Curr Biol, 1999 May 6, 9(9), 501 - 4 Activation of the yeast Arp2/3 complex by Bee1p, a WASP-family protein; Winter D et al.; The Arp2/3 complex is a highly conserved cytoskeletal component that has been implicated in the nucleation of actin filament assembly . Purified Arp2/3 complex has a low intrinsic actin nucleation activity, leading to the hypothesis that an unidentified cellular activator is required for the function of this complex . We showed previously that mutations in the Arp2/3 complex and in Bee1p/Las17p, a member of the Wiskott-Aldrich syndrome protein(WASP) family, lead to a loss of cortical actin structures (patches) in yeast . Bee1p has also been identified as an essential nucleation factor in the reconstitution of actin patches in vitro . Recently, it was reported that WASP-like proteins might interact directly with the Arp2/3 complex through a conserved carboxy-terminal domain . Here, we have shown that Bee1p and the Arp2/3 complex co-immunoprecipitate when expressed at endogenous levels, and that this interaction requires both the Arc15p and Arc19p subunits of the Arp2/3 complex . Furthermore, the carboxy-terminal domain of Bee1p greatly stimulated the nucleation activity of purified Arp2/3 complex in vitro, suggesting a direct role for WASP-family proteins in the activation of the Arp2/3 complex . Interestingly, deletion of the carboxy-terminal domain of Bee1p neither abolished the localization of the Arp2/3 complex, as had been suggested, nor resulted in a severe defect in cortical actin assembly . These results indicate that the function of Bee1p is not mediated entirely through its interaction with the Arp2/3 complex, and that factors redundant with Bee1p might exist to activate the nucleation activity of the Arp2/3 complex. Nat Genet, 1999 May, 22(1), 55 - 8 Action of BTN1, the yeast orthologue of the gene mutated in Batten disease; Pearce DA et al.; Neuronal ceroid-lipofuscinoses (NCL) are autosomal recessive disorders that form the most common group of progressive neurodegenerative diseases in children, with an incidence as high as 1 in 12,500 live births, and with approximately 440,000 carriers in the United States . Disease progression is characterized by a decline in mental abilities, increased severity of untreatable seizures, blindness, loss of motor skills and premature death . The CLN3 gene, which is responsible for Batten disease, has been positionally cloned . The yeast gene, denoted BTN1, encodes a non-essential protein that is 39% identical and 59% similar to human CLN3 . Strains lacking Btn1p, btn1-delta, are resistant to D-(-)-threo-2-amino-1-{p-nitrophenyl}-1,3-propanediol (ANP) in a pH-dependent manner . This phenotype was complemented by expression of human CLN3, demonstrating that yeast Btn1p and human CLN3 share the same function . Here, we report that btn1-delta yeast strains have an abnormally acidic vacuolar pH in the early phases of growth . Furthermore, DNA microarray analysis of BTN1 and btn1-delta strains revealed differential expression of two genes, with at least one, HSP30, involved in pH control . Because Btn1p is located in the vacuole, we suggest that Batten disease is caused by a defect in vacuolar (lysosomal) pH control . Our findings draw parallels between fundamental biological processes in yeast and previously observed characteristics of neurodegeneration in humans. Proc Natl Acad Sci U S A, 1999 May 11, 96(10), 5873 - 7 A highly conserved kinase is an essential component for stress tolerance in yeast and plant cells; Lee JH et al.; Osmotic stress (drought, salt stress) is a major limiting factor for crop productivity in the world . Because cellular responses to osmotic stress are thought to be conserved in eukaryotes and because yeast is much more amenable than plants to genetic research, a functional strategy has been performed to identify limiting steps in osmotolerance of plants based on the complementation of yeast with a plant library . A new plant cDNA that encodes a functional homologue of the yeast Dbf2 kinase enhances salt, drought, cold, and heat tolerance upon overexpression in yeast as well as in transgenic plant cells. J Bacteriol, 1999 May, 181(10), 3051 - 7 MHY1 encodes a C2H2-type zinc finger protein that promotes dimorphic transition in the yeast Yarrowia lipolytica; Hurtado CA et al.; The yeast-to-hypha morphological transition (dimorphism) is typical of many pathogenic fungi . Dimorphism has been attributed to changes in temperature and nutritional status and is believed to constitute a mechanism of response to adverse conditions . We have isolated and characterized a gene, MHY1, whose transcription is dramatically increased during the yeast-to-hypha transition in Yarrowia lipolytica . Deletion of MHY1 is viable and has no effect on mating, but it does result in a complete inability of cells to undergo mycelial growth . MHY1 encodes a C2H2-type zinc finger protein, Mhy1p, which can bind putative cis-acting DNA stress response elements, suggesting that Mhy1p may act as a transcription factor . Interestingly, Mhy1p tagged with a hemagglutinin epitope was concentrated in the nuclei of actively growing cells found at the hyphal tip. Biochim Biophys Acta, 1999 May 18, 1438(2), 223 - 38 A yeast strain defective in oleic acid utilization has a mutation in the RML2 gene; Trotter PJ et al.; The molecular mechanisms of cellular long-chain fatty acid assimilation and its regulation remain unclear . In an attempt to identify essential mediators of these processes, we have isolated mutant strains of the yeast Saccharomyces cerevisiae unable to utilize oleic acid as sole carbon source, while retaining the ability to utilize acetate . These strains are then subjected to several secondary screening assays to identify mutants of interest . Here we describe a mutant (denoted fat21) that, despite a temperature-sensitive inability to utilize oleic acid as sole carbon source, displays no general defect in oleic acid uptake or incorporation of oleic acid into glycerolipids . Oxidation of acetate after growth in acetate medium is increased similarly in the mutant and parent strains . Oleic acid beta-oxidation in acetate grown cells is also comparable between strains . Induction of oleic acid oxidation following exposure to oleic acid is, however, defective in the fat21 mutant . The fat21 mutant allele displays conditional synthetic lethality in combination with a null allele of the OLE1 gene, which encodes Delta9-desaturase and is required for proper mitochondrial segregation . Clones capable of complementing the fat21 defect contained the RML2 gene, encoding a yeast mitochondria ribosomal protein . Segregation analysis and gene replacement experiments demonstrate that RML2 is the gene defective in the fat21 mutant . These observations of a defect in a mitochondrial protein differentially affecting the adaptation to oleic acid and acetate as carbon sources suggest that the phenotype of fat21 is associated with a novel pathway of mitochondrial-nuclear-peroxisomal communication. Bioinformatics, 1999 Apr, 15(4), 267 - 77 DNA-binding requirements of the yeast protein Rap1p as selected in silico from ribosomal protein gene promoter sequences; Lascaris RF et al.; MOTIVATION: High-level transcriptional activation of most ribosomal protein (rp) genes in Saccharomyces cerevisiae is promoted by the global DNA-binding factor Rap1p . The creation of the complete database of yeast rp gene promoter sequences enabled us to develop a computational selection strategy aimed at acquiring detailed information concerning the DNA-binding specificity of Rap1p . RESULTS: Rap1p sites in rp gene promoters are often found in duplicate, exhibiting strong preferences in both spacing and orientation . Using these preferences, a weight matrix was selected that represents the in vivo binding requirements of Rap1p . The resulting matrix renders the identification of functional Rap1p binding sites more accurate and allowed us to re-evaluate previous in vitro data . Tandemly arranged Rap1p binding sites appear to be typical for rp gene promoters and differ in preferred spacing from sites occurring in (sub)telomeric repeats . The preferred spacing that is found in duplicate Rap1p binding sites of rp gene promoters is restricted to a small window between 15 and 26 bp . This is proposed to reflect the borders within which binding co-operativity operates . The data presented clearly illustrate that computational selection strategies provide information that reaches beyond experimental data . AVAILABILITY: The rp database is available at the url: chem.vu.nl/BMB/Database.html. Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 915 - 24 AFLP fingerprinting for analysis of yeast genetic variation; de Barros Lopes M et al.; Amplified fragment length polymorphism (AFLP) was used to investigate genetic variation in commercial strains, type strains and winery isolates from a number of yeast species . AFLP was shown to be effective in discriminating closely related strains . Furthermore, sufficient similarity in the fingerprints produced by yeasts of a given species allowed classification of unknown isolates . The applicability of the method for determining genome similarities between yeasts was investigated by performing cluster analysis on the AFLP data . Results from two species, Saccharomyces cerevisiae and Dekkera bruxellensis, illustrate that AFLP is useful for the study of intraspecific genetic relatedness . The value of the technique in strain differentiation, species identification and the analysis of genetic similarity demonstrates the potential of AFLP in yeast ecology and evolutionary studies. Proc Natl Acad Sci U S A, 1999 May 11, 96(10), 5651 - 6 Selection of functional T cell receptor mutants from a yeast surface-display library; Kieke MC et al.; The heterodimeric alphabeta T cell receptor (TCR) for antigen is the key determinant of T cell specificity . The structure of the TCR is very similar to that of antibodies, but the engineering of TCRs by directed evolution with combinatorial display libraries has not been accomplished to date . Here, we report that yeast surface display of a TCR was achieved only after the mutation of specific variable region residues . These residues are located in two regions of the TCR, at the interface of the alpha- and beta-chains and in the beta-chain framework region that is thought to be in proximity to the CD3 signal-transduction complex . The mutations are encoded naturally in many antibody variable regions, indicating specific functional differences that have not been appreciated between TCRs and antibodies . The identification of these residues provides an explanation for the inherent difficulties in the display of wild-type TCRs compared with antibodies . Yeast-displayed mutant TCRs bind specifically to the peptide/MHC antigen, enabling engineering of soluble T cell receptors as specific T cell antagonists . This strategy of random mutagenesis followed by selection for surface expression may be of general use in the directed evolution of other eukaryotic proteins that are refractory to display. Proc Natl Acad Sci U S A, 1999 May 11, 96(10), 5446 - 51 Oxygen sensing in yeast: evidence for the involvement of the respiratory chain in regulating the transcription of a subset of hypoxic genes; Kwast KE et al.; Oxygen availability affects the transcription of a number of genes in nearly all organisms . Although the molecular mechanisms for sensing oxygen are not precisely known, heme is thought to play a pivotal role . Here, we address the possibility that oxygen sensing in yeast, as in mammals, involves a redox-sensitive hemoprotein . We have found that carbon monoxide (CO) completely blocks the anoxia-induced expression of two hypoxic genes, OLE1 and CYC7, partially blocks the induction of a third gene, COX5b, and has no effect on the expression of other hypoxic or aerobic genes . In addition, transition metals (Co and Ni) induce the expression of OLE1 and CYC7 in a concentration-dependent manner under aerobic conditions . These findings suggest that the redox state of an oxygen-binding hemoprotein is involved in controlling the expression of at least two hypoxic yeast genes . By using mutants deficient in each of the two major yeast CO-binding hemoproteins (cytochrome c oxidase and flavohemoglobin), respiratory inhibitors, and cob1 and rho0 mutants, we have found that the respiratory chain is involved in the anoxic induction of these two genes and that cytochrome c oxidase is likely the hemoprotein "sensor." Our findings also indicate that there are at least two classes of hypoxic genes in yeast (CO sensitive and CO insensitive) and imply that multiple pathways/mechanisms are involved in modulating the expression of hypoxic yeast genes. J Biol Chem, 1999 May 14, 274(20), 14500 - 7 Characterization of the DOC1/APC10 subunit of the yeast and the human anaphase-promoting complex; Grossberger R et al.; The anaphase-promoting complex/cyclosome (APC) is a ubiquitin-protein ligase whose activity is essential for progression through mitosis . The vertebrate APC is thought to be composed of 8 subunits, whereas in budding yeast several additional APC-associated proteins have been identified, including a 33-kDa protein called Doc1 or Apc10 . Here, we show that Doc1/Apc10 is a subunit of the yeast APC throughout the cell cycle . Mutation of Doc1/Apc10 inactivates the APC without destabilizing the