing and signalling pathways are involved . For many years attention has focussed on the main glucose-repression pathway which is responsible for the downregulation of respiration, gluconeogenesis and the transport and catabolic capacity of alternative sugars during growth on glucose . The hexokinase 2- dependent glucose-sensing mechanism of this pathway is not well understood but the downstream part of the pathway has been elucidated in great detail . Two putative glucose sensors, the Snf3 and Rgt2 non-transporting glucose carrier homologs, control the expression of many functional glucose carriers . Recently, several new components of this glucose-induction pathway have been identified . The Ras-cAMP pathway controls a wide variety of cellular properties in correlation with cellular proliferation . Glucose is a potent activator of cAMP synthesis . In this case glucose sensing is carried out by two systems, a G-protein-coupled receptor system and a still elusive glucose-phosphorylation-dependent system . The understanding of glucose sensing and signalling in yeast has made dramatic advances in recent years and has become a strong paradigm for the elucidation of nutrient-sensing mechanisms in other eukaryotic organisms. FEMS Yeast Res, 2002 May, 2(2), 81 - 6 Metschnikowia arizonensis and Metschnikowia dekortorum, two new large-spored yeast species associated with floricolous beetles; Lachance MA et al.; Two new haplontic heterothallic species of Metschnikowia were discovered in flowers and associated beetles . Metschnikowia arizonensis was recovered from flowers of cholla cactus (Opuntia echinocarpa) and a specimen of Carpophilus sp . (Coleoptera: Nitidulidae) found in these flowers, in Arizona . Metschnikowia dekortorum was isolated in specimens of the nitidulid beetle Conotelus sp . captured in flowers of two species of Ipomoea in northwestern Guanacaste Province, Costa Rica . The sexual cycle of these yeasts is typical of the large-spored Metschnikowia species, but the asci and spores are intermediate in size between these and other members of the genus . The physiology is consistent with that of most Metschnikowia species except that both species fail to utilize lysine as sole nitrogen source . Also, M . arizonensis utilizes fewer carbon compounds than most species and exhibits considerable variability among strains at this level . Partial ribosomal DNA large-subunit (D1/D2) sequences suggest that M . arizonensis and M . dekortorum are moderately related sister species whose positions are intermediate between the large-spored species Metschnikowia and Metschnikowia hibisci . The type cultures are: M . arizonensis, strains UWO(PS)99-103.3.1=CBS 9064=NRRL Y-27427 (h(+), holotype) and UWO(PS)99-103.4=CBS 9065=NRRL Y-27428 (h(-), isotype); and M . dekortorum, strains UWO(PS)01-142b3=CBS 9063=NRRL Y-27429 (h(+), holotype) and UWO(PS)01-138a3=CBS 9062=NRRL Y-27430 (h(-), isotype). FEMS Yeast Res, 2002 Aug, 2(3), 371 - 9 Protein expression and secretion in the yeast Yarrowia lipolytica; Nicaud JM et al.; Strains and vectors for protein expression and secretion have been developed in the yeast Yarrowia lipolytica . Host strains were constructed with non-reverting auxotrophic markers, deletions of protease-encoding genes, and carrying a docking platform . To drive transcription, either the synthetic hp4d or the inducible POX2 promoter were used . Protein secretion is either directed by the targeting sequence of the alkaline extracellular protease or the extracellular lipase (LIP2p) signal sequence . We describe a set of vectors based on these promoters, targeting sequences and two URA3 alleles as selection markers . The wild-type URA3 allele, ura3d1, was used for single-copy integration and a mutant URA3 allele, ura3d4, was used to select for multi-copy integration into the genome . These vectors were used to express the Y . lipolytica extracellular lipase LIP2p and the Aspergillus oryzae leucine amino peptidase II . Lipase production under the control of the hp4d promoter by a strain containing a single copy reached 1000 U ml(-1) in shake flasks, while a strain containing multiple integrations reached 2000 U ml(-1) in shake flasks, 11500 U ml(-1) in batch and 90500 U ml(-1) in fed batch . Leucine amino peptidase production under the control of the hp4d promoter reached 320 mU ml(-1) in batch with a mono-copy lapA integrant and 28000 mU ml(-1) in fed batch with a multi-copy transformant. FEMS Yeast Res, 2002 Aug, 2(3), 307 - 14 Metabolically engineered methylotrophic yeast cells and enzymes as sensor biorecognition elements; Gonchar M et al.; An extended definition of the term metabolic engineering is given and its successful use in the construction of biorecognition elements of sensors is demonstrated . It is shown that genetic and chemical modifications of methylotrophic yeast cells provide directed changes in their physiological responses towards methanol, ethanol and formaldehyde resulting in enhanced selectivity and shorter time response of the corresponding potentiometric and amperometric biosensors. FEMS Yeast Res, 2003 Mar, 3(1), 97 - 103 Metschnikowia santaceciliae, Candida hawaiiana, and Candida kipukae, three new yeast species associated with insects of tropical morning glory; Lachance MA et al.; A new haplontic heterothallic species of Metschnikowia and two related asexual yeast species were discovered in morning glory flowers and associated insects . Metschnikowia santaceciliae came from Conotelus (Coleoptera: Nitidulidae) and other insect species associated with flowers of Ipomoea indica (purple morph) in Costa Rica . Candida hawaiiana and Candida kipukae were found in I . indica (syn . I . acuminata) and its insects in Hawai'i, and the former was also isolated in a specimen of Conotelus collected on Merremia tuberosa (Convolvulaceae) in Costa Rica . The three species have nearly identical physiological profiles, typical of the genus Metschnikowia . The sequences of the D1/D2 domains of their large subunit ribosomal DNA confirm that the species belong to the Metschnikowia clade, even though they share a very low degree of inter-relatedness . M . santaceciliae is a sister species to Metschnikowia continentalis . C . kipukae is a basal member of the large-spored Metschnikowia subclade, and C . hawaiiana has a weak affinity to Metschnikowia agaves . Two of the three species appear to be endemic . The type cultures are: Metschnikowia santaceciliae, strains UWO(PS)01-517a1=CBS 9148=NRRL Y-27475 (h(+, holotype) and UWO(PS)01-520a1=CBS 9149=NRRL Y-27476 (h-, isotype); Candida hawaiiana, strain UWO(PS)91-698.3=CBS 9146=NRRL Y-27473; Candida kipukae, strain UWO(PS)00-669.2=CBS 9147=NRRL Y-27474. FEMS Yeast Res, 2003 Mar, 3(1), 35 - 43 Functional analysis in yeast of the Brix protein superfamily involved in the biogenesis of ribosomes; Bogengruber E et al.; An extensive homology search based on the sequence of the yeast protein Brx1p (biogenesis of ribosomes in Xenopus, YOL077c) revealed that it is a member of a superfamily of proteins sharing remarkable sequence similarities . Previous work on Brx1p showed that this protein is involved in the process of ribosome biogenesis {Kaser et al., Biol . Chem . 382 (2001) 1637-1647} . Brx1p is the founding member of one of the five existing eukaryotic subfamilies which are all present in yeast . Four of them are represented by one essential gene each and one family is represented by two closely related genes which can functionally replace each other but are essential together for survival . We created conditional alleles of four of the five genes which allowed us to study the effect of depletion of the respective proteins on the ribosome profiles of the strains . In this study we show that not only Brx1p but also three additional superfamily members, namely YHR088w (Rpf1p), YKR081c (Rpf2p) and the homologous proteins Ssf1p (YHR066w)/Ssf2p (YDR312w) are all involved in the multistep process of the assembly of the large ribosomal subunit . This agrees well with the fact that these three proteins, like Brx1p, are located in the nucleolus . Moreover, all four proteins closely interact functionally, because all four mutants are suppressed by the same multicopy suppressor gene. EMBO J, 2003 Apr 15, 22(8), 1939 - 49 The yeast Sgs1 helicase is differentially required for genomic and ribosomal DNA replication; Versini G et al.; The members of the RecQ family of DNA helicases play conserved roles in the preservation of genome integrity . RecQ helicases are implicated in Bloom and Werner syndromes, which are associated with genomic instability and predisposition to cancers . The human BLM and WRN helicases are required for normal S phase progression . In contrast, Saccharomyces cerevisiae cells deleted for SGS1 grow with wild-type kinetics . To investigate the role of Sgs1p in DNA replication, we have monitored S phase progression in sgs1Delta cells . Unexpectedly, we find that these cells progress faster through S phase than their wild-type counterparts . Using bromodeoxyuridine incorporation and DNA combing, we show that replication forks are moving more rapidly in the absence of the Sgs1 helicase . However, completion of DNA replication is strongly retarded at the rDNA array of sgs1Delta cells, presumably because of their inability to prevent recombination at stalled forks, which are very abundant at this locus . These data suggest that Sgs1p is not required for processive DNA synthesis but prevents genomic instability by coordinating replication and recombination events during S phase. Proc Natl Acad Sci U S A, 2003 Apr 29, 100(9), 5154 - 9 Epub 2003 Apr 16. The role of side-chain interactions in the early steps of aggregation: Molecular dynamics simulations of an amyloid-forming peptide from the yeast prion Sup35; Gsponer J et al.; Understanding the early steps of aggregation at atomic detail might be crucial for the rational design of therapeutics preventing diseases associated with amyloid deposits . In this paper, aggregation of the heptapeptide GNNQQNY, from the N-terminal prion-determining domain of the yeast protein Sup35, was studied by 20 molecular dynamics runs for a total simulation time of 20 micros . The simulations generate in-register parallel packing of GNNQQNY beta-strands that is consistent with x-ray diffraction and Fourier transform infrared data . The statistically preferred aggregation pathway does not correspond to a purely downhill profile of the energy surface because of the presence of enthalpic barriers that originate from out-of-register interactions . The parallel beta-sheet arrangement is favored over the antiparallel because of side-chain contacts; in particular, stacking interactions of the tyrosine rings and hydrogen bonds between amide groups . No ordered aggregation was found in control simulations with the mutant sequence