FEBS Lett, 2003 Aug 14, 549(1-3), 135 - 40 The yeast deubiquitinating enzyme Ubp16 is anchored to the outer mitochondrial membrane; Kinner A et al.; We looked for membrane-associated Dubs (deubiquitinating enzymes) among the 16 yeast members of the ubiquitin-specific processing protease (Ubp) family to identify potential regulators of ubiquitin-dependent processes at membranes . For each of the Ubps examined, a certain fraction was found to be membrane associated . This fraction was only small for most Ubps but quite substantial for some Ubps . For Ubp4/Doa4 almost 40% of the protein was found in the membrane fraction suggesting that this protein performs a major function at membranes, probably at endosomes . Among the proteins tested, only one protein (Ubp16) was exclusively membrane associated . By cell fractionation and immunofluorescence experiments, we could show that Ubp16 is localized to mitochondria . Ubp16 contains an N-terminal hydrophobic domain that is similar to N-terminal sequences of other yeast outer mitochondrial membrane proteins . The presence of this putative signal sequence and the result of protease protection experiments suggest that Ubp16 is an integral membrane protein of the outer mitochondrial membrane with an N(in)-C(out) orientation . Phenotypic characterization of the Deltaubp16 mutant and overexpression studies further suggest that Ubp16 is probably not important for the general functioning of mitochondria, but that it rather performs a more specialized function at mitochondria. FEBS Lett, 2003 Aug 14, 549(1-3), 63 - 6 Functional homology between yeast piD261/Bud32 and human PRPK: both phosphorylate p53 and PRPK partially complements piD261/Bud32 deficiency; Facchin S et al.; Yeast piD261/Bud32 belongs to the piD261 family of atypical protein kinases structurally conserved, from Archaea to human . The disruption of its gene is causative of severely defective growth . Its human homologue, PRPK, interacts with and phosphorylates the oncosuppressor p53 protein, which is lacking in yeast . Here we show that on one hand piD261/Bud32 interacts with and phosphorylates human p53 in vitro, on the other hand PRPK can partially complement the phenotype of yeast lacking the gene encoding piD261/Bud32 . These data indicate that, despite considerable structural divergence, members of the piD261 family from distantly related organisms display a remarkable functional conservation. Traffic, 2003 Sep, 4(9), 607 - 17 Yeast functional analysis: identification of two essential genes involved in ER to Golgi trafficking; Belgareh-Touze N et al.; We screened for genes potentially involved in the secretory and vacuolar pathways a collection of 61 yeast strains, each bearing an essential orphan gene regulated by the tetO7-CYC1 promoter that can be down-regulated by doxycycline . After down-regulating the expression of these genes, we performed systematic Western blot analysis for markers of the secretory and vacuolar pathways that undergo post-translational modifications in their intracellular trafficking . Accumulation of protein precursors, revealed by Western immunoblot analysis, indicates defects in the secretory pathway or in associated biochemical modifications . After screening the whole collection, we identified two genes involved in ER to Golgi trafficking: RER2, a cis-prenyl transferase, and USE1, the function of which was unknown . We demonstrated that repression of USE1 also leads to BiP secretion, and therefore likely affects retrograde, in addition to anterograde, ER to Golgi trafficking . The collection also includes two essential genes involved in intracellular trafficking that were conveniently repressed without resulting growth or trafficking defects. Nat Biotechnol, 2003 Sep, 21(9), 1075 - 81 Epub 2003 Aug 10. Isolation of drugs active against mammalian prions using a yeast-based screening assay; Bach S et al.; We have developed a rapid, yeast-based, two-step assay to screen for antiprion drugs . The method allowed us to identify several compounds effective against budding yeast prions responsible for the {PSI+} and {URE3} phenotypes . These inhibitors include the kastellpaolitines, a new class of compounds, and two previously known molecules, phenanthridine and 6-aminophenanthridine . Two potent promoters of mammalian prion clearance in vitro, quinacrine and chlorpromazine, which share structural similarities with the kastellpaolitines, were also active in the assay . The compounds isolated here were also active in promoting mammalian prion clearance . These results validate the present method as an efficient high-throughput screening approach to identify new prion inhibitors and furthermore suggest that biochemical pathways controlling prion formation and/or maintenance are conserved from yeast to humans. Proc Natl Acad Sci U S A, 2003 Aug 19, 100(17), 9906 - 11 Epub 2003 Aug 08. ELG1, a yeast gene required for genome stability, forms a complex related to replication factor C; Ben-Aroya S et al.; Many overlapping surveillance and repair mechanisms operate in eukaryotic cells to ensure the stability of the genome . We have screened to isolate yeast mutants exhibiting increased levels of recombination between repeated sequences . Here we characterize one of these mutants, elg1 . Strains lacking Elg1p exhibit elevated levels of recombination between homologous and nonhomologous chromosomes, as well as between sister chromatids and direct repeats . These strains also exhibit increased levels of chromosome loss . The Elg1 protein shares sequence homology with the large subunit of the clamp loader replication factor C (RFC) and with the product of two additional genes involved in checkpoint functions and genome maintenance: RAD24 and CTF18 . Elg1p forms a complex with the Rfc2-5 subunits of RFC that is distinct from the previously described RFC-like complexes containing Rad24 and Ctf18 . Genetic data indicate that the Elg1, Ctf18, and Rad24 RFC-like complexes work in three separate pathways important for maintaining the integrity of the genome and for coping with various genomic stresses. J Biol Chem, 2003 Oct 24, 278(43), 42487 - 94 Epub 2003 Aug 08. Steroidogenic acute regulatory protein-binding protein cloned by a yeast two-hybrid system; Sugawara T et al.; Steroidogenic acute regulatory (StAR) protein plays a key role in the transport of cholesterol from the outer mitochondrial membrane to the inner membrane . A StAR mutant protein lacking the first 62 amino acids (N-62 StAR protein) has been reported to be as effective as wild-type StAR protein . In the present study, we examined the mechanism by which StAR protein stimulates steroidogenesis . A Gal4-based yeast two-hybrid system was used to identify proteins interacting with N-62 StAR protein . Nine positive clones were obtained from screening 1 x 106 clones . The results of pull-down assays and mammalian two-hybrid assays confirmed interaction between N-62 StAR protein and the clone 4 translated product . The clone 4 translated product was named StAR-binding protein (SBP) . We prepared an expression plasmid (pSBP) by inserting SBP cDNA into the pTarget vector . After cotransfection with the human cytochrome P450scc system, StAR expression vector, and pSBP, the amount of pregnenolone produced by COS-1 cells was increased . The amount of steroid hormones produced by steroidogenic cells subjected to small interfering RNA treatment was less than that produced by control cells . In conclusion, SBP binds StAR protein in cells and enhances the ability of StAR protein to promote syntheses of steroid hormones. Int J Radiat Oncol Biol Phys, 2003 Sep 1, 57(1), 79 - 83 Oropharyngeal candidiasis caused by non-albicans yeast in patients receiving external beam radiotherapy for head-and-neck cancer; Dahiya MC et al.; PURPOSE: To characterize non-albicans Candida oral infections in patients with head-and-neck cancer receiving external beam radiotherapy (EBRT) with or without concurrent chemotherapy . METHODS AND MATERIALS: Thirty-seven patients with head-and-neck cancer received EBRT in 2.0-Gy daily fractions to a median dose of 60.4 Gy (range 38-82.8, mean 64.6) . They were followed for oropharyngeal candidiasis (OPC) confirmed by positive examination, positive KOH smear, and/or positive swab or swish culture . Samples were identified and plated on chromogenic media to identify non-albicans yeasts . Colonies were plated on Sabouraud dextrose slants for microdilution antifungal susceptibility testing to fluconazole . DNA typing, including karyotyping, restriction fragment length polymorphism analysis, and Southern blot hybridization with the moderately repetitive Ca3 probe, was performed on selected isolates to confirm individual species . RESULTS: Of the 37 patients, 10 (27%) developed OPC, and 26 (70.3%) displayed Candida carriage state . The median EBRT dose at time of positive culture was 22.5 Gy and at time of OPC was 28.6 Gy . Of the 6 patients receiving chemotherapy and EBRT, 4 (66%) developed OPC at median dose of 27.6 Gy . Three (8%) of 37 patients were infected with non-albicans Candida, and 3 (30%) of all 10 infections were caused by these organisms . CONCLUSION: Non-albicans Candida is emerging as a relatively common cause of OPC in head-and-neck cancer patients . Chromogenic media are helpful to screen these infections . Our data also suggest a greater likelihood of developing OPC in patients receiving concomitant chemotherapy and EBRT. Nucleic Acids Res, 2003 Aug 15, 31(16), 4899 - 909 Sequence-structure-function relationships of Tgs1, the yeast snRNA/snoRNA cap hypermethylase; Mouaikel J et al.; The Saccharomyces cerevisiae Tgs1 methyltransferase (MTase) is responsible for conversion of the m(7)G caps of snRNAs and snoRNAs to a 2,2,7- trimethylguanosine structure . To learn more about the evolutionary origin of Tgs1 and to identify structural features required for its activity, we performed a structure-function study . By using sequence comparison and phylogenetic analysis, we found that Tgs1 shows strongest similarity to Mj0882, a protein related to a family comprised of bacterial rRNA:m(2)G MTases RsmC and RsmD . The structural information of Mj0882 was used to build a homology model of Tgs1p which allowed us to predict the range of the minimal globular MTase domain and the localization of other residues that may be important for enzyme function . To further characterize functional domains of Tgs1, mutants were constructed and tested for their effects on cell viability, subcellular localization and binding to the small nuclear ribonucleoproteins (snRNPs) and small nucleolar RNPs (snoRNPs) . We found that the N-terminal domain of the hypermethylase is dispensable for binding to the common snRNPs and snoRNPs proteins but essential for correct nucleolar localization . Site- directed mutagenesis of Tgs1 allowed also the identification of the residues likely to be involved in the formation of the m7G-binding site and the catalytic center. Microbiol Res, 2003, 158(2), 83 - 90 Prospore membrane formation: how budding yeast gets shaped in meiosis; Moreno-Borchart AC et al.; During meiosis in Saccharomyces cerevisiae four daughter cells, called spores, are generated within the boundaries of the mother cell . This cell differentiation process requires de novo synthesis of prospore membranes (PSMs), which are the precursors of the spore plasma membranes . Assembly of these membranes is initiated at the spindle pole bodies (SPBs) during meiosis II . At this stage of the cell cycle, 4 SPBs are present . Two different meiosis-specific structures are known to be required for PSM formation . At the SPBs, specialized attachments, called the meiotic plaques, provide the required functionality necessary for the recruitment and assembly of the membranes . During subsequent membrane elongation, a second structure becomes important . This proteinaceous assembly forms a coat, called the leading edge protein coat (LEP coat), which covers the boundaries of the membranes . Assembly of the coat occurs at sites next to the SPBs, whereas its disassembly is concomitant to the closure of the membranes . This mini review discusses our current understanding of how the meiotic plaque and the LEP coat might function during biogenesis of the prospore membrane. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2001 Apr, 23(2), 111 - 4 {Hunting for novel protein factors in G-protein pathway with yeast two-hybrid system}; Lu N et al.; OBJECTIVE: To explore the protein factors that could interact with G beta subunit within the G protein signal transducing pathway . METHODS: The highly sensitive protein-protein interaction system--Yeast Two-hybrid System was applied to screen the human cDNA library with constructed "Bite plasmid" containing G beta subunit gene fragment . And then the false positive test was adapted . RESULTS: Three positive gene fragments were obtained . One codes for "Actin bundling protein" . The other two are new ones and their Gene Bank accession numbers are AF288405 and AF288406 respectively . CONCLUSIONS: It is the first time to find that among human brain tissue, G beta subunit becomes a structural or functional unit interacts with actin bundling protein and the other two unknown protein factors to activate the following pathway . This result may be important to understand the relationship between the actin cytoskeleton and G proteins. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2002 Dec, 24(6), 582 - 7 {Study on the function of HSD-3.8 gene encoding a testis-specific protein with yeast two-hybrid system}; Lin W et al.; OBJECTIVE: To explore the protein factors that could interact with the testis-specific protein encoded by HSD-3.8 gene (GenBank Accession Number AF311312) related with female fertilization . METHODS: Yeast two-hybrid system was used to screen the human ovary MATCHMAKER cDNA library with constructed "bait plasmid" containing the 0.7 kb fragment (HSD-0.7) of HSD-3.8 . The interaction with the positive fragments using a series of truncated bait plasmids was investigated . RESULTS: One positive gene fragment was obtained, which coded for 144 amino acids of the C-terminus of human G protein beta subunit 1 . Truncated bait plasmids couldn't interact with the fish protein fragment in yeast . CONCLUSIONS: The protein encoded by HSD-3.8 gene may function through G protein signal transduction pathway and the interaction depends on the integration of the bait protein. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2000 Aug, 22(4), 388 - 91 {Yeast one-hybrid system--one effective method studying DNA-protein interaction}; Liao M et al.; Yeast one-hybrid system based on the principle of yeast two-hybrid system, is applied for isolating novel genes encoding proteins that bind to a target, cis-acting regulatory element . The system may also be applied to map the DNA-binding domain of previously known, or newly identified DNA-binding proteins . Yeast one-hybrid system offers maximal sensitivity because detection of the DNA-protein interaction occurs while proteins are in their native configurations . In addition, the gene encoding the DNA-binding proteins of interest is immediately available after a library screening . In this mini-review, we introduce the principle, the experimental procedures, the applications, and the merits of yeast one-hybrid system. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2000 Aug, 22(4), 317 - 21 {Screening the trans-action factors binding to the enhancer element of rat GST-P by yeast one-hybrid system}; Liao M et al.; OBJECTIVE: To investigate the diversity of the mechanisms of rat glutathione S-transferase P (GST-P) gene expression regulation and the relation between rat GST-P gene expression and carcinogenesis . To search the trans-action factors binding to the enhancer element of rat GST-P (glutathione S-transferase P enhancer, GPE I) . METHODS: We searched the trans-action factors binding to GPE I using yeast one-hybrid system, and analyzed the DNA sequence by sequencing and computer search . RESULTS: Two positive clones pYGPE1 and pYGPE2 were obtained . The homologies of the insert sequence of pYGPE1 nucleotide and amino acid sequences with rat c-jun cDNA were 99% and 100% respectively; the homologies of the insert sequence of pYGPE2 nucleotide and amino acid sequences with rat mitochondrial adenine nucleotide translocase cDNA were 99% and 100% respectively . CONCLUSIONS: Rat c-Jun and mitochondrial adenine nucleotide translocase can bind to the GPE I core sequence, and they perhaps are the trans-action factors of GPE I. Nucleic Acids Res Suppl, 2002, (2), 231 - 2 Effects of triplet repeat sequences on nucleosome positioning and gene expression in yeast minichromosomes; Tomita N et al.; Triplet repeat sequences that cause human hereditary diseases can form a variety of DNA conformations . Since DNA structures act as determinants of chromatin structure, chromatin may be involved in mechanisms of these diseases . To address this issue, we examined effects of triplet repeat sequences on chromatin structure and gene expression in Saccharomyces cerevisiae . We show here that (1) (CTG)12 promotes nucleosome formation, (2) (CGG)12 disrupts an array of positioned nucleosomes, and (3) (GAA)12 has little effect on nucleosome formation . Also, we show that insertion of (CGG)12 increases gene expression of a UAS-less promoter about 10-fold, while (CTG)12 and (GAA)12 have no effect . Thus, expansion of triplet repeat sequences may cause improper expression of disease related genes, through their effects on chromatin structure. Nucleic Acids Res Suppl, 2002, (2), 93 - 4 The role of nucleosome positioning in repression by the yeast alpha 2/Mcm1p repressor; Saito S et al.; In Saccharomyces cerevisiae, alpha 2/Mcm1p represses a-cell specific genes . Previous studies indicated that repression by alpha 2/Mcm1p is associated with organization of positioned nucleosomes in promoters of a-cell specific genes such as STE6 and BAR1 . Here, we examined the role of nucleosome positioning in repression of STE6-lacZ by alpha 2/Mcm1p . We showed that insertion of an A34 sequence, which is shown to disrupt an array of positioned nucleosomes, affects STE6-lacZ expression . Furthermore, artificial recruitment of TBP is facilitated by the insertion of A34 to derepress STE6-lacZ expression . These findings support the model that nucleosome positioning plays a regulatory role in repression by alpha 2/Mcm1p. Nucleic Acids Res Suppl, 2002, (2), 87 - 8 The role of deoxycytidyl transferase activity of yeast Rev1 protein in the bypass of abasic sites; Otsuka C et al.; We have studied the mutagenic specificity of abasic sites using the yeast oligonucleotides transformation assay . We introduced oligonucleotide containing a natural abasic site and a tetrahydrofuran abasic site into Rev1 mutants, rev1AA, which contains mutations of Asp467 and Glu468 residues of Rev1p to Ala in order to inactivate dCMP transferase activity, and rev1 delta, which lacks its whole coding sequence . The transformation efficiencies of rev1AA with abasic-containing oligonucleotides were lower than those of B7528, a strain proficient in REV1 gene, but much higher than rev1 delta mutant . Sequence analysis opposite the lesions showed that the mutation spectra were different among these three strains. J Biol Chem, 2003 Oct 17, 278(42), 40612 - 20 Epub 2003 Aug 05. A specific role of the yeast mitochondrial carriers MRS3/4p in mitochondrial iron acquisition under iron-limiting conditions; Muhlenhoff U et al.; The yeast genes MRS3 and MRS4 encode two members of the mitochondrial carrier family with high sequence similarity . To elucidate their function we utilized genome-wide expression profiling and found that both deletion and overexpression of MRS3/4 lead to up-regulation of several genes of the "iron regulon." We therefore analyzed the two major iron-utilizing processes, heme formation and Fe/S protein biosynthesis in vivo, in organello (intact mitochondria), and in vitro (mitochondrial extracts) . Radiolabeling of yeast cells with 55Fe revealed a clear correlation between MRS3/4 expression levels and the efficiency of these biosynthetic reactions indicating a role of the carriers in utilization and/or transport of iron in vivo . Similar effects on both heme formation and Fe/S protein biosynthesis were seen in organello using mitochondria isolated from cells grown under iron-limiting conditions . The correlation between MRS3/4 expression levels and the efficiency of the two iron-utilizing processes was lost upon detergent lysis of mitochondria . As no significant changes in the mitochondrial membrane potential were observed upon overexpression or deletion of MRS3/4, our results suggest that Mrs3/4p carriers are directly involved in mitochondrial iron uptake . Mrs3/4p function in mitochondrial iron transport becomes evident under iron-limiting conditions only, indicating that the two carriers do not represent the sole system for mitochondrial iron acquisition. Trends Genet, 2003 Aug, 19(8), 417 - 22 Evolution of cis-regulatory elements in duplicated genes of yeast; Papp B et al.; An increasing number of studies report that functional divergence in duplicated genes is accompanied by gene expression changes, although the evolutionary mechanism behind this process remains unclear . Our genomic analysis on the yeast Saccharomyces cerevisiae shows that the number of shared regulatory motifs in the duplicates decreases with evolutionary time, whereas the total number of regulatory motifs remains unchanged . Moreover, genes with numerous paralogs in the yeast genome do not have especially low number of regulatory motifs . These findings indicate that degenerative complementation is not the sole mechanism behind expression divergence in yeast . Moreover, we found some evidence for the action of positive selection on cis-regulatory motifs after gene duplication . These results suggest that the evolution of functional novelty has a substantial role in yeast duplicate gene evolution. Biochemistry, 2003 Aug 12, 42(31), 9431 - 7 Yeast pol eta holds a cis-syn thymine dimer loosely in the active site during elongation opposite the 3'-T of the dimer, but tightly opposite the 5'-T; Sun L et al.; Polymerase eta is a member of the Y family of DNA polymerases which is able to bypass thymine dimers efficiently and in a relatively error-free manner . To elucidate the mechanism of dimer bypass, the efficiency of dAMP and pyrene nucleotide insertion opposite the thymine dimer and its N3-methyl derivatives was determined . Pol eta inserts pyrene nucleotide with greater efficiency than dAMP opposite the 3'-T of an undimerized or dimerized T and is an effective inhibitor of DNA synthesis by pol eta . Substitution of the N3H of the 3'-T of an undimerized T or a dimerized T with a methyl group has little effect on the insertion efficiency of pyrene nucleotide but greatly inhibits the insertion of dAMP . Together, these results suggest that the error-free insertion of dAMP opposite the 3'-T of the cis-syn thymine dimer happens by way of a loosely held dimer in the active site which can be displaced from the active site by pyrene nucleotide . In contrast, pol eta cannot insert pyrene nucleotide opposite the 5'-T of the dimer, whereas it can insert dAMP with efficiency comparable to that opposite the 3'-T . The inability to insert pyrene nucleotide opposite the 5'-T of the dimer is consistent with the idea that while the polymerase binds loosely to a templating nucleotide, it binds tightly to the nucleotide to its 3'-side . Overall, the results show a marked difference from similar studies on pol I family polymerases, and suggest mechanisms by which this Y family polymerase can process damaged DNA efficiently. Yeast, 2003 Aug, 20(11), 985 - 93 New 'marker swap' plasmids for converting selectable markers on budding yeast gene disruptions and plasmids; Voth WP et al.; Marker swap plasmids can be used to change markers for genes disrupted with nutritional markers in the yeast Saccharomyces cerevisiae . We describe 18 new marker swap plasmids, and we also review other plasmids available for marker conversions . All of these plasmids have long regions of flanking sequence identity, and thus the efficiency of homologous recombination mediated by marker conversion is very high . Marker swaps allow one to easily perform crosses to construct double mutant strains even if each of the disrupted strains contains the same marker, as is the case with the KanMX marker used in the yeast knockout collection . Marker swaps can also be used to change the selectable marker on plasmids, eliminating the need for subcloning . Yeast, 2003 Aug, 20(11), 955 - 71 NET1 and HFI1 genes of yeast mediate both chromosome maintenance and mitochondrial rho(-) mutagenesis; Koltovaya NA et al.; An increase in the mitochondrial rho(-) mutagenesis is a well-known response of yeast cells to mutations in numerous nuclear genes as well as to various kinds of stress . Despite extensive studies for several decades, the biological significance of this response is still not fully understood . The genetic approach to solving this enigma includes a study of genes that are required for the high incidence of spontaneous rho(-) mutants . We have obtained mutations of a few nuclear genes of that sort and found that mutations in certain genes, including CDC28, the central cell-cycle regulation gene, result in a decrease in spontaneous rho(-) mutability and simultaneously affect the maintenance of the yeast chromosomes and plasmids . Two more genes resembling CDC28 in this respect are identified in the present work as a result of the characterization of four new mutants . These two genes are NET1 and HFI1 which mediate important regulatory protein-protein interactions in the yeast cell . The effects of four mutations, including net1-srm and hfi1-srm, on the maintenance of the yeast mitochondrial genome, chromosomes and plasmids, as well as on the cell's sensitivity to ionizing radiation, are also described . The data presented suggest that the pleiotropic srm mutations determining coordinate changes in the fidelity of mitotic transmission of chromosomes, plasmids and mtDNA molecules identify genes that most probably operate high up in the hierarchy of the general genetic regulation of yeast . Mol Genet Genomics, 2003 Sep, 269(6), 776 - 88 Epub 2003 Jul 30. Mutation of a conserved CDK site converts a metazoan Elongation Factor 1Bbeta subunit into a replacement for yeast eEF1Balpha; Pomerening JR et al.; Elongation factor subunit eEF1Bbeta (formerly EF-1beta in plants and EF-1delta in animals) was identified and cloned in a screen for proteins from pea that interact with a cyclin-dependent kinase (CDK) . CDKs are enzymes that regulate progression through meiotic and mitotic cell cycles in eukaryotes . eEF1Bbeta and the related protein eEF1Balpha (formerly EF-1beta' in plants and EF-1beta in animals and fungi) can catalyze GTP/GDP exchange on the G-protein eEF1A (formerly EF-1alpha in plants, animals and fungi) during the elongation phase of protein synthesis in eukaryotes . Recombinant Cdc2 and its native homologues from pea extracts associated both in vitro and in vivo with eEF1Bbeta . A Cdc2-cyclin B complex phosphorylated recombinant plant eEF1Bbetas, but not eEF1Balpha . These interactions between CDK and eEF1Bbeta prompted investigations into the in vivo consequences of this relationship . Expression of cDNAs encoding rice or pea eEF1Bbeta subunits failed to complement a Saccharomyces cerevisiae mutant deleted for the eEF1Balpha gene, as was previously observed for the human eEF1Bbeta . However, replacement of Thr91, the sole consensus CDK phosphorylation site in pea eEF1Bbeta, with alanine allowed the pea protein to substitute for eEF1Balpha function in vivo . In addition, this rescued strain was severely cold sensitive, and more sensitive to translational inhibitors than wild-type yeast . Taken together, these results suggest a physiological connection between the cyclin-dependent class of kinases and a translational elongation factor in mitotic cells, and provide the first in vivo evidence that an altered form of eEF1Bbeta can serve as the guanine nucleotide exchange factor for eEF1A. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Aug, 35(8), 747 - 51 Interaction of C17orf25 with ADP-ribose pyrophosphatase NUDT9 detected via yeast two-hybrid method; Zhang HT et al.; The gene C17orf25 was isolated from the liver by RACE PCR . nudt9 gene was screened by yeast two-hybrid method in MatchMaker human HeLa cDNA library . NUDT9 is an enzyme that has pyrophosphatase activity with ADP-ribose as its substrate . Fusion expression of C17orf25 and GFP and computer analysis showed that C17orf25 was probably located in mitochondria . Furthermore, C17orf25 may suppress the cell growth by interaction with NUDT9. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Aug, 35(8), 707 - 16 {Estimating coarse gene networks from yeast gene expression time series}; Xu XJ et al.; Gene networks is the collection of gene-gene regulatory relations at the expression level . In this study, a combined approach of the linear transcriptional modeling, identification of promoter elements and gene co-expression clustering is developed to decipher yeast gene networks from expression time series . The cell must reorganize the genomic expression to programs required for growth and survival in each environment . The expression of many genes is regulated by environmental stress . The products of many genes that induced in the environmental stress are involved in metabolism of carbohydrates, structural repairs and even sporulation . Interestingly, it is identified that transcription factors Mcm1 and Dal82 matched their binding sites TT{bond}CC{triple bond}T{double bond}GGAAA and TGAAAAWTTT in cell cycle progression and environmental stress response, respectively . These conclusions agree with the known observations . The results indicate that the approach may be useful for modeling gene networks from microarray data. Mol Cell Biol, 2003 Aug, 23(16), 5768 - 79 Cotranscriptional recruitment of the U1 snRNP to intron-containing genes in yeast; Kotovic KM et al.; Evidence that pre-mRNA processing events are temporally and, in some cases, mechanistically coupled to transcription has led to the proposal that RNA polymerase II (Pol II) recruits pre-mRNA splicing factors to active genes . Here we address two key questions raised by this proposal: (i) whether the U1 snRNP, which binds to the 5' splice site of each intron, is recruited cotranscriptionally in vivo and, (ii) if so, where along the length of active genes the U1 snRNP is concentrated . Using chromatin immunoprecipitation (ChIP) in yeast, we show that elevated levels of the U1 snRNP were specifically detected in gene regions containing introns and downstream of introns but not along the length of intronless genes . In contrast to capping enzymes, which bind directly to Pol II, the U1 snRNP was poorly detected in promoter regions, except in genes harboring promoter-proximal introns . Detection of the U1 snRNP was dependent on RNA synthesis and was abolished by intron removal . Microarray analysis revealed that intron-containing genes were preferentially selected by ChIP with the U1 snRNP . Thus, U1 snRNP accumulation at genes correlated with the presence and position of introns, indicating that introns are necessary for cotranscriptional U1 snRNP recruitment and/or retention. Genes Dev, 2003 Aug 1, 17(15), 1817 - 22 Differential DNA affinity specifies roles for the origin recognition complex in budding yeast heterochromatin; Palacios DeBeer MA et al.; The origin recognition complex (ORC) marks chromosomal positions as replication origins and is essential for replication initiation . At a few loci, the ORC functions in heterochromatin formation . We show that the ORC's two roles at the heterochromatic HMRa locus in Saccharomyces cerevisiae were regulated by differences in the ORC's interaction with its target site . At HMRa, a strong ORC-DNA interaction inhibited and delayed replication initiation but promoted heterochromatin formation, whereas a weak ORC-DNA interaction allowed for increased and earlier replication initiation but reduced heterochromatin formation . Therefore, the ORC's interaction with its target site could modulate ORC activity within a heterochromatin domain in vivo. Biochim Biophys Acta, 2003 Aug 7, 1614(2), 139 - 55 The role of ubiquitin in down-regulation and intracellular sorting of membrane proteins: insights from yeast; Horak J; Ubiquitination is a versatile tool used by all eukaryotic organisms for controlling the stability, function, and intracellular localization of a wide variety of proteins . Two of the best characterized functions of protein ubiquitination are to mark proteins for degradation by cytosolic proteasome and to promote the internalization of certain plasma membrane proteins via the endocytotic pathway, followed by their degradation in the vacuole . Recent studies of membrane proteins both in yeast and mammalian cells suggest that the role of ubiquitin may extend beyond its function as an internalization signal in that it also may be required for modification of some component(s) of the endocytotic machinery, and for cargo protein sorting at the late endosome and the Golgi apparatus level . In this review, I will attempt to bring together what is currently known about the role of ubiquitination in controlling protein trafficking between the yeast plasma membrane, the trans-Golgi network, and the vacuole/lysosome. J Biol Chem, 2003 Oct 10, 278(41), 39558 - 64 Epub 2003 Jul 31. A cytosolic domain of the yeast Zrt1 zinc transporter is required for its post-translational inactivation in response to zinc and cadmium; Gitan RS et al.; Nutrient metals such as zinc are both essential to life and potentially toxic if overaccumulated by cells . Non-essential toxic metals like cadmium can enter cells through the uptake transporters responsible for nutrient metal acquisition . Therefore, in the face of ever changing extracellular metal levels, organisms tightly control their intracellular levels of nutrient metals and prevent accumulation of toxic metals . We show here that post-translational inactivation of the yeast Zrt1 zinc uptake transporter is important for zinc homeostasis . During the transition from zinc-limiting to zinc-replete growth conditions (i.e . zinc shock), the Zrt1 transporter is ubiquitinated, endocytosed, and subsequently degraded in the vacuole . To further understand this process at a molecular level, we mapped a region of Zrt1 required for ubiquitination and endocytosis in response to zinc to a domain located on the intracellular surface of the plasma membrane . This domain is a critical cis-acting component of the metal signaling pathway that controls Zrt1 protein trafficking . Using mutant alleles defective for metal-responsive inactivation, we also show that Zrt1 inactivation may be an important mechanism for preventing cadmium uptake and toxicity in zinc-limited cells. FEMS Microbiol Lett, 2003 Jul 29, 224(2), 299 - 305 Chitin synthase III activity, but not the chitin ring, is required for remedial septa formation in budding yeast; Cabib E et al.; Chitin is a minor but essential component of the Saccharomyces cerevisiae cell wall . In wild-type, chitin synthase II is required for the formation of primary septa and chitin synthase III (CSIII) is not essential . However, in chs2 mutants CSIII becomes essential for the formation of aberrant septa . We examined which of two CSIII functions, the formation of a chitin ring at bud emergence or of chitin in the remedial septa, was required for viability . By using cell cycle synchronization in combination with nikkomycin Z, a specific inhibitor of CSIII, we inhibited chitin synthesis in a chs2 mutant, during formation of either the ring or the remedial septa . The results show that only synthesis of the chitin during aberrant septa formation is essential for viability . Thus, the unique function of the chitin ring seems to be maintenance of the integrity of the mother-bud neck, as we recently found, and the importance of chitin in septum closure, both in normal and abnormal situations, is underlined. J Biol Chem, 2003 Oct 3, 278(40), 38646 - 52 Epub 2003 Jul 30. Expression of the yeast PIS1 gene requires multiple regulatory elements including a Rox1p binding site; Gardocki ME et al.; The PIS1 gene is required for de novo synthesis of phosphatidylinositol (PI), an essential phospholipid in Saccharomyces cerevisiae . PIS1 gene expression is unusual because it is uncoupled from the other phospholipid biosynthetic genes, which are regulated in response to inositol and choline . Relatively little is known about regulation of transcription of the PIS1 gene . We reported previously that PIS1 transcription is sensitive to carbon source . To further our understanding of the regulation of PIS1 transcription, we carried out a promoter deletion analysis that identified three regions required for PIS1 gene expression (upstream activating sequence (UAS) elements 1-3) . Deletion of either UAS1 or UAS2 resulted in an approximately 45% reduction in expression, whereas removal of UAS3 yielded an 84% decrease in expression . A comparison of promoters among several Saccharomyces species shows that these sequences are highly conserved . Curiously, the UAS3 element region (-149 to -138) includes a Rox1p binding site . Rox1p is a repressor of hypoxic genes under aerobic growth conditions . Consistent with this, we have found that expression of a PIS1-cat reporter was repressed under aerobic conditions, and this repression was dependent on both Rox1p and its binding site . Furthermore, PI levels were elevated under anaerobic conditions . This is the first evidence that PI levels are affected by regulation of PIS1 transcription. J Biotechnol, 2003 Aug 15, 103(3), 213 - 25 Functional analysis of expressed peptides that bind yeast STE proteins; Caponigro G et al.; Peptides are potentially useful for target validation and other reverse genetic applications . For instance, if a specific protein is susceptible to peptide inhibition, it may have a higher probability of being vulnerable to small molecules . We used the yeast two-hybrid technique to identify and study peptide binders for three yeast proteins involved in pheromone response: Ste11p, Ste18p, and Ste50p . A subset of peptide binders was shown to inhibit pheromone response in cells using two different functional assays . In addition, we utilized a variant of the yeast two-hybrid method to examine relative binding affinities based on competitive interactions in yeast . Our results suggest that binding affinity and inhibitory potency of peptides do not correlate perfectly and that peptide-protein interactions can be complex and unpredictable . Taken together these results suggest that while peptides are useful as in vivo inhibitors of protein function, caution must be exercised when choosing peptides for further studies and when inferring affinities from expression phenotypes. J Mol Biol, 2003 Aug 8, 331(2), 375 - 83 Yeast aspartyl-tRNA synthetase binds specifically its own mRNA; Frugier M et al.; Dimeric class II aspartyl-tRNA synthetase (AspRS) from yeast has a modular architecture and includes an N-terminal appendix of 70 amino acid residues that protrudes from the anticodon-binding module . This extension, of predicted helical structure, is not essential for aminoacylation but contains an RNA-binding motif that promotes non-specific interactions with tRNAs . As shown here, this protein extension can also interact with the 5' end of the AspRS mRNA . In vitro, optimal binding occurs on an mRNA domain comprising part of the 87 nucleotide long 5'UTR and the sequence encoding the N-terminal appendix . At the protein side, only the appendix and the anticodon-binding module participate in the interaction between AspRS and the mRNA domain . Binding is specific, since only tRNA(Asp) can dissociate the complex . In vivo, AspRS also binds specifically this mRNA domain and in doing so triggers a reduced translation of a fused GFP mRNA . From that, a mechanism for the regulation of this eukaryotic aminoacyl-tRNA synthetase is proposed . Implications for aspartylation accuracy in yeast are given. J Mol Biol, 2003 Aug 8, 331(2), 315 - 20 The central PPT of the yeast retrotransposon Ty1 is not essential for transposition; Heyman T et al.; The yeast retrotransposon Ty1 has structural and functional similarities to retroviruses . We report here that, as in retroviruses, the plus-strand DNA of Ty1 is synthesized as two segments . A central DNA flap is formed during reverse transcription consecutive to elongation (with strand displacement) of the upstream segment beyond the central polypurine tract (cPPT) until the replication machinery is stopped at the central termination