complex . An ortholog of Doc1/Apc10, which we call APC10, is associated with the APC in different vertebrates, including humans and frogs . Biochemical fractionation experiments and mass spectrometric analysis of a component of the purified human APC show that APC10 is a genuine APC subunit whose cellular levels or association with the APC are not cell cycle-regulated . We have further identified an APC10 homology region, which we propose to call the DOC domain, in several protein sequences that also contain either cullin or HECT domains . Cullins are present in several ubiquitination complexes including the APC, whereas HECT domains represent the catalytic core of a different type of ubiquitin-protein ligase . DOC domains may therefore be important for reactions catalyzed by several types of ubiquitin-protein ligases. Yeast, 1999 Apr, 15(6), 527 - 31 A rapid technique for the determination of unknown plasmid library insert DNA sequence directly from intact yeast cells; Klebanow ER et al.; A polymerase chain reaction (PCR)-based technique is described which allows for the determination of library plasmid insert DNA sequence directly and rapidly from intact yeast cells . Yeast spheroplasts are used to template a PCR reaction to amplify the insert sequence . This PCR product is then purified and its sequence directly determined using thermal cycle sequencing . Readable sequence can reproducibly be obtained from multiple yeast colonies in just two days . Uses of this technique in yeast two-hybrid screening as well as other types of yeast library screens are discussed. Gene, 1999 Apr 29, 231(1-2), 41 - 7 Arabidopsis thaliana RNA polymerase II subunits related to yeast and human RPB5; Larkin RM et al.; Arabidopsis thaliana contains at least four genes that are predicted to encode polypeptides related to the RPB5 subunit found in yeast and human RNA polymerase II . This subunit has been shown to be the largest subunit common to yeast RNA polymerases I, II, and III (RPABC27) . More than one of these genes is expressed in Arabidopsis suspension culture cells, but only one of the encoded polypeptides is found in purified RNA polymerases II and III . This polypeptide has a predicted pI of 9.6, matches 14 of 16 amino acids in the amino terminus of cauliflower RPB5 that was microsequenced, and shows 42 and 53% amino acid sequence identity with the yeast and human RPB5 subunits, respectively. Eur J Biochem, 1999 May, 262(1), 26 - 35 Polyproline binding is an essential function of human profilin in yeast; Ostrander DB et al.; Structural analysis of human profilin has revealed two tryptophan residues, W3 and W31, which interact with polyproline . The codons for these residues were mutated to encode phenylalanine and the mutant proteins overexpressed in Eschericia coli . The isolated proteins were diminished in their ability to bind polyproline, whereas phosphatidylinositol 4,5-bisphosphate (PIP2) binding remained unchanged . In many strains of Saccharomyces cerevisiae, disruption of the gene encoding profilin, PFY1, is lethal . It was found that expression of the gene for human profilin is capable of suppressing this lethality . The polyproline-binding mutant alleles of the human gene were cloned into various yeast expression vectors . Each of the mutant genes resulted in suppression of the lethality of pfy1Delta . It was observed that the mutant protein expression levels paralleled the growth rates of the strains . The severity of various morphological abnormalities of the strains was also attenuated with increased protein levels, suggesting that profilin polyproline-binding mutations are deleterious to cell growth unless overexpressed . Both tryptophan mutations were combined to give a third mutant allele that was found both unable to bind polyproline and to suppress the lethality of a pfy1 deletion . Immunoprecipitation experiments suggested that the mutants were unaltered in their affinity for actin and PIP2 . These data strongly suggest that polyproline binding is an essential function of profilin. Bioorg Med Chem Lett, 1999 Apr 5, 9(7), 1029 - 32 Total synthesis of an antioxidant isolated from yeast via palladium-catalyzed coupling and its application for related compounds; Jinno S et al.; A total synthesis of an antioxidant (1) having a benzofuran skeleton was achieved in four steps via the palladium(0)-catalyzed cross-coupling reaction . We also prepared several related compounds bearing a variety of aromatic or heterocyclic rings . Some of these compounds demonstrate more potent than 1 for antioxidative activity using guinea pig liver microsomes. Mol Cell, 1999 Apr, 3(4), 447 - 55 Elimination of replication block protein Fob1 extends the life span of yeast mother cells; Defossez PA et al.; A cause of aging in yeast is the accumulation of circular species of ribosomal DNA (rDNA) arising from the 100-200 tandemly repeated copies in the genome . We show here that mutation of the FOB1 gene slows the generation of these circles and thus extends life span . Fob1p is known to create a unidirectional block to replication forks in the rDNA . We show that Fob1p is a nucleolar protein, suggesting a direct involvement in the replication fork block . We propose that this block can trigger aging by causing chromosomal breaks, the repair of which results in the generation of rDNA circles . These findings may provide a novel link between metabolic rate and aging in yeast and, perhaps, higher organisms. EMBO J, 1999 May 4, 18(9), 2593 - 609 The Mex67p-mediated nuclear mRNA export pathway is conserved from yeast to human; Katahira J et al.; Human TAP is an orthologue of the yeast mRNA export factor Mex67p . In mammalian cells, TAP has a preferential intranuclear localization, but can also be detected at the nuclear pores and shuttles between the nucleus and the cytoplasm . TAP directly associates with mRNA in vivo, as it can be UV-crosslinked to poly(A)+ RNA in HeLa cells . Both the FG-repeat domain of nucleoporin CAN/Nup214 and a novel human 15 kDa protein (p15) with homology to NTF2 (a nuclear transport factor which associates with RanGDP), directly bind to TAP . When green fluorescent protein (GFP)-tagged TAP and p15 are expressed in yeast, they localize to the nuclear pores . Strikingly, co-expression of human TAP and p15 restores growth of the otherwise lethal mex67::HIS3/mtr2::HIS3 double knockout strain . Thus, the human TAP-p15 complex can functionally replace the Mex67p-Mtr2p complex in yeast and thus performs a conserved role in nuclear mRNA export. EMBO J, 1999 May 4, 18(9), 2538 - 50 Limitations of silencing at native yeast telomeres; Pryde FE et al.; Silencing at native yeast telomeres, in which the subtelomeric elements are intact, is different from silencing at terminal truncations . The repression of URA3 inserted in different subtelomeric positions at several chromosome ends was investigated . Many ends exhibit very little silencing close to the telomere, while others exhibit substantial repression in limited domains . Silencing at native ends is discontinuous, with maximal repression found adjacent to the ARS consensus sequence in the subtelomeric core X element . The level of repression declines precipitously towards the centromere . Mutation of the ARS sequence or an adjacent Abf1p-binding site significantly reduces silencing . The subtelomeric Y' elements are resistant to silencing along their whole length, yet silencing can be re-established at the proximal X element . Deletion of PPR1, the transactivator of URA3, and SIR3 overexpression do not increase repression or extend spreading of silencing to the same extent as with terminally truncated ends . sir1Delta causes partial derepression at X-ACS, in contrast to the lack of effect seen at terminal truncations . orc2-1 and orc5-1 have no effect on natural silencing yet cause derepression at truncated ends . X-ACS silencing requires the proximity of the telomere and is dependent on SIR2, SIR3, SIR4 and HDF1 . The structures found at native yeast telomeres appear to limit the potential of repressive chromatin. EMBO J, 1999 May 4, 18(9), 2522 - 37 Cohabitation of insulators and silencing elements in yeast subtelomeric regions; Fourel G et al.; In budding yeast, the telomeric DNA is flanked by a combination of two subtelomeric repetitive sequences, the X and Y' elements . We have investigated the influence of these sequences on telomeric silencing . The telomere-proximal portion of either X or Y' dampened silencing when located between the telomere and the reporter gene . These elements were named STARs, for subtelomeric anti-silencing regions . STARs can also counteract silencer-driven repression at the mating-type HML locus . When two STARs bracket a reporter gene, its expression is no longer influenced by surrounding silencing elements, although these are still active on a second reporter gene . In addition, an intervening STAR uncouples the silencing of neighboring genes . STARs thus display the hallmarks of insulators . Protection from silencing is recapitulated by multimerized oligonucleotides representing Tbf1p- and Reb1p-binding sites, as found in STARs . In contrast, sequences located more centromere proximal in X and Y' elements reinforce silencing . They can promote silencing downstream of an insulated expressed domain . Overall, our results suggest that the silencing emanating from telomeres can be propagated in a discontinuous manner via a series of subtelomeric relay elements. EMBO J, 1999 May 4, 18(9), 2424 - 34 A Bub2p-dependent spindle checkpoint pathway regulates the Dbf2p kinase in budding yeast; Fesquet D et al.; Exit from mitosis in all eukaroytes requires inactivation of the mitotic kinase . This occurs principally by ubiquitin-mediated proteolysis of the cyclin subunit controlled by the anaphase-promoting complex (APC) . However, an abnormal spindle and/or unattached kinetochores activates a conserved spindle checkpoint that blocks APC function . This leads to high mitotic kinase activity and prevents mitotic exit . DBF2 belongs to a group of budding yeast cell cycle genes that when mutated prevent cyclin degradation and block exit from mitosis . DBF2 encodes a protein kinase which is cell cycle regulated, peaking in metaphase-anaphase B/telophase, but its function remains unknown . Here, we show the Dbf2p kinase activity to be a target of the spindle checkpoint . It is controlled specifically by Bub2p, one of the checkpoint components that is conserved in fission yeast and higher eukaroytic cells . Significantly, in budding yeast, Bub2p shows few genetic or biochemical interactions with other members of the spindle checkpoint . Our data now point to the protein kinase Mps1p triggering a new parallel branch of the spindle checkpoint in which Bub2p blocks Dbf2p function. J Mass Spectrom, 1999 Apr, 34(4), 311 - 29 Determination of N-linked glycosylation of yeast external invertase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Zeng C et al.; The extent of N-glycosylation of yeast external invertase at each of the 14 potential sites was determined by the combination of proteolytic digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) . The average molecular mass of the intact external invertase was determined as 97 kDa by MALDI/TOF-MS . The intact protein was digested with trypsin, Lys-C and Asp-N, followed by high-performance liquid chromatographic separation . The proteolytic digests were analyzed by MALDI/MS screening for the glycopeptides . The glycopeptides were then treated with peptide:N-glycosidase F (PNGase F) and/or endo-beta-N-acetylglucosaminidase (Endo H) and the molecular mass of the deglycosylated peptide was determined by MALDI/MS and matched with the peptide predicted by a computer program . The sequences of some peptides or deglycosylated peptides were identified by the MALDI post-source decay technique . The size of the oligosaccharide, the degree of glycosylation and the distribution of the oligosaccharides at each individual potential glycosylation site were characterized . This information goes for beyond previously published data and sometimes differs from them . During this study, the amino acid sequence originally derived from the DNA sequence of the gene coding for invertase was also verified and it was found that this protein when expressed from SUC2 gene might be created as more than one sequence which differ by a few amino acid substitutions (Asn58<-->Thr, Asn65-->His and Val412<-->Ala). Parasitol Res, 1999 May, 85(5), 417 - 20 Primary sequence of a putative non-ATPase subunit of the 26S proteasome from Entamoeba histolytica is similar to the human and yeast S2 subunit; Hellberg A et al.; The cDNA coding for a non-ATPase S2 subunit of the 26S proteasome from Entamoeba histolytica was cloned from a cDNA library (EhS2) . The open reading frame has 2529 bp and the deduced amino acid sequence encodes a protein with a calculated molecular mass of 92,000 Da . EhS2 has 29-35% identity with the three other known S2 subunit sequences of yeasts and humans. Arch Virol, 1999, 144(3), 621 - 6 The yeast two-hybrid system reveals no interaction between p73 alpha and SV40 large T-antigen; Reichelt M et al.; SV40 and/or DNA sequences indistinguishable from SV40 have been detected in several types of human tumours . The oncoprotein of Simian virus 40, SV40 large T-antigen (Tag), is known to bind and inactivate tumour suppressor proteins, such as members of the retinoblastoma