SQNGNQQRG in accord with experimental data and the strong sequence dependence of aggregation. Curr Biol, 2003 Apr 15, 13(8), 654 - 8 Septins have a dual role in controlling mitotic exit in budding yeast; Castillon GA et al.; In Saccharomyces cerevisiae, the spindle position checkpoint ensures that cells do not exit mitosis until the mitotic spindle moves into the mother/bud neck and thus guarantees that each cell receives one nucleus {1-6} . Mitotic exit is controlled by the small G protein Tem1p . Tem1p and its GTPase activating protein (GAP) Bub2p/Bfa1p are located on the daughter-bound spindle pole body . The GEF Lte1p is located in the bud . This segregation helps keep Tem1p in its inactive GDP state until the spindle enters the neck . However, the checkpoint functions without Lte1p and apparently senses cytoplasmic microtubules in the mother/bud neck {7-9} . To investigate this mechanism, we examined mutants defective for septins, which compose a ring at the neck {10} . We found that the septin mutants sep7Delta and cdc10Delta are defective in the checkpoint . When movement of the spindle into the neck was delayed, mitotic exit occurred, inappropriately leaving both nuclei in the mother . In sep7Delta and cdc10Delta mutants, Lte1p is mislocalized to the mother . In sep7Delta, but not cdc10Delta, mutants, inappropriate mitotic exit depends on Lte1p . These results suggest that septins serve as a diffusion barrier for Lte1p, and that Cdc10p is needed for the septin ring to serve as a scaffold for a putative microtubule sensor. Mol Cell Biol, 2003 May, 23(9), 3202 - 15 The yeast RSC chromatin-remodeling complex is required for kinetochore function in chromosome segregation; Hsu JM et al.; The accurate segregation of chromosomes requires the kinetochore, a complex protein machine that assembles onto centromeric DNA to mediate attachment of replicated sister chromatids to the mitotic spindle apparatus . This study reveals an important role for the yeast RSC ATP-dependent chromatin-remodeling complex at the kinetochore in chromosome transmission . Mutations in genes encoding two core subunits of RSC, the ATPase Sth1p and the Snf5p homolog Sfh1p, interact genetically with mutations in genes encoding kinetochore proteins and with a mutation in centromeric DNA . RSC also interacts genetically and physically with the histone and histone variant components of centromeric chromatin . Importantly, RSC is localized to centromeric and centromere-proximal chromosomal regions, and its association with these loci is dependent on Sth1p . Both sth1 and sfh1 mutants exhibit altered centromeric and centromere-proximal chromatin structure and increased missegregation of authentic chromosomes . Finally, RSC is not required for centromeric deposition of the histone H3 variant Cse4p, suggesting that RSC plays a role in reconfiguring centromeric and flanking nucleosomes following Cse4p recruitment for proper chromosome transmission. Mol Cell Biol, 2003 May, 23(9), 3186 - 201 Structural and functional analysis of mutations along the crystallographic dimer interface of the yeast TATA binding protein; Kou H et al.; The TATA binding protein (TBP) is a central component of the eukaryotic transcription machinery and is subjected to both positive and negative regulation . As is evident from structural and functional studies, TBP's concave DNA binding surface is inhibited by a number of potential mechanisms, including homodimerization and binding to the TAND domain of the TFIID subunit TAF1 (yTAF(II)145/130) . Here we further characterized these interactions by creating mutations at 24 amino acids within the Saccharomyces cerevisiae TBP crystallographic dimer interface . These mutants are impaired for dimerization, TAF1 TAND binding, and TATA binding to an extent that is consistent with the crystal or nuclear magnetic resonance structure of these or related interactions . In vivo, these mutants displayed a variety of phenotypes, the severity of which correlated with relative dimer instability in vitro . The phenotypes included a low steady-state level of the mutant TBP, transcriptional derepression, dominant slow growth (partial toxicity), and synthetic toxicity in combination with a deletion of the TAF1 TAND domain . These phenotypes cannot be accounted for by defective interactions with other known TBP inhibitors and likely reflect defects in TBP dimerization. Mol Cell Biol, 2003 May, 23(9), 3116 - 25 The Tap42-protein phosphatase type 2A catalytic subunit complex is required for cell cycle-dependent distribution of actin in yeast; Wang H et al.; In Saccharomyces cerevisiae, the Tor proteins mediate a wide spectrum of growth-related cellular processes in response to nutrients . The pleiotropic role of the Tor proteins is mediated, at least in part, by type 2A protein phosphatases (PP2A) and 2A-like protein phosphatases . Tor-mediated signaling activity promotes the interaction of phosphatase-interacting protein Tap42 with PP2A and 2A-like protein phosphatases . The distinct complexes formed between Tap42 and different phosphatases mediate various cellular events and modulate phosphorylation levels of many downstream factors in the Tor pathway in a Tor-dependent and rapamycin-sensitive manner . In this study, we demonstrate that the interaction between Tap42 and the catalytic subunits of PP2A (PP2Ac) is required for cell cycle-dependent distribution of actin . We show that mutations in PP2Ac and Tap42 that perturb the interaction cause random distribution of actin during the cell cycle and that overexpression of the Rho2 GTPase suppresses the actin defects associated with the mutants . Our findings suggest that the Tap42-PP2Ac complex regulates the actin cytoskeleton via a Rho GTPase-dependent mechanism . In addition, we provide evidence that PP2A activity plays a negative role in controlling the actin cytoskeleton and, possibly, in regulation of the G(2)/M transition of the cell cycle. BMC Genet . 2003 Apr 03;4(1):6. Fission yeast Rad26 responds to DNA damage independently of Rad3; Wolkow TD et al.; BACKGROUND: The Rad26/Rad3 complex in fission yeast detects genotoxic insults and initiates the cell cycle arrest and recovery activities of the DNA damage checkpoint . To investigate how the Rad26/Rad3 complex performs these functions, we constructed and characterized Rad26-GFP . RESULTS: Rad26-GFP localized to approximately six nuclear dots in cycling cells . Following treatment with a DNA damaging agent, Rad26-GFP localization changed . Damaged cells contained one or two bright Rad26-GFP spots, in addition to smaller, more numerous Rad26-GFP speckles . Genetic analyses demonstrated that these Rad26-GFP patterns (dots, spots and speckles) were unaffected by null mutations in other DNA damage checkpoint genes, including rad3+ . Data obtained with our Rad26.T12-GFP fusion protein correlate spots with cell cycle arrest activities and speckles with DNA repair activities . In addition, physiological experiments demonstrated that rad26Delta and rad3Delta alleles confer sensitivity to a microtubule-depolymerizing drug . CONCLUSION: We have discovered three distinct Rad26-GFP cellular structures . Formation of these structures did not require other checkpoint proteins . These data demonstrate that Rad26 can respond to genotoxic insult in the absence of Rad3 and the other checkpoint Rad proteins. J Agric Food Chem, 2003 Apr 23, 51(9), 2584 - 90 Impact of oxygen consumption by yeast lees on the autolysis phenomenon during simulation of wine aging on lees; Fornairon-Bonnefond C et al.; Potential oxygen consumption by lees, more precisely by nonviable yeasts, during wine aging was recently described . Additionally, yeast autolysis is described as the main mechanism of degradation of lees during wine aging . Thus, to understand the effect of oxygen consumption by yeast lees during wine aging, an accelerated wine aging methodology was tested . Wine aging in the presence of yeast lees was studied both in the presence and in the absence of oxygen . Different markers of yeast autolysis were followed to find a relationship between oxygen consumption by yeast lees and changes in the final wine composition after aging . No differences for compounds tested were found in the wine and in the lees except among sterol compounds in lees: in the presence of oxygen, the concentration of ergosterol in lees was significantly lower than that in the absence of oxygen . It was hypothesized that ergosterol could be oxidized under the influence of oxygen, but none of the known products of ergosterol oxidation were recovered in the corresponding yeast lees . In addition, the decrease of ergosterol content in yeast lees cannot account for the total amount of oxygen consumed by yeast lees during such wine aging. Curr Genet, 2003 May, 43(2), 71 - 8 Epub 2003 Mar 07. Role for lipid signaling and the cell integrity MAP kinase cascade in yeast septum biogenesis; Tahirovic S et al.; Polarized deposition of chitin at the bud neck is essential for cell separation in yeast . Chitin septum biogenesis is catalyzed by two distinct chitin synthase activities encoded by the CHS2 and CHS3 genes . The phosphoinositide phosphatase Sac1p is required for proper trafficking of the Chs3p chitin synthase . sac1 mutants also display a severe synthetic growth defect, with mutations in the SLT2 gene which encodes a MAP kinase involved in cell integrity . We characterized the defect that underlies this genetic interaction and found that sac1 Delta slt2 Delta cells arrest as large-budded cells because they fail to separate at the end of mitosis . This inability to complete cell division appears to be caused by an increased deposition of chitin at the septum area and correlates with a mislocalized accumulation of the Chs2p chitin synthase at the cell periphery . Our data therefore indicate that Sac1p and Slt2p have synergistic roles in regulating chitin septum biogenesis. Curr Genet, 2003 Jun, 43(3), 171 - 7 Epub 2003 Apr 15. Genetic background affects relative nonsense mRNA accumulation in wild-type and upf mutant yeast strains; Kebaara B et al.; The Saccharomyces cerevisiae nonsense-mediated mRNA decay (NMD) pathway targets mRNAs with premature stop codons and some wild-type mRNAs for accelerated decay . Upf1p, Upf2p and Upf3p are required for NMD . NMD-targeted mRNAs are degraded rapidly in wild-type cells and stabilized in upf1, upf2 or upf3 mutants . We report here that the relative CYH2 pre-mRNA/mRNA accumulation is enhanced in cells derived from a W303 background, compared with a variety of commonly used strains . The enhanced CYH2 pre-mRNA accumulation phenotype results from a larger difference in mRNA half-lives in the W303 strains than two previously used strains . This phenotype can be selected in crosses and is also seen in upf2 and upf3 mutants . These results suggest