sequence . Comparison of wild-type and cPPT-mutant Ty1 elements shows that the mutant element lacking the central DNA flap is only twofold defective in transposition. Mol Cell, 2003 Jul, 12(1), 221 - 32 Recruitment of the recombinational repair machinery to a DNA double-strand break in yeast; Wolner B et al.; Repair of DNA double-strand breaks (DSBs) by homologous recombination requires members of the RAD52 epistasis group . Here we use chromatin immunoprecipitation (ChIP) to examine the temporal order of recruitment of Rad51p, Rad52p, Rad54p, Rad55p, and RPA to a single, induced DSB in yeast . Our results suggest a sequential, interdependent assembly of Rad proteins adjacent to the DSB initiated by binding of Rad51p . ChIP time courses from various mutant strains and additional biochemical studies suggest that Rad52p, Rad55p, and Rad54p each help promote the formation and/or stabilization of the Rad51p nucleoprotein filament . We also find that all four Rad proteins associate with homologous donor sequences during strand invasion . These studies provide a near comprehensive view of the molecular events required for the in vivo assembly of a functional Rad51p presynaptic filament. Mol Cell, 2003 Jul, 12(1), 135 - 45 Silencing in yeast rDNA chromatin: reciprocal relationship in gene expression between RNA polymerase I and II; Cioci F et al.; About half of approximately 150 rRNA genes are transcriptionally active in Saccharomyces cerevisiae . Chromatin structures in the inactive, and not the active, copies were previously thought to silence both rRNA genes and reporter Pol II genes . Contrary to this belief, we found that silencing of reporters is much stronger in a mutant with approximately 25 rDNA copies, all of which are transcriptionally active . By integrating reporter gene mURA3 with an inactive rDNA copy missing the Pol I promoter, we found that mURA3 is not silenced in chromosomal rDNA repeats . Together with the demonstration of a requirement for active Pol I in silencing, these results show a reciprocal relationship in gene expression between Pol I and Pol II in rDNA. Appl Microbiol Biotechnol, 2003 Aug, 62(2-3), 226 - 32 Epub 2003 Mar 21. Display of a functional hetero-oligomeric catalytic antibody on the yeast cell surface; Lin Y et al.; A functional hetero-oligomeric protein was, for the first time, displayed on the yeast cell surface . A hetero-oligomeric Fab fragment of the catalytic antibody 6D9 can hydrolyze a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol . The gene encoding the light chain of the Fab fragment of 6D9 was expressed with the tandemly-linked C-terminal half of alpha-agglutinin . At the same time, the gene encoding the Fd fragment of the heavy chain of the Fab fragment was expressed as a secretion protein . The combined Fab fragment displayed and associated on the yeast cell surface had an intermolecular disulfide linkage between the light and heavy chains . This protein fragment catalyzed the hydrolysis of a chloramphenicol monoester derivative and exhibited high stability in binding with a transition-state analog (TSA) . The catalytic reaction was also inhibited by the TSA . The successful display of a functional hetero-oligomeric catalytic antibody provides a useful model for the display of hetero-oligomeric proteins and enzymes. J Biol Chem, 2003 Oct 17, 278(42), 40943 - 52 Epub 2003 Jul 25. Monitoring active site alterations upon mutation of yeast pyruvate kinase using 205Tl+ NMR; Susan-Resiga D et al.; The interaction of the monovalent cation with wild type (WT) yeast pyruvate kinase (YPK) and with the T298S, T298C, and T298A mutants was investigated by 205Tl+ NMR to monitor possible structural alterations at the active site by Thr-298 mutation . TlNO3 activates WT YPK with a kcat value similar to that obtained with KCl and an apparent Ka of 0.96 +/- 0.07 mm in the presence of Mn2+ and fructose 1,6-bisphosphate . With the three mutants, Tl+ is a better activator than is K+ based on kcat values . Tl+ activation and inhibition of YPK is affected by mutation of the active site Thr-298 . The effect of Mn2+ on the 1/T value of 205Tl+1 in the presence of the WT and mutant YPK complexes was determined at 173 MHz (300 MHz, 1H) and 346 MHz (600 MHz, 1H) . For each complex studied, 1/pT2p >> 1/pT1p and 1/pT1p is frequency-dependent suggesting fast exchange conditions . The values of 1/pT1p differ for each mutant . A correlation time of 0.65 +/- 0.35 ns was estimated for the Mn2+-205Tl+ interaction . The Tl+-Mn2+ distances at the active site of YPK were calculated from the paramagnetic contribution of Mn2+ to 1/T1M of YPK-bound 205Tl+ . The calculated Tl+-Mn2+ distance for the Thr-298 mutants is decreased by about 1 A from 6.0 +/- 0.2 A observed with WT . The results suggest conformational alterations at the active site of YPK where phosphoryl transfer occurs upon mutation of Thr-298 . These conformational changes may, in part, explain the alteration in kcat and kcat/Km,PEP observed with the Thr-298 mutants. J Biol Chem, 2003 Oct 31, 278(44), 43770 - 80 Epub 2003 Jul 25. Unique error signature of the four-subunit yeast DNA polymerase epsilon; Shcherbakova PV et al.; We have purified wild type and exonuclease-deficient four-subunit DNA polymerase epsilon (Pol epsilon) complex from Saccharomyces cerevisiae and analyzed the fidelity of DNA synthesis by the two enzymes . Wild type Pol epsilon synthesizes DNA accurately, generating single-base substitutions and deletions at average error rates of </=2 x 10-5 and </=5 x 10-7, respectively . Pol epsilon lacking 3' --> 5' exonuclease activity is less accurate to a degree suggesting that wild type Pol epsilon proofreads at least 92% of base substitution errors and at least 99% of frameshift errors made by the polymerase . Surprisingly the base substitution fidelity of exonuclease-deficient Pol epsilon is severalfold lower than that of proofreading-deficient forms of other replicative polymerases . Moreover the spectrum of errors shows a feature not seen with other A, B, C, or X family polymerases: a high proportion of transversions resulting from T.dTTP, T.dCTP, and C.dTTP mispairs . This unique error specificity and amino acid sequence alignments suggest that the structure of the polymerase active site of Pol epsilon differs from those of other B family members . We observed both similarities and differences between the spectrum of substitutions generated by proofreading-deficient Pol epsilon in vitro and substitutions occurring in vivo in a yeast strain defective in Pol epsilon proofreading and DNA mismatch repair . We discuss the implications of these findings for the role of Pol epsilon polymerase activity in DNA replication. Biotechnol Lett, 2003 Feb, 25(4), 311 - 4 By-product formation by a novel glycerol-producing yeast, Candida glycerinogenes, with different O2 supplies; Jin H et al.; Candida glycerinogenes is an aerobe which does not depend on sulphite for production of glycerol . With a sufficient O2 supply, up to 130 g glycerol l(-1) was produced with 2.6 g acetic acid l(-1) as by-product . However, with an insufficient O2 supply--with higher volumes of medium or at higher corn steep liquid concentrations--the glycerol concentration was lower because the by-products, ethanol, pyruvate and lactic acid, were produced in greater amounts, up to 45 g l(-1), 4.3 g l(-1), 1.6 g l(-1), respectively, whereas, less acetic acid (0.6 g l(-1)) was produced . In addition, ethanol decreased to 0.4 g l(-1) and the glycerol yield improved from 34 to 50% (w/w) by adding 50 g sulphite l(-1), nevertheless, acetic acid increased to 7.8 g l(-1). Aging Cell, 2002 Dec, 1(2), 149 - 57 The shortened replicative life span of prohibitin mutants of yeast appears to be due to defective mitochondrial segregation in old mother cells; Piper PW et al.; Prohibitin proteins have been implicated in cell proliferation, aging, respiratory chain assembly and the maintenance of mitochondrial integrity . The prohibitins of Saccharomyces cerevisiae, Phb1 and Phb2, have strong sequence similarity with their human counterparts prohibitin and BAP37, making yeast a good model organism in which to study prohibitin function . Both yeast and mammalian prohibitins form high-molecular-weight complexes (Phb1/2 or prohibitin/BAP37, respectively) in the inner mitochondrial membrane . Expression of prohibitins declines with senescence, both in mammalian fibroblasts and in yeast . With a total loss of prohibitins, the replicative (budding) life span of yeast is reduced, whilst the chronological life span (the survival of stationary cells over time) is relatively unaffected . This effect of prohibitin loss on the replicative life span is still apparent in the absence of an assembled respiratory chain . It also does not reflect the production of extrachromosomal ribosomal DNA circles (ERCs), a genetic instability thought to be a major cause of replicative senescence in yeast . Examination of cells containing a mitochondrially targeted green fluorescent protein indicates this shortened life span is a reflection of defective mitochondrial segregation from the mother to the daughter in the old mother cells of phb mutant strains . Old mother phb mutant cells display highly aberrant mitochondrial morphology and, frequently, a delayed segregation of mitochondria to the daughter . They often arrest growth with their last bud strongly attached and with the mitochondria adjacent to the septum between the mother and the daughter cell. Biotechnol Lett, 2003 May, 25(10), 767 - 71 Preparation of the red yeast, Xanthophyllomyces dendrorhous, as feed additive with increased availability of astaxanthin; An GH et al.; Xanthophyllomyces dendrorhous (Phaffia rhodozyma) is used as a colorant for aquaculture, egg yolks, and crustaceans but its carotenoids can only be absorbed by animals when its cell wall is degraded . Conditions that degraded the cell wall of X . dendrorhous were developed . To measure the degrees of cell wall degradation, the carotenoid extractability (extracted carotenoid by acetone/total carotenoid) unit was used . Treatment with HCl (0.2 M, 9 h, 90 degrees C) followed by neutralization to pH 3 by NaOH and spray-drying increased carotenoid extractability to 100% with minimal decomposition. EMBO J, 2003 Aug 1, 22(15), 3960 - 70 Nonsense-mediated mRNA decay in Drosophila: at the intersection of the yeast and mammalian pathways; Gatfield D et al.; The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature stop codons (PTCs) . In Caenorhabditis elegans, seven genes (smg1-7) playing an essential role in NMD have been identified . Only SMG2-4 (known as UPF1-3) have orthologs in Saccharomyces cerevisiae . Here we show that the Drosophila orthologs of UPF1-3, SMG1, SMG5 and SMG6 are required for the degradation of PTC-containing mRNAs, but that there is no SMG7 ortholog in this organism . In contrast, orthologs of SMG5-7 are encoded by the human genome and all three are required for NMD . In human cells, exon boundaries have been shown to play a critical role in defining PTCs . This role is mediated by components of the exon junction complex (EJC) . Contrary to expectation, however, we show that the components of the EJC are dispensable for NMD in Drosophila cells . Consistently, PTC definition occurs independently of exon boundaries in DROSOPHILA: Our findings reveal that despite conservation of the NMD machinery, different mechanisms have evolved to discriminate premature from natural stop codons in metazoa. EMBO J, 2003 Aug 1, 22(15), 3951 - 9 Interaction between Ski7p and Upf1p is required for nonsense-mediated 3'-to-5' mRNA decay in yeast; Takahashi S et al.; Aberrant mRNAs containing premature termination codons (PTC-mRNAs) are degraded by a conserved surveillance system, referred to as the nonsense- mediated decay (NMD) pathway . Although NMD is reported to operate on the decapping and 5'-to-3' exonucleolytic decay of PTC-mRNAs without affecting deadenylation, a role for an opposite 3'-to-5' decay pathway remains largely unexplored . In this study, we have characterized the 3'-to-5' directed mRNA degradation in the yeast NMD pathway . PTC-mRNAs are stabilized in yeast cells lacking the components of 3'-to-5' mRNA-decay machinery . The 3'-to-5' directed degradation of PTC-mRNAs proceeds more rapidly than that of the PTC-free transcript, in a manner dependent on the cytoplasmic exosome and Upf proteins . Moreover, Upf1p, but not Upf2p, interacts physically with an N-terminal domain of Ski7p, although the interaction requires Upf2p . The efficiency of 3'-to-5' directed degradation of PTC-mRNAs is impaired by overexpression of Ski7p N-domain fragments that contain a sequence of the Upf1p-interaction region . These data suggest that the activation of 3'-to-5' directed NMD is mediated through the interaction between Upf1p and the Ski7p N domain. Biochim Biophys Acta, 2003 Jul 21, 1633(2), 106 - 17 Enzymological properties of sterol-C4-methyl-oxidase of yeast sterol biosynthesis; Darnet S et al.; Despite genes of the sterol methyl-oxidase component (SMO) of the sterol-C4-demethylation multienzymatic complex have been identified in a variety of organisms and the key role played by SMO in yeast sterol biosynthesis, the enzymological properties of yeast SMO have not been investigated . An enzymatic assay for measuring specifically sterol 4alpha-methyl-oxidase activity in Saccharomyces cerevisiae has been developed for the first time by using {14C}-4,4-dimethyl-zymosterol as substrate . It allowed enzymatically formed C4 mono- and di-demethylated products to be characterized as well as two novel C4-hydroxymethyl-zymosterol derivatives to