family and p53, thereby promoting cell transformation . In this study, we used the yeast two-hybrid system to investigate whether the Simian virus 40 (SV40) large T-antigen is able to interact with p73, a noval discovered putative tumour suppressor, that is homologous both structurally and functionally to p53 . The yeast two-hybrid system is a genetic method to detect protein-protein-interactions in vivo . Our results suggest that the SV40 large T-antigen is not able to bind p73 in yeast although both proteins are expressed in the transformed yeast strain as was shown by western blot analysis. Anticancer Res, 1999 Jan-Feb, 19(1A), 625 - 8 Detection of p53 mutations in Hong Kong colorectal carcinomas by conventional PCR-SSCP analysis versus p53 yeast functional assays; Leung CS et al.; BACKGROUND: Previous reports indicate that the p53 yeast functional assay is a highly sensitive method of detection of p53 mutations in clinical specimens . Our earlier report (1) showed a 35.4% p53 mutation frequency in Hong Kong colorectal carcinoma (CRC) patients, when conventional molecular screening techniques were utilized to assess the mutation rate in the hot spots in exons 5-8 . MATERIALS AND METHODS: The yeast functional assay was used to determine if the previous mutation frequency determined by PCR-SSCP techniques was under-estimated, and if so, to see if other hot spots for mutations explain this difference . RESULTS: The p53 functional yeast assay results showed an increased mutation frequency . However, sequencing showed the mutations were confined to common hot spots for mutations in exons 6 and 7 . CONCLUSIONS: The mutation frequency in CRC patients observed with the yeast assay is higher than previously reported . Forty-five percent of 20 SSCP-negative specimens were positive by the yeast assay, which this study shows is superior for detection of p53 mutations directly in clinical specimens containing varying amounts of normal tissue contamination. Genetics, 1999 May, 152(1), 101 - 15 Maximal stimulation of meiotic recombination by a yeast transcription factor requires the transcription activation domain and a DNA-binding domain; Kirkpatrick DT et al.; The DNA sequences located upstream of the yeast HIS4 represent a very strong meiotic recombination hotspot . Although the activity of this hotspot requires the transcription activator Rap1p, the level of HIS4 transcription is not directly related to the level of recombination . We find that the recombination-stimulating activity of Rap1p requires the transcription activation domain of the protein . We show that a hybrid protein with the Gal4p DNA-binding domain and the Rap1p activation domain can stimulate recombination in a strain in which Gal4p-binding sites are inserted upstream of HIS4 . In addition, we find recombination hotspot activity associated with the Gal4p DNA-binding sites that is independent of known transcription factors . We suggest that yeast cells have two types of recombination hotspots, alpha (transcription factor dependent) and beta (transcription factor independent). Genetics, 1999 May, 152(1), 89 - 100 The REG1 gene product is required for repression of INO1 and other inositol-sensitive upstream activating sequence-containing genes of yeast; Ouyang Q et al.; A search was conducted for suppressors of the inositol auxotrophic phenotype of the ino4-8 mutant of yeast . The ino4-8 mutation is a single base pair change that results in substitution of lysine for glutamic acid at position 79 in the bHLH domain of the yeast regulatory protein, Ino4p . Ino4p dimerizes with a second bHLH protein, Ino2p, to form a complex that binds to the promoter of the INO1 gene, activating transcription . Of 31 recessive suppressors of ino4-8 isolated, 29 proved to be alleles of a single locus, identified as REG1, which encodes a regulatory subunit of a protein phosphatase involved in the glucose response pathway . The suppressor mutation, sia1-1, identified as an allele of REG1, caused constitutive INO1 expression and was capable of suppressing the inositol auxotrophy of a second ino4 missense mutant, ino4-26, as well as ino2-419, a missense mutation of INO2 . The suppressors analyzed were unable to suppress ino2 and ino4 null mutations, but the reg1 deletion mutation could suppress ino4-8 . A deletion mutation in the OPI1 negative regulator was incapable of suppressing ino4-8 . The relative roles of the OPI1 and REG1 gene products in control of INO1 expression are discussed. Genetics, 1999 May, 152(1), 47 - 59 Genetic factors affecting the impact of DNA polymerase delta proofreading activity on mutation avoidance in yeast; Tran HT et al.; Base selectivity, proofreading, and postreplication mismatch repair are important for replication fidelity . Because proofreading plays an important role in error correction, we have investigated factors that influence its impact in the yeast Saccharomyces cerevisiae . We have utilized a sensitive mutation detection system based on homonucleotide runs of 4 to 14 bases to examine the impact of DNA polymerase delta proofreading on mutation avoidance . The contribution of DNA polymerase delta proofreading on error avoidance was found to be similar to that of DNA polymerase epsilon proofreading in short homonucleotide runs (A4 and A5) but much greater than the contribution of DNA polymerase epsilon proofreading in longer runs . We have identified an intraprotein interaction affecting mutation prevention that results from mutations in the replication and the proofreading regions, resulting in an antimutator phenotype relative to a proofreading defect . Finally, a diploid strain with a defect in DNA polymerase delta proofreading exhibits a higher mutation rate than a haploid strain . We suggest that in the diploid population of proofreading defective cells there exists a transiently hypermutable fraction that would be inviable if cells were haploids. J Biol Chem, 1999 May 7, 274(19), 12990 - 5 SAC1-like domains of yeast SAC1, INP52, and INP53 and of human synaptojanin encode polyphosphoinositide phosphatases; Guo S et al.; The SAC1 gene product has been implicated in the regulation of actin cytoskeleton, secretion from the Golgi, and microsomal ATP transport; yet its function is unknown . Within SAC1 is an evolutionarily conserved 300-amino acid region, designated a SAC1-like domain, that is also present at the amino termini of the inositol polyphosphate 5-phosphatases, mammalian synaptojanin, and certain yeast INP5 gene products . Here we report that SAC1-like domains have intrinsic enzymatic activity that defines a new class of polyphosphoinositide phosphatase (PPIPase) . Purified recombinant SAC1-like domains convert yeast lipids phosphatidylinositol (PI) 3-phosphate, PI 4-phosphate, and PI 3,5-bisphosphate to PI, whereas PI 4,5-bisphosphate is not a substrate . Yeast lacking Sac1p exhibit 10-, 2.5-, and 2-fold increases in the cellular levels of PI 4-phosphate, PI 3,5-bisphosphate, and PI 3-phosphate, respectively . The 5-phosphatase domains of synaptojanin, Inp52p, and Inp53p are also catalytic, thus representing the first examples of an inositol signaling protein with two distinct lipid phosphatase active sites within a single polypeptide chain . Together, our data provide a long sought mechanism as to how defects in Sac1p overcome certain actin mutants and bypass the requirement for yeast phosphatidylinositol/phosphatidylcholine transfer protein, Sec14p . We demonstrate that PPIPase activity is a key regulator of membrane trafficking and actin cytoskeleton organization and suggest signaling roles for phosphoinositides other than PI 4,5-bisphosphate in these processes . Additionally, the tethering of PPIPase and 5-phosphatase activities indicate a novel mechanism by which concerted phosphoinositide hydrolysis participates in membrane trafficking. Arch Biochem Biophys, 1999 May 1, 365(1), 131 - 42 Mitochondrial superoxide decreases yeast survival in stationary phase; Longo VD et al.; Yeast lacking mitochondrial superoxide dismutase (MnSOD) display shortened stationary-phase survival and provide a good model system for studying mitochondrial oxidative damage . We observed a marked decrease in respiratory function preceding stationary-phase death of yeast lacking MnSOD (sod2Delta) . Agents (mitochondrial inhibitors) that are known to increase or decrease superoxide production in submitochondrial particles affected stationary-phase survival in a manner inversely correlated with their effects on superoxide production, implicating superoxide in this mitochondrial disfunction . Similar but less-dramatic effects were observed in wild-type yeast . The activities of certain mitochondrial enzymes were particularly affected . In sod2Delta yeast the activity of aconitase, a 4Fe-4S-cluster-containing enzyme located in the matrix, was greatly and progressively decreased as the cells established stationary phase . Succinate dehydrogenase activity also decreased in MnSOD mutants; cytochrome oxidase and ATPase activities did not . Aconitase could be reactivated by addition of materials required for cluster assembly (Fe3+ and a sulfur source), both in extracts and in vivo, indicating that inactivation of the enzyme was by disassembly of the cluster . Our results support the conclusion that superoxide is generated in the mitochondria in vivo and under physiological conditions and that MnSOD is the primary defense against this toxicity . When the balance between superoxide generation and MnSOD activity is disrupted, superoxide mediates iron release from mitochondrial iron-sulfur clusters, leading first to loss of mitochondrial function and then to death, independently of mtDNA damage . These results raise the possibility that similar processes may occur in higher eukaryotes . Yeast, 1999 Mar 30, 15(5), 397 - 407 Initial characterization of the nascent polypeptide-associated complex in yeast; Reimann B et al.; The three subunits of the nascent polypeptide-associated complex (alpha, beta1, beta3) in Saccharomyces cerevisiae are encoded by three genes (EGD2, EGD1, BTT1) . We found the complex bound to ribosomes via the beta-subunits in a salt-sensitive manner, in close proximity to nascent polypeptides . Estimation of the molecular weight of the complex of wild-type cells and cells lacking one or two subunits revealed that the composition of the complex is variable and that as yet unknown proteins might be included . Regardless of the variability, a certain balance of the subunits has to be maintained: the deletion of one subunit causes downregulation of the remaining subunits at physiological growth temperature . Cells lacking both beta-subunits are unable to grow at 37 degrees C, most likely due to a toxic effect of the alpha-subunit . Based on in vitro experiments, it has been proposed that the function of mammalian nascent-polypeptide associated complexes (NAC) is to prevent inappropriate targeting of non-secretory nascent polypeptides . In vivo, however, the lack of NAC does not cause secretion of signal-less invertase in yeast . This result and the lack of a drastic phenotype of cells missing one, two or three subunits at optimal conditions (28 degrees C, YPD-medium) suggest either the existence of a substitute for NAC or that cells tolerate or 'repair' the damage caused by the absence of NAC. Proc Natl Acad Sci U S A, 1999 Apr 27, 96(9), 4989 - 94 Bifurcation of the mitotic checkpoint pathway in budding yeast; Li R; The coordination of mitotic events is ensured through the spindle assembly checkpoint . BFA1 is required for this checkpoint in budding yeast because its disruption abolishes the mitotic arrest when spindle assembly is inhibited . Analysis of the genetic interaction of BFA1 with known mitotic checkpoint genes suggest that Bfa1 functions in the same pathway with Bub2 but not with Mad1 or Mad2 . Both Bfa1 and Bub2 localize to spindle poles, and overexpression of Bfa1 arrests the cell cycle in anaphase . These findings suggest a bifurcation of the spindle assembly checkpoint: whereas one branch of the pathway, consisting of Mad1-3, Bub1 and 3, and Mps1, may prevent premature disjunction of sister chromosomes, the other, consisting of Bfa1 and Bub2, may function at spindle poles to prevent cytokinesis before the completion of chromosome segregation. Proc Natl Acad Sci U S A, 1999 Apr 27, 96(9), 4868 - 73 Three proteins define a class of human histone deacetylases related to yeast Hda1p; Grozinger CM et al.; Gene expression is in part controlled by chromatin remodeling factors and the acetylation state of nucleosomal histones . The latter process is regulated by histone acetyltransferases and histone deacetylases (HDACs) . Previously, three human and five yeast HDAC enzymes had been identified . These can be categorized into two classes: the first class represented by yeast Rpd3-like proteins and the second by yeast Hda1-like proteins . Human HDAC1, HDAC2, and HDAC3 proteins are members of the first class, whereas no class II human HDAC proteins had been identified . The amino acid sequence of Hda1p was used to search the GenBank/expressed sequence tag databases to identify partial sequences from three putative class II human HDAC proteins . The corresponding full-length cDNAs were cloned and defined as HDAC4, HDAC5, and HDAC6 . These proteins possess certain features present in the conserved catalytic domains of class I human HDACs, but also contain additional sequence domains . Interestingly, HDAC6 contains an internal duplication of two catalytic domains, which appear to function independently of each other . These class II HDAC proteins have differential mRNA expression in human tissues and possess in vitro HDAC activity that is inhibited by trichostatin A . Coimmunoprecipitation experiments indicate that these HDAC proteins are not components of the previously identified HDAC1 and HDAC2 NRD and mSin3A complexes . However, HDAC4 and HDAC5 associate with HDAC3 in vivo . This finding suggests that the human class II HDAC enzymes may function in cellular processes distinct from those of HDAC1 and HDAC2. Proc Natl Acad Sci U S A, 1999 Apr 27, 96(9), 4862 - 7 Protein footprinting at cysteines: probing ATP-modulated contacts in cysteine-substitution mutants of yeast DNA topoisomerase II; Tu BP et al.; Cysteine-substitution mutants of yeast DNA topoisomerase II were used to test footprinting of the enzyme by 2-nitro-5-thiocyanobenzoate, which cyanylates exposed cysteines in a native protein for peptide cleavage at the cyanylated sites upon unfolding and incubating the protein at pH 9 . For a mutant enzyme containing a single cysteine, the extent of peptide cleavage was found to reflect the accessibility of the residue in the native protein . For proteins with multiple cysteines, however, such a correlation was obscured by the transfer of cyano groups from modified to unmodified cysteines during incubation of the unfolded protein at pH 9; accessibilities of the cysteinyl residues in a native protein could be assessed only if cyano shuffling was prevented by blocking uncyanylated sulfhydryls with a second thiol reagent . The successive use of two reagents in cysteine footprinting was applied in probing the ATP-modulated formation of contacts in yeast DNA topoisomerase II. Nucleic Acids Res, 1999 May 15, 27(10), 2126 - 34 A role for Ctr9p and Paf1p in the regulation G1 cyclin expression in yeast; Koch C et al.; Entry into the cell cycle in budding yeast involves transcriptional activation of G1cyclin genes and DNA synthesis genes when cells reach a critical size in late G1 . Expression of G1cyclins CLN1 and CLN2 is regulated by the transcription factor SBF (composed of Swi4p and Swi6p) and depends on the cyclin-dependent Cdc28 protein kinase and cyclin Cln3p . To identify novel regulators of SBF-dependent gene expression we screened for mutants that fail to activate transcription of G1cyclins . We found mutations in a gene called CTR9 . ctr9 mutants are inviable at 37 degrees C and accumulate large cells . CTR9 is identical to CDP1 . CTR9 encodes a conserved nuclear protein of 125 kDa containing several TPR repeats implicated in protein-protein interactions . We show that Ctr9p is a component of a high molecular weight protein complex . Using immuno-affinity chromatography we found that Ctr9p associates with polypeptides of 50 and 65 kDa . By mass spectrometry these were identified as Cdc73p and Paf1p . We show that Paf1p, like Ctr9p, is required for efficient CLN2 transcription, whereas Cdc73p is not . Paf1p and Cdc73p were previously reported to be RNA poly-merase II-associated proteins, suggesting that the Ctr9p complex may interact with the general transcription apparatus. Nucleic Acids Res, 1999 May 15, 27(10), 2072 - 9 Formation of the yeast Mre11-Rad50-Xrs2 complex is correlated with DNA repair and telomere maintenance; Chamankhah M et al.; The yeast Mre11 is a multi-functional protein and is known to form a protein complex with Rad50 and Xrs2 . In order to elucidate the relationship between Mre11 complex formation and its mitotic functions, and to determine domain(s) required for Mre11 protein interactions, we performed yeast two-hybrid and functional analyses with respect to Mre11 DNA repair and telomere maintenance . Evidence presented in this study indicates that the N-terminal region of Mre11 constitutes the core homo-dimerization and hetero-dimerization domain and is sufficient for Mre11 DNA repair and maintaining the wild-type telomere length . In contrast, a stretch of 134 amino acids from the extreme C-terminus, although essential for achieving a full level of self-association, is not required for the aforementioned Mre11 mitotic functions . Interestingly, deletion of these same 134 amino acids enhanced the interaction of Mre11 with Rad50 and Xrs2, which is consistent with the notion that this region is specific for meiotic functions . While Mre11 self-association alone is insufficient to provide the above mitotic activities, our results are consistent with a strong correlation between Mre11-Rad50-Xrs2 complex formation, mitotic DNA repair and telomere maintenance . This correlation was further strengthened by analyzing two mre11 phosphoesterase motif mutants ( mre11-2 and rad58S ), which are defective in DNA repair, telomere maintenance and protein interactions, and a rad50S mutant, which is normal in both complex formation and mitotic functions . Together, these results support and extend a current model regarding Mre11 structure and functions in mitosis and meiosis. FEBS Lett, 1999 Mar 19, 447(1), 53 - 7 Imaging fluorescence resonance energy transfer between two green fluorescent proteins in living yeast; Sagot I et al.; We show that fluorescence resonance energy transfer between two mutants of the green fluorescent protein (GFP) can be monitored by imaging microscopy in living yeast . This work is based on the constitutive expression of a GFP-containing fusion protein and the inducible expression of the tobacco etch virus (TEV) protease . In the fusion protein, the P4.3 GFP mutant is linked to the YS65T GFP mutant by a spacer bearing the TEV protease-specific cleavage site. Microbiology, 1999 Mar, 145 ( Pt 3), 703 - 14 Novel alleles of yeast hexokinase PII with distinct effects on catalytic activity and catabolite repression of SUC2; Hohmann S et al.; In the yeast Saccharomyces cerevisiae, glucose or fructose represses the expression of a large number of genes . The phosphorylation of glucose or fructose is catalysed by hexokinase PI (Hxk1), hexokinase PII (Hxk2) and a specific glucokinase (Glk1) . The authors have shown previously that either Hxk1 or Hxk2 is sufficient for a rapid, sugar-induced disappearance of catabolite-repressible mRNAs (short-term catabolite repression) . Hxk2 is specifically required and sufficient for long-term glucose repression and either Hxk1 or Hxk2 is sufficient for long-term repression by fructose . Mutants lacking the TPS1 gene, which encodes trehalose 6-phosphate synthase, can not grow on glucose or fructose . In this study, suppressor mutations of the growth defect of a tps1delta hxk1delta double mutant on fructose were isolated and identified as novel HXK2 alleles . All six alleles studied have single amino acid substitutions . The mutations affected glucose and fructose phosphorylation to a different extent, indicating that Hxk2 binds glucose and fructose via distinct mechanisms . The mutations conferred different effects on long- and short-term repression . Two of the mutants showed very similar defects in catabolite repression, despite large differences in residual sugar-phosphorylation activity . The data show that the long- and short-term phases of catabolite repression can be dissected using different hexokinase mutations . The lack of correlation between in vitro catalytic hexokinase activity, in vivo sugar phosphate accumulation and the establishment of catabolite repression suggests that the production of sugar phosphate is not the sole role of hexokinase in repression . Using the set of six hxk2 mutants it was shown that there is a good correlation between the glucose-induced cAMP signal and in vivo hexokinase activity . There was no correlation between the cAMP signal and the short- or long-term repression of SUC2, arguing against an involvement of cAMP in either stage of catabolite repression. FEBS Lett, 1999 Apr 1, 448(1), 57 - 61 Recombinant human uncoupling protein-3 increases thermogenesis in yeast cells; Hinz W et al.; The long form of human uncoupling protein-3 (hUCP3L) is highly homologous to thermogenin (UCPI), the uncoupling protein of brown fat mitochondria, but is expressed predominantly in skeletal muscle . Its putative role is to regulate the coupling efficiency of oxidative phosphorylation and thus thermogenesis in skeletal muscle, a major thermogenic tissue in higher mammals . To study the functional relevance of hUCP3L, the protein was expressed in yeast cells under the control of the galactose promoter . Expression of hUCP3L induced a series of phenotype changes in the yeast cells . The cellular growth and the mitochondrial membrane potential were both diminished . The portion of cellular respiration coupled to oxidative phosphorylation decreased from 57% to 11% (P<0.001) and the cellular heat production, as measured by direct microcalorimetry, was increased by 33.3 +/- 3.2% (P<0.001) after induction of UCP3L . These observations demonstrate for the first time the intrinsic thermogenic properties of hUCP3L in intact cells. Int J Biochem Cell Biol, 1999 Jan, 31(1), 163 - 73 Functional interaction of yeast elongation factor 3 with yeast ribosomes; Chakraburtty K; Elongation factor 3 (EF-3) is a unique and essential requirement of the fungal translational apparatus . EF-3 is a monomeric protein with a molecular mass of 116,000 . EF-3 is required by yeast ribosomes for in vitro translation and for in vivo growth . The protein stimulates the binding of EF-1 alpha :GTP:aa-tRNA ternary complex to the ribosomal A-site by facilitating release of deacylated-tRNA from the E-site . The reaction requires ATP hydrolysis . EF-3 contains two ATP-binding sequence motifs (NBS) . NBSI is sufficient for the intrinsic ATPase function . NBSII is essential for ribosome-stimulated activity . By limited proteolysis, EF-3 was divided into two distinct functional domains . The N-terminal domain lacking the highly charged lysine blocks failed to bind ribosomes and was inactive in the ribosome-stimulated ATPase activity . The C-terminally derived lysine-rich fragment showed strong binding to yeast ribosomes . The purported S5 homology region of EF-3 at the N-terminal end has been reported to interact with 18S ribosomal RNA . We postulate that EF-3 contacts rRNA and/or protein(s) through the C-terminal end . Removal of these residues severely weakens its interaction mediated possibly through the N-terminal domain of the protein. Eur J Biochem, 1999 Apr, 261(2), 444 - 51 Bioenergetic and structural consequences of allotopic expression of subunit 8 of yeast mitochondrial ATP synthase . The hydrophobic character of residues 23 and 24 is essential for maximal activity and structural stability of the enzyme complex; Roucou X et al.; Subunit 8 (Y8), a mitochondrially encoded subunit of the F0 sector of the F1F0-ATP synthase is essential for oxidative phosphorylation . We have previously introduced the technique of allotopic expression to study the structure/function of Y8, whereby an artificial Y8 gene is expressed in the nucleus of cells lacking a functional mitochondrial Y8, thus generating assembly of a functional F1F0-ATPase complex . In this paper we show that when a gene encoding an essentially unmodified version of Y8 is allotopically expressed, ATP synthesis and hydrolysis rates, as well as efficiency of oxidative phosphorylation, were similar to those of the parental wild-type strain in which Y8 is naturally expressed in mitochondria . We then tested the requirement for the hydrophobicity of the central domain (residues 14-32), which possibly represents a transmembrane stem, by introducing adjacent negative charges at different positions of Y8 . One of the variants thus generated, which carries the double substitution Leu23-->Asp, Leu24-->Asp, when expressed in a strain lacking endogenous Y8, gave rise to cells which grew very slowly by oxidative phosphorylation . Measurement of bioenergetic parameters showed two major defects in these cells relative to control cells allotopically expressing unmodified Y8 . First, the activity of the F1F0-ATP synthase was significantly decreased . ATP synthesis and state 3 of respiration were reduced by approximately 30-40% . ATP hydrolysis was reduced by approximately 30% and was almost insensitive to the F0 inhibitor oligomycin . Second, the physical coupling between the two sectors of the enzyme, as well as the stability of the F1 sector itself, were affected as shown by decreased recovery of F0 sector {8, 9, b, oligomycin sensitivity-conferring protein (OSCP), d, h and f} and F1 sector (alpha, gamma, delta) subunits in immunoprecipitates of ATP synthase . This study indicates that Y8 not only performs an important role in the structure of the mitochondrial complex but also in its activity . We conclude that the hydrophobic character of amino acids 23 and 24 in the middle of the putative transmembrane stem of Y8 is essential for coupling proton transport through F0 to ATP synthesis on F1. Eur J Biochem, 1999 Apr, 261(2), 371 - 8 Antibodies to yeast Sm motif 1 cross-react with human Sm core polypeptides; Bahia D et al.; Two regions common to all UsnRNP core polypeptides have been described: Sm motif 1 and Sm motif 2 . Rabbits were immunized with a 22 amino-acid peptide containing one segment of Sm motif 1 (YRGTLVSTDNYFNLQLNEAEEF, corresponding to residues 11-32) from yeast F protein . After immunization, the rabbit sera contained antibodies that