there are genes that influence the efficiency of NMD and that yeast strains derived from the W303 background may be useful for measurement of abundance and half-lives of low abundance, short-lived NMD substrates. Cell Cycle, 2003 Mar-Apr, 2(2), 120 - 2 Yeast POL5 is an evolutionarily conserved regulator of rDNA transcription unrelated to any known DNA polymerases; Yang W et al.; We show that yeast protein Yel055cp, which has been identified as the fifth essential DNA polymerase in Saccharomyces cerevisiae (POL5), is a member of a family of predicted rDNA transcription regulators (typified by human MYB-binding protein MYBBP1 A), which are represented by a single ortholog in all animals, fungi and plants with sequenced genomes . These proteins are confidently predicted to have an entirely a-helical structure and are unrelated to the B class DNA polymerases, as claimed for yeast POL5, or any other known polymerases. Mol Microbiol, 2003 May, 48(3), 713 - 24 Glucose triggers different global responses in yeast, depending on the strength of the signal, and transiently stabilizes ribosomal protein mRNAs; Yin Z et al.; Glucose exerts profound effects upon yeast physiology . In general, the effects of high glucose concentrations (>1%) upon Saccharomyces cerevisiae have been studied . In this paper, we have characterized the global responses of yeast cells to very low (0.01%), low (0.1%) and high glucose signals (1.0%) by transcript profiling . We show that yeast is more sensitive to very low glucose signals than was previously thought, and that yeast displays different responses to these different glucose signals . Genes involved in central metabolic pathways respond rapidly to very low glucose signals, whereas genes involved in the biogenesis of cytoplasmic ribosomes generally respond only to glucose concentrations of> 0.1% . We also show that cytoplasmic ribosomal protein mRNAs are transiently stabilized by glucose, indicating that both transcriptional and post-transcriptional mechanisms combine to accelerate the accumulation of ribosomal protein mRNAs . Presumably, this facilitates rapid ribosome biogenesis after exposure to glucose . However, our data indicate that yeast activates ribosome biogenesis only when sufficient glucose is available to make this metabolic investment worthwhile . In contrast, the regulation of metabolic functions in response to very low glucose signals presumably ensures that yeast can exploit even minute amounts of this preferred nutrient. Genes Cells, 2003 May, 8(5), 465 - 80 Budding yeast mcm10/dna43 mutant requires a novel repair pathway for viability; Araki Y et al.; BACKGROUND: MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae . Mcm10p functionally interacts with components of the pre-replicative complex (Mcm2-Mcm7 complex and origin recognition complex) as well as the pre-initiation complex component (Cdc45p) suggesting that it may be a component of the pre-RC as well as the pre-IC . Two-dimensional gel electrophoresis analysis showed that Mcm10p is required not only for the initiation of DNA synthesis at replication origins but also for the smooth passage of replication forks at origins . Genetic analysis showed that MCM10 interacts with components of the elongation machinery such as Pol delta and Pol epsilon, suggesting that it may play a role in elongation replication . RESULTS: We show that the mcm10 mutation causes replication fork pausing not only at potentially active origins but also at silent origins . We screened for mutations that are lethal in combination with mcm10-1 and obtained seven mutants named slm1-slm6 for synthetically lethal with mcm10 . These mutants comprised six complementation groups that can be divided into three classes . Class 1 includes genes that encode components of the pre-RC and pre-IC and are represented by SLM3, 4 and 5 which are allelic to MCM7, MCM2 and CDC45, respectively . Class 2 includes genes involved in the processing of Okazaki fragments in lagging strand synthesis and is represented by SLM1, which is allelic to DNA2 . Class 3 includes novel DNA repair genes represented by SLM2 and SLM6 . CONCLUSIONS: The viability of the mcm10-1 mutant is dependent on a novel repair pathway that may participate either in resolving accumulated replication intermediates or the damage caused by blocked replication forks . These results are consistent with the hypothesis that Mcm10p is required for the passage of replication forks through obstacles such as those created by pre-RCs assembled at active or inactive replication origins. Eur J Biochem, 2003 Apr, 270(8), 1875 - 84 The GxxxG motif of the transmembrane domain of subunit e is involved in the dimerization/oligomerization of the yeast ATP synthase complex in the mitochondrial membrane; Arselin G et al.; A conserved putative dimerization GxxxG motif located in the unique membrane-spanning segment of the ATP synthase subunit e was altered in yeast both by insertion of an alanine residue and by replacement of glycine by leucine residues . These alterations led to the loss of subunit g and the loss of dimeric and oligomeric forms of the yeast ATP synthase . Furthermore, as in null mutants devoid of either subunit e or subunit g, mitochondria displayed anomalous morphologies with onion-like structures . By taking advantage of the presence of the endogenous cysteine 28 residue in the wild-type subunit e, disulfide bond formation between subunits e in intact mitochondria was found to increase the stability of an oligomeric structure of the ATP synthase in digitonin extracts . The data show the involvement of the dimerization motif of subunit e in the formation of supramolecular structures of mitochondrial ATP synthases and are in favour of the existence in the inner mitochondrial membrane of associations of ATP synthases whose masses are higher than those of ATP synthase dimers. Proc Natl Acad Sci U S A, 2003 Apr 29, 100(9), 5113 - 8 Epub 2003 Apr 11. Yeast DNA polymerase eta makes functional contacts with the DNA minor groove only at the incoming nucleoside triphosphate; Washington MT et al.; DNA polymerase eta (Pol eta) functions in the proficient bypass of a variety of DNA lesions . Relative to the replicative polymerases, Pol eta has a greater tolerance for distorted DNA geometries and possesses a low fidelity . X-ray crystal structures and studies with nucleotide analogs have implicated interactions with the DNA minor groove as being crucial for the high fidelity of replicative DNA polymerases . To determine whether Pol eta also makes such functionally important contacts with the DNA minor groove, here we examine the effects on Pol eta-catalyzed nucleotide incorporation when 3-deazaguanine, a base analog that lacks the ability to form minor-groove hydrogen bonds with the protein, is substituted for guanine at various positions in the DNA . From these studies, we conclude that Pol eta makes only a single functional contact with the DNA minor groove at the position of the incoming nucleotide; in this regard, Pol eta differs from high-fidelity DNA polymerases that are unable to replicate through DNA lesions . These results help explain the proficient ability of Pol eta for bypassing distorting DNA lesions. J Cell Sci, 2003 Jun 1, 116(Pt 11), 2169 - 78 Epub 2003 Apr 08. New N-RAP-binding partners alpha-actinin, filamin and Krp1 detected by yeast two-hybrid screening: implications for myofibril assembly; Lu S et al.; N-RAP, a muscle-specific protein concentrated at myotendinous junctions in skeletal muscle and intercalated disks in cardiac muscle, has been implicated in myofibril assembly . To discover more about the role of N-RAP in myofibril assembly, we used the yeast two-hybrid system to screen a mouse skeletal muscle cDNA library for proteins capable of binding N-RAP in a eukaryotic cell . From yeast two-hybrid experiments we were able to identify three new N-RAP binding partners: alpha-actinin, filamin-2, and Krp1 (also called sarcosin) . In vitro binding assays were used to verify these interactions and to identify the N-RAP domains involved . Three regions of N-RAP were expressed as His-tagged recombinant proteins, including the nebulin-like super repeat region (N-RAP-SR), the N-terminal LIM domain (N-RAP-LIM), and the region of N-RAP in between the super repeat region and the LIM domain (N-RAP-IB) . We detected significant alpha-actinin binding to N-RAP-IB and N-RAP-LIM, filamin binding to N-RAP-SR, and Krp1 binding to N-RAP-SR and N-RAP-IB . During myofibril assembly in cultured chick cardiomyocytes, N-RAP and filamin appear to co-localize with alpha-actinin in the earliest myofibril precursors found near the cell periphery, as well as in the nascent myofibrils that form as these structures fuse laterally . In contrast, Krp1 is not localized until late in the assembly process, when it appears at the periphery of myofibrils that appear to be fusing laterally . The results suggest that sequential recruitment of N-RAP binding partners may serve an important role during myofibril assembly. J Biol Chem, 2003 Jun 27, 278(26), 24189 - 99 Epub 2003 Apr 11. Intrinsic transcript cleavage in yeast RNA polymerase II elongation complexes; Weilbaecher RG et al.; Transcript elongation can be interrupted by a variety of obstacles, including certain DNA sequences, DNA-binding proteins, chromatin, and DNA lesions . Bypass of many of these impediments is facilitated by elongation factor TFIIS through a mechanism that involves cleavage of the nascent transcript by the RNA polymerase II/TFIIS elongation complex . Highly purified yeast RNA polymerase II is able to perform transcript hydrolysis in the absence of TFIIS . The "intrinsic" cleavage activity is greatly stimulated at mildly basic pH and requires divalent cations . Both arrested and stalled complexes can carry out the intrinsic cleavage reaction, although not all stalled complexes are equally efficient at this reaction . Arrested complexes in which the nascent transcript was cleaved in the absence of TFIIS were reactivated to readthrough blocks to elongation . Thus, cleavage of the nascent transcript is sufficient for reactivating some arrested complexes . Small RNA products released following transcript cleavage in stalled ternary complexes differ depending upon whether the cleavage has been induced by TFIIS or has occurred in mildly alkaline conditions . In contrast, both intrinsic and TFIIS-induced small RNA cleavage products are very similar when produced from an arrested ternary complex . Although alpha-amanitin interferes with the transcript cleavage stimulated by TFIIS, it has little effect on the intrinsic cleavage reaction . A mutant RNA polymerase previously shown to be refractory to TFIIS-induced transcript cleavage is essentially identical to the wild type polymerase in all tested aspects of intrinsic cleavage. Receptors