be identified as immediate oxidative metabolites by the yeast 4,4-dimethyl-zymosterol 4alpha-methyl-oxidase (ScSMO) . The properties of microsomal ScSMO have been established with respect to cofactor requirements and kinetics and the substrate selectivity examined with a number of 4,4-dimethyl- and 4alpha-methyl-sterols . Remarkably, ScSMO showed very low activity with 24-methylene-24-dihydrocycloartenol, the natural substrate of maize 4,4-dimethyl-sterol-C4-methyl-oxidase . Conversely, maize sterol-C4-methyl-oxidases showed extremely reduced activity with the natural substrate of ScSMO . The previously described antifungal agent, 6-amino-2-n-pentylbenzothiazole was shown to directly inhibit the microsomal ScSMO activity in vitro . The yeast system was more than 500 times more sensitive to this derivative than the maize systems . These distinct substrate specificities and inhibitor sensitivities between yeast and plant sterol-4alpha-methyl-oxidases probably reflect diversity in the structure of their active sites in relation to the distinct sterol biosynthetic pathways. Zhonghua Yu Fang Yi Xue Za Zhi, 2003 May, 37(3), 158 - 60 {Effect of yeast RNA on physical functions, morphology of hepatic cells and brain neurons in aged rats}; Pan HZ et al.; OBJECTIVE: To study the effect of exogenous nucleic acid on physical functions, morphology of hepatic cells and brain neurons in aged rats . METHODS: Thirty two aged Wistar rats (20 month-old) were divided randomly into four groups (one aged control group and three aged experimental groups) and eight young rats (3 month-old) was set as young control group . Control groups were fed on standard chow and experimental groups were fed on standard chow supplemented with 93.75 mg/kg (high-dosage group), 46.88 mg/kg (middle-dosage group) and 9.38 mg/kg (low-dosage group) of yeast RNA respectively . SOD, MDA, HDL, sex hormone and growth hormone were determined at the end of a 4-week observation . The microcosmic images of the hepatic cells and brain neurons using the image-pro plus (V.4.0) were also observed . RESULTS: SOD, serum HDL and growth hormone levels in the high dosage group were significantly higher (P < 0.05) than that in the aged control group, and the levels were not different from that in the young control group . MDA level of all yeast RNA supplemented groups was significantly lower than that of aged control group (P < 0.05) and that was not different from the young control group . Serum testosterone of the high and middle dosage groups reached the level of young control group, and that was much higher than the aged control and low dosage group (P < 0.05) . Estradiol levels among the aged rats were not different, and those were much lower than the young control group (P < 0.05) . Much more number of brain neurons were observed in the high-dose group than other aged rats (P < 0.05) . Brain neurons, hepatic cells and karyons in the high-dose group were bigger than that in other aged rats (P < 0.05) . CONCLUSION: Exogenous yeast RNA might play an important role in physical functions, the morphology of brain neurons and hepatic cells in natural aged rats . There might have a dose-effect relationship in the process. Folia Microbiol (Praha), 2003, 48(3), 361 - 2 The yeast flora of the coast redwood, Sequoia sempervirens; Middelhoven WJ; Only four yeast species could be isolated from young and perannual shoots of the coast redwood tree, Sequoia sempervirens, and from soil beneath the trees, viz . both varieties of Debaryomyces hansenii, Trichosporon pullulans, T . porosum and an unidentified red basidiomycetous yeast. Brain Res Mol Brain Res, 2003 Jul 23, 115(2), 104 - 10 The effect of lithium on expression of genes for inositol biosynthetic enzymes in mouse hippocampus; a comparison with the yeast model; Shamir A et al.; In the de novo synthesis of inositol, the conversion of D-glucose-6-phosphate to L-myo-inositol-1-phosphate (MIP) is catalyzed by MIP synthase . Little is known about mammalian MIP synthase and nothing is known about its regulation . The second step in inositol biosynthesis is the conversion of MIP to inositol by inositol-monophosphatase (IMPase), a common step to inositol production via the de novo pathway and its recycling from inositol phosphates . Because lithium inhibits IMPase both in yeast and in mammals, and the drug upregulates yeast MIP synthase (INO1) and downregulates IMPase (INM1), the present study was undertaken to determine whether chronic in vivo therapeutic lithium concentrations affect MIP synthase and IMPase expression in mouse frontal cortex and hippocampus . Mice were treated with food containing LiCl (2.5 g/kg) for 10 days . RNA was purified from the brain areas and mRNA amplified using RT-PCR . Expression of MIP synthase and IMPA1 (one of the genes coding for IMPase) but not IMPA2 was upregulated in mouse hippocampus . None of the genes were affected in the frontal cortex . In yeast, when inositol is limiting, the heterodimeric transcriptional activator Ino2p/Ino4p derepresses expression of INO1 by binding to the upstream activation sequence UAS(INO) . Using the TFSEARCH program, we found that the promoter of the virtual human MIP synthase gene contains upstream stimulating factor (USF) elements with a similar core binding sequence . The fact that lithium treatment upregulates both MIP synthase and IMPA1 mRNA levels in mouse hippocampus may reflect a compensatory response of both genes to inositol depletion. Cell Physiol Biochem, 2003, 13(3), 123 - 34 Characterisation of mammalian GLUT glucose transporters in a heterologous yeast expression system; Wieczorke R et al.; We have developed a new heterologous expression system for mammalian glucose transporters . The system is based on a Saccharomyces cerevisiae strain completely deleted for all its endogenous hexose transporters and unable to take up and to grow on hexoses . To target the heterologous glucose transporters into the yeast plasma membrane in a fully active form, additional mutations had to be introduced into the hexose transport-deficient strain . Although GLUT1 was localized at the cell surface already in the parent strain, it supported uptake of glucose only in an deltaHXT FGY1-1 mutant strain . Moreover, various mutations within the first half of the second predicted transmembrane helix converted GLUT1 into a form able to support uptake of glucose into yeast cells . GLUT4 was trapped in intracellular structures but became functionally expressed in the plasma membrane in deltaHXT FGY1-1 FGY4X mutant strains . Glucose transport kinetics were determined with intact yeast cells by zero-trans influx measurements with a Km of 3.2 mM for human GLUT1 and of 12.6 mM for human GLUT4 . Cytochalasin B inhibited these activities . Growth tests revealed that both transporter proteins are able to mediate uptake of glucose, mannose and galactose, but not of fructose . The new heterologous expression system should be a valuable tool to develop cell based high-throughput screening assays for identifying pharmaceutical compounds influencing the transporters . Proc Natl Acad Sci U S A, 2003 Aug 5, 100(16), 9286 - 91 Epub 2003 Jul 22. Importance of low-oligomeric-weight species for prion propagation in the yeast prion system Sup35/Hsp104; Narayanan S et al.; The {PSI+} determinant of Saccharomyces cerevisiae, consisting of the cytosolic translation termination factor Sup35, is a prion-type genetic element that induces an inheritable conformational change and converts the Sup35 protein into amyloid fibers . The molecular chaperone Hsp104 is required to maintain self-replication of {PSI+} . We observe in vitro that addition of catalytic amounts of Hsp104 to the prion-determining region of the NM domain of Sup35, Sup355-26, results in the dissociation of oligomeric Sup35 into monomeric species . Several intermediates of Sup355-26 could be detected during this process . Strong interactions are found between Hsp104 and hexameric/tetrameric Sup355-26, whereas the intermediate and monomeric "release" forms show a decreased affinity with respect to Hsp104, as monitored by saturation transfer difference and diffusion-ordered NMR spectroscopic experiments . Interactions are mediated mostly by the side chains of Gln, Asn, and Tyr residues in Sup355-26 . No interaction can be detected between Hsp104 and higher oligomeric states (>/=8) of Sup355-26 . Taking into account the fact that Hsp104 is required for maintenance of {PSI+}, we suggest that low-oligomeric-weight species of Sup35 are important for prion propagation in yeast. Cancer Gene Ther, 2003 Aug, 10(8), 603 - 10 Human-yeast chimeric repair protein protects mammalian cells against alkylating agents: enhancement of MGMT protection; Roth TJ et al.; Chemotherapeutic DNA alkylating agents are common weapons employed to fight both pediatric and adult cancers . In addition to cancerous cells, nontarget tissues are subjected to the cytotoxicity of these agents, and dose-limiting toxicity in the form of myelosuppression is a frequent result of treatment . One approach to prevent myelosuppression that results from the use of chemotherapeutic agents is to increase the levels of DNA repair proteins in bone marrow cells . Here we report our second successful attempt to create a fusion protein that possesses both direct reversal and base excision repair pathway DNA repair activities . The chimeric protein is composed of the human O(6)-Methylguanine-DNA Methyltransferase (MGMT) and the yeast Apn1 proteins and retains both MGMT and AP endonuclease activities as determined by biochemical analysis . We have also demonstrated that the chimeric protein is able to protect mammalian cells from the DNA alkylating agents 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) and methyl methanesulfonate (MMS) . The protection by the chimeric protein against BCNU is even greater than MGMT alone, which has potential translational significance given that MGMT is currently in clinical trials . Additionally, we show that the chimeric MGMT-Apn1 protein can protect mammalian cells from dual treatments of BCNU and MMS and that this effect is greater than that provided by MGMT alone . The data support our previous finding that a protein with multiple DNA repair activities can be constructed and that this and other constructs may play an important clinical role in guarding against dose-limiting effects of chemotherapy, particularly in situations of multiple drug use. J Biol Chem, 2003 Oct 3, 278(40), 38723 - 30 Epub 2003 Jul 18. Genome expression analysis in yeast reveals novel transcriptional regulation by inositol and choline and new regulatory functions for Opi1p, Ino2p, and Ino4p; Santiago TC et al.; In Saccharomyces cerevisiae, genes encoding phospholipid-synthesizing enzymes are regulated by inositol and choline (IC) . The current model suggests that when these precursors become limiting, the transcriptional complex Ino2p-Ino4p activates the expression of these genes, whereas repression requires Opi1p and occurs when IC are available . In this study, microarray-based expression analysis was performed to assess the global transcriptional response to IC in a wild-type strain and in the opi1delta, ino2delta, and ino4delta null mutant strains . Fifty genes were either activated or repressed by IC in the wild-type strain, including three already known IC-repressed genes . We demonstrated that the IC response was not limited to genes involved in membrane biogenesis, but encompassed various metabolic pathways such as biotin synthesis, one-carbon compound metabolism, nitrogen-containing compound transport and degradation, cell wall organization and biogenesis, and acetyl-CoA metabolism . The expression of a large number of IC-regulated genes did not change in the opi1delta, ino2delta, and ino4delta strains, thus implicating new regulatory elements in the IC response . Our studies revealed that Opi1p, Ino2p, and Ino4p have dual regulatory activities, acting in both positive and negative transcriptional regulation of a large number of genes, most of which are not regulated by IC and only a subset of which is involved in membrane biogenesis . These data provide the first global response profile of yeast to IC and reveal novel regulatory mechanisms by these precursors. Genetics, 2003 Jul, 164(3), 895 - 907 Genetic interactions with CLF1 identify additional pre-mRNA splicing factors and a link between activators of yeast vesicular transport and splicing; Vincent K et al.; Clf1 is a conserved spliceosome assembly factor composed predominately of TPR repeats . Here we show that the TPR elements are not functionally equivalent, with the amino terminus of Clf1 being especially sensitive to change . Deletion and add-back experiments reveal that the splicing defect associated with TPR removal results from the loss of TPR-specific sequence information . Twelve mutants were found that show synthetic growth defects when combined with an allele that lacks TPR2 (i.e., clf1Delta2) . The identified genes encode the Mud2, Ntc20, Prp16, Prp17, Prp19, Prp22, and Syf2 splicing factors and four proteins without established contribution to splicing (Bud13, Cet1, Cwc2, and Rds3) . Each synthetic lethal with clf1Delta2 (slc) mutant is splicing defective in a wild-type CLF1 background . In addition to the splicing factors, SSD1, BTS1, and BET4 were identified as dosage suppressors of clf1Delta2 or selected slc mutants . These results support Clf1 function through multiple stages of the spliceosome cycle, identify additional genes that promote cellular mRNA maturation, and reveal a link between Rab/Ras GTPase activation and the process of pre-mRNA