not only reacted specifically with the peptide from yeast F protein but also cross-reacted with Sm polypeptides from mammals; that is, with purified human U1snRNPs . The results suggest that the peptide used and human Sm polypeptides contain a common feature recognized by the polyclonal antibodies . A large collection of human systemic lupus erythematosus sera was assayed using the yeast peptide as an antigen source . Seventy per cent of systemic lupus erythematosus sera contain an antibody specificity that cross-reacts with the yeast peptide. FEBS Lett, 1999 Mar 26, 447(2-3), 247 - 50 Hypothetical protein KIAA0079 is a mammalian homologue of yeast Sec24p; Tani K et al.; The Sec23p-Sec24p complex is a component of COPII-coated vesicles that mediate protein transport from the endoplasmic reticulum in yeast . The mammalian hypothetical protein KIAA0079 (KIAA0079p) exhibits sequence similarity to yeast Sec24p . KIAA0079p was co-eluted with mammalian Sec23p on gel filtration . In vitro binding experiments revealed that the C-terminal region of KIAA0079p binds to the N-terminal region of mammalian Sec23p . Overexpression of KIAA0079p caused a defect in protein export from the endoplasmic reticulum . These results support the idea that KIAA0079p is a functional homologue of yeast Sec24p. Diagn Microbiol Infect Dis, 1999 Apr, 33(4), 223 - 9 Comparison of three commercial systems for the identification of germ-tube negative yeast species isolated from clinical specimens; Sand C et al.; Three commercial systems were evaluated for their ability to identify 171 germ-tube negative yeasts isolated from clinical specimens . The Yeast Biochemical Card and Analytical Profile Index 20 AUX identified 97% of 171 strains tested . The Biolog system had poor clinical utility: only 48% of strains were identified . For Yeast Biochemical Card and Analytical Profile Index 20 AUX, 9% and 6%, respectively, required repeat testing and both systems required supplemental tests for 28% of the strains . These observations indicate that considerable expertise and a battery of reagents in addition to the basic systems are required for accurate identification of germ-tube negative yeasts. J Biol Chem, 1999 Apr 30, 274(18), 12201 - 4 Base excision repair of N-methylpurines in a yeast minichromosome . Effects of transcription, dna sequence, and nucleosome positioning; Li S et al.; Base excision repair of dimethyl sulfate induced N-methylpurines (NMPs) was measured in a yeast minichromosome that has a galactose-inducible GAL1:URA3 fusion gene, a constitutively expressed HIS3 gene, and varied regions of chromatin structure . Removal rates of NMPs varied dramatically (>20-fold) at different sites along three selected fragments encompassing a total length of 1775 base pairs . Repair of NMPs was not coupled to transcription, because the transcribed strands of HIS3 and induced GAL1:URA3 were not repaired faster than the nontranscribed strands . However, the repair rate of NMPs was significantly affected by the nearest neighbor nucleotides . Slow repair occurred at NMPs between purines, especially guanines, whereas fast repair occurred at NMPs between pyrimidines . NMPs between a purine and pyrimidine were repaired at moderate rates . Moreover, a rough correlation between nucleosome positions and repair rates exists in some but not all regions that were analyzed. J Cell Sci, 1999 May, 112 ( Pt 10), 1553 - 65 XAIP1: a Xenopus homologue of yeast actin interacting protein 1 (AIP1), which induces disassembly of actin filaments cooperatively with ADF/cofilin family proteins; Okada K et al.; We carried out affinity column chromatography using Xenopus ADF/cofilin (XAC), identified several polypeptides in oocytes specifically bound to this column with actin, and isolated a full-length cDNA clone for a 65 kDa protein in this fraction . The predicted amino acid sequence revealed that the 65 kDa protein has seven obvious WD repeats and exhibits striking homology with yeast actin interacting protein 1 (AIP1) . Thus, we designated this protein Xenopus AIP1 (XAIP1) . We purified XAIP1 from Xenopus oocytes, and its interaction with actin was characterized by a pelleting assay, photometrical analysis and electron microscopy . Although XAIP1 itself cosedimented with F-actin and increased unsedimented actin to some extent, it induced a rapid, drastic disassembly of actin filaments associated with XAC . Electron microscopic observation revealed that XAIP1 severs actin filaments in the presence of XAC . To elucidate the in vivo effects of XAIP1, the purified protein was injected into blastomeres at the two-cell stage . Although the localization of XAIP1 was similar to that of XAC, at the cortical cytoskeleton and diffusely in the cytoplasm, injection of a large amount of XAIP1 arrested development and abolished the strong cortical staining of both actin and XAC . From these results, we concluded that XAIP1 regulates the dynamics of the cortical actin cytoskeleton cooperatively with XAC in eggs. J Cell Sci, 1999 May, 112 ( Pt 10), 1511 - 8 Association of the class V myosin Myo4p with a localised messenger RNA in budding yeast depends on She proteins; Munchow S et al.; Asymmetric distribution of messenger RNAs is a widespread mechanism to localize synthesis of specific protein to distinct sites in the cell . Although not proven yet there is considerable evidence that mRNA localisation is an active process that depends on the activity of cytoskeletal motor proteins . To date, the only motor protein with a specific role in mRNA localisation is the budding yeast type V myosin Myo4p . Myo4p is required for the localisation of ASH1 mRNA, encoding a transcriptional repressor that is essential for differential expression of the HO gene and mating type switching in budding yeast . Mutations in Myo4p, in proteins of the actin cytoskeleton, and in four other specific genes, SHE2-SHE5 disrupt the daughter-specific localisation of ASH1 mRNA . In order to understand if Myo4p is directly participating in mRNA transport, we used in situ colocalisation and coprecipitation of Myo4p and ASH1 mRNA to test for their interaction . Our results indicate an association of Myo4p and ASH1 mRNA that depends on the activity of two other genes involved in ASH1 mRNA localisation, SHE2 and SHE3 . This strongly suggests a direct role of Myo4p myosin as a transporter of localised mRNAs, convincingly supporting the concept of motor-protein based mRNA localisation. Mol Microbiol, 1999 Mar, 31(6), 1835 - 44 The contribution of the O-glycosylated protein Pir2p/Hsp150 to the construction of the yeast cell wall in wild-type cells and beta 1,6-glucan-deficient mutants; Kapteyn JC et al.; The cell wall of yeast contains a major structural unit, consisting of a cell wall protein (CWP) attached via a glycosylphosphatidylinositol (GPI)-derived structure to beta 1,6-glucan, which is linked in turn to beta 1, 3-glucan . When isolated cells walls were digested with beta 1,6-glucanase, 16% of all CWPs remained insoluble, suggesting an alternative linkage between CWPs and structural cell wall components that does not involve beta 1,6-glucan . The beta 1,6-glucanase-resistant protein fraction contained the recently identified GPI-lacking, O-glycosylated Pir-CWPs, including Pir2p/Hsp150 . Evidence is presented that Pir2p/Hsp150 is attached to beta 1,3-glucan through an alkali-sensitive linkage, without beta 1,6-glucan as an interconnecting moiety . In beta 1,6-glucan-deficient mutants, the beta 1,6-glucanase-resistant protein fraction increased from 16% to over 80% . This was accompanied by increased incorporation of Pir2p/Hsp150 . It is argued that this is part of a more general compensatory mechanism in response to cell wall weakening caused by low levels of beta 1,6-glucan. Ultrasonics, 1999 Jan, 37(1), 79 - 83 Mathematical description of combined action of ultrasound and hyperthermia on yeast cells; Petin VG et al.; The inactivation of diploid yeast cells of Saccharomyces cerevisiae was studied after simultaneous treatment of ultrasound and hyperthermia . The existence of a definite temperature range was proved within which a synergistic interaction was determined . An optimal temperature that maximized the synergy could be identified . A simple mathematical model of synergistic interaction of damages produced by ultrasound and high temperature has been proposed . The model suggests that synergism is expected from the additional lethal damage arising from the interaction of sub-lesions induced by both agents . The model allows quantitative analysis of the combined action of two agents used to be performed, and predicts the greatest value of the synergistic effect and conditions under which it can be achieved. Biochim Biophys Acta, 1999 Apr 14, 1418(1), 147 - 57 Potassium ion efflux induced by cationic compounds in yeast; Enriquez-Freire E et al.; Potassium efflux in yeast induced by several cationic compounds showed different characteristics . All of the observed efflux required glucose as substrate at the concentrations used . For most of them, the phenomenon required binding of the cationic compound to the cell surface and increased with the negative cell surface charge, and for all the compounds tested, it depended on a metabolizable substrate . Efflux induced with terbium chloride appeared more likely due to the function of a K+/H+ antiporter . With DEAE-dextran and dihydrostreptomycin, potassium efflux was dependent on the cell potassium content and was also sensitive to osmotic changes of the medium . DEAE-dextran-provoked efflux was not due to cell disruption . Dihydrostreptomycin seemed to activate a potassium efflux system which could not be studied in isolation, but its inhibition of potassium uptake may also be involved . Except for cells treated with ethidium bromide, no appreciable cell disruption was observed . The potassium efflux observed appears to be a membrane phenomenon reversible after washing with magnesium chloride. Curr Biol, 1999 Apr 8, 9(7), 365 - 8 The Pds1 anaphase inhibitor and Mec1 kinase define distinct checkpoints coupling S phase with mitosis in budding yeast; Clarke DJ et al.; In most eukaryotic cells, DNA replication is confined to S phase of the cell cycle {1} . During this interval, S-phase checkpoint controls restrain mitosis until replication is complete {2} . In budding yeast, the anaphase inhibitor Pds1p has been associated with the checkpoint arrest of mitosis when DNA is damaged or when mitotic spindles have formed aberrantly {3} {4}, but not when DNA replication is blocked with hydroxyurea (HU) . Previous studies have implicated the protein kinase Mec1p in S-phase checkpoint control {5} . Unlike mec1 mutants, pds1 mutants efficiently inhibit anaphase when replication is blocked . This does not, however, exclude an essential S-phase checkpoint function of Pds1 beyond the early S-phase arrest point of a HU block . Here, we show that Pds1p is an essential component of a previously unsuspected checkpoint control system that couples the completion of S phase with mitosis . Further, the S-phase checkpoint comprises at least two distinct pathways . A Mec1p-dependent pathway operates early in S phase, but a Pds1p-dependent pathway becomes essential part way through S phase. Curr Biol, 1999 Mar 25, 9(6), 337 - 40 ASH1 mRNA localization in yeast involves multiple secondary structural elements and Ash1 protein translation; Gonzalez I et al.; Localization of ASH1 mRNA to the distal cortex of daughter but not mother cells at the end of anaphase is responsible for the two cells' differential mating-type switching during the subsequent cell cycle . This localization depends on actin filaments and a type V myosin (She1/Myo4) . The 3' untranslated region (3' UTR) of ASH1 mRNA is reportedly capable of directing heterologous RNAs to a mother cell's bud {1} {2} . Surprisingly, however, its replacement has little or no effect on the localisation of ASH1 mRNA . We show here that, unlike all other known localization sequences that have been found in 3' UTRs, all the elements involved in ASH1 mRNA localization are located at least partly within its coding region . A 77 nucleotide region stretching from 7 nucleotides 5' to 67 nucleotides 3' of the stop codon of ASH1 mRNA is sufficient to localize mRNAs to buds; the secondary structure of this region, in particular two stems, is important for its localizing activity . Two regions entirely within coding sequences, both sufficient to localize green fluorescent protein (GFP) mRNA to growing buds, are necessary for ASH1 mRNA localization during anaphase . These three regions can anchor GFP mRNA to the distal cortex of daughter cells only inefficiently . The tight anchoring of ASH1 mRNA to the cortex of the daughter cell depends on translation of the carboxy-terminal sequences of Ash1 protein. Biochem Biophys Res Commun, 1999 Apr 21, 257(3), 871 - 3 Yeast one-hybrid assay identifies YY1 as a binding factor for a proacrosin promoter element; Schulten HJ et al.; The proacrosin gene is specifically expressed in the testis and encodes an acrosomal enzyme . Previously, footprint analyses have shown binding of nuclear extracts from testis and brain to a highly conserved 17 bp motif (F1 element: 5'-AACTTCAAAATGGCTCC/T-3') located in the proacrosin promoter . By using this DNA-element as a target in a yeast one-hybrid assay, a cDNA fragment coding for the C-terminal part of the transcription factor YY1 was