Channels, 2002, 8(5-6), 343 - 52 Yeast assays for G-protein-coupled receptors; Dowell SJ et al.; Yeast assays for G-protein-coupled receptors have many attractions due to their simplicity, low cost, and lack of endogenous receptors . Since the first report of functional coupling of the human beta 2 adrenergic receptor to the yeast pheromone-response pathway in 1990, the technology has developed to a point at which more than 30 heterologous GPCRs are now published to couple . Major breakthroughs have come from an understanding of receptor-G protein interactions, alongside advances in knowledge of the structure of heterotrimeric G proteins . Yeast screens have been used to identify ligands both from compound collections and through the autocrine expression of peptide libraries . Yeast genetics has also been applied to a functional analysis of GPCRs and peptide ligands . In this review we describe the historical development of yeast GPCR assay systems and their current applications. Int J Vitam Nutr Res, 2003 Feb, 73(1), 19 - 23 Ergosterol (major sterol of baker's and brewer's yeast extracts) inhibits the growth of human breast cancer cells in vitro and the potential role of its oxidation products; Subbiah MT et al.; The derivation of chemopreventive agents from dietary sources has been the subject of considerable attention in recent years . Yeast extracts have been used as nutritional supplements for a number of years . In this communication we show that ergosterol (a 28-carbon sterol found in baker's and brewer's yeast) can prevent growth of breast cancer cells in vitro in the presence of estradiol-17 beta . Estrogen receptor (+) MCF-7 cells appear to be more sensitive to ergosterol than estrogen receptor (-) MDA-231 cells . However, MDA-231 cells were more sensitive to ergosterol in terms of apoptotic effects than MCF-7 cells, indicating that other mechanisms (antiestrogenic activity) may also be operative in estrogen receptor (+) cells . Compared to freshly prepared ergosterol, stored preparations were more potent in inhibiting growth of cancer cells, indicating that oxidation product(s) of ergosterol may be responsible for the noted effects . Further studies on in vivo effects of ergosterol and lipid extracts of yeast in animal models are warranted to determine their potential for use as supplements in humans. Nucleic Acids Res, 2003 Apr 15, 31(8), 2025 - 34 DNA binding by yeast Mlh1 and Pms1: implications for DNA mismatch repair; Hall MC et al.; The yeast Mlh1-Pms1 heterodimer required for mismatch repair (MMR) binds to DNA . Here we map DNA binding to N-terminal fragments of Mlh1 and Pms1 . We demonstrate that Mlh1 and Pms1 N-terminal domains (NTDs) independently bind to double-stranded and single-stranded DNA, in the absence of dimerization and with different affinities . Full-length Mlh1p alone, which can homodimerize, also binds to DNA . Substituting conserved positively charged amino acids in Mlh1 produces mutator phenotypes in a haploid yeast strain characteristic of reduced MMR . These substitutions strongly reduce DNA binding by the Mlh1 NTD and, to a lesser extent, they also reduce DNA binding by full-length Mlh1 and the Mlh1-Pms1 heterodimer . Replacement of a homologous Pms1 residue has a much smaller effect on mutation rate and does not reduce DNA binding . The results demonstrate that NTDs of yeast Mlh1 and Pms1 contain independent DNA binding sites and they suggest that the C-terminal region of Mlh1p may also contribute to DNA binding . The differential mutator effects and binding properties observed here further suggest that Mlh1 and Pms1 differ in their interactions with DNA . Finally, the results are consistent with the hypothesis that DNA binding by Mlh1 is important for MMR. Biometals, 2003 Sep, 16(3), 379 - 82 Does 13C-or 15N-labeling affect Cu(I)-thiolate cluster arrangement in yeast copper-metallothionein? Hartmann HJ, Kaup Y, Weser U. It was attempted to examine whether or not isotope labeling may possibly affect an oligonuclear metal-thiolate cluster . Cu-metallothioneins are known to contain strongly distorted Cu-thiolate clusters and seemed appropriate for this study . Thus, yeast 13C-and 15N-Cu-metallothioneins were isolated from Saccharomyces cerevisiae cells grown in a minimal synthetic medium and some physicochemical parameters were compared with those of the unlabeled Cu-thionein . Surprisingly, the 13C- and 15N- labeled Cu7-thioneins are distinctly different in their characteristic spectroscopic properties . The electronic absorption was blue-shifted while both luminescence emission and chiroptic features display a distinct red shift with markedly diminished intensities, respectively . Contrary to common knowledge that isotope labeling does not affect the molecular architecture of a protein the present results support such a phenomenon . Attributable to the fortunate happenstance that there is a strongly distorted structural situation in the oligonuclear Cu-thiolate cluster this isotope effect came to light. Microbiology, 2003 Apr, 149(Pt 4), 865 - 76 Analysis of microtubules and F-actin structures in hyphae and conidia development of the opportunistic human pathogenic black yeast Aureobasidium pullulans; Kopecka M et al.; Organization of the cytoskeleton was studied in the ascomycetous black yeast Aureobasidium pullulans, an opportunistic human pathogen, in an effort to present it as a potential target of antifungal therapy . Long cytoplasmic microtubules, extending along the hyphae from the base to the growing apex, were the dominant structures in multinucleate interphase cells . Before mitosis these microtubules disappeared and were replaced by intranuclear spindles . This reorganization of microtubules occurred along the whole length of hypha before synchronous division of the nuclei . Actin cytokinetic rings were rarely seen . Cortical actin in the form of patches accumulated in areas of cell wall growth, i.e . in the hyphal apex and near the occasionally formed septum . Actin cables were not seen . During synchronous conidiogenesis, the cytoplasmic microtubules extended along developing conidia, and actin patches lined their subcortical areas . Actin rings were formed regularly at the base of uninuclear conidia . Microtubule inhibitor methyl benzimidazol-2-ylcarbamate disintegrated the microtubules, and inhibited nuclear division, development of hyphae and conidiogenesis . Actin inhibitor Cytochalasin D induced swelling of hyphal apexes and developing conidia . This inhibitory activity ceased after 5 to 12 h when the occasional septa appeared and conidiogenesis was completed . The lack of unicellular organization in multinucleate hyphae of A . pullulans seems be related to a rarity of F-actin structures: i.e . absence of actin cables, the lack of actin cytokinetic rings in particular, resulting in the uncoupling of the nuclear division from cytokinesis; the association of both processes is, however, retained during conidiogenesis. Mol Biol Cell, 2003 Apr, 14(4), 1664 - 76 An early function during transcription for the yeast mRNA export factor Dbp5p/Rat8p suggested by its genetic and physical interactions with transcription factor IIH components; Estruch F et al.; The yeast DEAD-box protein Dbp5p/Rat8p is an essential factor for mRNA export and shuttles between the nucleus and the cytoplasm . It is concentrated at the cytoplasmic fibrils of the nuclear pore complex where it interacts with several nucleoporins . On the basis of this localization, it has been suggested that it might participate in a terminal step of RNA export, the release from the mRNA of proteins that accompany the mRNA during translocation through nuclear pores . In this report, we present evidence linking Dbp5p to transcription . Two different screens identified genetic interactions between DBP5 and genes involved in early transcription events, initiation and promoter clearance . Mutations of transcription proteins expected to impair transcription act as suppressors of dbp5 mutants, whereas those that may act to increase transcription are synthetically lethal with dbp5 mutations . We also show that growth and mRNA export in dbp5 mutant strains are dependent on the carboxy-terminal domain of the RNA pol II largest subunit . Finally, we show that Dbp5p associates physically with components of transcription factor IIH . Because these interactions affect not only growth but also mRNA export, they are likely to reflect a functional relationship between Dbp5p and the transcription machinery . Together, our results suggest a nuclear role for Dbp5 during the early steps of transcription. Mol Biol Cell, 2003 Apr, 14(4), 1319 - 33 The synaptojanin-like protein Inp53/Sjl3 functions with clathrin in a yeast TGN-to-endosome pathway distinct from the GGA protein-dependent pathway; Ha SA et al.; Yeast TGN resident proteins that frequently cycle between the TGN and endosomes are much more slowly transported to the prevacuolar/late endosomal compartment (PVC) than other proteins . However, TGN protein transport to the PVC is accelerated in mutants lacking function of Inp53p . Inp53p contains a SacI polyphosphoinositide phosphatase domain, a 5-phosphatase domain, and a proline-rich domain . Here we show that all three domains are required to mediate "slow delivery" of TGN proteins into the PVC . Although deletion of the proline-rich domain did not affect general membrane association, it caused localization to become less specific . The proline-rich domain was shown to bind to two proteins, including clathrin heavy chain, Chc1p . Unlike chc1 mutants, inp53 mutants do not mislocalize TGN proteins to the cell surface, consistent with the idea that Chc1p and Inp53p act at a common vesicular trafficking step but that Chc1p is used at other steps also . Like mutations in the AP-1 adaptor complex, mutations in INP53 exhibit synthetic growth and transport defects when combined with mutations in the GGA proteins . Taken together with other recent studies, our results suggest that Inp53p and AP-1/clathrin act together in a TGN-to-early endosome pathway distinct from the direct TGN-to-PVC pathway mediated by GGA/clathrin. Curr Genet, 2003 Apr, 43(1), 1 - 10 Epub 2003 Feb 08. Functional analysis of the human orthologue of the RSP5-encoded ubiquitin protein ligase, hNedd4, in yeast; Gajewska B et al.; hNedd4 and Rsp5p are orthologous ubiquitin ligases that contain a catalytic Hect domain, a C2 domain and multiple WW domains that mediate interactions with proteins . hNedd4 associates with the epithelial sodium channel and mutations disrupting this interaction lead to Liddle's syndrome, a heritable hypertension . Yeast Rsp5p ubiquitinates plasma membrane receptors and transporters and regulates their endocytosis . To determine whether the human enzyme has activity in yeast, hNEDD4 was expressed in yeast from the RSP5 or GAL1/10 promoters . Ectopic hNedd4 improved the growth and partially suppressed the endocytosis defect of rsp5 mutant cells, although it did not restore the viability of the rsp5-delta strain . Wild-type cells harboring hNedd4 grew better at elevated temperature and on media containing cycloheximide . In contrast, hNedd4 WW domain mutants inhibited the growth of yeast when expressed at high levels . Our results show that hNedd4 affects cell growth, endocytosis and cycloheximide tolerance of yeast cells. J Biol Chem, 2003 Jun 20, 278(25), 22492 - 7 Epub 2003 Apr 08. Role of yeast glutaredoxins as glutathione S-transferases; Collinson EJ et al.; The yeast Saccharomyces cerevisiae contains two glutaredoxins, encoded by GRX1 and GRX2, that are required for resistance to reactive oxygen species . We recently reported that Grx1 is active as a glutathione peroxidase and can directly reduce hydroperoxides (Collinson, E . J., Wheeler, G . L., Garrido, E . O., Avery, A . M., Avery, S . V., and Grant, C . M . (2002) J . Biol . Chem . 