splicing. Genetics, 2003 Jul, 164(3), 855 - 65 The mitotic DNA damage checkpoint proteins Rad17 and Rad24 are required for repair of double-strand breaks during meiosis in yeast; Shinohara M et al.; We show here that deletion of the DNA damage checkpoint genes RAD17 and RAD24 in Saccharomyces cerevisiae delays repair of meiotic double-strand breaks (DSBs) and results in an altered ratio of crossover-to-noncrossover products . These mutations also decrease the colocalization of immunostaining foci of the RecA homologs Rad51 and Dmc1 and cause a delay in the disappearance of Rad51 foci, but not of Dmc1 . These observations imply that RAD17 and RAD24 promote efficient repair of meiotic DSBs by facilitating proper assembly of the meiotic recombination complex containing Rad51 . Consistent with this proposal, extra copies of RAD51 and RAD54 substantially suppress not only the spore inviability of the rad24 mutant, but also the gamma-ray sensitivity of the mutant . Unexpectedly, the entry into meiosis I (metaphase I) is delayed in the checkpoint single mutants compared to wild type . The control of the cell cycle in response to meiotic DSBs is also discussed. J Biol Chem, 2003 Sep 26, 278(39), 37083 - 91 Epub 2003 Jul 17. Human homologues of LAG1 reconstitute Acyl-CoA-dependent ceramide synthesis in yeast; Guillas I et al.; Lag1p and Lac1p are two highly homologous membrane proteins of the endoplasmic reticulum . lag1delta lac1delta double mutants in Saccharomyces cerevisiae lack an acyl-CoA-dependent ceramide synthase and are either very sick or nonviable, depending on the genetic background . LAG1 and LAC1 are members of a large eukaryotic gene family that shares the Lag1 motif, and some members of this family additionally contain a DNA-binding HOX homeodomain . Here we show that several human LAG1 homologues can rescue the viability of lag1delta lac1delta yeast cells and restore acyl-CoA-dependent ceramide and sphingolipid biosynthesis . When tested in a microsomal assay, Lac1p and Lag1p had a strong preference for C26:0-CoA over C24:0-CoA, C20-CoA, and C16-CoA, whereas some human homologues preferred C24:0-CoA and CoA derivatives with shorter fatty acids . This suggests that LAG1 proteins are related to substrate recognition and to the catalytic activity of ceramide synthase enzymes . CLN8, another human LAG1 homologue implicated in ceroid lipofuscinosis, could not restore viability to lag1delta lac1delta yeast mutants. Eur J Biochem, 2003 Aug, 270(15), 3189 - 95 Weak organic acid stress inhibits aromatic amino acid uptake by yeast, causing a strong influence of amino acid auxotrophies on the phenotypes of membrane transporter mutants; Bauer BE et al.; The ability of yeasts to grow in the presence of weak organic acid preservatives is an important cause of food spoilage . Many of the determinants of acetate resistance in Saccharomyces cerevisiae differ from the determinants of resistance to the more lipophilic sorbate and benzoate . Interestingly, we show in this study that hypersensitivity to both acetate and sorbate results when the cells have auxotrophic requirements for aromatic amino acids . In tryptophan biosynthetic pathway mutants, this weak acid hypersensitivity is suppressed by supplementing the medium with high levels of tryptophan or, in the case of sorbate sensitivity, by overexpressing the Tat2p high affinity tryptophan permease . Weak acid stress therefore inhibits uptake of aromatic amino acids from the medium . This allows auxotrophic requirements for these amino acids to strongly influence the resistance phenotypes of mutant strains . This property must be taken into consideration when using these phenotypes to attribute functional assignments to genes . We show that the acetate sensitivity phenotype previously ascribed to yeast mutants lacking the Pdr12p and Azr1p plasma membrane transporters is an artefact arising from the use of trp1 mutant strains . These transporters do not confer resistance to high acetate levels and, in prototrophs, their presence is actually detrimental for this resistance. Eur J Biochem, 2003 Aug, 270(15), 3133 - 45 Subcellular localization of yeast Sec14 homologues and their involvement in regulation of phospholipid turnover; Schnabl M et al.; Sec14p of the yeast Saccharomyces cerevisiae is involved in protein secretion and regulation of lipid synthesis and turnover in vivo, but acts as a phosphatidylinositol-phosphatidylcholine transfer protein in vitro . In this work, the five homologues of Sec14p, Sfh1p-Sfh5p, were subjected to biochemical and cell biological analysis to get a better view of their physiological role . We show that overexpression of SFH2 and SFH4 suppressed the sec14 growth defect in a more and SFH1 in a less efficient way, whereas overexpression of SFH3 and SFH5 did not complement sec14 . Using C-terminal yEGFP fusions, Sfh2p, Sfh4p and Sfh5p are mainly localized to the cytosol and microsomes similar to Sec14p . Sfh1p was detected in the nucleus and Sfh3p in lipid particles and in microsomes . In contrast to Sec14p, which inhibits phospholipase D1 (Pld1p), overproduction of Sfh2p and Sfh4p resulted in the activation of Pld1p-mediated phosphatidylcholine turnover . Interestingly, Sec14p and the two homologues Sfh2p and Sfh4p downregulate phospholipase B1 (Plb1p)-mediated turnover of phosphatidylcholine in vivo . In summary, Sfh2p and Sfh4p are the Sec14p homologues with the most pronounced functional similarity to Sec14p, whereas the other Sfh proteins appear to be functionally less related to Sec14p. Biochem J, 2003 Oct 15, 375(Pt 2), 287 - 95 SCP1 encodes an actin-bundling protein in yeast; Winder SJ et al.; The association of F-actin (filamentous actin) with a large number of binding proteins is essential for cellular function . Actin-binding proteins control the dynamics of actin filaments, nucleate new filaments and facilitate formation of higher-order structures such as actin bundles . The yeast gene SCP1 encodes a small protein with significant homology to mammalian SM22/transgelin . We have investigated the role of Scp1p in budding yeast to probe the fundamental role of this family of proteins . Here, we demonstrate that Scp1p binds to F-actin and induces the formation of tight F-actin bundles in vitro . Deletion of SCP1 in yeast lacking the actin-bundling protein, fimbrin (Sac6p), exacerbates the disrupted actin phenotype and enhances latrunculin-A sensitivity . Furthermore, Scp1p co-localizes with actin in cortical patches and its localization is lost in the presence of latrunculin-A . Our data support a role for Scp1p in bundling actin filaments and, in concert with Sac6p, acting as a second actin-bundling activity crucial to the stability of the yeast actin cytoskeleton. J Electron Microsc (Tokyo), 2003, 52(2), 133 - 43 Quantitative three-dimensional structural analysis of Exophiala dermatitidis yeast cells by freeze-substitution and serial ultrathin sectioning; Biswas SK et al.; The morphologies, numbers, sizes and volumes of all organelles and cell components identified on ultrathin sections of aerobically grown exponential phase yeast cells of Exophiala dermatitidis in G1 phase were examined by freeze-substitution fixation and serial ultrathin sectioning . The cell wall consisted of three layers and occupied approximately 22% of the cell volume . The nucleus was approximately 1.8 microm in diameter and occupied approximately 7% of the cell volume . There was only one nucleolus in the nucleus and it occupied approximately 16% of the nuclear volume . There were 17-52 mitochondria per cell, occupying 7-12% of the cell volume . Five to ten endoplasmic reticula were present per cell; these occupied only 0.2% of the cell volume and did not form a network . There were 1-4 vacuoles per cell and they occupied 4-10% of the cell volume . Storage material was round and electron transparent and occupied 4-11% of the cell volume . The cytosol occupied 43-53% of the cell volume . The Golgi apparatus, spindle pole body, autophagosomes, multivesicular bodies, lipid bodies, microtubules and microfilaments occupied approximately 1% of the cell volume in total . About 200,000 ribosome particles, 1000 glycogen granules and several tens of microtubules (average length 0.78 microm) were present per yeast cell . The membranes of this yeast could be classified into three groups by their appearance and thickness . This is the first report, to our knowledge, that analysed all the components in the yeast cell quantitatively and in three dimensions, and provides fundamental information for understanding various aspects of cell biology. Yeast, 2003 Jul 30, 20(10), 913 - 20 A genetic screen for yeast genes induced by sustained osmotic stress; Runner VM et al.; The budding yeast, Saccharomyces cerevisiae, responds to changes in external osmolarity through the activation of an osmosensing signal transduction pathway . Using lacZ-reporter gene fusions, clonal cell lines were screened for levels of beta-galactosidase activity in the presence or absence of osmotic stress . A screen of 9,000 transformants displayed 663 (7%) gene fusions that were active in rich medium . Each of the transformants were also assayed for gene activity 24 h following a transfer to high osmolarity medium (0.6 M NaCl) and of the 9,000 clonal cell lines, 86 (1%) displayed a decrease in expression, and seven (0.1%) displayed a reproducible increase in gene expression during primary screening . The chromosomal loci of the lacZ insertions were determined, and the gene(s) associated with that site was examined for osmotically induced expression using RNA blot analysis . Five stress-activated genes were analysed by RNA blot: YDL222C, NMD2, PTC7, FAA4 and YRF1 . The genes identified by this screen encompass cellular adaptations to stress including signal transduction, protein myristoylation and fatty acid/sphingolipid content in the cell membrane . Yeast, 2003 Jul 30, 20(10), 893 - 903 A diverse set of nuclear RNAs transfer between nuclei of yeast heterokaryons; Olson BL et al.; Small nuclear RNAs and small nucleolar RNAs function in the nucleus of eukaryotic cells during pre-mRNA splicing and ribosomal RNA processing, respectively . In metazoan cells, the small nuclear RNAs shuttle between the nucleus and the cytoplasm during ribonucleoprotein particle assembly . Nuclear export of these small RNAs in yeast, however, has not been demonstrated . Therefore, we have attempted to visualize internuclear RNA movements by in situ hybridization in heterokaryon yeast cells . Using the kar1Delta15 mutation to block karyogamy, we mated two strains, each expressing a unique allele of U1 snRNA . In these heterokaryons, we observed a time-dependent transfer of U1 snRNA from one nucleus to the other . This transfer was reduced two-fold by the addition of the Crm1p-inhibitor leptomycin B . Interestingly, however, we observed identical transfer of the U2 and U6 snRNAs and SNR4, SNR8, SNR9 and SNR11 snoRNAs . Remarkably, when the U2, U6 or SNR4 RNAs were observed in the same heterokaryon as the U1 snRNA, both RNAs always transferred simultaneously . These data suggest a global leaking or transport of material between nuclei of yeast heterokaryons . Our results suggest that caution must be taken when testing nuclear envelope shuttling in yeast heterokaryons . Biol Cell, 2003 May-Jun, 95(3-4), 157 - 67 Yeast RNA helicases of the DEAD-box family involved in translation initiation; Linder P; RNA helicases of the DEAD-box and related families have been found to be required for all processes involving RNA molecules . Biochemical and genetic analyses have shown that at least two RNA helicases are required for translation initiation in yeast . Although it is generally believed that these enzymes are necessary to unwind secondary structures in the 5' untranslated region of mRNAs, their exact role has not been convincingly shown . We discuss here our present knowledge of the function of eIF4A and Ded1p, two DEAD-box proteins required for translation in eukaryotic cells. Biochem J, 2003 Oct 15, 375(Pt 2), 373 - 83 Isolation and identification of L-dopa decarboxylase as a protein that binds to and enhances transcriptional activity of the androgen receptor using the repressed transactivator yeast two-hybrid system; Wafa LA et al.; The AR (androgen receptor) is a ligand-regulated transcription factor, which belongs to the steroid receptor family and plays an essential role in growth and development of the prostate . Transcriptional activity of steroid receptors is modulated by interaction with co-regulator proteins and yeast two-hybrid analysis is commonly used to identify these steroid receptor-interacting proteins . However, a limitation of conventional two-hybrid systems for detecting AR protein partners has been that they only allow for analysis of the ligand- and DNA-binding domains of the receptor, as its NTD (N-terminal domain) possesses intrinsic transactivation activity . To identify AR N-terminus-interacting proteins, its NTD was used in the RTA (repressed transactivator) system, which is specifically designed for transactivator bait proteins and was shown to be suitable for two-hybrid analysis with the AR NTD . DDC (L-dopa decarboxylase) was detected multiple times as a novel AR-interacting protein, which was subsequently confirmed in vitro and in vivo . Furthermore, transient transfection of DDC in prostate cancer cells strongly enhanced ligand-dependent AR transcriptional activity, an effect that was antagonized using high concentrations of the anti-androgen bicalutamide . Glucocorticoid receptor activity was also strongly enhanced with DDC co-transfection, while oestrogen