isolated . The binding of YY1 to this F1 element was confirmed by immunocompetition in EMSA . Because putative YY1 binding sites were also found in the promoters of other testis-specific genes, the YY1 transcription factor could play an important role in testicular gene expression . Yeast, 1999 Mar 15, 15(4), 351 - 60 Basic phenotypic analysis of six novel yeast genes reveals two essential genes and one which affects the growth rate; Sanjuan R et al.; Phenotypic analysis was performed on six mutants of Saccharomyces cerevisiae deleted in one of the following open reading frames (ORFs), located on chromosome II: YBR254c, YBR255w, YBR257w, YBR258c, YBR259w and YBR266c . Disruption of the ORFs was carried out in the diploid strain FY1679 using the kanMX4 marker flanked by short sequences homologous to the target locus . Tetrad analysis following sporulation of the heterozygous disruptants showed that YBR254c and YBR257w are essential genes . YBR257w was later characterized and renamed POP4, its gene product being involved in 5.8S rRNA and tRNA processing (Chu et al., 1997) . The tetrad analysis performed for the heterozygous disruptant for YBR266c showed that two of the four viable spores gave colonies of smaller size, reflecting a slower growth rate . Growth analysis of the disruptant haploids confirmed this defect at 30 degrees C and also at 15 degrees C . However, complementation tests failed to confirm that the deletion of YBR266c is responsible for this growth defect . Growth analysis of the disruptant haploid ybr255w(delta) showed a slower growth rate on YPD and minimal medium at 15 degrees C . Finally, no phenotypic effect could be detected associated to the disruption of ORFs YBR258c and YBR259w. Mol Cell Biol, 1999 May, 19(5), 3588 - 99 Yeast mutants affecting possible quality control of plasma membrane proteins; Li Y et al.; Mutations gef1, stp22, STP26, and STP27 in Saccharomyces cerevisiae were identified as suppressors of the temperature-sensitive alpha-factor receptor (mutation ste2-3) and arginine permease (mutation can1(ts)) . These suppressors inhibited the elimination of misfolded receptors (synthesized at 34 degrees C) as well as damaged surface receptors (shifted from 22 to 34 degrees C) . The stp22 mutation (allelic to vps23 {M . Babst and S . Emr, personal communication} and the STP26 mutation also caused missorting of carboxypeptidase Y, and ste2-3 was suppressed by mutations vps1, vps8, vps10, and vps28 but not by mutation vps3 . In the stp22 mutant, both the mutant and the wild-type receptors (tagged with green fluorescent protein {GFP}) accumulated within an endosome-like compartment and were excluded from the vacuole . GFP-tagged Stp22p also accumulated in this compartment . Upon reaching the vacuole, cytoplasmic domains of both mutant and wild-type receptors appeared within the vacuolar lumen . Stp22p and Gef1p are similar to tumor susceptibility protein TSG101 and voltage-gated chloride channel, respectively . These results identify potential elements of plasma membrane quality control and indicate that cytoplasmic domains of membrane proteins are translocated into the vacuolar lumen. Mol Cell Biol, 1999 May, 19(5), 3338 - 48 Oxidative stress-induced destruction of the yeast C-type cyclin Ume3p requires phosphatidylinositol-specific phospholipase C and the 26S proteasome; Cooper KF et al.; The yeast UME3 (SRB11/SSN3) gene encodes a C-type cyclin that represses the transcription of the HSP70 family member SSA1 . To relieve this repression, Ume3p is rapidly destroyed in cells exposed to elevated temperatures . This report demonstrates that Ume3p levels are also reduced in cultures subjected to ethanol shock, oxidative stress, or carbon starvation or during growth on nonfermentable carbons . Of the three elements (RXXL, PEST, and cyclin box) previously shown to be required for heat-induced Ume3p destruction, only the cyclin box regulates Ume3p degradation in response to these stressors . The one exception observed was growth on nonfermentable carbons, which requires the PEST region . These findings indicate that yeast cells contain multiple, independent pathways that mediate stress-induced Ume3p degradation . Ume3p destruction in response to oxidative stress, but not to ethanol treatment, requires DOA4 and UMP1, two factors required for 26S proteasome activity . This result for the first time implicates ubiquitin-mediated proteolysis in C-type cyclin regulation . Similarly, the presence of a membrane stabilizer (sorbitol) or the loss of phosphatidylinositol-specific phospholipase C (PLC1) protects Ume3p from oxidative-stress-induced degradation . Finally, a ume3 null allele suppresses the growth defect of plc1 mutants in response to either elevated temperature or the presence of hydrogen peroxide . These results indicate that the growth defects observed in plc1 mutants are due to the failure to downregulate Ume3p . Taken together, these findings support a model in which Plc1p mediates an oxidative-stress signal from the plasma membrane that triggers Ume3p destruction through a Doa4p-dependent mechanism. Mol Cell Biol, 1999 May, 19(5), 3328 - 37 A novel mechanism of ion homeostasis and salt tolerance in yeast: the Hal4 and Hal5 protein kinases modulate the Trk1-Trk2 potassium transporter; Mulet JM et al.; The regulation of intracellular ion concentrations is a fundamental property of living cells . Although many ion transporters have been identified, the systems that modulate their activity remain largely unknown . We have characterized two partially redundant genes from Saccharomyces cerevisiae, HAL4/SAT4 and HAL5, that encode homologous protein kinases implicated in the regulation of cation uptake . Overexpression of these genes increases the tolerance of yeast cells to sodium and lithium, whereas gene disruptions result in greater cation sensitivity . These phenotypic effects of the mutations correlate with changes in cation uptake and are dependent on a functional Trk1-Trk2 potassium transport system . In addition, hal4 hal5 and trk1 trk2 mutants exhibit similar phenotypes: (i) they are deficient in potassium uptake; (ii) their growth is sensitive to a variety of toxic cations, including lithium, sodium, calcium, tetramethylammonium, hygromycin B, and low pH; and (iii) they exhibit increased uptake of methylammonium, an indicator of membrane potential . These results suggest that the Hal4 and Hal5 protein kinases activate the Trk1-Trk2 potassium transporter, increasing the influx of potassium and decreasing the membrane potential . The resulting loss in electrical driving force reduces the uptake of toxic cations and improves salt tolerance . Our data support a role for regulation of membrane potential in adaptation to salt stress that is mediated by the Hal4 and Hal5 kinases. J Biol Chem, 1999 Apr 23, 274(17), 11469 - 72 Identification and characterization of receptor for mammalian hepatopoietin that is homologous to yeast ERV1; Wang G et al.; Hepatopoietin (HPO) is a novel polypeptide mitogen specific for hepatocytes and hepatoma cell lines, which is derived from liver and supports its regeneration . To determine whether HPO acts via a receptor-based signal transduction, recombinant human hepatopoietin was labeled by iodination and used to characterize its binding activity by specific displacement test and Scatchard analysis in primarily cultured rat hepatocytes and human hepatoma Hep-G2 cells . The binding was saturable and specific because it was replaceable by HPO but not by epidermal growth factor, transforming growth factor-alpha, or insulin . Scatchard analysis indicated the presence of a single class of high affinity receptor with dissociation constant (Kd) of 2 and 0.7 pM, and a receptor density of about 10, 000 sites/cell and 55,000 sites/cell in the rat hepatocytes and human hepatoma cells, respectively . The Kd values were consistent with the half-maximum dose of HPO activity . Affinity cross-linking of the receptor with 125I-HPO revealed a polypeptide of molecular mass approximately 90 kDa by SDS-polyacrylamide gel electrophoresis . Thus, the molecular mass of the HPO receptor was calculated to be about 75 kDa . These data demonstrated the existence of an HPO receptor in hepatocytes and hepatoma cells, which may account for biological effect. Plant J, 1999 Mar, 17(5), 569 - 74 Potato StubSNF1 interacts with StubGAL83: a plant protein kinase complex with yeast and mammalian counterparts; Lakatos L et al.; StubSNF1 is a potato cDNA that encodes a protein kinase similar to the yeast SNF1 gene involved in transcriptional regulation of glucose-repressible genes . The yeast SNF1 functions in a complex with GAL83/SIP1/SIP2 and SNF4 proteins . We have used StubSNF1 as bait in a yeast two-hybrid system to screen for potato cDNAs encoding proteins that bind to StubSNF1 . Three overlapping cDNAs, two different in size, were isolated . DNA sequence analysis revealed that they were orthologues of the yeast GAL83/SIP1/SIP2 genes and their mammalian counterparts, AMPK beta-subunits . The direct interaction between the potato proteins StubGAL83 and StubSNF1 was shown by an in vitro binding assay . Southern and Northern hybridisations revealed that StubGAL83 exists in a low copy number in the potato genome and is highly (but organ-specifically) expressed in potato . In contrast, StubSNF1 possesses low transcript levels in each organ, except in flowers where high amounts of StubSNF1 mRNA could be detected . We demonstrate here that StubGAL83 can also interact with yeast SNF4 in a yeast two-hybrid system suggesting that plant SNF1 kinases may function in complexes similar to those detected in yeast and mammals. Plant J, 1999 Mar, 17(5), 501 - 10 Functional complementation of yeast vma1 delta cells by a plant subunit A homolog rescues the mutant phenotype and partially restores vacuolar H(+)-ATPase activity; Kim W et al.; The ability of a vacuolar H(+)-ATPase (V-ATPase) subunit homolog (subunit A) from plants to rescue the vma mutant phenotype of yeast was investigated as a first step towards investigating the structure and function of plant subunits in molecular detail . Heterologous expression of cotton cDNAs encoding near-identical isoforms of subunit A in mutant vma1 delta yeast cells successfully rescued the mutant vma phenotype, indicating that subunit A of plants and yeast have retained elements essential to V-ATPases during the course of evolution . Although vacuoles become acidified, the plant-yeast hybrid holoenzyme only partially restored V-ATPase activity (approximately 60%) in mutant yeast cells . Domain substitution of divergent N- or C-termini only slightly enhanced V-ATPase activity, whereas swapping both domains acted synergistically, increasing coupled ATP hydrolysis and proton translocation by approximately 22% relative to the native plant subunit . Immunoblot analysis indicated that similar amounts of yeast, plant or plant-yeast chimeric subunits are membrane-bound . These results suggest that subunit A terminal domains contain structural information that impact V-ATPase structure and function. EMBO J, 1999 Apr 15, 18(8), 2229 - 40 Role of the essential yeast protein PSU1 in p6anscriptional enhancement by the ligand-dependent activation function AF-2 of nuclear receptors; Gaudon C et al.; Nuclear receptors (NRs) can function as ligandinducible transregulators in both mammalian and yeast cells, indicating that important features of transcriptional control have been conserved throughout evolution . We report here the isolation and characterization of an essential yeast protein of unknown function, PSU1, which exhibits properties expected for a co-activator/mediator of the ligand-dependent activation function AF-2 present in the ligand-binding domain (LBD, region E) of NRs . PSU1 interacts in a ligand-dependent manner with the LBD of several NRs, including retinoic acid (RARalpha), retinoid X (RXRalpha), thyroid hormone (TRalpha), vitamin D3 (VDR) and oestrogen (ERalpha) receptors . Importantly, both in yeast and in vitro, these interactions require the integrity of the AF-2 activating domain . When tethered to a heterologous DNA-binding domain, PSU1 can activate transcription on its own . By using yeast reporter cells that express PSU1 conditionally, we show that PSU1 is required for transactivation by the AF-2 of ERalpha . Taken together these data suggest that in yeast, PSU1 is involved in ligand-dependent transactivation by NRs . Sequence analysis revealed that in addition to a highly conserved motif found in a family of MutT-related proteins, PSU1 contains several alpha-helical leucine-rich motifs sharing the consensus sequence LLxPhiL (x, any amino acid; Phi, hydrophobic amino acid) in regions that elicit either transactivation or NR-binding activity. Mol Microbiol, 1999 Mar, 31(5), 1499 - 511 Overexpression of yeast karyopherin Pse1p/Kap121p stimulates the mitochondrial import of hydrophobic proteins in vivo; Corral-Debrinski M et al.; During evolution, cellular processes leading to the transfer of genetic information failed to send all the mitochondrial genes into the nuclear genome . Two mitochondrial genes are still exclusively located in the mitochondrial genome of all living organisms . They code for two highly hydrophobic proteins: the apocytochrome b and the subunit I of cytochrome oxidase . Assuming that the translocation machinery could not efficiently transport long hydrophobic fragments, we searched for multicopy suppressors of this physical blockage . We demonstrated that overexpression of Pse1p/Kap121p or Kap123p, which belong to the superfamily of karyopherin beta proteins, facilitates the translocation of chimeric proteins containing several stretches of apocytochrome b fused to a reporter mitochondrial gene . The effect of PSE1/KAP121 overexpression (in which PSE1 is protein secretion enhancer 1) on mitochondrial import of the chimera is correlated with an enrichment of the corresponding transcript in cytoplasmic ribosomes associated with mitochondria . PSE1/KAP121 overexpression also improves the import of the hydrophobic protein Atm1p, an ABC transporter of the mitochondrial inner membrane . These results suggest that in vivo PSE1/KAP121 overexpression facilitates, either directly or indirectly, the co-translational import of hydrophobic proteins into mitochondria. Wien Med Wochenschr, 1999, 149(1), 13 - 8 {Effect if 6-week nutritional intervention with enzymatic yeast cells and antioxidants on exercise stress and antioxidant status}; Konig D et al.; The aim of the present study was to investigate the influence of a yeast cell preparation rich in antioxidant vitamins, antioxidant enzymes, trace elements and minerals on the exercise-induced stress reaction and antioxidant status . The study was carried out in a non-blinded, controlled design . After a 6 week pre-ingestion period, 9 highly endurance-trained athletes took the preparation twice daily for another 6 weeks . After each phase venous blood samples were drawn in the resting state after an overnight fast followed by a strenuous 15 km cross country race . 