277, 16712-16717) . We now show that Grx2 is also a general hydroperoxidase, and kinetic data indicate that both enzymes have a similar pattern of activity, which is highest with hydrogen peroxide, followed by cumene hydroperoxide and tert-butyl hydroperoxide . Furthermore, both Grx1 and Grx2 are shown be active as glutathione S-transferases (GSTs), and their activity with model substrates such as 1-chloro-2,4-dinitrobenzene is similar to their activity with hydroperoxides . Analysis of the Grx1 active site residues shows that Cys-27, but not Cys-30, is required for both the peroxidase and transferase activities, indicating that these reactions proceed via a monothiol mechanism . Deletion analysis shows that Grx1 and Grx2 have an overlapping function with yeast GSTs, encoded by GTT1 and GTT2, and are responsible for the majority of cellular GST activity . In addition, multiple mutants lacking GRX1, GRX2, GTT1, and GTT2 show increased sensitivity to stress conditions, including exposure to xenobiotics, heat, and oxidants . In summary, glutaredoxins are multifunctional enzymes with oxidoreductase, peroxidase, and GST activity, and are therefore ideally suited to detoxify the wide range of xenobiotics and oxidants that can be generated during diverse stress conditions. Eukaryot Cell, 2003 Apr, 2(2), 274 - 83 Budding yeast CTDK-I is required for DNA damage-induced transcription; Ostapenko D et al.; CTDK-I phosphorylates the C-terminal domain (CTD) of the large subunit of yeast RNA polymerase II in a reaction that stimulates transcription elongation . Mutations in CTDK-I subunits-Ctk1p, Ctk2p, and Ctk3p-confer conditional phenotypes . In this study, we examined the role of CTDK-I in the DNA damage response . We found that mutation of individual CTDK-I subunits rendered yeast sensitive to hydroxyurea (HU) and UV irradiation . Treatment with DNA-damaging agents increased phosphorylation of Ser2 within the CTD repeats in wild-type but not in ctk1Delta mutant cells . Using microarray hybridization, we identified genes whose transcription following DNA damage is Ctk1p dependent, including several DNA repair and stress response genes . Following HU treatment, the level of Ser2-phosphorylated RNA polymerase II increased both globally and on the CTDK-I-regulated genes . The pleiotropic phenotypes of ctk mutants suggest that CTDK-I activity is essential during large-scale transcriptional repatterning under stress and unfavorable growth conditions. J Mol Biol, 2003 Apr 18, 328(1), 235 - 54 Relationship between stability of folding intermediates and amyloid formation for the yeast prion Ure2p: a quantitative analysis of the effects of pH and buffer system; Zhu L et al.; The dimeric yeast protein Ure2 shows prion-like behaviour in vivo and forms amyloid fibrils in vitro . A dimeric intermediate is populated transiently during refolding and is apparently stabilized at lower pH, conditions suggested to favour Ure2 fibril formation . Here we present a quantitative analysis of the effect of pH on the thermodynamic stability of Ure2 in Tris and phosphate buffers over a 100-fold protein concentration range . We find that equilibrium denaturation is best described by a three-state model via a dimeric intermediate, even under conditions where the transition appears two-state by multiple structural probes . The free energy for complete unfolding and dissociation of Ure2 is up to 50 kcal mol(-1) . Of this, at least 20 kcal mol(-1) is contributed by inter-subunit interactions . Hence the native dimer and dimeric intermediate are significantly more stable than either of their monomeric counterparts . The previously observed kinetic unfolding intermediate is suggested to represent the dissociated native-like monomer . The native state is stabilized with respect to the dimeric intermediate at higher pH and in Tris buffer, without significantly affecting the dissociation equilibrium . The effects of pH, buffer, protein concentration and temperature on the kinetics of amyloid formation were quantified by monitoring thioflavin T fluorescence . The lag time decreases with increasing protein concentration and fibril formation shows pseudo-first order kinetics, consistent with a nucleated assembly mechanism . In Tris buffer the lag time is increased, suggesting that stabilization of the native state disfavours amyloid nucleation . Tsitologiia, 2002, 44(12), 1205 - 11 {Morphometric study of yeast cells of Torulopsis sphaerica after He-Ne-laser irradiation}; Manteifel' VM et al.; A study was made of structural organization of Torulopsis sphaerica cells irradiated with He-Ne (lambda = 632.8 nm; dose--460 J/m2) and then cultured in the nutrient with 1% glucose and O2 for 6 h . The computer analysis of electron images of cell sections was carried out . Evidences of stimulation of cell proliferation were found, including decrease in the areas of cell and chondriome profiles, decrease in the number of mitochondria on sections, elongation of cells and mitochondria, and increased variability of cell parameters . In addition, cells of irradiated cultured were characterized by the increase in the number of mitochondria contacting the endoplasmic reticulum (in this case the outer mitochondrial membrane presumably associates with the ER membrane), which may suggest the activation of ATP synthesis . Thus, He-Ne laser irradiation activates cell metabolism even at the early stage of culture growth. J Cell Sci, 2003 May 15, 116(Pt 10), 2005 - 14 Epub 2003 Apr 01. Spatial and temporal dynamics of budding yeast mitochondria lacking the division component Fis1p; Jakobs S et al.; The mitochondrial compartment of budding yeast (Saccharomyces cerevisiae) is a highly dynamic net-like structure of tubules that constantly undergo fusion and fission . The outer membrane protein Fis1p plays a crucial role in mitochondrial fission . Here we report on the temporal and spatial dynamics of this organelle in wild-type cells and in fis1Delta mutants . Mitochondria of fis1Delta mutants adapt their mitochondrial network to a change in carbon source . We find that the frequencies of apparent matrix separation and fusion events decrease in both wild-type cells and in mutants lacking Fis1p upon glucose repression . Matrix separation could be caused by matrix constriction and does not necessarily require fission of the inner or outer membrane . Double-labelling experiments demonstrated that some of these matrix separations in fis1 mutants are due to genuine tubule fissions, whereas others do not involve fission of the outer membrane . The rates of matrix separation in fis1Delta mutants almost approach those of the wildtype, demonstrating that, although apparently involved in outer membrane fission, Fis1p is not crucial for the separation of the mitochondrial matrix . In mutants lacking the GTPase Dnm1p no complete tubule fissions were recorded, although dnm1Delta mutants display matrix separations as well . The data suggest that different molecular machineries are responsible for the separation of the matrix and the fission of the outer membrane in budding yeast. J Biol Chem, 2003 Jun 13, 278(24), 21550 - 8 Epub 2003 Apr 04. Yeast epiarginase regulation, an enzyme-enzyme activity control: identification of residues of ornithine carbamoyltransferase and arginase responsible for enzyme catalytic and regulatory activities; El Alami M et al.; In the presence of ornithine and arginine, ornithine carbamoyltransferase (OTCase) and arginase form a one-to-one enzyme complex in which the activity of OTCase is inhibited whereas arginase remains catalytically active . The mechanism by which these nonallosteric enzymes form a stable complex triggered by the binding of their respective substrates raises the question of how such a cooperative association is induced . Analyses of mutations in both enzymes identify residues that are required for their association, some of them being important for catalysis . In arginase, two cysteines at the C terminus of the protein are crucial for its epiarginase function but not for its catalytic activity and trimeric structure . In OTCase, mutations of putative ornithine binding residues, Asp-182, Asn-184, Asn-185, Cys-289, and Glu-256 greatly reduced the affinity for ornithine and impaired the interaction with arginase . The four lysine residues located in the SMG loop, Lys-260, Lys-263, Lys-265, and Lys-268, also play an important role in mediating the sensitivity of OTCase to ornithine and to arginase and appear to be involved in transducing and enhancing the signal given by ornithine for the closure of the catalytic domain. Curr Med Chem, 2003 Apr, 10(8), 663 - 9 Evaluation of the inhibiting effects from exposure to microwaves on the respiratory activity of yeast cells or on enzyme activity; Campanella L et al.; Microwaves are used in medical applications, so their eventual toxicity effects must be carefully evaluated . An integral toxicity test, based on the monitoring of the respiratory activity of yeast cells, is proposed to evaluate the damage from microwave exposure . Different exposure times and microwave powers were considered . On supposing that the damages occur at enzymatic levels, the inhibiting effects of microwave exposure on two enzymes (glucose oxidase (GOD) and superoxide dismutase (SOD)), assumed like models as present in the human organism, was evaluated. Protein Eng, 2003 Feb, 16(2), 147 - 56 Directed evolution of a single-chain class II MHC product by yeast display; Starwalt SE et al.; Many autoimmune diseases have been linked to the class II region of the major histocompatibility complex (MHC) . The linkage is thought to be a result of autoreactive T cells that recognize self-peptides bound to a product of this locus . For example, T cells from non-obese diabetic mice recognize specific 