receptor activity was only mildly affected . Together, our data demonstrate that DDC interacts with AR to enhance steroid receptor transactivation, which may have important implications in prostate cancer progression. Mol Cell Biol, 2003 Aug, 23(15), 5431 - 45 The yeast eukaryotic initiation factor 4G (eIF4G) HEAT domain interacts with eIF1 and eIF5 and is involved in stringent AUG selection; He H et al.; Eukaryotic initiation factor 4G (eIF4G) promotes mRNA recruitment to the ribosome by binding to the mRNA cap- and poly(A) tail-binding proteins eIF4E and Pap1p . eIF4G also binds eIF4A at a distinct HEAT domain composed of five stacks of antiparallel alpha-helices . The role of eIF4G in the later steps of initiation, such as scanning and AUG recognition, has not been defined . Here we show that the entire HEAT domain and flanking residues of Saccharomyces cerevisiae eIF4G2 are required for the optimal interaction with the AUG recognition factors eIF5 and eIF1 . eIF1 binds simultaneously to eIF4G and eIF3c in vitro, as shown previously for the C-terminal domain of eIF5 . In vivo, co-overexpression of eIF1 or eIF5 reverses the genetic suppression of an eIF4G HEAT domain Ts(-) mutation by eIF4A overexpression . In addition, excess eIF1 inhibits growth of a second eIF4G mutant defective in eIF4E binding, which was also reversed by co-overexpression of eIF4A . Interestingly, excess eIF1 carrying the sui1-1 mutation, known to relax the accuracy of start site selection, did not inhibit the growth of the eIF4G mutant, and sui1-1 reduced the interaction between eIF4G and eIF1 in vitro . Moreover, a HEAT domain mutation altering eIF4G moderately enhances translation from a non-AUG codon . These results strongly suggest that the binding of the eIF4G HEAT domain to eIF1 and eIF5 is important for maintaining the integrity of the scanning ribosomal preinitiation complex. Mol Cell Biol, 2003 Aug, 23(15), 5208 - 16 Specificity and regulation of DNA binding by the yeast glucose transporter gene repressor Rgt1; Kim JH et al.; Rgt1 is a glucose-responsive transcription factor that binds to the promoters of several HXT genes encoding glucose transporters in Saccharomyces cerevisiae and regulates their expression in response to glucose . Rgt1 contains a Zn(2)Cys(6) binuclear cluster responsible for DNA binding . Most proteins that contain this sequence motif bind as dimers to regularly spaced pairs of the sequence CGG . However, there are no CGG pairs with regular spacing in promoters of genes regulated by Rgt1, suggesting that Rgt1 binds as a monomer to CGG or to another sequence . We identified the Rgt1 consensus binding site sequence 5'-CGGANNA-3', multiple copies of which are present in all HXT promoters regulated by Rgt1 . Rgt1 binds in vivo to multiple sites in the HXT3 promoter in a nonadditive, synergistic manner, leading to synergistic repression of HXT3 transcription . We show that glucose inhibits the DNA-binding ability of Rgt1, thereby relieving repression of HXT gene expression . This regulation of Rgt1 DNA-binding activity is caused by its glucose-induced phosphorylation: the hyperphosphorylated Rgt1 present in cells growing on high levels of glucose does not bind DNA in vivo or in vitro; dephosphorylation of this form of Rgt1 in vitro restores its DNA-binding ability . Furthermore, an altered Rgt1 that functions as a constitutive repressor remains hypophosphorylated when glucose is added to cells and binds DNA under these conditions . These results suggest that glucose regulates the DNA-binding ability of Rgt1 by inducing its phosphorylation. J Biol Chem, 2003 Oct 3, 278(40), 39104 - 13 Epub 2003 Jul 14. The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting; Katoh K et al.; Alix (ALG-2-interacting protein X) is a 95-kDa protein that interacts with an EF-hand type Ca(2+)-binding protein, ALG-2 (apoptosis-linked gene 2), through its C-terminal proline-rich region . In this study, we searched for proteins that interact with human AlixDeltaC (a truncated form not containing the C-terminal region) by using a yeast two-hybrid screen, and we identified two similar human proteins, CHMP4a and CHMP4b (chromatin-modifying protein; charged multivesicular body protein), as novel binding partners of Alix . The interaction of Alix with CHMP4b was confirmed by a glutathione S-transferase pull-down assay and by co-immunoprecipitation experiments . Fluorescence microscopic analysis revealed that CHMP4b transiently expressed in HeLa cells mainly exhibited a punctate distribution in the perinuclear area and co-localized with co-expressed Alix . The distribution of CHMP4b partly overlapped the distributions of early and late endosomal marker proteins, EEA1 (early endosome antigen 1) and Lamp-1 (lysosomal membrane protein-1), respectively . Transient overexpression of CHMP4b induced the accumulation of ubiquitinated proteins as punctate patterns that were partly overlapped with the distribution of CHMP4b and inhibited the disappearance of endocytosed epidermal growth factor . In contrast, stably expressed CHMP4b in HEK293 cells was observed diffusely in the cytoplasm . Transient overexpression of AlixDeltaC in stably CHMP4b-expressing cells, however, induced formation of vesicle-like structures in which CHMP4b and AlixDeltaC were co-localized . SKD1(E235Q), a dominant negative form of the AAA type ATPase SKD1 that plays critical roles in the endocytic pathway, was co-immunoprecipitated with CHMP4b . Furthermore, CHMP4b co-localized with SKD1(E235Q) as punctate patterns in the perinuclear area, and Alix was induced to exhibit dot-like distributions overlapped with SKD1(E235Q) in HeLa cells . These results suggest that CHMP4b and Alix participate in formation of multivesicular bodies by cooperating with SKD1. FEBS Lett, 2003 Jul 17, 547(1-3), 69 - 74 A defect in the yeast plasma membrane urea transporter Dur3p is complemented by CpNIP1, a Nod26-like protein from zucchini (Cucurbita pepo L.), and by Arabidopsis thaliana delta-TIP or gamma-TIP; Klebl F et al.; Dur3 encodes the yeast plasma membrane urea transporter and Deltadur3 mutants are unable to grow on media containing low concentrations of urea as sole nitrogen source . Complementation of the Deltadur3 mutant line with expression libraries generated from whole Arabidopsis thaliana seedlings or from zucchini (Cucurbita pepo L.) vascular tissue yielded numerous lines that had regained the capacity to grow on low urea as sole nitrogen source . Analysis of several of these yeast lines revealed that the Deltadur3 mutation was complemented either by delta-TIP (TIP=tonoplast intrinsic protein) or gamma-TIP from Arabidopsis or by CpNIP1, a new NOD26-like protein from zucchini . delta-TIP (At3g16240) and gamma-TIP (At2g36830) had previously been characterized as proteins facilitating the transport of water across the tonoplast membrane, and Nod26-like proteins were characterized as glycerol transporters . So far, transport of urea has not been described for any of the proteins described in this paper . Further analyses support this function of TIPs and nodulin 26-like intrinsic proteins in urea transport. J Mol Biol, 2003 Jul 25, 330(5), 917 - 24 Communication between eukaryotic translation initiation factors 1 and 1A on the yeast small ribosomal subunit; Maag D et al.; We have used expressed protein ligation to site-specifically label eukaryotic translation initiation factors (eIFs) 1 and 1A at their C termini with tetramethyl rhodamine . These fluorescent proteins were used in steady-state anisotropy-based binding experiments to measure the dissociation constants of the factors and the yeast small (40S) ribosomal subunit for the first time . These studies demonstrate that both eIF1 and eIF1A are capable of binding to the 40S subunit in the absence of any other initiation factors or mRNA, arguing against previous suggestions that eIF3 is required for recruitment of eIF1 to the small ribosomal subunit . Strikingly, the data also demonstrate that there is approximately ninefold thermodynamic coupling in the binding of the two factors to the 40S subunit . This indicates that eIF1 and eIF1A communicate with one another when bound to the 40S subunit . Communication between these two factors is likely to be important for coordinating their functions during the initiation process . The data presented here provide a foundation on which to build a quantitative understanding of the network of interactions between these essential factors and the rest of the initiation machinery. Shi Yan Sheng Wu Xue Bao, 2003 Apr, 36(2), 130 - 6 {Reconstruction of large genomic segment coding for human immunoglobulin kappa chain by meiotic homologous recombination of yeast artificial chromosome}; Nie ZY et al.; Meiotic homologous recombination between overlapping yeast artificial chromosome (YAC) can be used to reconstruct larger genomic segment in vivo . We have screened CEPH and Olson human genomic YAC libraries and obtained two YAC clones containing the large genomic segments coding for partial human Ig kappa . In order to reconstruct whole gene cluster of human Ig kappa, two yeast cells containing the different YAC described over were mated and induced to undergo meiotic division and sporulation . Analyzing the results of PCR and Southern blotting, we obtained a clone containing a single recombinant YAC with 400 kb in length, which spans almost the entire gene cluster of human Ig kappa, including 32 V kappa, 5 J kappa, constant domain genes . Besides, we have improved and simplified the traditional method of meiotic recombination between overlapping YACs, and the results indicate the feasibility of this method to reconstruct any larger intact genomic segments. Plant Physiol, 2003 Jul, 132(3), 1678 - 87 Mitochondrial phosphatidylserine decarboxylase from higher plants . Functional complementation in yeast, localization in plants, and overexpression in Arabidopsis; Rontein D et al.; Plants are known to synthesize ethanolamine (Etn) moieties by decarboxylation of free serine (Ser), but there is also some evidence for phosphatidyl-Ser (Ptd-Ser) decarboxylation . Database searches identified diverse plant cDNAs and an Arabidopsis gene encoding 50-kD proteins homologous to yeast (Saccharomyces cerevisiae) and mammalian mitochondrial Ptd-Ser decarboxylases (PSDs) . Like the latter, the plant proteins have putative mitochondrial targeting and inner membrane sorting sequences and contain near the C terminus a Glycine-Serine-Threonine motif corresponding to the site of proteolysis and catalytic pyruvoyl residue formation . A truncated tomato (Lycopersicon esculentum) cDNA lacking the targeting sequence and a chimeric construct in which the targeting and sorting sequences were replaced by those from yeast PSD1 both complemented the Etn requirement of a yeast psd1 psd2 mutant, and PSD activity was detected in the mitochondria of the complemented cells . Immunoblot analysis of potato (Solanum tuberosum) mitochondria demonstrated that PSD is located in mitochondrial membranes, and mRNA analysis in Arabidopsis showed that the mitochondrial PSD gene is expressed at low levels throughout the plant . An Arabidopsis knockup mutant grew normally but had 6- to 13-fold more mitochondrial PSD mRNA and 9-fold more mitochondrial PSD activity . Total membrane PSD activity was, however, unchanged in the mutant, showing mitochondrial activity to be a minor part of the total . These results establish that plants can synthesize Etn moieties via a phospholipid pathway and have both mitochondrial and extramitochondrial PSDs . They also indicate that mitochondrial PSD is an important housekeeping enzyme whose expression is strongly regulated at the transcriptional level. Plant Physiol, 2003 Jul, 132(3), 1405 - 14 Arabidopsis UVH6, a homolog of human XPD and yeast RAD3 DNA repair genes, functions in DNA repair and is essential for plant growth; Liu Z et al.; To evaluate the genetic control of stress responses in Arabidopsis, we have analyzed a mutant (uvh6-1) that exhibits increased sensitivity to UV light, a yellow-green leaf coloration, and mild growth defects . We have mapped the uvh6-1 locus to chromosome I and have identified a candidate gene, AtXPD, within the corresponding region . This gene shows sequence similarity to the human (Homo sapiens) XPD and yeast (Saccharomyces cerevisiae) RAD3 genes required for nucleotide excision repair . We propose that UVH6 is equivalent to AtXPD because uvh6-1 mutants carry a mutation in a conserved residue of AtXPD and because transformation of uvh6-1 mutants with wild-type AtXPD DNA suppresses both UV sensitivity and other defective phenotypes . Furthermore, the UVH6/AtXPD protein appears to play a role in repair of UV photoproducts because the uvh6-1 mutant exhibits a moderate defect in the excision of UV photoproducts . This defect is also suppressed by transformation with UVH6/AtXPD DNA . We have further identified a T-DNA insertion in the UVH6/AtXPD gene (uvh6-2) . Plants carrying homozygous insertions were not detected in analyses of progeny from plants heterozygous for the insertion . Thus, homozygous insertions appear to be lethal . We conclude that the UVH6/AtXPD gene is required for UV resistance and is an essential gene in Arabidopsis. Plant Mol Biol, 2003 May, 52(2), 273 - 84 Arabidopsis RecQsim, a plant-specific member of the RecQ helicase family, can suppress the MMS hypersensitivity of the yeast sgs1 mutant; Bagherieh-Najjar MB et al.; The Arabidopsis genome contains seven genes that belong to the RecQ family of ATP-dependent DNA helicases . RecQ members in Saccharomyces cerevisiae (SGS1) and man (WRN, BLM and RecQL4) are involved