1 h after the exercise venous blood sampling was repeated . Compared to the pre-ingestion period, the following significant alterations could be detected after 6 weeks of ingestion: 1) In the resting state, soluble interleukin-2 receptor (p < 0.05) and plasma fibrinogen (p < 0.01) were shown to be lower whereas plasma fibronectin was increased (p < 0.01) . 2) 1 h after the race the significant difference in fibrinogen and fibronectin was confirmed . In addition myoglobin (p < 0.01) and CKMM3 (p < 0.05) as well as mangan superoxid dismutase (p < 0.05) were reduced . In conclusion, the 6-week ingestion-period resulted in an improvement of the systemic and muscular stress reaction . In addition, the decrease in mangan superoxide dismutase concentration can be interpreted as a sign of reduced free radical stress . However, these preliminary results have to be confirmed in larger trials in blinded designs . In addition, there seems to be a rationale for testing the effects of yeast-cell preparations in patients with chronic degenerative diseases as some of the investigated parameters are involved in the pathophysiology of such illnesses. Crit Rev Food Sci Nutr, 1999 Mar, 39(2), 189 - 202 Effect of beta-glucan from oats and yeast on serum lipids; Bell S et al.; Heart disease is the leading cause of death in the U.S . One way to reduce the risk of developing the disease is to lower serum cholesterol levels by making dietary changes . In addition to reducing intake of total fat, saturated fat, and dietary cholesterol, serum cholesterol can be further reduced by added fiber, especially from sources rich in beta-glucan . In this review, two sources of beta-glucan are described; one source is oats and the other yeast . Their chemical structures and physical properties are compared, and their effect on serum lipid levels is described . Oat beta-glucans are found in various breakfast cereals and snacks . Usually, several servings of these products are required to meet the Food and Drug Administration's claim of reducing the risk of heart disease . The yeast-derived fiber is a more concentrated source of beta-glucan than the oat product . It is currently being tested in a wide variety of food products. Nucleic Acids Res, 1999 May 1, 27(9), 2043 - 50 The yeast inositol-sensitive upstream activating sequence, UASINO, responds to nitrogen availability; Griac P et al.; The INO1 gene of yeast is expressed in logarithmically growing, wild-type cells when inositol is absent from the medium . However, the INO1 gene is repressed when inositol is present during logarithmic growth and it is also repressed as cells enter stationary phase whether inositol is present or not . In this report, we demonstrate that transient nitrogen limitation also causes INO1 repression . The repression of INO1 in response to nitrogen limitation shares many features in common with repression in response to the presence of inositol . Specifically, the response to nitrogen limitation is dependent upon the presence of a functional OPI1 gene product, it requires ongoing phosphatidylcholine biosynthesis and it is mediated by the repeated element, UASINO, found in the promoter of INO1 and other co-regulated genes of phospholipid biosynthesis . Thus, we propose that repression of INO1 in response to inositol and in response to nitrogen limitation occurs via a common mechanism that is sensitive to the status of ongoing phospholipid metabolism. Nucleic Acids Res, 1999 May 1, 27(9), 2022 - 8 A role for the yeast SWI/SNF complex in DNA replication; Flanagan JF et al.; The yeast SWI/SNF complex is required for expression of many genes and for the full functioning of several transcriptional activators . Genetic and biochemical studies indicate that SWI/SNF uses the energy of ATP hydrolysis to antagonize chromatin-mediated transcriptional repression . We have tested the possibility that SWI/SNF might also play a role in DNA replication . A mitotic minichromosome stability assay was used to investigate the replication efficiency of a variety of autonomous replication sequences (ARSs) in the presence and absence of SWI/SNF . The stability of minichromosomes that contain ARS1, ARS309 or ARS307 is not altered by lack of SWI/SNF, whereas the functioning of ARS121 is crippled when SWI/SNF is inactivated . The SWI/SNF dependence of ARS121 does not require the replication enhancer factor, ABF1, and thus, it appears to be a property of a minimal ARS121 origin . Likewise, a minimal derivative of ARS1 that lacks the ABF1 replication enhancer acquires SWI/SNF dependence . Replacing the ABF1 binding site at ARS1 with a binding site for the LexA-GAL4 chimeric activator also creates a SWI/SNF-dependent ARS . Our studies suggest that the SWI/SNF chromatin remodeling complex can play a role in both replication and transcription and, furthermore, that SWI/SNF dependence of ARS elements is a property of both an ARS-specific replication enhancer and the overall organization of ARS sequence elements. J Cell Sci, 1999 Apr, 112 ( Pt 7), 1085 - 92 Mitotic control in the absence of cdc25 mitotic inducer in fission yeast; Sveiczer A et al.; Fission yeast cells tolerate the total absence of the cdc25 mitotic inducer in two cases, either in cdc2-3w or in wee1 genetic backgrounds . In the cdc2-3w cdc25Delta double mutant, the rate-limiting step leading to mitosis is reaching a critical size . However, the size control of this mutant operates in late G2, which is different from wild-type (WT) cells . This fact suggests that in WT the rate-limiting molecular process during the G2 timer is the Tyr15 dephosphorylation of cdc2, for which the cdc25 phosphatase (together with its back-up, pyp3) is dependent . In the wee1-50 cdc25Delta mutant, the population splits into different clusters, all lacking mitotic size control . This strain maintains size homeostasis by a novel method, which is random movement of the cells from one cluster to another in the successive generations . These cells should normally have a 'minimal cycle', a 'timer' with short G1 and G2 phases . However, very often the cells abort mitosis, possibly at an early event and return back to early G2, thus lengthening their cycles . The inability of these cells to start anaphase might be caused by the absence of the main mitotic regulators (wee1 and cdc25) and the improper regulation of their back-up copies (mik1 and pyp3, respectively). Biochem Biophys Res Commun, 1999 Apr 13, 257(2), 561 - 6 The yeast Npi1/Rsp5 ubiquitin ligase lacking its N-terminal C2 domain is competent for ubiquitination but not for subsequent endocytosis of the gap1 permease; Springael JY et al.; The yeast ubiquitin ligase Npi1/Rsp5 and its mammalian homologue Nedd4 are involved in ubiquitination of various cell surface proteins, these being subsequently internalized by endocytosis and degraded in the vacuole/lysosome . Both enzymes consist of an N-terminal C2 domain, three to four successive WW(P) domains, and a C-terminal catalytic domain (HECT) containing a highly conserved cysteine residue involved in ubiquitin thioester formation . In this study, we show that the conserved cysteine of the HECT domain is required for yeast cell viability and for ubiquitination and subsequent endocytosis of the Gap1 permease . In contrast, the C2 domain of Npi1/Rsp5 is not essential to cell viability . Its deletion impairs internalization of Gap1, without detectably affecting ubiquitination of the permease . This suggests that Npi1/Rsp5 participates, via its C2 domain, in endocytosis of ubiquitinated permeases . Biochem Biophys Res Commun, 1999 Apr 13, 257(2), 273 - 8 Electrostatic interaction between cytochrome P450 and NADPH-P450 reductase: comparison of mixed and fused systems consisting of rat cytochrome P450 1A1 and yeast NADPH-P450 reductase; Kondo S et al.; The electrostatic interaction between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase was analyzed by using recombinant yeast microsomes containing both native enzymes or their fused enzyme . The Vmax of the 7-ethoxycoumarin O-deethylation in the recombinant microsomes containing both rat cytochrome P4501A1 and yeast NADPH-P450 reductase (the mixed system) was maximal when the ionic strength of the reaction mixture was 0.1-0.15 . However, on the fused enzyme between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase (the fused system), the activity was uniformly reduced with increasing ionic strength . The pH profiles of Vmax were also different between the mixed and the fused systems . Based on these results, we propose a hypothesis that cytochrome P450 and NADPH-P450 reductase have more than one binding mode . The maximal activity of the mixed system at ionic strength of 0.1-0.15 is explained by change of the binding mode . On the other hand, the fused enzyme appears to have only one binding mode due to the limited topology of cytochrome P450 and NADPH-P450 reductase domains . Plant Physiol, 1999 Apr, 119(4), 1527 - 34 An Arabidopsis GSK3/shaggy-like gene that complements yeast salt stress-sensitive mutants is induced by NaCl and abscisic acid; Piao HL et al.; GSK3/shaggy-like genes encode kinases that are involved in a variety of biological processes . By functional complementation of the yeast calcineurin mutant strain DHT22-1a with a NaCl stress-sensitive phenotype, we isolated the Arabidopsis cDNA AtGSK1, which encodes a GSK3/shaggy-like protein kinase . AtGSK1 rescued the yeast calcineurin mutant cells from the effects of high NaCl . Also, the AtGSK1 gene turned on the transcription of the NaCl stress-inducible PMR2A gene in the calcineurin mutant cells under NaCl stress . To further define the role of AtGSK1 in the yeast cells we introduced a deletion mutation at the MCK1 gene, a yeast homolog of GSK3, and examined the phenotype of the mutant . The mck1 mutant exhibited a NaCl stress-sensitive phenotype that was rescued by AtGSK1 . Also, constitutive expression of MCK1 complemented the NaCl-sensitive phenotype of the calcineurin mutants . Therefore, these results suggest that Mck1p is involved in the NaCl stress signaling in yeast and that AtGSK1 may functionally replace Mck1p in the NaCl stress response in the calcineurin mutant . To investigate the biological function of AtGSK1 in Arabidopsis we examined the expression of AtGSK1 . Northern-blot analysis revealed that the expression is differentially regulated in various tissues with a high level expression in flower tissues . In addition, the AtGSK1 expression was induced by NaCl and exogenously applied ABA but not by KCl . Taken together, these results suggest that AtGSK1 is involved in the osmotic stress response in Arabidopsis. Mol Biol Cell, 1999 Apr, 10(4), 1077 - 92 The Yck2 yeast casein kinase 1 isoform shows cell cycle-specific localization to sites of polarized growth and is required for proper septin organization; Robinson LC et al.; Casein kinase 1 protein kinases are ubiquitous and abundant Ser/Thr-specific protein kinases with activity on acidic substrates . In yeast, the products of the redundant YCK1 and YCK2 genes are together essential for cell viability . Mutants deficient for these proteins display defects in cellular morphogenesis, cytokinesis, and endocytosis . Yck1p and Yck2p are peripheral plasma membrane proteins, and we report here that the localization of Yck2p within the membrane is dynamic through the cell cycle . Using a functional green fluorescent protein (GFP) fusion, we have observed that Yck2p is concentrated at sites of polarized growth during bud morphogenesis . At cytokinesis, GFP-Yck2p becomes associated with a ring at the bud neck and then appears as a patch of fluorescence, apparently coincident with the dividing membranes . The bud neck association of Yck2p at cytokinesis does not require an intact septin ring, and septin assembly is