'diabetogenic' peptides bound to a class II MHC allele called I-A(g7) . The I-A(g7) molecule is noted for being unstable and difficult to work with, especially in soluble form . In this work, yeast surface display combined with fluorescence-activated cell sorting was used as a means of directed evolution to engineer stabilized variants of a single-chain form of I-A(g7) . A library containing mutations at two residues (positions 56 and 57 of the I-A(g7) beta-chain) that are important in the class II disease associations yielded stabilized mutants with preferences for a glutamic acid at residue 56 and a leucine at residue 57 . Random mutation of I-A(g7) followed by selection with an anti-I-A(g7) antibody also yielded stabilized variants with mutations in other residues . The methods described here allow the discovery of novel MHC complexes that could facilitate structural studies and provide new opportunities in the development of diagnostics or antagonists of class II MHC-associated diseases. Curr Biol, 2003 Apr 1, 13(7), 568 - 74 A human homolog of yeast Est1 associates with telomerase and uncaps chromosome ends when overexpressed; Reichenbach P et al.; Telomeres protect the eukaryotic chromosome ends from degradation and fusion . They are maintained by the ribonucleoprotein telomerase, the core of which is composed of a reverse transcriptase (TERT) and a RNA subunit . In the yeast Saccharomyces cerevisiae, a third critical telomerase subunit, the Ever Shorter Telomeres 1 (EST1) gene product, recruits or activates telomerase at the 3' end of telomeres . Est1p has so far only been known in budding yeast, and mechanisms that mediate telomerase access and activation in other eukaryotes have remained elusive . Here, we use iterative profile searches to identify homologs of yeast Est1p in a large variety of eukaryotes, including human . One of three human homologs, designated human EST1A (hEST1A), is shown to be associated with most or all active telomerase in HeLa cell extracts . Overexpression of hEST1A induces anaphase bridges due to chromosome-end fusions, and telomeric DNA persists at the fusion points . Thus, overexpression of hEST1A affects telomere capping . The identification of EST1 homologs in a large variety of eukaryotes may indicate that the mechanisms of telomere extension are more conserved than anticipated previously. Biotechnol Prog, 2003 Mar-Apr, 19(2), 631 - 8 Rapid method for measuring ScFv thermal stability by yeast surface display; Orr BA et al.; We have characterized a simplified method to determine the relative thermal stability of single-chain antibodies by following the irreversible denaturation of scFv fusions on the surface of yeast by flow cytometry . The method was highly reproducible and correlated well with other methods used to monitor thermal denaturation of the soluble proteins . We found a range of thermal stabilities for wild-type single-chain antibodies with half-maximum denaturation temperatures between 43 and 61 degrees C . The ability to quantitate thermal stability of antibodies or other proteins that are immobilized on the surface of yeast allows rapid comparisons of primary structural information with stability . Thermal denaturation could be a useful parameter to consider in the choice of scFv fragments for various applications. Biotechnol Prog, 2003 Mar-Apr, 19(2), 389 - 95 Asymmetric synthesis with immobilized yeast in organic solvents: equilibrium conversion and effect of reactant partitioning on whole cell biocatalysis; Gervais TR et al.; A newly isolated strain of the yeast Saccharomyces cerevisiae is investigated for the biocatalytic reduction of ketones and the oxidation of alcohols in organic solvents . The yeast cells are immobilized by entrapment within calcium alginate beads and are found to possess the ability to stereoselectively reduce prochiral ketones and oxidize chiral alcohols to equilibrium conversions . The effect of reactant partitioning on the initial rate of the reactions is also investigated . The observed initial rates are found to vary inversely with reactant partitioning between the organic solvent and the biocatalyst beads . A kinetic model is developed to describe the initial reaction rate of hexanone reduction as a function of substrate concentration within the alginate beads. Biotechnol Prog, 2003 Mar-Apr, 19(2), 273 - 80 Yeast metallothionein in transgenic tobacco promotes copper uptake from contaminated soils; Thomas JC et al.; Metallothioneins (MTs) are metal-binding proteins that confer heavy metal tolerance and accumulation in yeast . To augment higher plant metal sequestration, the yeast metallothionein (CUP 1) was introduced into tobacco plants . The CUP 1 gene expression and copper and cadmium phytoextraction were determined . To confirm transformation, selfed and kanamycin-resistant third generation plants were subjected to DNA blot and polymerase chain reaction (PCR) analysis . A 4 mM CuSO(4) stress for 7 days resulted in a decline in CUP 1 transcripts versus nonstress conditions . Despite low mRNA levels, CUP 1 transformants accumulated up to seven times more copper in older versus younger leaves during copper stress . Pooled leaves of transgenic plants grown in soils from copper stamp-sands contained two to three times the copper content as that of the control plants . Unlike some previous reports featuring MT overexpression in plants, CUP 1 seedlings did not significantly sequester or demonstrate tolerance to CdCl(2) . Using this transgenic approach, yeast CUP 1 expression under nonstressed conditions contributed to copper metal phytoextraction during a subsequent copper challenge . This strategy could be incorporated into plants designed for enhanced phytoremediation of metal contaminants. J Biol Chem, 2003 May 23, 278(21), 19171 - 5 Epub 2003 Apr 02. Opposite role of yeast ING family members in p53-dependent transcriptional activation; Nourani A et al.; The inhibitor-of-growth (ING) family of proteins was founded by human ING1, a tumor suppressor interacting with p53 in vivo and required for its function in transcription/apoptosis . There are five different ING genes in humans, three of which have been linked to p53 function . In this study, we analyzed the three ING family members present in yeast . We demonstrate that each one is purified as a key component of a specific histone-modifying complex . Pho23 is part of Rpd3/Sin3 histone deacetylase complex, while Yng1 and Yng2 are subunits of the NuA3 and NuA4 histone acetyltransferase complexes, respectively . We also show that the three different ING proteins have opposite roles in transcriptional activation by p53 in vivo . These effects are linked to the presence of each ING in its respective chromatin modifying complex, since mutation of the corresponding catalytic subunit gave similar results . Depletion of Pho23/Rpd3 leads to increased p53-dependent transcription in vivo while depletion of Yng2 abrogates it . Surprisingly, deletion of YNG1 or SAS3 leads to increased transcriptional activation by p53 . These data suggest that the NuA3 complex can function in gene-specific repression, an unusual role for a histone acetyltransferase complex . They also demonstrate the key specific role of ING proteins in different chromatin modifying complexes and their opposite functions in p53-dependent transcription. J Biol Chem, 2003 Jun 20, 278(25), 22303 - 8 Epub 2003 Apr 02. Correlation between checkpoint activation and in vivo assembly of the yeast checkpoint complex Rad17-Mec3-Ddc1; Giannattasio M et al.; Rad17-Mec3-Ddc1 forms a proliferating cell nuclear antigen-like complex that is required for the DNA damage response in Saccharomyces cerevisiae and acts at an early step of the signal transduction cascade activated by DNA lesions . We used the mec3-dn allele, which causes a dominant negative checkpoint defect in G1 but not in G2, to test the stability of the complex in vivo and to correlate its assembly and disassembly with the mechanisms controlling checkpoint activation . Under physiological conditions, the mutant complex is formed both in G1 and G2, although the mutant phenotype is detectable only in G1, suggesting that is not the presence of the mutant complex per se to cause a checkpoint defect . Our data indicate that the Rad17-Mec3-Ddc1 complex is very stable, and it takes several hours to replace Mec3 with Mec3-dn within a wild type complex . On the other hand, the mutant complex is rapidly assembled when starting from a condition where the complex is not pre-assembled, indicating that the critical factor for the substitution is the disassembly step rather than complex formation . Moreover, the kinetics of mutant complex assembly, starting from conditions in which the wild type form is present, parallels the kinetics of checkpoint inactivation, suggesting that the complex acts in a stoichiometric way, rather than catalytically. Chembiochem, 2003 Apr 4, 4(4), 319 - 32 Synthesis of novel acceptor substrates for the dolichyl phosphate mannose synthase from yeast; Sprung I et al.; Dolichols are polyisoprenoid lipid components of mammalian membranes consisting of an average of 20 head-to-tail linked isoprene units of which the first isoprene is fully saturated . The unusual size of these lipids is intriguing and poses questions about the role of dolichol structure in biological processes . In order to probe structure and function we have synthesised potential dolichyl analogues that retain only the first two isoprene units and carry a second functional group within the terminal lipid chain . Such analogues were evaluated as substrates for a key enzyme in the dolichyl-dependent pathway of glycan biosynthesis, dolichyl phosphate mannose (Dol-P-Man) synthase . It was shown that some functional groups, including labels such as biotin, could be tolerated . When the synthetic analogues were attached to a solid support they were still substrates for the Dol-P-Man system and thus allowed the enzymatic solid-phase synthesis of glycolipids. Nat Rev Mol Cell Biol, 2003 Apr, 4(4), 276 - 84 Histone acetylation and deacetylation in yeast; Kurdistani SK et al.; Histone acetylation and deacetylation in the yeast Saccharomyces cerevisiae occur by targeting acetyltransferase and deacetylase enzymes to gene promoters and, in an untargeted and global manner, by affecting most nucleosomes . Recently, new roles for histone acetylation have been uncovered, not only in transcription but also in DNA replication, repair and heterochromatin formation . Interestingly, specific acetylatable lysines can function as binding sites for regulatory factors . Moreover, histone deacetylation is not only repressive but can be required for gene activity. Biochem Biophys Res Commun, 2003 Apr 11, 303(3), 771 - 6 Yeast Fms1 is a FAD-utilizing polyamine oxidase; Landry J et al.; In this report we