in DNA recombination, repair and genome stability maintenance, but little is known about the function of their plant counterparts . The Arabidopsis thaliana RecQsim gene is remarkably different from the other RecQ-like genes due to an insertion in its helicase domain . We isolated the AtRecQsim orthologues from rice and rape and established the presence of a similar insertion in their helicase domain, which suggests a plant specific function for the insert . The expression pattern of the AtRecQsim gene was compared with the other Arabidopsis RecQ-like members in different tissues and in response to stress . The transcripts of the AtRecQsim gene were found in all plant organs and its accumulation was higher in roots and seedlings, as compared to the other AtRecQ-like members . In contrast to most AtRecQ-like genes, the examined environmental cues did not have a detectable effect on the accumulation of the AtRecQsim transcripts . The budding yeast sgs1 mutant, which is known to be hypersensitive to the DNA-damaging drug MMS, was transformed with the AtRecQsim cDNA . The AtRecQsim gene suppressed the MMS hypersensitivity phenotype of the sgs1 cells . We propose that the Arabidopsis RecQsim gene, despite its unusual structure, exhibits an evolutionary conserved function. Endocr Res, 2003 May, 29(2), 237 - 55 Mutation of serines 104, 106, and 118 inhibits dimerization of the human estrogen receptor in yeast; Sheeler CQ et al.; Ligand-dependent dimerization and phosphorylation participate in regulating transcriptional activation of the estrogen receptor-alpha (ER) . We investigated the role of serines 104, 106, and 118 located in the activation function-1 (AF-1) domain of ER in ligand-induced receptor dimerization . These serines, previously documented as important sites for transactivation, were mutated to alanine, and yeast genetic systems were used to determine their effect on receptor dimerization and transcriptional activity . The serine to alanine mutants resulted in 50-80% decreased dimerization in response to 17beta-estradiol, while having modest effects on ER-mediated transactivation . We further demonstrated that ER expressed in yeast became hyperphosphorylated in the presence of estradiol, most likely at a site(s) different than the serines under investigation . Ligand-induced phosphorylation was inhibited by U0126 indicating that the ER was phosphorylated via the MAPK pathway . Taken together, these data indicate that serines 104, 106, and 118 are important for ligand-dependent ER dimerization, and that MAP kinase mediated phosphorylation may be important for ER function, in yeast model systems. Appl Microbiol Biotechnol, 2004 Feb, 63(5), 527 - 36 Epub 2003 Jul 11. Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombopoietin in suspension cultures of Chinese hamster ovary cells; Sung YH et al.; To enhance the performance of a serum-free medium (SFM) for human thrombopoietin (hTPO) production in suspension cultures of recombinant Chinese hamster ovary (rCHO) cells, several low-cost hydrolysates such as yeast hydrolysate (YH), soy hydrolysate, wheat gluten hydrolysate and rice hydrolysate were tested as medium additives . Among various hydrolysates tested, the positive effect of YH on hTPO production was most significant . When 5 g l(-1) YH was added to SFM, the maximum hTPO concentration in batch culture was 40.41 microg ml(-1), which is 11.5 times higher than that in SFM without YH supplementation . This enhanced hTPO production in YH-supplemented SFM was obtained by the combined effect of enhanced q(hTPO) (the specific rate of hTPO production) . The supplementation of YH in SFM increased q(hTPO) by 294% and extended culture longevity by >2 days if the culture was terminated at a cell viability of 50% . Furthermore, cell viability throughout the culture using YH-supplemented SFM was higher than that using any other hydrolysate-supplemented SFM tested, thereby minimizing degradation of hTPO susceptible to proteolytic degradation . In addition, YH supplementation did not affect in vivo biological activity of hTPO . Taken together, the results obtained demonstrate the potential of YH as a medium additive for hTPO production in serum-free suspension cultures of rCHO cells. Nature, 2003 Jul 10, 424(6945), 194 - 7 Dosage sensitivity and the evolution of gene families in yeast; Papp B et al.; According to what we term the balance hypothesis, an imbalance in the concentration of the subcomponents of a protein-protein complex can be deleterious . If so, there are two consequences: first, both underexpression and overexpression of protein complex subunits should lower fitness, and second, the accuracy of transcriptional co-regulation of subunits should reflect the deleterious consequences of imbalance . Here we show that all these predictions are upheld in yeast (Saccharomyces cerevisiae) . This supports the hypothesis that dominance is a by-product of physiology and metabolism rather than the result of selection to mask the deleterious effects of mutations . Beyond this, single-gene duplication of protein subunits is expected to be harmful, as this, too, leads to imbalance . As then expected, we find that members of large gene families are rarely involved in complexes . The balance hypothesis therefore provides a single theoretical framework for understanding components both of dominance and of gene family size. Nucleic Acids Res, 2003 Jul 15, 31(14), 4157 - 61 On the number of protein-protein interactions in the yeast proteome; Grigoriev A; Using two different approaches, we estimated that on average there are about five interacting partners per protein in the proteome of the yeast Saccharomyces cerevisiae . In the first approach, we used a novel method to model sampling overlap by a Bernoulli process, compared the results of two independent yeast two-hybrid interaction screens and tested the robustness of the estimate . The most stable estimate of five interactors per protein was obtained when the three most highly connected nodes in the protein interaction network were removed from the analysis (eight interactors per protein if those nodes were kept) . In the second approach, we analysed a published high-confidence subset of putative interaction data obtained from multiple sources, including large-scale two-hybrid screens, complex purifications, synthetic lethals, correlated gene expression, computational predictions and previous annotations . Strikingly, the estimate was again five interactors per protein . These estimates suggest a range of approximately 16,000-26,000 different interaction pairs in the yeast, excluding homotypic interactions . We also discuss the approaches to estimating the rate of homotypic interactions. Nucleic Acids Res, 2003 Jul 15, 31(14), 4071 - 84 CADLIVE for constructing a large-scale biochemical network based on a simulation-directed notation and its application to yeast cell cycle; Kurata H et al.; The further understanding of the mechanisms of gene regulatory networks requires comprehensive tools for both the representation of complicated signal transduction pathways and the in silico identification of genomic signals that govern the regulation of gene expression . Consequently, sophisticated notation must be developed to represent the signal transduction pathways in a form that can be readily processed by both computers and humans . We propose the regulator-reaction equations combined with detailed attributes including the associated cellular component, molecular function, and biological process and present the simulation-directed graphical notation that is derived from modification of Kohn's method . We have developed the software suite, CADLIVE (Computer-Aided Design of LIVing systEms), which features a graphical user interface (GUI) to edit large-scale maps of complicated signal transduction pathways using a conventional XML-based representation . The regulator-reaction equations represent not only mechanistic reactions, but also semantic models containing ambiguous and incomplete processes . In order to demonstrate the feasibility of CADLIVE, we constructed a detailed map of the budding yeast cell cycle, which consists of 184 molecules and 152 reactions, in a really compact space . CADLIVE enables one to look at the whole view of a large-scale map, to integrate postgenomic data into the map, and to computationally simulate the signal transduction pathways, which greatly facilitates exploring novel or unexpected interactions. Nucleic Acids Res, 2003 Jul 15, 31(14), 4006 - 16 Initiation-mediated mRNA decay in yeast affects heat-shock mRNAs, and works through decapping and 5'-to-3' hydrolysis; Heikkinen HL et al.; The degradation of mRNA in the yeast Saccharomyces cerevisiae takes place through several related pathways . In the most general mRNA-decay pathway, that of poly(A)-dependent decay, the normal shortening of the poly(A) tail on an mRNA molecule by deadenylation triggers mRNA decapping by the enzyme Dcp1p, followed by exonucleolytic digestion by Xrn1p . A specialized mRNA-decay pathway, termed nonsense-mediated decay, comes into play for mRNAs that contain an early nonsense codon . This pathway operates through the Upf proteins in addition to Dcp1p and Xrn1p . Previously, we identified a different specialized mRNA-decay pathway, the initiation-mediated decay pathway, and showed that it affects two Hsp70 heat-shock mRNAs under conditions of slowed translation initiation . Here we report that initiation-mediated mRNA decay also works through the Dcp1 and Xrn1 enzymes, and requires ongoing transcription by RNA polymerase II . We show that several other heat-shock mRNAs, including two from the Hsp90 gene family and three more from the Hsp70 gene family, are also subject to initiation-mediated decay, whereas a variety of non-heat-shock mRNAs are not affected. Nucleic Acids Res, 2003 Jul 15, 31(14), 3936 - 45 The role of the yeast cleavage and polyadenylation factor subunit Ydh1p/Cft2p in pre-mRNA 3'-end formation; Kyburz A et al.; Cleavage and polyadenylation factor (CPF) is a multi-protein complex that functions in pre-mRNA 3'-end formation and in the RNA polymerase II (RNAP II) transcription cycle . Ydh1p/Cft2p is an essential component of CPF but its precise role in 3'-end processing remained unclear . We found that mutations in YDH1 inhibited both the cleavage and the polyadenylation steps of the 3'-end formation reaction in vitro . Recently, we demonstrated that an important function of CPF lies in the recognition of poly(A) site sequences and RNA binding analyses suggesting that Ydh1p/Cft2p interacts with the poly(A) site region . Here we show that mutant ydh1 strains are deficient in the recognition of the ACT1 cleavage site in vivo . The C-terminal domain (CTD) of RNAP II plays a major role in coupling 3'-end processing and transcription . We provide evidence that Ydh1p/Cft2p interacts with the CTD of RNAP II, several other subunits of CPF and with Pcf11p, a component of CF IA . We propose that Ydh1p/Cft2p contributes to the formation of important interaction surfaces that mediate the dynamic association of CPF with RNAP II, the recognition of poly(A) site sequences and the assembly of the polyadenylation machinery on the RNA substrate. EMBO J, 2003 Jul 15, 22(14), 3664 - 74 Use1p is a yeast SNARE protein required for retrograde traffic to the ER; Dilcher M et al.; SNAREs on transport vesicles and target membranes are required for vesicle targeting and fusion . Here we describe a novel yeast protein with a typical SNARE motif but with low overall amino acid homologies to other SNAREs . The protein localized to the endoplasmic reticulum (ER) and was therefore named Use1p (unconventional SNARE in the ER) . A temperature-sensitive use1 mutant was generated . use1 mutant cells accumulated the ER forms of carboxypeptidase Y and invertase . More specific assays revealed that use1 mutant cells were defective in retrograde traffic to the ER . This was supported by strong genetic interactions between USE1 and the genes encoding SNAREs in retrograde traffic to the ER . Antibodies directed against Use1p co-immunoprecipitated the SNAREs Ufe1p, myc-Sec20p and Sec22p, which form a SNARE complex required for retrograde traffic from the Golgi to the ER, but neither Bos1p nor Bet1p (members of the SNARE complex in anterograde traffic to the Golgi) . Therefore, we conclude that Use1p is a novel SNARE protein that functions in retrograde traffic from the Golgi to the ER. EMBO J, 2003 Jul 15, 22(14), 3624 - 34 A docking site determining specificity of Pbs2 MAPKK for Ssk2/Ssk22 MAPKKKs in the yeast HOG pathway; Tatebayashi K et al.; Mitogen-activated protein kinase (MAPK) cascades are conserved signaling modules composed of three sequentially activated kinases (MAPKKK, MAPKK and MAPK) . Because individual cells contain multiple MAPK cascades, mechanisms are required to ensure the fidelity of signal transmission . In yeast, external high osmolarity activates the HOG (high osmolarity glycerol) MAPK pathway, which consists of two upstream branches (SHO1 and SLN1) and common downstream elements including the Pbs2 MAPKK and the Hog1 MAPK . The Ssk2/Ssk22 MAPKKKs in the SLN1 branch, when activated, exclusively phosphorylate the Pbs2 MAPKK . We found that this was due to an Ssk2/Ssk22-specific docking site in the Pbs2 N-terminal region . The Pbs2 docking site constitutively bound the Ssk2/Ssk22 kinase domain . Docking site mutations drastically reduced the Pbs2-Ssk2/Ssk22 interaction and hampered Hog1 activation by the SLN1 branch . Fusion of the Pbs2 docking site to a different MAPKK, Ste7, allowed phosphorylation of Ste7 by Ssk2/Ssk22 .