altered in a Yck-deficient mutant . The sites of GFP-Yck2p concentration and the defects observed for Yck-deficient cells together suggest that Yck plays distinct roles in morphogenesis and cytokinesis that are effected by differential localization. Mol Biol Cell, 1999 Apr, 10(4), 1001 - 17 The tail of a yeast class V myosin, myo2p, functions as a localization domain; Reck-Peterson SL et al.; Myo2p is a yeast class V myosin that functions in membrane trafficking . To investigate the function of the carboxyl-terminal-tail domain of Myo2p, we have overexpressed this domain behind the regulatable GAL1 promoter (MYO2DN) . Overexpression of the tail domain of Myo2p results in a dominant-negative phenotype that is phenotypically similar to a temperature-sensitive allele of myo2, myo2-66 . The tail domain of Myo2p is sufficient for localization at low- expression levels and causes mislocalization of the endogenous Myo2p from sites of polarized cell growth . Subcellular fractionation of polarized, mechanically lysed yeast cells reveals that Myo2p is present predominantly in a 100,000 x g pellet . The Myo2p in this pellet is not solubilized by Mg++-ATP or Triton X-100, but is solubilized by high salt . Tail overexpression does not disrupt this fractionation pattern, nor do mutations in sec4, sec3, sec9, cdc42, or myo2 . These results show that overexpression of the tail domain of Myo2p does not compete with the endogenous Myo2p for assembly into a pelletable structure, but does compete with the endogenous Myo2p for a factor that is necessary for localization to the bud tip. Mol Biol Cell, 1999 Apr, 10(4), 875 - 90 Distinct domains within Vps35p mediate the retrieval of two different cargo proteins from the yeast prevacuolar/endosomal compartment; Nothwehr SF et al.; Resident membrane proteins of the trans-Golgi network (TGN) of Saccharomyces cerevisiae are selectively retrieved from a prevacuolar/late endosomal compartment . Proper cycling of the carboxypeptidase Y receptor Vps10p between the TGN and prevacuolar compartment depends on Vps35p, a hydrophilic peripheral membrane protein . In this study we use a temperature-sensitive vps35 allele to show that loss of Vps35p function rapidly leads to mislocalization of A-ALP, a model TGN membrane protein, to the vacuole . Vps35p is required for the prevacuolar compartment-to-TGN transport of both A-ALP and Vps10p . This was demonstrated by phenotypic analysis of vps35 mutant strains expressing A-ALP mutants lacking either the retrieval or static retention signals and by an assay for prevacuolar compartment-to-TGN transport . A novel vps35 allele was identified that was defective for retrieval of A-ALP but functional for retrieval of Vps10p . Moreover, several other vps35 alleles were identified with the opposite characteristics: they were defective for Vps10p retrieval but near normal for A-ALP localization . These data suggest a model in which distinct structural features within Vps35p are required for associating with the cytosolic domains of each cargo protein during the retrieval process. Genes Dev, 1999 Apr 1, 13(7), 798 - 803 Yeast calcineurin regulates nuclear localization of the Crz1p transcription factor through dephosphorylation; Stathopoulos-Gerontides A et al.; Calcineurin, a Ca2+/calmodulin dependent protein phosphatase, regulates Ca2+-dependent processes in a wide variety of cells . In the yeast, Saccharomyces cerevisiae, calcineurin effects Ca2+-dependent changes in gene expression through regulation of the Crz1p transcription factor . We show here that calcineurin dephosphorylates Crz1p and that this results in translocation of Crz1p to the nucleus . We identify a region of Crz1p that is required for calcineurin-dependent regulation of its phosphorylation, localization, and activity, and show that this region has significant sequence simlarity to a portion of NF-AT, a family of mammalian transcription factors whose localization is also regulated by calcineurin . Thus, the mechanism of Ca2+/calcineurin-dependent signaling shows remarkable conservation between yeast and mammalian cells. Urology, 1999 Apr, 53(4), 835 - 42 Now what do we do with all these genes? Yeast-based two-hybrid analysis: an emerging technology for molecular urology in the post-genome era; Gaston SM et al.; By the year 2005, the Human Genome Project is expected to have mapped and sequenced all of the estimated 100,000 genes that encode the various proteins found in human cells . Defining the role of each gene, and using that information to redirect its action when therapeutic intervention is required, is one of the major assignments for molecular medicine and molecular urology in the post-genome era . This challenge to determine gene function, and to do it cost-effectively and on a large scale, has driven development of new technologies that can more efficiently flag genes that are likely targets for therapeutic intervention . Yeast-based genetic assays that detect protein-protein interactions in vivo offer many of the features required of a practical "gene-flagging" strategy for identifying genes that might be functionally manipulated to achieve therapeutic goals . In the past few years, the yeast-based assays collectively referred to as "two-hybrid interaction traps" or simply "two-hybrid systems" have become increasingly important tools for experimental analysis of gene function . This review presents an overview of the principles of yeast-based two-hybrid analysis, examines some specific applications of the technique of interest to urologic investigators, and discusses some key points that a basic urologic investigator new to the technology would want to consider when designing or evaluating a yeast-based two-hybrid project. Biol Chem, 1999 Feb, 380(2), 147 - 50 Being at the right place at the right time: the role of nuclear transport in dynamic transcriptional regulation in yeast; Gorner W et al.; As a consequence of compartmentalization in eukaryotic cells, the transcriptional response to extracellular stimuli requires signals to be transmitted from the cytoplasm to the nucleus . Thus, nuclear import and export of signal transduction and transcription factors provide an important level of transcriptional control in eukaryotes . Recent investigations have illustrated the importance of this control for dynamic gene expression, as will be discussed on transcriptional activators and repressors (Mac1p, Mig1p, Msn2p/4p, Swi5p) and protein kinases (Hog1p, Sty1/Spc1, protein kinase A). J Cell Sci, 1999 May, 112 ( Pt 9), 1313 - 24 nimO, an Aspergillus gene related to budding yeast Dbf4, is required for DNA synthesis and mitotic checkpoint control; James SW et al.; The nimO predicted protein of Aspergillus nidulans is related structurally and functionally to Dbf4p, the regulatory subunit of Cdc7p kinase in budding yeast . nimOp and Dbf4p are most similar in their C-termini, which contain a PEST motif and a novel, short-looped Cys2-His2 zinc finger-like motif . DNA labelling and reciprocal shift assays using ts-lethal nimO18 mutants showed that nimO is required for initiation of DNA synthesis and for efficient progression through S phase . nimO18 mutants abrogated a cell cycle checkpoint linking S and M phases by segregating their unreplicated chromatin . This checkpoint defect did not interfere with other checkpoints monitoring spindle assembly and DNA damage (dimer lesions), but did prevent activation of a DNA replication checkpoint . The division of unreplicated chromatin was accelerated in cells lacking a component of the anaphase-promoting complex (bimEAPC1), consistent with the involvement of nimO and APC/C in separate checkpoint pathways . A nimO deletion conferred DNA synthesis and checkpoint defects similar to nimO18 . Inducible nimO alleles lacking as many as 244 C-terminal amino acids supported hyphal growth, but not asexual development, when overexpressed in a ts-lethal nimO18 strain . However, the truncated alleles could not rescue a nimO deletion, indicating that the C terminus is essential and suggesting some type of interaction among nimO polypeptides. Biotechnol Bioeng, 1998 Apr 20-May 5, 58(2-3), 321 - 4 Yeast cell permeabilizing beta-1,3-glucanases: A tool for the integration of downstream processes and metabolic engineering applications to yeast; Ferrer P et al.; In this article, we consider the impact on downstream process design resulting from the use of metabolically engineered yeast strains . We address the issue of how manipulation of cell wall permeability can improve the release and subsequent recovery of heterologous products produced in yeast . Mol Genet Metab, 1999 Apr, 66(4), 320 - 3 Studies of pH regulation by Btn1p, the yeast homolog of human Cln3p; Pearce DA et al.; Although the gene responsible for Batten disease, CLN3, was positionally cloned in 1995, the function of Cln3p and the molecular basis of the disease still remain elusive . We previously reported that the yeast Saccharomyces cerevisiae contains a homolog to Cln3p, designated Btn1p, and that the human Cln3p complemented the pH-dependent resistance to D-(-)-threo-2-amino-1-{p-nitrophenyl}-1, 3-propanediol in btn1-Delta yeast mutants . We have determined that yeast lacking Btn1p have an elevated ability to acidify media during growth that correlates with an elevated plasma membrane ATPase activity . Btn1p may be involved in maintaining pH homeostasis of yeast cells . Nucleic Acids Res . 1999 Apr 15;27(8):e1. Targeted deletions created in yeast vectors by recombinational excision Duno M, Bendixen C, Krejci L, Thomsen B. We have developed a simple method for creating defined deletions in yeast vectors by utilizing the ability of Saccharomyces cerevisiae to perform homologous recombination . Two complementary single-stranded oligonucleotides are designed so that the 5' and 3' halves of the resulting double-stranded oligonucleotide are homologous to the 5' and 3' side of a desired deletion junction, respectively . The sequence to be deleted is cleaved by restriction endonuclease digestion, followed by co-transformation of the linearized plasmid and the oligonucleotide into yeast . By homologous recombination in vivo, a subset of the plasmids will recircularize and simultaneously acquire the deletion as defined by the oligonucleotide. Cell Mol Life Sci, 1999 Feb, 55(2), 284 - 96 The cdk-activating kinase (CAK): from yeast to mammals; Kaldis P; Cell cycle progression is regulated by cyclin-dependent kinases (cdks) . The activity of cdks is tightly controlled by several mechanisms, including binding of subunits to cdks (cyclins and inhibitors), and phosphorylation events . This review focuses on the activating phosphorylation of cdks by an enzyme termed cdk-activating kinase (CAK) . Two classes of CAKs have been identified: monomeric Cak1p from budding yeast and the p40MO15 (cdk7)/cyclin H/MAT1 complex from vertebrates . Cak1p is the physiological CAK in budding yeast and localizes to the cytoplasm . p40MO15(cdk7)/cyclin H/MAT1 localizes to the nucleus, is a subunit of the general transcription factor IIH and activates cdks as well as phosphorylates several components of the transcriptional machinery . Functions, substrate specificities, regulation, localization, effects on cdk structure and involvement in transcription are compared for Cak1p and p40MO15(cdk7). J Burn Care Rehabil, 1999 Mar-Apr, 20(2), 155 - 62 Topical application of yeast extract accelerates the wound healing of diabetic mice; Crowe MJ et al.; Alcoholic extracts of yeast have been used as the active ingredient in medications under names such as "tissue or skin respiratory factor," Biodyne (Sperti Drug Co, Cincinnati, Ohio--now defunct), and live yeast cell derivative (LYCD) . Beneficial clinical results from the use of LYCD have been reported for the treatment of burns, wounds, and hemorrhoids . The medicinal effects of LYCD have recently been localized to a protein fraction containing a mixture of several peptides . The effects of topical application of the peptide mixture on wounds were examined in diabetic mice, an animal model in which the healing process is disrupted and delayed . Full-thickness wounds were created on the backs of diabetic (DB) and nondiabetic (non-DB) mice . Half of the DB and non-DB mice were treated with 0.05 mL of LYCD after wounding and for 4 successive days . All other mice received vehicle . Wound areas were measured at Day 0 and at 2-day intervals . Mice were sacrificed at 3, 7, 10, 21, and 28 days postinjury . Differences in the extent and quality of healing appeared between DB mice receiving LYCD and DB mice receiving vehicle by day 10 (P < .0001) . By 24 days postinjury, DB mice receiving LYCD had achieved 100% wound closure, whereas DB mice receiving vehicle had achieved only 31.4% wound closure . Histologic examination of wounds reflected improved wound healing in DB mice receiving LYCD as compared with those receiving vehicle . A topically applied yeast extract peptide mixture significantly attenuates wound closure and the degree of cellular reorganization of full-thickness excisional wounds of DB mice. Biochemistry (Mosc), 1999 Feb, 64(2), 213 - 6 Effect of 2,2'-dipyridyl on accumulation of protoporphyrin IX and its derivatives in yeast mitochondria and plasma membranes; Strakhovskaya MG et al.; The iron chelator 2,2'-dipyridyl (0.2 mM) more than fourfold increased the concentration of protoporphyrin IX and also of its zinc-containing complex in mito