show that recombinant Saccharomyces cerevisiae Fms1 protein is a polyamine oxidase that binds FAD with an FAD:Fms1 stoichiometry of 1:1 . Biochemical characterization of Fms1 shows that it can oxidize spermine, N(1)-acetylspermine, N(1)-acetylspermidine, and N(8)-acetylspermidine, but not spermidine . The products of spermine oxidation are spermidine and 3-aminopropanal . A kinetic analysis revealed that spermine, N(1)-acetylspermine, and N(1)-acetylspermidine are oxidized with similar efficiencies, while N(8)-acetylspermidine is a poor substrate . The data support a previous report, suggesting that Fms1 is responsible for the production of beta-alanine from spermine for the synthesis of pantothenic acid. J Biol Chem, 2003 Jun 6, 278(23), 20673 - 80 Epub 2003 Mar 31. Phosphorylation of the yeast phospholipid synthesis regulatory protein Opi1p by protein kinase A; Sreenivas A et al.; The Opi1p transcription factor plays a negative regulatory role in the expression of UASINO-containing genes involved in phospholipid synthesis in the yeast Saccharomyces cerevisiae . The phosphorylation of Opi1p by protein kinase A (cAMP-dependent protein kinase) was examined in this work . Using a maltose-binding protein-Opi1p fusion protein as a substrate, protein kinase A activity was time- and dose-dependent and dependent on the concentrations of Opi1p and ATP . Protein kinase A phosphorylated Opi1p on multiple serine residues . The synthetic peptides SCRQKSQPSE and SQVRESLLNL containing the protein kinase A motif for Ser31 and Ser251, respectively, within Opi1p were substrates for protein kinase A . Phosphorylation of S31A and S251A mutant maltose-binding protein-Opi1p fusion proteins by protein kinase A was reduced when compared with the wild type protein, and phosphopeptides present in wild type Opi1p were absent from the S31A and S251A mutant proteins . In vivo labeling experiments showed that the extent of phosphorylation of the S31A and S251A mutant proteins was reduced when compared with the wild type protein . The physiological consequence of the phosphorylation of Opi1p at Ser31 and Ser251 was examined by measuring the effects of the S31A and S251A mutations on the expression of the UASINO-containing gene INO1 . The beta-galactosidase activity driven by an INO1-CYC-lacZ reporter gene in opi1Delta mutant cells expressing the S31A and S251A mutant Opi1p proteins was elevated 42 and 35%, respectively, in the absence of inositol and 55 and 52%, respectively, in the presence of inositol when compared with cells expressing wild type Opi1p . These data supported the conclusion that phosphorylation of Opi1p at Ser31 and Ser251 mediated the stimulation of the negative regulatory function of Opi1p on the expression of the INO1 gene. J Cell Biol, 2003 Mar 31, 160(7), 1093 - 103 An anillin homologue, Mid2p, acts during fission yeast cytokinesis to organize the septin ring and promote cell separation; Tasto JJ et al.; Anillin is a conserved protein required for cell division (Field, C.M., and B.M . Alberts . 1995 . J . Cell Biol . 131:165-178; Oegema, K., M.S . Savoian, T.J . Mitchison, and C.M . Field . 2000 . J . Cell Biol . 150:539-552) . One fission yeast homologue of anillin, Mid1p, is necessary for the proper placement of the division site within the cell (Chang, F., A . Woollard, and P . Nurse . 1996 . J . Cell Sci . 109(Pt 1):131-142; Sohrmann, M., C . Fankhauser, C . Brodbeck, and V . Simanis . 1996 . Genes Dev . 10:2707-2719) . Here, we identify and characterize a second fission yeast anillin homologue, Mid2p, which is not orthologous with Mid1p . Mid2p localizes as a single ring in the middle of the cell after anaphase in a septin- and actin-dependent manner and splits into two rings during septation . Mid2p colocalizes with septins, and mid2 Delta cells display disorganized, diffuse septin rings and a cell separation defect similar to septin deletion strains . mid2 gene expression and protein levels fluctuate during the cell cycle in a sep1- and Skp1/Cdc53/F-box (SCF)-dependent manner, respectively, implying that Mid2p activity must be carefully regulated . Overproduction of Mid2p depolarizes cell growth and affects the organization of both the septin and actin cytoskeletons . In the presence of a nondegradable Mid2p fragment, the septin ring is stabilized and cell cycle progression is delayed . These results suggest that Mid2p influences septin ring organization at the site of cell division and its turnover might normally be required to permit septin ring disassembly. Hum Mol Genet, 2003 Apr 15, 12(8), 879 - 89 Iron use for haeme synthesis is under control of the yeast frataxin homologue (Yfh1); Lesuisse E et al.; The YFH1 gene is the yeast homologue of the human FRDA gene, which encodes the frataxin protein . Saccharomyces cerevisiae cells lacking the YFH1 gene showed very low cytochrome content . In Deltayfh1 strains, the level of ferrochelatase (Hem15p) was very low, as a result of transcriptional repression of HEM15 . However, the low amount of Hem15p was not the cause of haeme deficiency in Deltayfh1 cells . Ferrochelatase, a mitochondrial protein, able to mediate insertion of iron or zinc into the porphyrin precursor, made primarily the zinc protoporphyrin product . Zinc protoporphyrin instead of haeme accumulated during growth of Deltayfh1 mutant cells and, furthermore, preferential formation of zinc protoporphyrin was observed in real time . The method for these studies involved direct presentation of porphyrin to mitochondria and to ferrochelatase of permeabilized cells with intact architecture, thereby specifically testing the iron delivery portion of the haeme biosynthetic pathway . The studies showed that Deltayfh1 mutant cells are defective in iron use by ferrochelatase . Mossbauer spectroscopic analysis showed that iron was present as amorphous nano-particles of ferric phosphate in Deltayfh1 mitochondria, which could explain the unavailability of iron for haeme synthesis . A high frequency of suppressor mutations was observed, and the phenotype of such mutants was characterized by restoration of haeme synthesis in the absence of Yfh1p . Suppressor strains showed a normal cytochrome content, normal respiration, but remained defective in Fe-S proteins and still accumulated iron into mitochondria although to a lesser extent . Yfh1p and Hem15p were shown to interact in vitro by Biacore studies . Our results suggest that Yfh1 mediates iron use by ferrochelatase. Mol Cell Biol, 2003 Apr, 23(8), 2762 - 77 Molecular dissection of a yeast septin: distinct domains are required for septin interaction, localization, and function; Casamayor A et al.; The septins are a family of cytoskeletal proteins present in animal and fungal cells . They were first identified for their essential role in cytokinesis, but more recently, they have been found to play an important role in many cellular processes, including bud site selection, chitin deposition, cell compartmentalization, and exocytosis . Septin proteins self-associate into filamentous structures that, in yeast cells, form a cortical ring at the mother bud neck . Members of the septin family share common structural domains: a GTPase domain in the central region of the protein, a stretch of basic residues at the amino terminus, and a predicted coiled-coil domain at the carboxy terminus . We have studied the role of each domain in the Saccharomyces cerevisiae septin Cdc11 and found that the three domains are responsible for distinct and sometimes overlapping functions . All three domains are important for proper localization and function in cytokinesis and morphogenesis . The basic region was found to bind the phosphoinositides phosphatidylinositol 4-phosphate and phosphatidylinositol 5-phosphate . The coiled-coil domain is important for interaction with Cdc3 and Bem4 . The GTPase domain is involved in Cdc11-septin interaction and targeting to the mother bud neck . Surprisingly, GTP binding appears to be dispensable for Cdc11 function, localization, and lipid binding . Thus, we find that septins are multifunctional proteins with specific domains involved in distinct molecular interactions required for assembly, localization, and function within the cell. Mol Cell Biol, 2003 Apr, 23(8), 2623 - 32 A yeast homologue of Hsp70, Ssa1p, regulates turnover of the MFA2 transcript through its AU-rich 3' untranslated region; Duttagupta R et al.; Many eukaryotic mRNAs exhibit regulated decay in response to cellular signals . AU-rich elements (AREs) identified in the 3' untranslated region (3'-UTR) of several such mRNAs play a critical role in controlling the half-lives of these transcripts . The yeast ARE-containing mRNA, MFA2, has been studied extensively and is degraded by a deadenylation-dependent mechanism . However, the trans-acting factors that promote the rapid decay of MFA2 have not been identified . Our results suggest that the chaperone protein Hsp70, encoded by the SSA family of genes, is involved in modulating MFA2 mRNA decay . MFA2 is specifically stabilized in a strain bearing a temperature-sensitive mutation in the SSA1 gene . Furthermore, an AU-rich region within the 3'-UTR of the message is both necessary and sufficient to confer this regulation . Stabilization occurs as a result of slower deadenylation in the ssa1(ts) strain, suggesting that Hsp70 is required for activation of the turnover pathway. Cell Biol Int, 2003, 27(2), 135 - 46 Effect of melanins from black yeast fungi on proliferation and differentiation of cultivated human keratinocytes and fibroblasts; Blinova MI et al.; The effects of melanin preparations from black yeast fungi (BYF) on the proliferation and differentiation of normal cultivated human skin keratinocytes and embryonic pulmonary fibroblasts have been investigated . Melanin preparations in the range of 5-0.1 microg/ml were optimally active, with a more pronounced effect on keratinocyte than on fibroblast proliferation . Of 17 dihydroxynaphthalene (DHN) natural melanin preparations and two commercial dihydroxyphenylalanine (DOPA) melanin preparations, only one preparation--DOPA melanin (of animal origin) significantly stimulated proliferation of keratinocytes at 5 microg/ml; four preparations (DHN melanin from BYF) significantly inhibited proliferation of these cells at 5 or 1 microg/ml . The remaining preparations had no significant effect . Similarly, of the 17 preparations of DHN melanin from BYF, one preparation significantly stimulated fibroblast proliferation, and four significantly inhibited proliferation at 5 microg/ml, one at all the concentrations, and three from 1 down to 0.1 microg/ml . These melanin preparations were also shown to affect the in vitro differentiation of keratinocytes . J Mol Biol, 2003 Apr 11, 327(5), 985 - 1000 Sequence dependence of substrate recognition and cleavage by yeast RNase III; Lamontagne B et al.; Yeast Rnt1p is a member of the double-stranded RNA (dsRNA) specific RNase III family of endoribonucleases involved in RNA processing and RNA interference (RNAi) . Unlike other RNase III enzymes, which recognize a variety of RNA duplexes, Rnt1p cleaves specifically RNA stems capped with the conserved AGNN tetraloop . This unusual substrate specificity challenges the established dogma for substrate selection by RNase III and questions the dsRNA contribution to recognition by Rnt1p . Here we show that the dsRNA sequence adjacent to the tetraloop regulates Rnt1p cleavage by interfering with RNA binding . In context, sequences surrounding the cleavage site directly influence the cleavage efficiency . Introduction of sequences that stabilize the RNA helix enhanced binding while reducing the turnover rate indicating that, unlike the tetraloop, Rnt1p binding to the dsRNA helix may become rate-limiting . These results suggest that Rnt1p activity is strictly regulated by a combination of primary and tertiary structural elements allowing a substrate-specific binding and cleavage efficiency. Helicobacter, 2003 Apr, 8(2), 81 - 9 Molecular cloning of p53 cDNA of Mongolian gerbil and establishment of yeast p53 functional assay system; Ishizuka J et al.; BACKGROUND: Epidemiological studies have shown a correlation between Helicobacter pylori infection and human gastric carcinogenesis . A Mongolian gerbil model has demonstrated that H . pylori infection induced gastric carcinoma . However, the disadvantage of this animal model is a lack of information regarding the cellular genes involved in oncogenesis . Mutation of the p53 gene is one of the most common steps in gastric carcinogenesis . In this study, we aimed to clone the p53 gene of the Mongolian gerbil and detect the functional mutations in H . pylori-infected animals . MATERIALS AND METHODS: The p53 complementary DNA (cDNA) of Mongolian gerbil was cloned by the methods of reverse-transcribed polymerase chain reaction and rapid amplification of cDNA ends . RESULTS: The p53 cDNA of Mongolian gerbil has a 78.8% homology to that of humans . A novel yeast p53 assay system was established and enabled to detect the functional mutations of the p53 gene in the stomach of the Mongolian gerbil . CONCLUSIONS: This is the first report of the complete sequence of wild-type p53 cDNA of the Mongolian gerbil . This genetic information and an assay system designed to detect the functional mutations of the p53 gene are useful for further investigations of gastric oncogenesis in this animal model. J Biol Chem, 2003 Jun 13, 278(24), 21798 - 804 Epub 2003 Mar 26. Gbetagamma recruits Rho1 to the site of polarized growth during mating in budding yeast; Bar EE et al.; In mating mixtures of Saccharomyces cerevisiae, cells polarize their growth toward their conjugation partners along a pheromone gradient . This chemotropic phenomenon is mediated by structural proteins such as Far1 and Bem1 and by signaling proteins such as Cdc24, Cdc42, and Gbetagamma . The Gbetagamma subunit is thought to provide a positional cue that recruits the polarity establishment proteins, and thereby induces polarization of the actin cytoskeleton . We identified RHO1 in a screen for allele-specific high-copy suppressors of Gbetagamma overexpression, suggesting that Rho1 binds Gbetagamma in vivo . Inactivation of Rho1 GTPase activity augmented the rescue phenotype, suggesting that it is the activated form of Rho1 that binds Gbetagamma . We also found, in a pull-down assay, that Rho1 associates with GST-Ste4 and that Rho1 is localized to the neck and tip of mating projections . Moreover, a mutation in STE4 that disrupts Gbetagamma-Rho1 interaction reduces the projection tip localization of Rho1 and compromises the integrity of pheromone-treated cells deficient in Rho1 activity . In addition to its roles as a positive regulator of 1,3-beta-glucan synthase and of the cell integrity MAP kinase cascade, it was recently shown that Rho1 is necessary for the formation of mating projections . Together, these results suggest that Gbetagamma recruits Rho1 to the site of polarized growth during mating. EMBO J, 2003 Apr 1, 22(7), 1697 - 706 The number of vertebrate repeats can be regulated at yeast telomeres by Rap1-independent mechanisms; Brevet V et al.; The number of telomeric DNA repeats at chromosome ends is maintained around a mean value by a dynamic balance between elongation and shortening . In particular, proteins binding along the duplex part of telomeric DNA set the number of repeats by progressively limiting telomere growth . The paradigm of this counting mechanism is the Rap1 protein in Saccharomyces cerevisiae . We demonstrate here that a Rap1-independent mechanism regulates the number of yeast telomeric repeats (TG(1-3)) and of vertebrate repeats (T(2)AG(3)) when TEL1, a yeast ortholog of the human gene encoding the ATM kinase, is inactivated . In addition, we show that a T(2)AG(3)-only telomere can be formed and maintained in humanized yeast cells carrying a template mutation of the gene encoding the telomerase RNA, which leads to the synthesis of vertebrate instead of yeast repeats . Genetic and biochemical evidences indicate that this telomere is regulated in a Rap1-independent manner, both in TEL1 and in tel1Delta humanized yeast cells . Altogether, these findings shed light on multiple repeat-counting mechanisms, which may share critical features between lower and higher eukaryotes. EMBO J, 2003 Apr 1, 22(7), 1688 - 96 Rap1p telomere association is not required for mitotic stability of a C(3)TA(2) telomere in yeast; Alexander MK et al.; Telomeric DNA usually consists of a repetitive sequence: C(1-3)A/TG(1-3) in yeast, and C(3)TA(2)/T(2)AG(3) in vertebrates . In yeast, the sequence-specific DNA- binding protein Rap1p is thought to be essential for telomere function . In a tlc1h mutant, the templating region of the telomerase RNA gene is altered so that telomerase adds the vertebrate telomere sequence instead of the yeast sequence to the chromosome end . A tlc1h strain has short but stable telomeres and no growth defect . We show here that Rap1p and the Rap1p-associated Rif2p did not bind to a telomere that contains purely vertebrate repeats, while the TG(1-3) single-stranded DNA binding protein Cdc13p and the normally non-telomeric protein Tbf1p did bind this telomere . A chromosome with one entirely vertebrate-sequence telomere had a wild-type loss rate, and the telomere was maintained at a short but stable length . However, this telomere was unable to silence a telomere-adjacent URA3 gene, and the strain carrying this telomere had a severe defect in meiosis . We conclude that Rap1p localization to a C(3)TA(2) telomere is not required for its essential mitotic functions. EMBO J, 2003 Apr 1, 22(7), 1588 - 98 Ssu72 is a phosphatase essential for transcription termination of snoRNAs and specific mRNAs in yeast; Ganem C et al.; Ssu72 is an essential yeast protein that is involved in transcription . It physically interacts with transcription initiation and termination complexes . In this report, we provide evidence that Ssu72 is a phosphatase that physically interacts with the CTD kinase Kin28 and functionally interacts with the CTD phosphatase Fcp1 . A genome-wide expression analysis of mutant ssu72-ts69 during growth in complete medium revealed a number of defects, including the accumulation of a limited number of mRNAs and the read-through transcription of small nucleolar RNAs and of some mRNAs . We hypothesize that Ssu72 plays a key role in the transcription termination of certain transcripts, possibly by promoting RNA polymerase pausing and release . The possibility that the CTD of the largest subunit of RNA polymerase II is a substrate of Ssu72 is discussed. Gene, 2003 Mar 13, 306, 45 - 55 The ATP-dependent Lon protease of Mus musculus is a DNA-binding protein that is functionally conserved between yeast and mammals; Lu B et al.; The ATP-dependent Lon protease is a multi-functional enzyme that is conserved from archae to mammalian mitochondria, which not only degrades protein substrates but also binds DNA . As a starting point toward understanding Lon function in development, the mouse Lon cDNA was cloned and the encoded protein was characterized in cultured mammalian cells, in yeast and in vitro . Mouse Lon shows 87, 40 and 33% amino acid similarity with the human, yeast and bacterial homologs, respectively . Expression of a single mouse Lon transcript is detected in liver>heart>kidney>testis and is present during early embryonic development . Endogenous as well as transiently overexpressed mouse Lon co-localize with mitochondrial markers and have half-lives greater than 24 h as determined by pulse-chase studies . Enzymatically active mouse Lon that hydrolyses ATP and degrades protein and peptide substrates in an ATP-dependent manner also specifically binds to single-stranded but not to double-stranded DNA oligonucleotides . We propose that binding to TG-rich DNA sequences has been conserved between the mouse and human proteins . In addition, the evolutionary conservation of mitochondrial Lon function is demonstrated by the ability of mouse Lon to substitute for the yeast protein in vivo. Mol Microbiol, 2003 Apr, 48(1), 53 - 65 A C-terminal EGF-like domain governs BAD1 localization to the yeast surface and fungal adherence to phagocytes, but is dispensable in immune modulation and pathogenicity of Blastomyces dermatitidis; Brandhorst T et al.; BAD1, an adhesin and immune modulator of Blastomyces dermatitidis, is an essential virulence factor that is released extracellularly before association with the yeast surface . Here, deletion of the C-terminal EGF-like domain profoundly affected BAD1 function, leading to non-association with yeast, extracellular accumulation and impaired yeast adherence to macrophages . In equilibrium binding assays, DeltaC-term BAD1, lacking an EGF-like domain, bound poorly to BAD1 null yeast, yielding a low affinity (Kd, 3 x 10(-7) M versus 5 x 10(-8) M) and Bmax (1.9 x 10(5) versus 7.9 x 10(5)) compared with BAD1 . Similar protein binding profiles were observed using chitin particles, reinforcing the notion that chitin fibrils are a receptor for BAD1, and that the EGF-like domain is critical for BAD1 interactions with chitin on yeast . DeltaC-term strains bound poorly to macrophages, compared with parental or BAD1-reconstituted null strains . However, DeltaC-term strains and the purified protein itself sharply suppressed tumour necrosis factor (TNF)-alpha release by phagocytes in vitro and in lung in vivo, and the strains retained pathogenicity in a murine model of blastomycosis . Our results illustrate the previously undefined role of the EGF-like domain for BAD1 localization to yeast surfaces during cell wall biogenesis . They also demonstrate th